key: cord-023346-8sqbqjm1
authors: nan
title: MONDAY: POSTERS
date: 2005-06-08
journal: Vox Sang
DOI: 10.1111/j.1423-0410.2005.00652.x
sha: 
doc_id: 23346
cord_uid: 8sqbqjm1

nan

From the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (RCTs). In addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. RCTs are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. If randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. RCTs should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. Because the allocation of subjects to the treatment and control arms of an RCT is random, the play of chance should distribute all confounding factors equally between these two arms. Thus, the treatment and control arms of an RCT should be equivalent with respect to all confounding factors except for the treatment under study. Randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. Moreover, if an RCT is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. When the undertaking of RCTs is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small RCTs may allow investigators to address issues of possible biases 2ED-01-01 Standardizing blood components would allow better monitoring of the effectiveness of transfusion D DAVENPORT University of Michigan, Ann Arbor, MI, USA In routine transfusion of red blood cells (RBC) or platelet concentrates (PC), we have only a very rough idea of the dose we administer. The minimum expected hemoglobin content of RBC should be 50 g (FDA criteria). However, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. The effect of storage senescence can magnify these differences. The resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. Apheresis collection of RBC can help standardized unit contents, but this technology is relatively expensive and time consuming. Knowledge of actual hemoglobin content can be used to optimize red cell transfusion. One trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dL. For PC transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. Without knowledge of the actual content of PC, determination of refractoriness and response to matched PC is problematic. Standardization and labeling of RBC and PC units for content will permit accurate dosage and quantification of transfusion practice.

Are transfusion guidelines evidence-based? JP AUBUCHON Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA Application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. Unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. However, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. In the intensive care setting, transfusing red cells at a trigger point of 7 g/dL rather than 10 g/dL not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. Subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. Following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. An observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. Most would agree that transfusion is necessary below a Hb of 7 g/dL and unlikely to be necessary at a Hb above 10 g/dL, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. Sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from RCTs. Meta-analysis is the structured and systematic integration of information from different studies of a given problem. When the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. When the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. This measure is referred to as the 'summary' effect of the treatment under study. Meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance.

2ED-02-04 Biology of stem cell mobilization TH PAPAYANNOPOULOU University of Washington, Seattle, WA, USA At baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (BM), whereas fully mature cells emigrate to peripheral circulation. A small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. However, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. The introduction of G-CSF as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. From these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within BM post mobilization, and less so by studying cells mobilized in blood. Several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. A prominent role of the SDF-1/CXCR4 pathway has been emphasized, either by down regulation of CXCR4, changes in SDF-1 gradients, or by disruption of SDF-1/CXCR4 signaling. Whether this pathway is disrupted by a proteolytic mechanism prevailing within BM post G-CSF or by other protease-independent mechanisms has not yet been settled. In addition to SDF-1/CXCR4, disruption of other pathways responsible for the retention of primitive cells in BM can lead to mobilization. For example, disengagement of the VLA4/VCAM-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. Mobilization is also seen following the use of other cytokines (i.e. kit-L, Flt-3 L, IL-3) or chemokines (i.e. IL-8, Gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. Finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with G-CSF, either by adding another cytokine (i.e. Growth Hormone), agonistic SDF-1 molecules (i.e. CTCE0021), or CXCR4 antagonists (i.e. AMD3100) which have yielded synergistic increments, and these will be discussed. A full understanding of the principles of mobilization, as well as the BM homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future.

The possibility of deriving all kinds of mature cell types from embryonic stem (ES) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available HLA types for an ever growing population in demand. One possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (HSC) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. However cells with the repopulating ability of HSCs are still elusive for tissues such as muscle, heart, CNS and pancreas. It was therefore exciting news when it was shown that HSCs could repopulate other non-haematopoietic tissues arguing for a general role of BMderived cells in tissue regeneration. The term Plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. This in turn implied that a certain degree of developmental plasticity was still available in the adult HSC, or their derived progeny, that allowed them to present with novel phenotypes. How exactly this was accomplished was a matter of speculation. In order to address the mechanism we used an animal model of liver failure where BMT with normal (wild type, wt) HSC was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the BM compartment. By studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant DNA was present in the nodules, a finding that was consistent with BM-derived cells fusing with host hepatocytes. The mechanism of plasticity was therefore directly related to the exposure of the donor BM-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. This fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to Purkinje cells in the cerebellum and to cardiomyocytes. However, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. What these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. If this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate HSCs to diverse lineages opening the way for realistic cell replacement therapies.

Collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported Blood Programs. The development, maintenance, and ultimate success of such Programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. As with all endeavors that directly affect human life, safety is a central, unifying theme of this process. Because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of Risk Management (RM). RM involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. Specific approaches to RM and quality assurance in transfusion medicine will be presented. Examples from Blood Programs currently in effect in selected countries will be discussed. RM efforts within the corresponding blood program in Greece will then be examined. The lack of data to adequately assess the status of this program suggests that at least one of the elements of RM -monitoring -can still be improved. A preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the Greek population. The findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective RM program in Greece.

2ED-03-08 The use of haemovigilance data to increase safety in transfusion medicine

Haemovigilance is a surveillance of all the procedures in the transfusion chain. The intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. In Europe haemovigilance was introduced as a concept in the middle of the nineties. The first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. Later on other kind of events like 'near miss' events were added. Nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. The state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. The aim of the different national haemovigilance systems has been to improve safety in the transfusion line. After the first 10 years with haemovigilance in the European countries, what has been the outcome of this big effort? Data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. However, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. Systems which can prevent most of the failures are on the market. The cost of these equals the cost of NAT screening for virus introduced in the same period of time. The common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. Therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks.

The role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding G DESPOTIS Washington University School of Medicine, St Louis, MO, USA Excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. Several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. Transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. In addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. This highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. Patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vWD, Hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. In specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). Patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. CPB-related abnormalities). In addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. Plavix, Reopro, low molecular weight heparin etc) are at increased risk for bleeding. Clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. On this basis, the use of point-of-care (POC) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. Five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. DDAVP has bee shown to be beneficial with uremia-induced platelet dysfunction and with Type I von Willbrand's disease. Although previous studies have not been able to conclusively show that DDAVP can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. Recombinant activated factor VII (rFVIIa) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. Although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rFVIIa as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with DIC or after cardiac surgery). Therefore, large clinical trials evaluating the efficacy and safety of rFVIIa are needed before any widespread use can be recommended.

In recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. They have proved especially valuable for the determination of fetal blood groups. The ability to detect RHD in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. Furthermore, the occurrence of fetal DNA in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (Lo YMD. (1999) Ann Med 31:308-312). Determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. By correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (Daniels GL (2002) Human Blood Groups, 2nd ed. Blackwell Science). The polymorphic antigens of most systems other than ABO and Rh, are defined by single nucleotide substitutions (SNPs) which effect a single amino acid sequence change in the gene product. Numerous methods, both manual and automated, are available to determine SNPs and these have been applied to the determination of blood groups. Useful applications of SNPs detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (Rozman P, Dove T, Gassner C et al. (2000) Transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (Castilho L, Rios M, Bianco C et al. (2002) Transfusion 42:232-238). Other applications of molecular methods include zygosity determination for RHD, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. The molecular bases of ABO and Rh antigens are complex and cannot be determined reliably by a single SNP. Nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to ABO and Rh typing. Whether or not this will ever be a cost-effective procedure is a moot point. It is clear that molecular methods are a useful addition to the range of diagnostic procedures available to Transfusion Medicine practitioners but it is important to remember that methods based on DNA analysis determine phenotype by inference and not by direct measurement. The results of molecular tests should be interpreted with this caveat in mind.

2ED-06-18

The HLA system G STAVROPOULOS General Hospital 'G. Gennimatas', Athens, Greece Since its discovery in the mouse in 1936 the major histocompatibility complex (MHC) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. MHC primary function is to provide protection against pathogens. This is achieved through sophisticated pathways in which MHC class I molecules present endogenous antiges to CD8+ T cells and class II molecules present exogenous antigens to CD4+ T cells. An increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class III complement proteins, also map to the MHC. The MHC molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. Nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of MHC compatibility between the donor and patient. Thus, for patients who lack matched donors, the rules that govern permissibility of MHC mismatching still need to be identified. For patients with high risk disease who lack matched donors, use of donors with a single MHC mismatch may permit early treatment before disease progression.

Scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. Despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. This evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). This policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. Several national and international organizations endorse the above policy. Although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in J Clin Oncol 2001;19:1519-1538. Additional data on lumbar puncture are reported in Ann Hematol 2003;82:570-573. Another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. With regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in Europe and current object of increased interest in Canada, as compared to the platelet-rich plasma method traditionally used in the US, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. In addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. As far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of CMV transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. Finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients.

2ED-07-20 FFP: appraisal of the evidence for the clinical use of FFP

Although the indications for transfusion of plasma (Fresh Frozen Plasma; FFP) are limited, the use of FFP continues to rise in the United Kingdom and has risen by over 20% in the past few years. Local UK audits continue to document that a significant proportion of FFP transfusions are not consistent with indications reported in guidelines. A systematic review of the evidence base for the effectiveness of FFP was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of FFP. In the systematic review of FFP, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study;

• allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include FFP or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented.

The main analysis was qualitative, and differentiated between:

1. studies of interventions comparing FFP with no FFP; 2. studies of interventions comparing FFP with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing FFP with a different blood product; 4. studies comparing different formulations of FFP, e.g. solvent detergent and methodine blue treated.

An evaluation of studies comparing FFP with no FFP would be expected to provide the clearest direct evidence for a positive effect of FFP. Studies comparing FFP with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. Studies comparing different formulations of FFP do not directly evaluate effectiveness of FFP but were included because there is now a UK national policy to use these products in younger patients and therefore these trials might identify negative outcomes. The search strategy identified a total 57 publications. Although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. Few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). The sample size of many included studies was very small (range 8-78 patients per arm). Few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with FFP. Many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. However, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic FFP, irrespective of clinical setting. There is a pressing need to develop new trials to determine the effectiveness of FFP, including for those clinical situations in which it has become an accepted part of current transfusion practice.

A law decided upon by the Riksdag (the Swedish parliament) often sets the general standards as a framework and authorises the Central Government Authority concerned to elaborate and decide upon the more detailed regulations. Laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. Guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. Making a law is complicated and time-consuming. The ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the Council on Legislation for consideration before it is submitted to the Riksdag. A parliamentary committee deals with the bill before it is put to the chamber of the Riksdag for approval. When adopted, the bill becomes law. Regulations elaborated by Central Government Authorities are handled in a principally similar way. However, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. The Ministry of Health and Social Affairs is responsible for elaborating the bill to be submitted to the Riksdag on the Blood Safety Law. Two Central Government Authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments:

• the National Board of Health and Welfare (NBHW), responsible for requirements related to blood components intended for transfusion, and • the Medical Product Agency (MPA), responsible for requirements related to blood components intended for the manufacture of medicinal products.

Sweden became a member of the EU ten years ago. Since then, experts in transfusion medicine have been appointed by the Ministry, NBHW and MPA for advice on scientific and technical issues and for representing Sweden at expert meetings arranged by the Commission. Some of these experts are also members of the Handbook Committee of the National Society for Transfusion Medicine, preparing and revising the national guidelines. Consequently, the requirements of the Directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new Blood Safety Law and the revised NBHW and MPA regulations can be promulgated. To a great extent, requirements of the blood directives are covered by existing Swedish legislation. No major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. A few detailed requirements have been found difficult to follow precisely in the routine work. These minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. The landscape for blood collection and distribution includes availability and safety. True international availability of blood has not been reached. The landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. The reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. Mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. Increasing demands on safety, availability and economy force the health care providers to reorganise blood services. National systems are winning ground. This may make it easier to achieve international collaboration. The Council of Europe has pioneered in creation of international recommendations for blood collection and distribution. In 1958 a European agreement on the exchange of therapeutic substances of human origin, including human blood, was published. Its purpose was to make blood available to other parties of the agreement in case of urgent need. Many countries ratified the agreement rapidly, some did it first in the nineties. In practice little exchange of blood components has taken place. The Council of Europe recommendations lack legal power. The European Union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the Community (Commission Communication 1994) . The agenda is based on an agreement reached at a high level EU meeting in Adare, Ireland in 1994. First in the Commission agenda was a recommendation on donor selection criteria, given in 1998. Then came the European blood directive 2002/98/EC, the aim of which is to improve the safety of blood and blood components within the Union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. Being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in Europe. Hopefully the European Commission has the resources to follow its implementation. There are concerns, however. The epidemiological differences among the EU member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. Blood collection and preparation of components are already the same in EU member countries and the directive helps in their further standardisation. There has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the US from Switzerland and the Netherlands. The European legislation opens new horizons and widens the European landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the EU Commission blood agenda.

The EU directive implemented into the legal act for polish blood transfusion service

Blood Transfusion Service (BTS) in Poland is an integral part of the public Polish health service. In the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. Blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. The legal basis for the activity of Polish BTS is Polish Blood Transfusion Act of 22nd August 1997 which came into force as of January 1st 1999. This Act was then updated in November 2003 according to EU Directive 2002/98/EC and came into force as of January 13th 2004. This Act introduced the principles for organization, collection, processing, storage, transport and quality assurance in BTS. It specified -among others -the system of accreditation, haemovigilence, as well as requirements for BTS employees. According to this act the Polish BTS is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. Polish BTS consists of 21 Regional Blood Transfusion Centers (RBTC), one Military Center and one Ministry of Internal Affairs and Administration Center as well as the Institute of Hematology and Blood Transfusion (IHBT) which acts as supervisor. The 21 RBTCs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by IHBT. They have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. The Polish Blood Transfusion Act of 22nd August 1997, in force since January 1st 1999, has been supplemented by 8 Decrees:

1. procedures for external BTS audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for BTS personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a National Committee for Blood and Blood Transfusion.

Introduction: Several technical aspects must be considered in pediatric apheresis due to the size of the patient. Factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. Adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. Aim of the study: In this study we show our experience using Fresenius Hemocare COM.TEC for PBSC collection in children.

Methods: Twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the P1Y kit of the Fresenius Hemocare COM.TEC blood cell separator. Our CD34+ cells target was 10 ¥ 10 e 6 /kg. Collections were started if a peak of at least 0.02 10e9/l CD34+ cells (20 per microlitre) were reached in the peripheral blood. In all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. ACD ratio 1 : 14-1 : 16 was combined with heparin 40 U/kg. In 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. Results: Twenty-five procedures were performed. A median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). The median product weight was 108 g (range 37-258 g) and yield of CD34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). Three poor mobilizing patients (peripheral blood CD34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). All collection procedures were well tolerated. Children never required sedation to perform the leukapheresis. Only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. No circulatory side effects were observed. Blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. Conclusion: In summary, leukapheresis in children can be safely and effectively performed with the Fresenius Hemocare COM.TEC separator with minimal technical difficulties even in patients under 15 kg.

M-PA-007 Alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. However, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor VIII, protein S and other labile proteins. For methods involving pooling, there are also concerns about the risks due to the pool sizes. The major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and TRALI must be evaluated. The study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (QALY) are used as the effectiveness endpoint. Qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. A complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. In literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. Concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. However, the conclusions of different authors have been conflicting. In 1994, AuBuchon and Birkmeyer published a paper (JAMA 1994; 272(15) :1210-1214) where they concluded that the cost was USD 340 000 per QALY, which is far above the 'acceptable limit' of USD 50 000. This estimate was adjusted to USD 9.7 mill. per QALY in a 1999 letter to JAMA (Jackson, JAMA 282). In 2003 , Riedler et al. published (Vox Sang 2003 85:88-95 ) that the discounted cost/life year saved for SD-FFP use in the UK was GBP 22 278 for neonates and GBP 98 465 for patients aged 70. The main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. The papers cited above underline the difficulties of the cost-benefit considerations. The age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. In addition, how much are we willing to pay for protection against emerging viruses? This paper will not provide the answers, but Introduction: The photodynamic treatment of therapeutic plasma using methylene blue (MB) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. Aim of the study: Evaluation of the quality and stability of MB/light-treated plasma (MB plasma) prepared under worst case conditions for routine processing. Methods: Single donor units (n = 12) were treated using the Macopharma Theraflex MB-Plasma system which includes plasma membrane filtration (PLAS4) and addition of MB prior to illumination followed by MB and photoproduct filtration (Blueflex). Samples were taken before treatment and from the final product. Additionally MB-treated plasma was prepared from four different plasma pools and stored for up to 9 months. Treatment was done under worst case conditions for the preservation of coagulation factors: Maximum MB concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°C, 17 h), maximum storage time of MB plasma before freezing (1 h).

Several plasma parameters and the concentration of MB and its photoproducts (azure A, azure B, azure C and thionine) were determined. Results: MB/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (Clauss) (-20 .4%), factor V (-15.6%), factor VIII (-22.3%), factor XI (-13.3%) and protein C (-10.3%). No significant changes were detected for AT III, VWF : RCo, VWF cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. The entire virus inactivation procedure including the filtration steps for leukocyte depletion and MB and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, D-dimers). No further essential loss of coagulation factor activity was observed during storage of the plasma at 30°C for 9 months. MB and its photoproducts (azure A, azure B, azure C) were depleted to a final concentration of <0.1 mmol/l. Thionine was undetectable in all samples. Conclusion: Photodynamic treatment of Fresh Frozen Plasma (FFP) using the Theraflex MB-Plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. MB and its photoproducts were effectively removed from the plasma by the Blue-Flex-filter integrated in the Theraflex-System. The quality of MB plasma is well preserved during storage. Pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. TRALI is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. Other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. All blood products, except for albumin and solvent/detergent plasma, have been associated with TRALI, but Red Blood Cells, platelets and FFP are the most common. The incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure.

In 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. Death occurs in 6-23%. The profile of the at-risk recipient has not been identified. Recurrent cases have been infrequently described. There are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. Evidence supports both concepts and neither is mutually exclusive. More than 60% of reported cases are associated with blood components containing either HLA-specific (Class I or II) or HNAspecific antibodies. In 50% these antibodies correspond to at least one epitope in the recipient. Most implicated components are donated by multiparous women. Five percent of patients have HLA or HNA antibodies in their pre-transfusion serum. Treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. Because TRALI has a much better prognosis than ARDS, it is an important diagnosis. Some blood collectors are diverting plasma from multiparous donors away from FFP production or routinely screening for HLA antibodies. The most effective method for identifying 'high risk' components has not been identified. Introduction: TRALI is a life threatening adverse reaction of blood transfusion. It is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. In Japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including TRALI cases since 1997. Since the diagnostic criteria of TRALI have not been established until recently and no specific diagnostic markers for TRALI have been discovered so far, it is very difficult to make proper diagnosis of TRALI in each reported case. We have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of TRALI. We reevaluated each report whether it meet the recommended diagnostic criteria for TRALI published in Transfusion journal in Dec. 2004 . Aim of the study: Purpose of this study is to select TRALI cases which met internationally recognized criteria so that it will reveal the possible causes of TRALI with laboratory testing for anti-leukocyte antibodies in donors and recipients. Methods: The cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to Japanese Red Cross. In order to make a proper diagnosis of TRALI, we have been utilizing the respiratory distress questionnaire which has recently revised. This helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized TRALI cases properly. For laboratory testing, FlowPRA and LabScreen for HLA antibodies and GIFT-FCM for HNA antibodies are performed. The cross-matching test is also performed if possible. Results: During past 8 years, 95 cases of TRALI and 29 cases of possible TRALI have been confirmed by critical review of each questionnaire. Of 95 cases of definite TRALI, donor specimens were obtained in 84 cases. Of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). Of 43 cases of antibody positive donors, anti HLA antibodies were detected in 33 cases, anti HNA antibodies were detected in 18 cases, and both were detected in 8 cases. Of 33 cases of positive anti HLA antibodies, class I antibodies were detected in 22 cases, class II antibodies were detected in 28 cases, and both were detected in 17 cases. On the other hand, the anti-leukocyte antibodies were detected in 38% of TRALI recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%).

These results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing TRALI from the antibody-hypothesis-oriented point of view. Other cases with no detectable antibodies should be investigated in more detail in the future. Thus, reevaluating TRALI cases based on recommended TRALI criteria will allow us to reveal new information about TRALI.

Of 2087 adverse events analysed by the Serious Hazards of Transfusion (SHOT) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (HTRs). 303 were due to incorrect blood component transfused (IBCT): 226/303 were ABO incompatible and 77/303 caused by other red cell antibodies. A further 40 cases were reported as acute HTRs (AHTRs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed HTRs (DHTR). 21/556 (4%) patients died and 58 suffered major morbidity. HTRs associated with IBCT result from clinical or laboratory errors and are all preventable. It has been assumed that other HTRs are unavoidable. Closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 AHTRs occurred in group A (8/9) or group B (1/9) patients given group O platelets. Of the 31 AHTRs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. At least 24/213 DHTRs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. Two patient deaths related to DHTR might have been avoided by earlier diagnosis and clinical involvement. 14% HTRs reported to SHOT would have been avoided by compliance with pretransfusion testing guidelines and provision of group A platelets for all group A recipients. HTRs can be clinically overlooked and inadequately investigated. National guidelines are needed for the investigation and management of HTR, with focus on the identification of underlying causes to guide the choice of future component therapy. Reference laboratories can provide valuable support in elucidating complex serological problems.

Patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. We analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. Aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. In 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. We followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. No significant difference in survival was demonstrated for our two patient groups.

Background: The use of adequate number of peripheral blood stem cells (PBSC), collected by MNC apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. Aims: The goals of this study were: (a) to obtain an effective apheretic protocol for MNC harvesting, and (b) to compare the hematopoietic reconstitution after HSPC transplantations in different clinical settings. Methods: In this study, PBSC transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (ALL, ANLL, CML), multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. PBSC mobilization was achieved with rhG-CSF (5-16 g/kgbm/day). MNC-apheresis procedures (generally one and occasionally two) were performed using blood cell separator COBE-Spectra. The first MNC-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10E9/L (autologous setting) or on the 5th day 4-5 hour after the last rhG-CSF administration (allogeneic setting). The processed blood volume during one MNC-apheresis was 12. 7-22.2 L, and 24 .3 L for one PBSC transplantation in average. Results: Using a minimal target dose of CD34+ cell count (3 ¥ 10E6/kgbm), performing one MNC-apheresis procedure for 86% recipients sufficient number of PBSCs were obtained. The MNC yield was 10.1 ¥ 10E8/kgbm in allogeneic and 8.1 ¥ 10E8/kgbm in autologous setting in average. The mean CD34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10E6/kgbm and 6.5 ¥ 10E6/kgbm, respectively. Hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when PBSC transplantation was applied. Summary/conclusion: Improved MNC and CD34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) The intention of auditing any clinical practice is, ostensibly, to improve patient care. The term 'audit' implies comparison against a standard. Although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. Assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. There are several common ways of performing transfusion audits. Retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. Furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. To speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. This appears to be at least as effective as the traditional retrospective audit system. Prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. However, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. Both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. Providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. Extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (Studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). Increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. The public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. Carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program.

Informed consent: The term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. Numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. Initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. Current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . What is however the meaning of informed consent? Is it a mutual decision making between physician and patient? In 'Principles of Biomedical Ethics' Beauchamp and Childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. The elements of consent include: disclosure, voluntariness, decision and authorization. When applying the concept of informed consent in Transfusion Medicine one can distinguish it into Donors' and Patients' consent. Donor informed consent: Information to blood donors constitutes a sensitive issue. It refers to their protection from side-effects of the donation, as well as to protection of the recipient. With regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. For repeat donors one needs mainly an updated history. Because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. Donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. With first time apheresis donors more time is needed in order to explain the procedure and potential side effects. Since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. The fact that all donors sign the informed consent does not mean that they are all adequately informed! Interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information.

Misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. In medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). These events happen also in transfusion medicine and focus on safety is not unique. Haemovigilance which is defined in the EU Blood Directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (EU Directive 2002/98/EC Off. J. European Union.8.2.2003:L33/30-L33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. The causes or reasons should be studied in order to prevent re-occurrence. Adverse event reporting in blood transfusion and transfusion medicine is complex. It depends on the cooperation between blood establishments with clinicians and hospitals. It implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. An adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. In almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. Blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. The products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. In case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of FFP prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. Haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. It should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. It is quite worrisome that underreporting is a general problem in medical care. It might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. Although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. Many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. It seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. For problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. Attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). Lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. It should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. Setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. It will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. The confidentiality of the information should be guarded sufficiently. For a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. For blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way.

M-PA-032 8 years of SHOT data 1996-2004: a view of transfusion safety in the UK and reactions. This will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. Transfusion-transmitted infections and serious immunological reactions are rare; SHOT has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. From the inception of SHOT it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. Only a minority of these events results in patient harm and is reportable under the terms of the Directive. Haemovigilance schemes such as SHOT, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the UK between 1996 and 2003 . Analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . Clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. Strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. Onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in SHOT in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. However, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. If haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. Barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. Transfusion Practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active Hospital Transfusion Committee. *Provisional.

M-PA-033 Passing the borders: when, how and where D PIRC-TILJAK Croatian Institute of Transfusion Med., Zagreb, Croatia

There may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. In order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. Once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . Methods/results: Reality checking, personal experiences and observations studying the path of serious error report. Although the QC system functions, yet omissions happen . . . The possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . How strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. There is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. Who will hear it? Transfusion transmitted infections (TTI) are a major source of concern given the repercussions of HIV, hepatitis C, bacteria and vCJD transmission by blood components. Large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. Identifying emerging infections of concern is a major activity for many Transfusion Services.

Of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for TTI requires, amongst other things, that:

• The agent is identifiable.

• It is present in blood • It causes a disease of concern.

• It is transmitted by transfusion.

• It is present at relatively low frequency.

• If a test is available (NAT, serology or immunoassay) what the infection window period is.

Once an agent has been identified various approaches are possible, including:

• donor selection by testing, geography or lifestyle e.g. WNV;

• product selection e.g. erythrovirus (B19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. CMV matching, immune status.

Against this background where should our attentions focus? Some agents of initial concern are now known to be ubiquitous and have minimal disease association (TTV, GBV) although transmitted by transfusion. For others (coronavirus -SARS or the possible Kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vCJD, HIV).

• enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for HIV, and recently, for HCV); • awareness of HBsAg vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or Chagas' disease infection (for retrieval of otherwise wasted blood); • European Union's in vitro diagnostics directive: this has caused some problems and reduced flexibility.

Nucleic acid testing (NAT): NAT continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. Trends include:

• reduction in sample pool size;

• increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by NAT (varies in different countries); • introduction of rapid and flexible NAT to detect West Nile Virus, in North America.

Bacterial screening of platelet preparations: Several countries have introduced (or will introduce) routine screening of platelet concentrates either with Biomerieux, BacTAlert or PALL eBDS (02 depletion assessment). Other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing.

M-PA-036 Evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions E NELSON*, H TAYLOR † , P WHITLEY † and T LIEU* *Pall Medical, Covina, CA, † American Red Cross And EVMS, Norfolk, VA, USA Background: A filter, called the Leukotrap Affinity Prion Reduction Filter (PRF1B filter, Pall Medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant Creuztfeldt-Jakob Disease (vCJD), from leukocyte-reduced red cell products. Aim: The objective of this study was to evaluate the quality of leukocyte-reduced red cells (LR-RBC) processed through this filter and stored for 42 days. Red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. Storage hemolysis and ATP were also determined. Methods: Units of blood (450 ml) were collected from normal volunteers into the Leukotrap WB System containing CP2D/AS-3 anticoagulant/preservative solutions (Pall Medical). Units were either processed to LR-RBC within 8 hours at room temperature (RT units), or after 24 hours at 1-6°C (cold units). The prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. Samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and ATP determinations. Post-storage samples were taken for labeling with 51-Cr radioisotope, re-infusion, and determination of the 24-hour in vivo RBC recovery. A donor sample was also labeled with 99m-Tc to allow for red cell mass determination. Thus, both single-and double-label 24-hour recovery values were calculated. Results: Twelve units were collected. In vitro testing was completed on all units. In vivo testing was completed on 11 units. The mean single-label 24-hour recoveries were 84.4% and 85.7% for the RT and cold units, respectively. The mean double-label recoveries were 81.9% and 84.1% for the RT and cold units, respectively. The overall combined mean in vivo and in vitro results are shown in the Table. Conclusion: The 24-hour in vivo red cell recovery means are well above the FDA and Council of Europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed Leukotrap RC System with CP2D/AS-3 (Pall Medical Introduction: To reduce the risk of platelet transfusion-associated sepsis (TAS), methods to routinely screen for bacterial contamination have been implemented. Pathogen inactivation treatment of labile blood components provides an alternative means to prevent TAS.

The INTERCEPT Blood System for platelets (Baxter Healthcare) has received the CE Mark and has been introduced into clinical practice. Aims: This study compared the efficacy of bacterial screening using a culture method (BacT/Alert System, Biomerieux) with pathogen inactivation (INTERCEPT Blood System) for prevention of transfusion of platelet components contaminated with bacteria. Methods: Seven strains of bacteria associated with TAS, including Gram-positive Staphylococcus epidermidis, Streptococcus agalactiae, and Staphylococcus aureus, Gram-negative Escherichia coli, and Klebsiella pneumoniae, and the anaerobes Propionibacterium acnes and Clostridium perfringens were studied. For each strain, three double-dose platelet concentrates (~6 ¥ 10E 11 platelets in 600 mL of 35% plasma and 65% InterSol) were collected using the Amicus® Cell Separator. On day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. Each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. The Control platelet concentrate was not treated. The Test platelet concentrate was treated with the INTERCEPT process (150 mM amotosalen + 3 J/sq cm UVA). Both units were cultured using the BacT/Alert System at the time of bacterial inoculation and on days 1, 2 and 5 of storage. Samples (10 mL) were taken for both the aerobic and anaerobic cultures. A platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. Results: For Control platelet concentrates, cultures failed to detect low-dose inocula. The time to positive culture varied with the bacterial strain, contamination level, and time of sampling. At 10 and 100 cfu per product, 4 strains (S. epidermidis, E. coli, C. perfringens, S. agalactiae) and 2 strains (E. coli, C. perfringens) tested negative after 5 days of platelet storage, respectively. K. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. At 1000 cfu per product, P. acnes tested negative in aerobic culture and C perfringens tested negative in anaerobic culture after 5 days of platelet storage. The anaerobic cultures of P. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. Of the 7 strains studied, only S. aureus consistently tested positive after 7-16 hours of culture. In contrast, all Test platelet concentrates treated with INTERCEPT remained negative by BacT/Alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. Conclusions: Bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. Failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. In contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. Background: Since NAT implementation for HIV-1 and HCV RNA in France, the residual risk (RR) of transfusion-transmitted infections (TTI) has dramatically decreased. The RR estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before NAT implementation to 1/3 150 000 and 1/10 000 000 after NAT implementation for HIV and HCV respectively. As for HBV, the serological screening is only based on both HBsAg and anti-HBc assays. For the same period, the RR estimate for HBV is 1/640 000, five times higher than HIV one and 16 times higher than HCV one. Aims: As the overall RR of TTI is mainly related to HBV, and given the availability of HBV NAT assays, a study was conducted to determine whether HBV NAT has the ability to further reduce the HBV RR and then should be implemented in blood donor screening in France.

We have estimated the WP reduction by NAT in comparison with one of the most sensitive HBsAg screening assays, on 10 commercial seroconversion panels (Bioclinical Partners, Franklin, MA, USA). The NAT test was the Procleix Ultrio assay (Genprobe/Chiron, San Diego, USA). The HBs Ag test was the Prism HBsAg (Abbott, France). The comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. Then, we have calculated the yield of HBV-infected donations detected by NAT relative to Prism HBsAg assay. Results: On the basis of a window period (WP) of 56 days, Ultrio Assay is projected to close the WP by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. The projected yield calculated on the basis of 2.4 million donations collected per year in France, would be 0.65 unit per Year for minipool-NAT and 2 to 3 units per year for individual donation NAT. Conclusion: Introduction of minipool-NAT will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both HBsAg and anti-HBc assays. HBV minipool-NAT is then unsuitable for HBV screening in French blood donors. Single-sample NAT or minipool-NAT with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. Automation when technologically and practically feasible is a prerequisite for single-donation NAT. Therefore, decision has been made not to implement HBV NAT in the French transfusion network until fully automated systems will be available. However, as the prevalence of HBV infections is higher in the overseas territories than in continental France, and as NAT is performed on individual donations in these sites, HBV-NAT has been implemented since December 2004 in these territories.

Combined detection of hepatitis C virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. Significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and CAT, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. Testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. To demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual WBCs (rWBC) following leukoreduction of blood components. There were no significant differences in accuracy and precision when rWBC in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the LeucoCOUNT method performed manually. Given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform.

Noninvasive prenatal genotyping on cell free fetal DNA in maternal plasma CE VAN DER SCHOOT Sanquin Research, Amsterdam, Netherlands In 1997 Lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal DNA are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. Most likely this DNA is derived fom apoptotic syncitiorophoblasts. The human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. The cell free DNA is very rapidly cleared from the circulation, the T1/2 being only 15 minutes. In 1998 we have shown that this cell free fetal DNA could be used for RHD genotyping. In the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, Duchenne's disease, adrenogenital syndrome etc. on this source of DNA. Importantly, no false positive result have been described due to the presence of fetal DNA from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. At present prenatal RHD genotyping has been introduced in routine diagnostics in the United Kingdom, France and the Netherlands. In large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal RHD genotyping is over 99%, and it is to be expected that in the Netherlands this screening will soon be introduced to restrict the antenatal anti-D immunoprophylaxis to women carrying RhD-positive fetuses. In a large European project (SAFE, co-ordinator Maj Hulten, Warwick UK) many researchers collaborate to further explore the possibilities of cell free fetal DNA for future diagnostics. Standard operating procedures for the isolation of plasma DNA have been established. Control PCRs for the presence of fetal DNA have been developed. Recent findings on differences in methylation status of placental genes in fetal DNA opens new possibilities. The main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal DNA, only 1-5% of the cell free DNA in plasma is from fetal origin. This makes diagnostic assays on numerical chromosomal abnormalities impossible. And also for many single nucleotide polymorphisms (SNPs) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal DNA. Our own preliminary results indicate that this latter problem can be solved by PNA clamping. The addition of a PNA probe specific for the k-allele Partial D feature D antigen alteration, often identified as distinct 'partial' D epitope loss. The clinical impact of partial Ds is due to the ability of their carriers to form anti-D antibodies upon confrontation with regular D after transfusion, or pregnancy. This leads to the -naively spoken -contradictory finding of an allo anti-D antibody in a D positive individual in connection with a negative autocontrol. The antibodies themselves include the same fatal clinical potential as anti-D antibodies of D negative individuals, but may be even more hazardous since unexpected in D positive individuals a priori. D categories (II to VII) represent a nomenclatorily defined subgroup of partial Ds. The molecular cause of partial D lies within single (caused by point mutation in the respective RHD gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to RHD-RHCE-RHD hybrid genes) which determines a qualitative D antigen alteration, rendering them distinguishable from regular D by a partial D carriers immune system. Nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. Most partial D exhibit decreased D antigen density, enabling principal recognition of them. However, routine serological methods may not properly recognise all partial Ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. This actively prognostic proceeding with respect to early detection of partial Ds became widely feasible by RHD DNA typing techniques. Currently, routine RHD DNA typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial Ds and their reliable discrimination from weak D types, not at risk for allo anti-D immunisation. A reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine RHD DNA typing of all weakly expressed Ds as defined by serology, since most partial Ds also meet this phenotype. RHD allele frequencies and their geographical and regional prevalence will certainly have an important impact on DNA typing strategies and their (mandatory) specificities.

Fluorescence cytometry for completely automated immunohematology testing D ROBACK*, B BARCLAY † and D HILLYER † *Emory University School of Medicine, Atlanta, † Transfusion & Transplantation Technologi, Decatur, GA, USA

We previously described a methodology for accurate immunohematology testing by fluorescence cytometry [Roback, J.D. et al. (2004) Transfusion 44 (2), 187]. This system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. When authentic clinical samples from hospitalized patients were tested for ABO group, the presence of D antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (CAT). This system determined the correct ABO group and D type for 98.7% of 520 samples, compared to 98.8% for CAT (P > 0.05). When samples were tested for unexpected alloantibodies, FC determined the correct result for 98.6% of 215 samples, as compared to 96.3% for CAT (P > 0.05). This novel method was better than CAT at detecting weak anti-A (P < 0.0001) and alloantibodies. Based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal K-allele in the presence of excess of maternal k-alleles. Furthermore, it has been shown that fetal DNA is in the plasma present in shorter fragments (<300 bp) than maternal DNA. Size separation of cell free fetal DNA can therefore be used to increase the relative concentration of fetal DNA, which will help the development of new genotyping assays. In conclusion, cell free fetal DNA in maternal plasma is nowadays routinely used for prenatal RHD typing and fetal sexing. New technical developments will make it possible to extend these indications to other blood group antigens in the near future. More insight in the characteristics of fetal DNA might finally lead to wider applications, including numerical chromosomal aberrations. Furthermore, it might become possible to apply genomic DNA microarrays for the screening on many different inherited diseases, including hemoglobinopathies.

Determination of the affinity of anti-D present in the serum of immunized subjects and in anti-D Ig preparations by a method using unlabeled antibodies P LAMBIN*, M DEBBIA* and Y BROSSARD † *Institut National de la Transfusion, † CHP Hopital Saint Antoine, Paris, France

Introduction: Few data are available concerning the affinity of maternal anti-D responsible for the hemolytic disease of the fetus and the newborn (HDN), and the affinity of anti-D immunoglobulin used for the prophylaxis of that disease. We recently described a method to measure the affinity (Ka) of untagged anti-D monoclonal antibodies. Aims of the study: In this work, a similar method was applied to determine the affinity constant (Ka) of polyclonal anti-D present in the serum from D-immunized mothers and donors and from anti-D Ig preparations. Methods: A constant amount of O R1r RBCs was sensitized with increasing concentrations of anti-D present in the sera from immunized subjects, and in anti-D Ig preparations. At equilibrium, the amount of anti-D bound to RBCs was measured by ELISA. The Scatchard equation (linear regression) and the Langmuir equation (hyperbolic regression) were used to determine the Ka of anti-D.

The experimental data fitted well with the Scatchard equation (mean r † = 0.95) but a better correlation was observed with the Langmuir equation (mean r † = 0.99). In 11 maternal sera, the mean Ka of anti-D was 5.6 ¥ 10 to the 8 M-1 (from 2.8 to 12 ¥ 10 to the 8 M-1). In the sera from 6 immunized donors, the mean Ka was 3.9 ¥ 10 to the 8 M-1 (from 1.5 to 6.8 ¥ 10 to the 8 M-1) and in 5 lots of anti-D Ig, the mean Ka was 3.4 ¥ 10 to the 8 M-1 (from 3.1 to 4.2 ¥ 10 to the 8 M-1). The comparison of anti-D affinity measured in 5 cases of HDN in which infants presented a fetal anemia and in 6 cases of HDN in which infants presented only a postnatal anemia showed no significant difference. The mean value of Ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 M-1 whereas in the cases of postnatal anemia the mean value of Ka was 5.8 ¥ 10 to the 8 M-1. Conclusion: The method previously described for monoclonal anti-D was applied to polyclonal anti-D present in the serum of D-immunized subjects and in Ig preparations. The experimental data fitted well with the Langmuir equation, and the affinity of polyclonal of anti-D was measured with accuracy. In addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-D measured in the most severe cases of HDN (fetal anemia) and in the less severe cases of HDN (post-natal anemia).

Introduction: Cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of HPA antibodies. But recovered platelets do not express the HPA-15 alloantigens. Aim of the study: Here we describe a method for the successful preservation of platelets by lyophilization. We report the value of this new reagent for the detection of HPA alloantibodies and especially anti HPA-15 alloantibodies. Methods: Rehydrated lyophilised platelets (Lyo P) were tested for their reactivity with monoclonal antibodies against GPIIbIIIa, GPIbIX, GPIaIIa and CD109 by flow cytometry. The levels of reactivity were comparable with the ones obtained with fresh platelets. The rehydrated platelets were used in the MAIPA with a panel of HPA antibodies (anti-HPA-1a, 30; anti-HPA-1b, 3; anti-HPA-3a, 3; anti-HPA-5a, 3; anti-HPA-5b, 50; anti-HPA-15a, 2 and anti-HPA-15b, 4). Results: All HPA antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. Absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). Interestingly, we used lyophilized platelet with a high expression of CD 109 bearing the HPA-15 system and we have detected 24 anti HPA 15 antibodies among 1000 sera previously negative with fresh platelets. Nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. Conclusion: Lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of HPA antibodies. Moreover, this work bring new insights on the HPA-15 system in platelet transfusion. We are now pursuing more extensive validation studies with a larger number of samples representing all known HPA specificities and several diseases. The diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. One of these options is transfusion of blood and blood products. Transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. Guidelines that direct the indications for transfusion differ from those in adults. Non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. Metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. Evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. A restrictive approach to RBC transfusion that maintained the Hb concentration between 70 and 90 g/L was found to be as effective as and possibly superior to a more liberal strategy of maintaining the Hb concentration between 100 and 120 g/L. There are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher Hb levels.

RBCs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. It is important to realise that blood is not a uniform product and the clinical efficacy of RBC transfusion may vary. One factor that may have a considerable effect on the quality of the RBC product is the storage time. RBCs undergo marked changes during refrigerated storage. The implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. There is insufficient evidence to support such practice. It is of great practical importance to determine if, or when, fresh RBCs could be superior to stored RBCs. Background: With a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (FUWB) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. Aims: To outline the use of FUWB in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in FUWB versus platelet components. Methods: Outcomes of FUWB and traditional blood component use were examined for 20 cases of cardiac bypass surgery. In addition, exclusive use of FUWB for 23 burns debridement cases was analysed. An evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters.

Results: There was a decreased requirement for blood components following administration of whole blood post cardiac surgery. Whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. Platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings.

Conclusion: FUWB appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss.

M-PA-051 Transfusion practice for coronary artery bypass surgery in Greece S LAKOUMENTA, M VASSILI, G HATZIDIMITRIOU, T ASTERI, P STRATIGI, S KANELLAS and G PALATIANOS Hellenic Society of Blood Transfusion, Athens, Greece

Cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. The impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (CABG) in US attracted great attention 1. The present study is conducted in order to reveal the transfusion practice in Greece on a similar population i.e. patients undergoing CABG operations. Methods: Five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time CABG procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (CPB) etc. The total estimated blood loss was calculated as the sum of red blood cell volume reduction [(Body Weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). Results: Results are shown in Table 1 : Means, standard deviations, and p-values of the Wilcoxon test comparisons between the hospitals. A preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). There is a statistically significant difference (P < 0.0001) between the five centers in duration of the operation, CPB, estimated blood loss and volume of transfused plasma. Red cell use showed also a variation which however did not reach statistical significance (P < 0.02). The center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. Conclusions: Although variations, as those observed in Greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. Our study is in progress and additional data are being collected and will be presented. Introduction: Premature infants and at term newborns have an higher circulating blood volume per Kilogram than the adults (100 mL/kg in premature; 80 mL/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high Platelet concentration, needs for transfusion therapy. The laboratory criteria for Platelet transfusion are the following: (A) a PLT count < 150 ¥ 10 9 cells/L if bleeding is observed; (B) a PLT count < 20 ¥ 10 9 cells/L without bleeding; (C) a PLT count < 50 ¥ 10 9 cells/L in newborns showing critical clinical conditions. Aim of this study: In this study, we have monitored the PLT transfusion therapy in our Neonatal Intensive Care Unit (NICU) in the last four years. Methods: Effects of PLT transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). The weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. Gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. For every Platelet transfusion in these newborns, the volume of Platelet Concentrate has been of 10-20 mL/kg, with a PLT count < 700 ¥ 10 9 cells/L. Results : In the study period, 77 PLT transfusions have been performed: 23 children have been only transfused one time, while  multiple PLT transfusions (ranged 2-10) needed for 14 children  according to clinical conditions. The observed clinical indications  for transfusions have been the sepsis with haemorrhagic syndrome  (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). After 24 hours from transfusion therapy, the absolute PLT count and the Correct Count Increment have increased in all 37 little patients. The highest increase in PLT count was 45 ¥ 10 9 cells/L, while the lowest 5 ¥ 10 9 cells/L. No difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls.

In conclusion, we can affirm that PLT transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. However a collaboration between NICU and Transfusion Center is necessary to choice the adequate Platelet Concentrate's volume for transfusion and the best PLT donor for the newborn.

Developing Transfusion Strategies 27

fusion Society of Turkey (BBTST) in 2004 with contribution of 253 blood transfusion centers. According to these figures 51% of centers attended operates apheresis procedures. Two centers informed us that apheresis in the hospital was carried out at blood bank. There was not enough information from one center, so it was excluded.

Of the 51 blood banks performing apheresis, 30 were university hospital blood banks. Another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. One center did not respond. All centers reported to prepare and separate erythrocyte and plasma. However only 48 centers reported to prepare random platelets as well. Each center had apheresis machines between 8-15. A total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). Of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. At 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. A total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. Productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. Around 68% of all apheresis procedures were carried out by large well run blood banks. Conclusion: As the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • Planning of resources for the financing of the BTS, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • Achieving recognition of real costs of products and services from the Health Insurance Fund and Ministry of Health. • Harmonization of low level of acclaimed costs and real costs of basic transfusion activities.

Results: Acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. The prices of health services in the official Price list are much lower than the proportion of costs of material resources needed for the realization of these services. This especially affects the management of independent blood establishments (BTI's in Serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. The 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the Health Insurance Fund, while independent blood establishments are financed through the price of products and services they provide. Conclusion: In order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole Blood Transfusion Service in Serbia. This can be achieved only by continuous cooperation of the Health Insurance Fund, Ministry of Health and independent blood establishments.

sion Centers (RBTC). The activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the RBTC and in 24 Departments of Blood Transfusion (DBT), part of the district hospitals. The collected units in DBT are transported by special cars to the NCHT and the RBTC for processing, testing and control. The same transport is used for the requested by DBT blood components for storage and distribution to hospital departments. Thus the issued components are with an equal quality and safety for all patients throughout the country. LBBDBT introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. It includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the Blood Transfusion Service. . Seventy hospitals are exclusively users of blood, blood products and services. The current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of IT system, lack of planned, continuous skill upgrading. As a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. Aim of study: To reorganize blood collection activities in Serbia to increase collection of safe blood up to 4% (304 000 blood units).

Methods: Division of responsibilities between blood establishments and hospital based transfusion services by:

• Optimizing organizational structure • Implementing blood collection standards to enhance blood safety and donor care • Gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • Creating and implementing a training strategy.

Results: Through the EU funded Project Support to a national Blood Transfusion Service in Serbia, we are in the effort of integrating the services and standardizing their work. The Blood Collection working group began by dividing Serbia into 3 blood collection regions: North, Central, and South. Each region is divided into sub regions covering approximately half a million population (4 in the North, 8 in the Central and 4 in the South region). Each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. The 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in Serbia. To this effect, the following has been achieved:

• Blood collection activities in Serbia analyzed • Performance analysis of BTE and HBTS mobile teams in place • Two model standard mobile teams tested in the field • National blood collection SOP's written • National Donor Questionnaire Form prepared • National set of blood collection standards prepared • List of donor deferral criteria prepared • Blood collection equipment renewed • Regional reorganization plans in progress.

The objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. P-005 60 years of the national blood transfusion institute in Serbia N NEDELJKOVIC National Blood Transfusion Institute, Belgrade, Yugoslavia NBTI was founded in 1944 . Since 1953 and unpaid blood donation is mandatory, organized in cooperation with Red Cross. Blood donation is regulated by the law in 1974, 1993/94. Codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. In the past 60 years, there was over 3 million blood donations, performed in accordance with WHO regulations. Over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in FR Yugoslavia with 10.3 million inhabitants in Serbia, Montenegro and Kosovo. Plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. Now, they have been regulated by tender. In 1951, test to lues was introduced, to HBsAg in 1971, to a-HIV in 1985 and to a-HCV in 1993. Information system was introduced in 1987. NBTI includes: National Haemovigilance Coordinatoin center, Center for Medical Care of Haemophiliacs, Tissue typing Center, Center for Prenatal and Perinatal protection of pregnant women and newborns. Activities of NBTI are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 West Balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. NBTI is the third year of GMP, SOP, YUS ISO 9002 implementation. In the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. NBTI is the publisher of the National Bulletin of Transfusion Medicine and it is included in the education system of the Belgrade University Medical Faculty and the ESTM in Belgarde 2003.

The problems of blood service in Russia EA SELIVANOV and T DANILOVA Russian Inst. of Hematol. & Transfusiol., St. Petersburg, Russian Federation Background: The Blood Transfusion Service (BTS) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. Aims: Russian blood service assessment with international comparison. Methods: A study was conducted on the base of the reports from all regions of Russia followed by a computer statistical analysis. Results: Blood and blood components were collected in the Russian Federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. Amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. The average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. The average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. An average volume of one blood donation is 396 ml. Blood was collected into plastic bags containing domestic or foreign anticoagulants. About 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. Amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. At present in Russia all donations are tested for ABO blood group, Rh(D) type, anti-HIV-1/2, HbsAg, anti-HCV and Syphilis. The total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. Blood components collected are as FFP, RBC, frozen RBC, 1eukocyte-and platelet-depleted RBC, RBC suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. As to blood safety measures -implementation of blood components leucodepletion and FFP and RBC quarantine in process. The new national strategy of BTS reorganization has been developed. It includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional BTS establishments, appropriate blood and blood components usage.

Calculating the cost of blood in Turkey N SOLAZ, S KEMAHLI and S CIN Ankara University, Ankara, Turkey Background: Like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. Since last 5 years even the most developed countries started to discuss about the cost of blood. In Turkey Ministry of Health determines the cost of blood annually. Aim: To establish a safe, cost effective and reliable prices for blood components. Methods: Turkish Ministry of Health (MoH) started to determine the cost of blood components as 'all inclusive' principle. This means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. This system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. There might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. Conclusion: Current blood product pricing system looks generally reasonable and reliable but MoH should establish close follow up systems for avoiding any abuse on the safety of blood. Background: A positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. The incidence of a positive DAT was expected to increase since more sensitive techniques (gel test) were installed. The aim of our study was to examine whether DAT positive otherwise healthy donors presented any clinical or laboratory abnormalities. Methods: In the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to DAT positivity of blood donors' red cells (0.02%). DAT positive [(1+)-(3+)] samples were only IgG positive in 32 cases, only C3d positive in 5 and IgM positive and C3d positive in 1 case. All blood donors were notified and thirty two of them responded to a request for a further sample. A complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ANA, anti-DNA, anti-ENA, RF, anticardiolipin antibodies), quantitative assessment of immunoglobulins, APTT and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. DAT and IAT were performed by gel test (ID-DiaMed) according to the manufacturer's instructions. DAT were performed with polyvalent and monovalent reagents (anti-IgG, -IgM, -IgA, -C3c, -C3d). The blood donors were also examined clinically. The donors who had positive immunological tests were referred to a rheumatologist for further investigation. Results: Among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis B recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ANA, two were positive for anticardiolipin antibodies only and two had a positive ANA test only. In six blood donors we did not find any abnormality that might be interrelated to DAT positivity. Conclusions: All blood donors with positive DAT should be requested to undergo further investigation. Some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ANA and/ or anticardiolipin antibodies. The eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated.

tions: usefulness, frequency and sincerity in answering questions. Donors could choose one of the offered answers and elaborate in writing the answer they have chosen. Results: Of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. That the survey was useful thought 91% and 9% that it was not. Opinions were elaborated by 61.1%. That the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. The answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. Conclusion: Most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. They are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers.

Analysis of blood donor's deferral in national institute for transfusion medicine -Skopje for the last five years (2000) (2001) (2002) (2003) (2004) P BLAGOEVSKA*, I NIKOLOVSKA † and R GRUBOVIK* *National Institute for Transfusion Medic, Skopje, † Medical Center, Prilep, Macedonia Introduction: Safety of blood and blood products depends on many different factors, starting with selection of blood donors. The aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in NITM and their correlation (voluntary/family donors). Materials and methods: This is a retrospective, epidemiological study and data were taken from the Blood Donor's Registry in NITM from 01.01.2000 till 31.12.2004. Statistical mass includes blood donors who came to NITM to donate blood in the mentioned period. Results: There were total 14 229 donors in NITM and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). The most common reason for deferral is low Hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. Relation voluntary/family donors is almost equal (49. 6 : 50.4 ). In the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. Conclusion: Percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). Reasons for deferral are predominantly from temporary character (98.9%). Permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. We should establish the National Registry for Deferred Donors, as well as for the donors with positive markers for TTI. We should design a strategy for returning of temporary deferred donors.

Regruting blood donors in multiethnical environment P BLAGOEVSKA*, R GRUBOVIK* and K ELEZI † *National Institute for Transfusion Medic, Skopje, † Medical Center, Gostivar, Macedonia Introduction: Population in R.Macedonia consists of 75% Macedonians, 22% Albanians and 3% others (Serbs, Gypsies, Turks). Over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. Transfusion service and Red Cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. The aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention.

The study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. Results: There were 3704 blood donations for the mentioned period. Predominant blood donors are employed and high school students in 75%. Family blood donors are ~20%; between them 70% are from Albanian population. The ratio between blood donors Macedonians vs. Albanians is 80 : 20. Woman blood donors are presented with 14%. First time blood donors are 53%, and regular donors arẽ 13%. Conclusion: First step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. For a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. Background: Pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. Decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. Regulations have been formulated by governmental agencies with limited input from the medical community. The decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. Policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. Discussion: In the U.S., the FDA's implementation of donor exclusions to prevent possible transfusion transmitted vCJD has reduced the donor base by more than 2%. Given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. To compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. One study has indicated that two-thirds of donors have no intention of donating more frequently. New donors have higher rates of infectious disease markers with positivity for HIV and HCV twice as high as repeat donors. Despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. Another area of concern is the aggressive use of inducements to attract new donors. Some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. Most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donat- Conclusion: (a) Blood donors who were patients' relatives were many more than volunteers as well as more were men than women. Also people of young ages were more than those from older ages. (b) The frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. Specifically as concerns HCV, it seems that transmission frequency has been reduced after the obligatory testing of HCV in Blood Transfusion Centres and Stations. 

Genotype 1B of hepatitis C virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. These are differently distributed in the world: types 1 and 3 are the most common in Europe and in USA. Aims: Considering that, in our region, anti-HCV antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of HCV genotypes in blood donors. Methods: In period from May 2003 to December 2004, 83 362 blood units were analyzed by NAT for viral RNA research. NAT has been performed on single sample by TMA technique. On RNA-positive samples, the HCV genotype has been identified by reverse hybridisation with line probe assay. Results: 143 blood donors have resulted HCV-RNA positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. We have also analyzed the differences between the two sexes in HCV-genotypes distribution. HCV-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women.

Although an accurate pre-donation selection, discharging all subjects with ALT >40 IU, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted HCV-positive. Analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. The high frequency of genotype 1 in blood donors is explained by the observation that HCV 1 is usually associated with low ALT levels, for this reason affected subjects may escape to donor's screening only based on dosage of ALT. On the contrary subjects affected by other HCV types, associated with high ALT levels, may be deferred increasing the HCV 1b relative prevalence. At the end, the different distribution of HCV genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. In fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in Europe. P-023 Kell blood group system and rare blood donors V FAKITSA*, P KARYDA*, S GIANNOULEA † , C ANTONIOU*, J FLESIOPOULOU*, E HALIOU*, M PAPAKONSTANTINOU*, H DESSILLA † , E KATSADOROU*, G LYRAKOS* and K SOFRONIADOU* *General Hospital of Nikea, Pireas, † Blood Transfusion Center, Athens, Greece

Background: The Kell blood group system is a compound antigen system exclusively of red blood cells. Some of the Kell antigens are highly immunogenic. The commoner Kell antibody is anti-KEL1. The KEL2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. The blood donors who have not factor cellano are classified in the rare blood donors. Rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. There are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%).

Motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). They want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( The mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. The mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. The blood donors who were female, first time blood donor low wt the rate of vasovegal RX was higher in female, first time, low weight, younger blood donors (P < 0.005). The rate of vasovegal RX was higher in blood donor (P < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. When each variable was adjusted for other variable by regression analysis. Young age, first time blood donation, anxiety, fatigue, starvation were significant (P < 0.005) and the others were not. Conclusion: Donation -related vasovegal syncopal reactions are a multi factorial process. These reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. These reactions might be predicted vasovegal reaction and these some facth donors need more care. With better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. To avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. We present 3 studies dealing with different dose and duration of iron compensation. In the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females.

In the second open study we reduced iron dose to 20 mg daily over one month for both genders. This iron dose was sufficient for compensation of iron loss. A further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. In all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. Aim of the study: One of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). The aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. Methods: The donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . The percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . Results please see Tables 1 and 2. In the tables there is shown observed data in relation to the total number of births in the Czech Republic in reviewed years.

The study showed that number of donation from donors of age from 18 to 20 decreased during objected years. Unfavourable state of total number of births in the Czech Republic (153 801 birth in 1980 Republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. Despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. We should find new ways and methods to attract new blood donors and keep the regular ones, too. The aim of the research was to investigate women's attitudes towards blood donation in Cyprus. A statistical sample was selected using stratified sampling and consisted of 369 women from the district of Limassol (the second largest urban center of Cyprus) between the ages of 19 and 60. Using Linear Logistic Regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. The percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. In addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. The research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. Even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the Discussion: It is about small group of students. The impression is that the altruistic behaviour is present at most of the questioned students. The fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. Families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. Conclusion: Including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). Active participation of the Department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results.

Adverse reactions in 2 blood donors taking betablocking antihypertensive medications L PAESANO*, M D'ONOFRIO*, S MISSO † , G FRATELLANZA* and E D'AGOSTINO* *University Federico II, Naples, † Hospital San Sebastiano, Caserta, Italy

One aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). Blood donation is surely contraindicated in various pharmacologic therapies, but not in all. In fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. According to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. On the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. Nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. A possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not Specialist of Transfusion Medicine young doctor). Another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. In fact, in this last case, the risk/benefit balance justify the blood letting procedure. In the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. The reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. No prodromic symptoms have been observer or referred. Cardiac Frequencies (CF) before donation were respectively 60 and 64 beat per minutes and Blood Pressures (BP) were both in the normal range (130/80 and 120/80 mmHg). After donation, during adverse reaction, CF showed no substantial variations, while BP have been decreased respectively to 80/45 and 60/30 mmHg. Immediate treatment has consisted in putting the donors in the Trendeleburg's position and in applying a dolorous stimulation. In the first case this treatment has been sufficient to report the BP to 100/65 mmHg (with disappearing of all symptoms) in only half hour time. In the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. These two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. Our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. In fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. This effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the Transfusion Center.

Introduction and aim of the study: In society under transition privatisation and marketisation probe all areas of life. Transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. The aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in Serbia (ideas about privatization of some parts of National Blood Transfusion Institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from VBD, stories about tradition of paid blood donations in some European countries). Publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. Table. Conclusion: Surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. Information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. SKAE has built up such procedures working along the lines of the European Haemovigilance Network. Improvement of existing national haemovigilance systems is expected to follow from the implementation of the EU Directive. Although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. To estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from Regional Blood Centers using standardized criteria for temporary and permanent blood donor deferrals. Within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases).

For Regional Blood Centers the temporary deferral rates varied widely (see the Table below ). In the case of individual Regional Centers, the differences as well as the most common causes were often difficult to explain. According to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. To some extent such losses could be avoided. Further studies are recommended to elucidate the problem and eliminate unnecessary deferrals.

Caption 1: Percentage of deferrals Aims: From our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of Blood donation. Methods: Many first time Blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. This is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. Those potential donors will never even approach again Blood donation Centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. Results: One of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). As we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. Therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. Even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. In fact 85-90% of those donors rejected for hypotension are readmitted in Blood donation after meeting the above mentioned criteria. Another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. Since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. The doctor must explain the donor the reasons for iron depletion, so Blood donation should not be considered as the only cause for this situation from the donor. There are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. It is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in Blood donation in the future (75-80% of those rejected are readmitted in our Centre). Summary/conclusions: At our Blood Centre we have created a program of regular tests (blood tests-physical examination) for all our Blood donors. Our experienced and well taught personnel offers advice and provides useful information in every aspect of Blood donation and more. We have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. Introduction: An innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. Aims: The purpose of this study is to find methods and ideas that can help Blood donation centers throughout our country to create new Blood donors, give a motive and inspiration for Blood donation by adopting new trends of society and finally accomplish national need. Methods: By having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, Cholesterol levels. We investigated whether they believe that Blood donation has, if any role towards a more hygienic life. Results: We divided blood donor volunteers according to their age, educational level, and number of blood donations per year. Our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. This dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. Besides the yearly run lab tests that are done by our Blood Centre they also seek advice and discuss any matter concerning their health with the Blood Centre doctor. It appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. In our blood Centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. Summary/conclusions: This approach has already shown some positive results in our Blood Centre as many people especially young educated women have joined our Blood donorship program and donate blood at scheduled intervals. In order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. We have to move with society and modernize the way we attract various groups of people.

Blood donation against prejudice AS SALTAMAVROS*, S DIMITRAKOPOULOS † , V ZACHARAKI*, P GIANNAROS*, S MARKOU* and P TSELIOU* *St. Andrews Hospital Patras Greece, Patras, † Pyrgos General Hospital, Pyrgos, Greece Introduction: In order to achieve a greater population to be admitted in Blood donation we have to provide information concerning any obscure issues that presents in selecting donors.

To examine the accuracy of Hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (Cell-Dyn 1600, Abbott Laboratories, USA), in a blood donor setting. Methods: The NBM-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (O-RNIRS) to detect and analyze the Hb/Hct levels. The clinical trials were conducted at two blood donor centers (Israel and USA). Studies were carried out on a group of 159 subjects (86 Females, 73 Males) aged 19-64. Subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. After obtaining informed consent, Hb/Hct levels of all the study volunteer participants was tested non-invasively, using the NBM-100 device, followed by a venous blood sample. Additionally, the USA center tested a capillary blood sample using the HemataStat Hct measurement device (71 donors). Hb levels were considered normal when readings were equal to or >12.5 g/dL. Results: Venous Hb measurements ranged from 9.9-16.2 g/dL. The mean NBM-100 Hb level was 13.20 ± 1.35 g/dL, only 0.28 g/dL lower than the mean Hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dL. The standard deviation of the difference between the invasive and noninvasive Hb readings was found to be ±1.04 g/dL. The mean absolute error (MAE) of their difference was 0.81 g/dL. When checked against the Cell-Dyn in the USA center, where 21 subjects had Hb of 12.5 g/dL or lower, the NBM-100 and HemataStat devices showed comparable sensitivity results.

The NBM-100 using O-RNIRS is a promising noninvasive technique for Hb screening in blood donors. The device is easy to use and agreeable for both blood donors and personnel. The technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation.

The effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations

Background: Blood donors are deferred for numerous reasons. Some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. The majority of donor deferrals, however, are short-term temporary deferrals (STTDs) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. The effect of STTDs on blood donor return rates and subsequent blood donations is studied. Materials and methods: Donors given STTDs during the 15 December 1999 to 15 March 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). Computer records were evaluated during the next 3 years (21 March 2000 to 21 March 2003 to determine donor return rates. Significance for comparison between the two groups was based on Chi-square analysis. Results: The most common reasons STTDs were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). Non deferred donors were a little more likely than donors with STTDs to return over the next 3 years (36.6% vs. 34.8% PV = 0.0001) and non deferred donors donated more whole blood units. . According to ethnic structure, women -ethnic Macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. The most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). Conclusion: Having in mind that 50% of the population in Macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. This is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. Incorporating them in education and organization would contribute to their more extensive participating in blood donation.

Comparison of serum beta 2-microglobulin (b2-MG) between HBsAg positive donor and healthy control F TARABADI*, M SHAEIGAN*, G BABAEE † , A TALABIEAN* and M KHADIR* *Iranian Blood Transfusion Center, † Tarbiat Modarrs University, Tehran, Iran Background: Beta 2-microglobulin (b2-MG) is a low molecular weight protein (11 800 Daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. In the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-HLA antigen plays a role in the elimination of the virus. Method & samples: Beta 2-micro globulin was measured in serum drawn from 45 Hbs Ag positive blood donors Include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-MG by Enzyme immunoassay (ELA). Results: Our studies showed b2 MG level increased in 7 (15.6%) Hbs Ag positive donor that was significant differences with healthy control (P = 0.0001). Conclusions: It seems that serum b2MG is a good marker for Hbs Ag replication. The role of b2MG in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by VD, RD and DD respectively. Over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as RD (44.9% to 38.4%) and DD (0.9% to 0.8%) have shown a declining trend. The percentage of female donors was maximum in voluntary group as compared to RD and DD (11.01% vs. 2.02% vs. 6.17%) respectively. The rates of all TTI markers reactivity were significantly higher in RD as compared to others donors. The HBsAg and anti HCV reactivity in VD and DD is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). HIV antibodies was found more frequently in VD as compared to DD [0.15% vs. 0.06% (P < 0.05)] whereas, VDRL reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (P < 0.05)]. Conclusion: Voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. These strategies need to be strengthened to increase the female donor base. The safety of DD is equivalent to VD when the rates of TTI are compared. Thus, DD should be advised to donate blood regularly as voluntary blood donors. Blood safety depends on a number of factors. The chain of safe blood starts with the donor. One of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. Donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. Aim: To present the most frequent reasons for declining volunteer blood donors. Material: The materials used for analysis were the questionnaires completed by all the potential blood donors at the Transfusion Department of the Medical Center in Strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. These donors donated blood in the period between 2001 and 2003. Results: During this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. Out of the total number of blood donors 3313 were male and 627 female donors. The reasons for declining potential donors were the following: 42.3% had low levels of Hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. Conclusion: Donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. The obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion.

Questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation F VLADAREANU, A BUGNER and S SIRIAN National Institute of Heamatology Transf, Bucharest, Romania

The research theme of this questionnaire is as follows: 'What is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' We also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. The purpose of this questionnaire was not dissimulated. The general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. Students had never been an investigated lot up to now. The hypothesis referring to this problem is as follows: Students are not informed either on the act of donation, or on the crisis of blood.

1. The lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. The lack of motivation of the studied lot is another cause.

The questionnaire was applied on a 2700 lot of students from seven different cities: Bucharest, Iasi, Constanta, Cluj, Sibiu, Brasov, Timisoara. The number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. As a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. Not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. On a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level.

The donor data and the details of blood application of the North West Transdanubian region of Hungary K VÖRÖS*, C BERCSÉNYI † , O PETRÓ † , R JÁGER † and E MISKOVITS ‡ *Hungarian National Blood Transfusion S., Györ, † Blood Bank, Tatabánya, ‡ Headquarters Hungarian N.B.S., Budapest, Hungary

The ongoing fundamental reorganization of the Blood Service began on the 01.07.1998 in Hungary. As the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the HHNBTS. The working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. Virus screening, blood group serology and processing will be made in the 6 regional centers. One of the regions is the 'North West Transdanubian Region' (NWTR, city Györ as the Center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (Tatabánya, Sopron, and Szombathely) are belonging to NWTR. The regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. Annually 56 000 donors donate blood in this region. This donation activity covers about the 6% of all inhabitants. The acceptance ratio of the donors is good (6-8% of the donors were deferred). There are 14 hospitals in our region. The regional demand on RBCC is 30-45.000 U/year, on FFP is 5.500-7.000 U/year and on PC is 8-10.000 U/year. The poster shows the donor data and the details of blood application of this region since 1999. P-054 Implementation of 2RBC collection using Haemonetics MCS ® +: medical staff training, donor recruitment and acceptance G WOIMANT, C FRETZ, D PUYDUPIN, E PÉLISSIER and JL BEAUMONT EFS Ile de France, Paris, France Background: Single donor 2RBC collection is an approved apheresis technique in France. Aims: Our goal was to evaluate the implementation of 2RBC collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. Methods: Donors were selected according to the French requirements for 2RBC collection (weight ≥ 65 kg, height ≥ 165 cm, Hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2RBC donors). All personnel were trained on adequate communication with donors. Eligible donors were contacted by mail, by phone or during pre-donation interview. Among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. The medical staff was trained on 2RBC collection with the SDR protocol and disposable set LN 948PF on the MCS ® +. Most of the medical staff was already used to autologous RBC donation with similar apheresis devices. Blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. Donors were asked to fill out a post-donation survey for assessing donor comfort and information. Results: Donor profile and clinical follow-up are summarized in Table 1 . Six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. The collections were well tolerated and no changes in vital signs were noted. Four reactions were reported: 2 hematomas and 2 citrate reactions. No reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. The technique was easily implemented by the medical staff and fitted well in the existing blood center processes. The medical staff as well as the donors found collection duration short (average of 35 min). The results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. Most of them agreed to donate again and several actually donated twice during the evaluated period. Conclusion: The implementation of 2RBC collection in our center, using Haemonetics MCS ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. We now plan to evaluate donor loyalty in the longer term.

Risk from first-time blood donors E ZHIBURT, S GOLOSOVA and P REIZMAN Federal Blood Center, Moscow, Russian Federation Introduction: Each third dose of whole blood in Russia is donated by first-time blood donor. There are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. Aim of the study: We investigated role of regional deferred donors registry (RDDR) in by first-time donor selection. Methods: Moscow RDDR includes parts: HIV, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. RDDR was complete and our center began actively work with it since last year. Each donor has to be registered in RDDR and automatically checked for deferral reason. Effectiveness of RDDR was investigated. Results: First-time donors donate less than 10% blood in our center. About a quarter of them are deferred before possible donation. Part of donors deferred by RDDR has been significantly increase in 2003 (c2 = 19.6; P < 0.001) at the expense of seropositive people. Conclusion: RDDR is effective for blood donor selection and decreases necessity in laboratory screening. First-time blood donors have to be examined before blood donation. If they have not contraindications, donation can be performed up to 10 days before examination and screening. The double unite platelet production is important especially if the relatives of patient find the donors. We evaluated the effectiveness two apheresis machine for platelet collection. In our blood bank, one Fenvall Amicus and one CS3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. Including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /L. Donors firstly was enrolled to amicus. If amicus was busy, then it was enrolled to CS. The properties of our donor populations were given in Blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. Modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. This involves new solutions to achieve higher yields and better quality of such components. The aim of our study was to estimate the efficacy of blood cell separator Cobe Trima in obtaining platelet concentrates (PCs) as compared to older-generation Cobe Spectra blood separator. Apheresis procedures were performed on both these blood cell separators. The quality of platelet concentrates was tested during 5 day storage period (see Table below ). We have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. The tolerance of both separators was satisfactory except for more frequent hypocalcemia when Trima separator was used. Most donors were more satisfied with Trima procedure because of single venipuncture although it involved special donor selection (good vein access). In general we may say that Trima is undoubtedly a more modern and more friendly separator. However, Cobe Spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). Methods and results: TLs (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10E9/L or blast count >50 ¥ 10E9/L) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. Performed TL procedures were rapidly reduced both the white cell count and the whole blood viscosity. Average fall in white cell count after treatment was 73.3%.

TP-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10E9/L) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . The TPs performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. TEs (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. It was shown that TE procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. Average fall in hemoglobin and red blood cell concentrations after TE treatments was from 20.5% till 35.7%. RBCX treatment (8 procedures on five patients with malaria and two with severe AIHA crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected RBCs and Summary/conclusion: The effects of TCs depended on the nature and stage of the basic HDs, of adequate selection of patients and of timely applied apheresis. Rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. Thus, TC should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. The present study indicates that the best therapeutic effects were obtained by RBCX. were carried out with continuous flow blood cell separator COBE Spectra and all patients underwent large volume leukapheresis (LVL). In all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. Six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. Seven children had vital signs and ECG continuously monitored during the procedure. Results: Each patient underwent a median of 2 collections (range 1-6). The inlet blood flow ranged between 16.0 and 95.4 mL/min (median 54.5 mL/min). The median blood volume processed was 11 754 ml (range 2700-22 500). Leukapheresis lasted a median of 240 min (range 120-270). The median total nucleated cell yield was 11.86 ¥ 10E8/kg (range 1.94-21.21), mononuclear cell (MNC) yield was 6.01 ¥ 10E8/kg (range 0.97-12.73) and CD34+ cell yield was 3.5 ¥ 10E6/kg (range 0.19-28.01). The median of MNC collection efficiencies was 56.11% (range 12.34-158.09). In 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10E6 CD34+ cell/kg were collected. During 6 (9.09%) procedures patients had experienced apheresis-related side effects. The citrate-induced reactions were most commonly observed. The reactions were mild and cessation of collection was required only in one case, because of catheter related complication. Mild sedation was required only in few very small children. Post-donation platelet count was less than 30 ¥ 10E9/L in 3 cases and these patients required platelet transfusion before subsequent procedure.

Our results show that LVL in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. Close monitoring of blood counts, especially platelets, between PBSC collections is necessary. The cessation of procedure was required in only one case and no life threatening side effects occurred. Neonatal alloimmune thrombocytopenia (NATP) caused by fetomaternal mismatch for human platelet (PLT) alloantigens (HPAs) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ICH) and sometimes death of the fetus or newborns. We describe the successful management of a 37-year-old pregnant woman, alloimmunized to the HPA-1a (P1A1, Zwa) antigen, with a history of two previously children with severe thrombocytopenia and ICH. The pregnant woman was at her terminal pregnancy and was suddenly admitted.

To evaluate the risk of ICH in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (PLT 4.000/mmc). To ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. Plateletapheresis was performed using COM.TEC separator, Fresenius. Blood processed was 1.100 ml in a short time procedure (35 minutes). No significant adverse effects were observed in the mother and fetus, during and after the procedure. Platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in Octaplas AB. Then we separated the platelets into two units containing 0.7 ¥ 10e11 each. The day after the donation, the mother gave birth to a girl by caesarean section. After the transfusion, the PLT account increased from 4.000/mmc to 35.000/mmc and after a week the child had PLT 75.000/mmc without hemorrhagic complication.

According to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. In the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. The aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). These women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). In all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. The discrete centrifugation plasmapheresis was included in the complex of medical treatment. The woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. Six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. Three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. The system heparin was not used. In all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. Four women are discharged from the hospital, the other patients are observed with progressive pregnancy. Thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications.

Extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen C DEL FANTE*, C PEROTTI*, GL VIARENGO*, P BERGAMASCHI*, P PEDRAZZOLI † and L SALVANESCHI* *IRCCS Policlinico S. Matteo, Pavia, † Ospedale Niguarda Cà Granda, Milano, Italy Background: Extracorporeal photochemotherapy (ECP) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (GVHD) after allotransplants for oncohematological diseases. Reduced intensity conditioning regimen (RICR) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. The use of immunosuppressive therapy (IST) to control GVHD is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (GVT) effect. Aims: to evaluate the effectiveness and safety of ECP in treating pts affected with GVHD post RCR and the possibility to taper, at the same time, the IST. Methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with aGVHD grade II (1) and extensive cGVHD (5) GTX with median total granulocyte doses of 55 (21-150) ¥ 10 9 per GTX corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults.

The WBC counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) G/L for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 G/L (children) and 0.9 (0.3-9.4) ¥ 10 12 G/L (adults) at one hour after GTX and to 1.4 (1.1-13.6) ¥ 10 12 G/L (children) and 0.4 (0.3-8.6) ¥ 10 12 G/L (adults) at 24 hours after GTX. In 42 out of 54 patients (80%), the CRP levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. Thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and GTX. Patients without CRP response to GTX (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. Background: In recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. Plateletpheresis donation is considered to be a safe procedure with modern instruments. So far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. Aim: To evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. Materials and methods: Eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with MCS3p and 100 with MCSplus (Haemonetics), according to automatic standard protocols A3p and LDPLP, respectively (with the collection of an additional plasma unit). ACD-A was used as the anticoagulant in all apheresis procedures (ACD-A: blood ratio was 1 : 11). Most of the donors (84% men and 16% women) were patient´s relatives. Half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. The mean platelet yield was 3.2E11 /unit.

The overall rate of the donor related adverse events was 3.4%. Feeling faint was the most frequent event, which was occurred in 1.62% of donations. Hypotension and citrate related rates were 0.77% and 0.73%, respectively. All citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. Donor unconsciousness was the only observed severe event, the rate of which was 0.25%. Other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%).

(1) Plateletpheresis using the MCS3p and the MCSplus automated cell separators is a safe procedure, with a low risk of serious adverse effects.

(2) With the used ACD-A-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events.

(3) The peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms.

Quality assessment of FFP collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation.

In vitro platelet response to the aggregation-inducing agonists ADP, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (PAP-4C, Bio/Data). Results: There were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis WBC and PLT counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. Our findings are shown in the following Table. Mildly decreased response to all agonists was observed (mainly to ADP and Arachidonic Acid) in the samples taken right after the initiation of the procedure, in both groups. Platelets from the final component showed a further slight decrease in response to ADP, which was more prominent in the MCS3p device (P = 0.025). On the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. Conclusions: Reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. Literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. In our study, such traumatisation was observed only in the case of ADP in the MCS3p obtained collections and this could be correlated with the technological differences between the two devices. Recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. Background: Lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. In pregnancy cytostatic therapy affects gestation. On the other hand the course of disease can be refractory to corticosteroid therapy. Elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms.

A 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. Treatment was initially 1 mg/kg BW Prednisolone for 3 weeks and subsequently 8 mg/kg BW for 2 weeks. Plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( Table 1) . The eliminated plasma was substituted by fresh frozen plasma. The medium volume was 2.951 mL per apheresis. After day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. Results: Prednisolone therapy alone brought no effect even after changing to high-dose treatment. A significant amelioration of all symptoms could be observed after the first plasmapheresis. Good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. Intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. Apheresis No. 6 again lead to a recovery of the patient which held on until day 11. Conclusions: Treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. Exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. Background: the collection of MNC represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. In this work we compare the cell yield of two collection programs on Cobe Spectra device: the MNC versus the AutoPbsc program using the ECP procedure modified by Andreu. Methods: 39 procedures were carried out with MNC program and 19 procedures with AutoPbsc on 4 patients with cGVHD. Both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/TU, K+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, LDH from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, pH from 7.50 ± 0.14 to 6.74 0.08. The volume of apheresis units was lower than WB-RBC, the leucocyte count was normal in all units. The RBC loss by filtration was 24.9 ± 3.1 ml/TU and was lower than at WB-RBC. In apheresis RBC there were the differences in Hb and Ht value between the day of storage 0 and 40, in WB-RBC there were no differences. During the storage period we found no differences in K+ increasing value and no change in pH value between apheresis RBC and WB-RBC, the increasing of lactate was higher in WB -RBC, increasing of LDH correlated to hemolysis. The plasma Hb value increase was higher at apheresis RBC in contradistinction to literature. Hb and Ht correlation in apheresis units according to predonate value in donors was lower than at WB -RBC.

The method is a useful alternative to conventional whole blood donation, we get RBC units with high standard of quality and low correlation according to predonate Hb and Ht value in donors. Acknowledgement: The study is supported by grant IGA Ministry of Healthy CR N. NR/8015-3.

Background: In life-threatening exacerbations of SLE a satisfying efficient therapy is lacking. Despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. In particular circulating antibodies and immune complexes play an important role in the pathogenesis of SLE and MCTD. An extracorporeal removal of these pathological substances may be effective in the treatment of active disease. Methods: Five patients with severe therapy-resistant SLE/MCTD underwent immunoadsorption onto protein A. Blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. Anticoagulation was performed with ACD-A and heparine or ACD-A and r-hirudine. Plasma was separated by centrifugation. The 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. The columns were floated with a maximal plasma flow of 20 ml/min. The procedure was carried out every second day. Additionally supplementary intravenous immunglobulin therapy was given only once. Results: Remission of the disease was achieved in four patients. See Table below . Conclusion: PA-IA is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with SLE/MCTD. It is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. Background: Purification of bone marrow from erythrocytes is used to prevent early hemolysis in major ABO incompatible allogeneic hemopoietic cell transplantations. Erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and DMSO toxicity that might develop after thawing. Centrifugation, sedimentation with HES, and cell separating devices are methods for erythrocytes depletion. Aim: In our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. Success of the process is retrospectively analyzed for high and low volumes. Method: Erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with Cobe Spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. Fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with COBE PBSC coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). Results: The mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. Regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. No complication developed related to the device or product, and waste bag never had to be re-used. In none of the patients early massive intravascular hemolysis was observed. Conclusion: Erythrocyte depletion and volume reducing with cell separation device is a reliable method. This process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and CD34+ cells.

Platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration V SREJIC*, G BOGDANOVIC*, Z GARIC*, N VAVIC* and B BALINT † *National Blood Transfusion Institute, † Military Medical Academy, Belgrade, Serbia Apheresis team of the National Blood Transfusion Institute processed and classified data of 100 donors who donated platelets by apheresis procedure from January till April 2004. Procedures were performed in accordance with the LDPLP protocol, using Haemonetics MCS+. Initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. Donors' initial platelet count was not less than 191 ¥ 10 9 /L and the volume of the processed blood was not less than 2300 ml. According to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /L to 308 ¥ 10 9 /L (70%). Final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. Regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. Student's T test showed P < 0.0011. Leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /L to 0.4 ¥ 10 9 /L. Presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /L to 0.02 ¥ 10 12 /L. P-077 Therapeutic apheresis (TA) in Croatian hospitalsadherence to respectable guidelines Z ZIVKOVIC*, B JEREN STRUJIC*, S BORAS † , I BOJANIC † , B GOLUBIC CEPULIC † and Z IVANKOVIC † *Clinical Hospital Dubrava, † Clinical Hospital Center Zagreb, Zagreb, Croatia Introduction: Besides considerable resources, TA requires high costs and risk for patients. Therefore, indication for TA often considers interests of patient, hospital and requesting physician. The most respectable guidelines for the implementation of TA were defined by AABB and ASFA, classifying total of 58 diseases into 4 categories (Ctg), ranging from 'standard therapy' to 'lack of efficacy' . The objective of this study was to determine indications for TA performed in 6 Croatian hospitals in the period 2001-2004, respecting AABB/ASFA guidelines. Results: During the observed period in Croatia, TA was performed in 777 patients suffering from 26 various diseases. In 514 (66%) patients TA was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (Table 1) . According to the 4 Ctg, s of the AABB/ASFA guidelines, TA was performed in 573 (74%) diseases from Ctg I, 126 (17%) Ctg II, 54 (6%) Ctg III, and 25 (3%) Ctg IV of patients. The most frequent indications included in Ctg I were: myasthenia gravis (32%), collection of PBPCs (31%), Sy. Guillain-Barré (17%), and plasmacytoma (6%). In Ctg II frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in Ctgs III and IV: cytoreduction-polycythaemia (52%), thyroid storm (15%), GVHD (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) MTP 0/0, (3) discarded blood units 10/4. III group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. The consequences are: (1) the time spent for resolving h 35/40, (2) MTP 2.5/3.5, (3) discarded blood units 9/18. Conclusion: The analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate SOP, effective organization, QMP for equipment. The conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units.

An ISO standard for blood transfusion?

Background: In our search for an independent, objective assessment at Western Province Blood Transfusion Service, we were unable to find one single model that met the specific requirements of blood transfusion. We therefore resorted to developing our own model but ask the question: Why not have one international standard for blood transfusion? Aims: Our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. We wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. Methods: Having moved away from accreditation by the American Association of Blood Banks in mid 1990's, we evaluated various other options such as inspection by our government Department of Health or the World Health Organisation but neither organisation had trained inspectors or systems in place. We also investigated ISO 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. Results: We therefore developed our own model for accreditation that consists of three parts:

• a quality management section incorporating ISO 9000 principles; • a technical section incorporating specifications from the South African Standards of Practice (we also consulted the European, American, Canadian and Australian guides); • a laboratory section incorporating ISO 17025 parameters (soon to be updated with ISO 15189).

We then chose to be accredited by the South African National Accreditation System, SANAS, an internationally recognised institution. Once we had written a national accreditation checklist, SANAS submitted this to two international accreditation bodies (IAF and ILAC) for approval. The system has been in place for three years now during which time we have had four successful assessments. Summary/conclusions: In developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there P-078 Software for the management of the Scansystem bacterial detection method

The Scansystem TM was developed for bacterial detection in blood products. To be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. The software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (Product Bar Code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, ID) and to validate some specific results, an operator level for routine testing. The software assess sample traceability when testing pools of samples from 1 to more than 20. Indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. Each sample in the pool is traced through its barcode until the final result. The system is compatible with most of the barcode standard including ISBT 128. In addition, the system checks each barcode protecting the sample against duplicate testing. The software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. For a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. For a 'negative' pool, each sample constituting the pool are determined as 'negative' . For an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . Data transfer may be manual or automatic. A final technical validation is necessary before the transfer through an active selection of results to download. Final results are provided in a compliant format for an easy import into the blood bank database. All necessary information are displayed: 'machine ID' 'product/sample barcode ID' 'date' 'time of transfer' 'operator ID' 'result' ('negative' or 'positive').

The main advantage of this software is a continuous check of each step reducing the risk of error in testing. It makes the Scansystem TM test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. We feel it would be of benefit to establish an international working group to investigate the feasibility of writing an ISO Standard for Blood Transfusion. The standard would harmonise quality management parameters based on ISO principles and technical/laboratory parameters specific to blood transfusion. Minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. This ISO standard could then be used for the purposes of certification/accreditation or government inspections. This would ensure global standardisation of world-class best practices. First world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term.

Residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry 

Web-based outcome review: do you know how productive your Trima® can be?

Background: Web-based Outcome review is a new software tool developed by Gambro BCT for the management and the interpretation of data from the Trima and Trima Accel TM automated blood collection systems. Aim: Does the interpretation of reports obtained through Outcome Review lead to an increase in the number of products per run and the overall productivity of the apheresis center? Method: Run Data Files (RDF) from Trimaᮀ were collected and transferred onto the Outcome Review server. These RDF do not contain any donor related data that can lead to possible donor identification. The reports were generated on the Outcome Review website (Gambro BCT intranet), interpreted by a Gambro BCT employee and presented and discussed with the management of blood centers. Results: A total of 21 different reports can be generated on the website as a PDF file. For this study, 9 reports were investigated.

They are: Doses per collection, Doses per collection trend, Platelet collection trend, Platelet procedure performance, Platelet procedure performance trend, Product distribution, Average procedure time, Procedure time, Machine productivity trend. The results obtained for a center can be compared to word-wide, national, regional or to other individual Trima devices in the centre (benchmarks). This benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. Implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of Trima. The reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. Reaching and maintaining the optimal production capabilities of Trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. Conclusion: Web-based Outcome Review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of Trima are reached and maintained.

Introduction: To examine the cell vitality of packed RBC's during storage several parameters like ATP, free Hb or 2,3-DPG are used. Less kits for the determination of ATP are available and they need either a large sample volume and/or are time consuming. Here we present the modification of a commercial testkit for a time-and cost saving detection of ATP. Methods: Samples were analysed with (a) 3-phosphoglyceratekinase reaction according to Bergmeyer, H. Methods of Enymatic Analysis 2nd. Edition. Academic Press, New York 1965; (b) detection via HPLC and c) using a commercial testkit (R. Greiner Bio-Chemica, Germany). The ATP-Kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. Results: 245 samples were analysed in HPLC and modificated commercial testkits, another 20 samples have been examined in all tests. Comparison of the results showed no discrepancies in the above mentioned methods. Standard curves have been performed (range 5-1000 mM ATP) and statistical analysis demonstrated a given linearity (r = 9.97). Variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). The hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. At least the costs of ATP-determination have been reduced from €3.45 to €0.22 per sample. Conclusion: Performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of ATP in packed RBC's. Background: In order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. Aims: (1) To monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) To asses the relative contribution of different defective materials (DM) to the need to discard valuable blood components. Materials and methods: About 831 000 whole blood units were collected and processed by MDA National Blood Services in [2002] [2003] [2004] . As part of the routine Quality Control activities, derangements of the DM used were recorded and analyzed. Some 40 different types of DM were defined. Out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. About 70% of DM were detected during blood collection. Manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. Conclusion: Defective Materials are one of the major causes of the infringements of blood collection and blood component preparation processes. Analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. Establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. Such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. Results: Table 1 Analysis and characteristics of t 

(1) Comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital.

(2) Monitor, measure and analyze these processes, in order to improve their effectiveness continuously.

(3) Implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) Identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. The QMS of our blood bank was certified by TUV Rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in Thalassaemia patients' .

The implementation of the QMS based on ISO 9001:2000 standards ensures the improvement of services provided by the Blood bank and the increase in customer satisfaction, whether donors or patients are concerned. The former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. Finally, we shall not underestimate the positive impact of QMS in the motivation of Blood Bank personnel. Quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work.

Equipment management in the national blood transfusion service in Serbia

Introduction: New equipment was urgently needed in three blood transfusion establishments (BTE) in Serbia. Equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. Also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. Further more, coordination on equipment issues with the QA Department was not recognized.

Service funded by the European Agency for Reconstruction, provided various equipment for the three blood transfusion establishments. The new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and IT equipment of 1.6 million euros value. Before the new equipment is installed the BTE's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. This will ensure that the equipment can be properly installed and validated before use.

The Project has provided training on validation to the Working Group (WG) on Quality. The WG has created national procedures related to the equipment, including quite new term validation. The same problems in implementation of procedures were present in all three BTE's. Significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. QA Managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (Master cards, instruction for use) and designing the validation protocols. The same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between BTE's. The QA Managers also prepared an introduction of equipment requirements to the Heads of Departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment.

Results: National standards in equipment management in Serbia are set and are being implemented. QA Managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. The additional, positive effect is that validation is performed in one establishment for all BTE in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three BTE's.

Organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. Background: The Vista Information System (Vista TM ) is used in centers in Europe as an apheresis management system. With Vista TM it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. Aim: A calculation tool (Microsoft Excel) was developed to evaluate the added value of Vista TM . Three Blood Centers in different European countries completed a questionnaire using their local data. Summarizing these figures gives us an idea about the impact of Vista TM on the daily work and budget of a Blood Centre. Method: The Excel calculation tool that was used investigated 3 major areas where Vista TM could show added value: Improvement of regulatory compliance, increase the efficiency in operations and improve productivity. Results: Regulatory Compliance: The number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. The time spent on regulatory reviews decreased with a mean value of 35%. Operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. Because Vista TM tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. The percent time reduction in reporting is therefore very variable. However the % of errors related to these reports decreased considerably with a mean value of 30%. Efficiency in operations was also obtained because of the number of reports that are available in Vista TM . Productivity, management and process reports allow verifying and correcting the daily operations of the blood center. Increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. The number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted Trima settings. The percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). But because with Vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis.

The use of Vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. The financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure.

Establishment of national quality system in blood transfusion service in Serbia Introduction: The production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. Aim of the study: We introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products.

Methods: The blood products tested for statistical process control were Red cells in additive solution, buffy coat removed, and leukodepleted (LD) Platelet pools prepared from 4 buffy coats. The products were collected in T&B triple Opti-pac from Baxter and the Platelet pools were LD using PLX-5 filters from Asahi and stored in platelet bags from Baxter. Using control charts, namely X-mrchart, exponentially weighted moving average EWMA chart and for autocorrelated stationary data the EWMAST chart, we examined if time series of quality control values were in statistical control. If not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. Data included approximately biweekly measurements of volume, Haemoglobin (HB) concentration, HB/unit, haematocrit and log leukocyte count (WBC)/unit of 352 units of Red cells, measurements of volume, platelet concentration and platelet count/pool of 79 LD Platelet pools produced by a team of 13 technologists and of 79 LD Platelet pools produced by a specially trained team of four technologists. Results: Log WBC/unit was out of statistical control due to systematic differences between technologists. Apparent lack of control of volume, HB-concentration, HB/unit caused by autocorrelation disappeared when the EWMAST chart was used. Platelet concentration and volume of the Platelet pools produced by the 13 technologists were out of control. In that some technologists systematically produced low values. This could be explained by inappropriate handling of the platelet product between centrifugation and separation. Systematic differences between the four specially trained technologists could not be demonstrated and they produced Platelet pools with a significantly higher platelet count/pool. However, standard deviations of the four technologists differed significantly causing occasional outlying values. Conclusion: Training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended.

Background: One important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. This study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by Fort Portal Regional Blood Bank in Western Uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients.

There was sufficient reporting on the data requested by the blood bank. These results suggest that it is possible to establish effective 'look back' and haemovigilance systems. Capture of data on outcomes and adverse effects will be necessary to fully establish the system. Further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation.

External quality assessment of blood grouping were misinterpreted as RhD-positive samples without the use of control reagent. RBC phenotyping was made correctly by 79.7% of participants. The remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. Polyspecific human sera and monoclonal antibodies were used for ABO, Rh and antigens typing. The reason of errors in antigen detection was low quality of reagents. Antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-D-K-c alloantibody correctly. The rest of participants did not found alloantibodies or detected their specificity incorrectly. Results of testing depended on quality of screening cells. Thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using DiaMed AG cell panel. Consequently, the results of the first Federal External Quality Assessment Scheme show the necessity of improving the quality of red cell reagents produced in Russia. In addition, the more appropriate training of staff is required.

The importance of ISO 15189-quality system in increasing safety of blood transfusion

Introduction: Hadassah hospital transfusion medicine department received on 3/2004 ISO 15189-quality system accreditation. An essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. Aim of the study: Assessment of events and corrective actions following implementation of ISO 15189 quality system. Methods: Events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. All events were recorded and classified into two categories; quality system and technical. The latter were further classified into preanalytical (sample receipt), analytical (ABO Rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). All events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. Events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event.

Results: During the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). Most of the events were technical. All events were detected before causing harm to the patients. Results are summarized in the table. *The percent analytical value is a summary of rates of events per test types included in this category. Events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. Analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. The main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events.

The implementation of ISO 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. Understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. Evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. These results are in agreement with data in the literature. Creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. The explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. Those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. Growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. Aim: The goal of this document is to explain how ISBT intends to provide guidance to the blood banking community on implementation of effective information security policy. When using information for critical activities, blood banks should consider information security as an important aspect of their management policies. Evaluation of existing standards, such as ISO 17799 and HIPAA, allows us to establish a framework for information security without regard to the type of organization. It remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. Method: The ISBT information task force was created to provide guidelines on information security for blood banking organizations of all sizes. The intent is to help them understand existing standards as well as provide tools for implementing information security policy. These guidelines are based on existing standards that are followed by most worldwide countries: ISO 17999 and HIPAA. Results: Information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . Understanding existing information security standards was the first step for establishing a structure for the guidelines. The core is organized within an implementation framework and presented under the three following layers:

1. Administrative for defining the IT security organization, the information security policy, and information security awareness and training. 2. Physical for providing solutions that relate to physical environment protection and access, equipment and IT infrastructure security, and control for accessing computerized equipment. 3. Technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.).

Strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. Further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. Conclusions: Information security standards are prerequisite to understanding the issues involved when considering information vulnerability. International guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. The ISBT task force is committed to providing such guidelines.

Introduction: The important part of quality planning and quality assurance within production of blood components is measurement system analysis (MSA). Measurement system analysis was performed on microscopic counting of blood cells in our study.

Aim of the study: Aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. Methods: We practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. The counting performed two lab technicians in ten samples of plasma from whole blood. Leukocytes and erythrocytes were measured in Naggeotte counting chamber. We used the method of mean and range for the determination of reproducibility and repeatability (R&R) and analysis of variance for complex measurement system analysis. Regulation diagrams were applied for the graphic statement.

We determined the value of repeatability, reproducibility, coefficient R&R, variability among samples of plasma and total variability of measurement system. The important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in Guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l).

Our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. Aim of the study: To provide Transfusion Services with a tool for proper QMS implementation, an international collaborative study on QMS applications, employing the 'process approach', has been undertaken by a group of Transfusion Services of varying sizes and structures with experience of QMS, in collaboration with a University Institute offering a Master in QMS implementation in Health Services, and an expert from a Quality Association.

The 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. Guide-Lines have been produced based upon this principle and will be published (Volume and CD-ROM) and distributed at no cost to all Transfusion Services of Nations participating in the Study. The main contents of the Guide-Lines' chapters are: Through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the Transfusion Centre's functioning Units completely interdependent, eliminates process interface barriers, provides Personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of Transfusion Service quality, organization and performance through efficient control of processes' interactions.

The Blood Screening System 400 automated high-throughput NAT system for simultaneous screening of HCV, HBV and HIV nucleic acids: full process surveillance E PFEIFER*, B ALESSANDRI † , HR BACHMANN † , T BARKER † , Y OHHASHI*, C PARKHOUSE*, J PINSL-OBER ‡ , P WENZIG ‡ and G ZIEGLER ‡ *Roche Molecular Systems, Inc., Pleasanton, USA, † Roche Instrument Center AG, Rotkreuz, Switzerland, ‡ Roche Diagnostics GmbH, Penzberg, Germany Introduction: The Blood Screening System 400 combines on one deck both automation of DNA/RNA extraction from blood/plasma samples and multiplex PCR amplification and detection of nucleic acid targets. The System is designed for high-throughput single unit testing and pooled specimen processing. A Data Management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. The objective of this project was to present the System 400 assay and device built-in quality control measures that guarantee process safety and reliability. The study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability.

Method: Failure Modes and Effects Criticality Analysis (FMECA) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. This method makes use of a system risk objective-function (SROF), which provides a multivariate description of sample processing, amplification and detection steps. The method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. This provides objective means for directing system design leading to risk minimization.

The SROF responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. The System 400 liquid flow, airpressure-based (pLLD) and capacity-coupled liquid (cLLD) sensors detect aspiration and dispensing inaccuracies. Sensor signal tolerance band widths studied for fluid classes representative of System 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with SROF multivariate modelling. The impact of surveillance design elements on the SROF response demonstrates that risk is functionally dependent on design elements. Additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. Treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. The negative external control (NC) is used to check for contamination of reagents. Five low concentration armored external controls, i.e. HIV-1 group M aRNA, HIV-1 group O aRNA, HIV-2 aRNA, HCV aRNA, and protected HBV DNA are run at the beginning of a batch as a run-control measure. In addition to these Roche controls the System 400 supports running of user-defined external controls for co-validating the batch. The internal control (IC) is based on armored HIV-1 group M RNA that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. Lastly, the System 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes.

The FMECA methodology provides a risk-minimized, comprehensive system design. Redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. The System 400 brings a new level of surveillance and throughput for automated PCR testing, and emphasizes Roche's commitment to increasing the safety of the global blood supply.

Technical standards for safe storage and transport of blood components and blood samples

Objective: To implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. Methods: In the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (SOPs) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with EU Directives 2002/98/EC and 2004/33/EC and the recommendations of the Council of Europe and WHO. Procedures are validated and relevant records are kept. A statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation.

Blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, HLA-typed units, anti-CMV negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. Packing and transport conditions of red cells, platelets and FFP are submitted to the tests and criteria of ADR (European Agreement Concerning the International Carriage of Dangerous Goods by Road). In Greece, the ADR legal framework has applied since 1999. Blood samples are classified according to ADR in division 6.2 under UN 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. Our blood establishment has a contract with BIOTRANS, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. Assurance System). The approach is specified in the form of requirements of art. 4.1 of the Standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. Regional Centre for Transfusion Medicine in Biaĺystok was the first transfusion service in Poland which was certified according to ISO 9001:2000 Standard. Our expected profits from the implementation QMS were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification.

Our one year of experience with ISO 9001:2000 Standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. The implementation and certification of the internationally nor-malized Quality Management System simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. The implementation of QMS facilitates the implementation of other quality systems and simplifies procedures required for the CE certification for the products. Red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. The ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. The chemical alterations are mainly changes in levels of Na+, K+, Cl-concentrations, pH and 2,3 DPG levels and they affect the electrical impedance of blood. The electrical impedance, Cole-Cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (Re), the resistance of the intracellular fluid (Ri) and the capacitance of the cell membranes (Cm).

In this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. All parameters were measured on 51 erythrocyte suspension (ES) samples during 42 days of storage at 4oC on days 0, 10, 21, 35 and 42. For 31 whole blood (WB) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. The measurement of the complex impedance of blood samples were performed in the frequency range from 100 kHz to 1 MHz. By using the 4284A HP LCR Meter, the impedance Z, and the phase angle a for each sample were read. These values were corrected according to the gain and phase characteristics of the amplifier; and the resistance R and the reactance X were calculated. Each data was first fit to the Cole-Cole model; hence the Cole-Cole parameters, intracellular resistance Ri, extracellular resistance Re, characteristic frequency Fc and phase angle a were obtained by using LMS software. Afterwards, Cm was calculated by using Ri, Re, a and Fc. Whereas Ri and Cm decreased progressively with time on both WB and ES, Re changes showed some differences. The electrical impedance alterations were explained by measurements of Na+, K+, Clconcentrations, pH and 2,3 DPG by indicating days. Storage of red cells resulted in a rise in extracellular K+ and a fall in extracellular Na+, Cl-, pH and 2,3 DPG. ANOVA was used to evaluate differences in blood measures in relation to storage time. The results were presented as the mean ± SD. According to the regression analysis in SPSS, the intracellular resistance (Ri) on both ES and WB was affected more efficiently from all blood parameters among all other electrical parameters. Although Ri and Re were correlated with Na+, K+, Cl-, pH and 2,3 DPG more significantly, Cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and Fc. The best relationship between the parameters mentioned above on both ES and WB was Ri and K+. pH changes were the same for Ri and Re both for ES and WB. The correlation between parameters on ES was better than those on WB, because whole blood consists of several particles that may affect our measurements. Our study showed that 2,3 DPG has an effect on Ri and Re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications.

Background: Issue of quality blood products and donor safety are the main aims of blood transfusion services. A comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (SOPs). The Drugs and Cosmetics Act of India, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. Aim: We therefore established our own SOP and operational flow chart for plateletpheresis that can be easily followed by other centers in India. Methods: A total of 100 plateletpheresis procedures performed using two cell separators (CS3000 Baxter, USA, MCS3p, Hemonetics, USA) were evaluated following our established SOP.

The mean platelet yield in CS3000 was 2.9 ± 0.84 ¥ 10 11 and in MCS3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of SDPs showed WBC levels <5 ¥ 10 6 . Six of 100 donors complained of hypocalcemic symptoms. The operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country.

The first advanced Quality Management Training course for blood transfusion services in the Western Pacific Region MK TAN*, JP YU † and D TEO* *Health Sciences Authority, Singapore, † WHO Regional Office for WPR, Manila, Singapore

Background: Blood transfusion is a key part of modern medicine. A well-organised Blood Transfusion Service (BTS) is a prerequisite for the safe and effective use of blood and blood products. In 2000, the World Health Organization (WHO) introduced a new initiative of Quality Management Project (QMP) to achieve the goal of safe and adequate global supply of blood. Through QMP, regional training centers were identified and Quality Management Training (QMT) courses were established. In 2001, Centre for Transfusion Medicine (CTM) of Health Sciences Authority Singapore was appointed as a collaborating center for QMT in the Western Pacific Region (WPR 

Background: Spectrophotometric method according to Harboe was traditionally used at our Institute for determination of free haemoglobin in supernatants of RBC blood components at the end of storage time.

In the year 2002 HemoCue Plasma Low Hb system (HemoCue, Sweden) was introduced as a replacement method. Aim: The aim of the study was to examine reliability and suitability of the HemoCue method for measurement of free haemoglobin in supernatants of RCCs. Methods: HemoCue plasma Low Hb method was validated and compared with Harboe method. Supernatants of 36 RBC products were tested by two methods and results compared using regression analysis. Additional testing was performed to investigate precision of HemoCue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling.

Results: Regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with HemoCue method. Immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. Coefficient of variation (CV) calculated from 6 consecutive measurements was 6.1%. To investigate between-day imprecision of the HemoCue method one sample was divided in 5 aliquots and frozen. One sample was thawed each day and measured. CV of five measurements was 1.95%. During the period of validation 37 measurements of reference material (low, medium and high) were performed. CV calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). In order to investigate possibility of batch testing, supernatants of 5 different RBC products were divided in aliquots and measured periodically during 1-month period. CVs calculated from 7 measurements of each sample were in range 3.1-9.9%. Conclusion: HemoCue Plasma Low Hb method appears to be an excellent replacement for the Harboe method. It is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations.

Background: Determination of haemolysis in red cell concentrates (RCCs) at the end of storage time is routine method in quality control of blood components at our Institute. For this purpose, haemoglobin concentration in supernatant of RCCs is measured using HemoCue Plasma/Low Hb system (HemoCue, Sweden). According to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. Because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. Aim: The aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. Methods: Haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using HemoCue Plasma/Low Hb photometer (7 WB Negative controls were used for each blood component.

In all blood components tested bacterial contamination was detected using both types of bottles. In comparison with SA/SN bottles, nearly all enrolled microorganisms were detected faster in BPA/BPN bottles (see Table 1 ). All negative controls were negative (no false positive).

The results of the study performed support the use of BacT/Alert BPA and BPN plastic bottles in quality control testing of different blood components.

Objective: Management of returned blood products is important part of quality assurance activities in transfusion medicine. Blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received.

Methods: QA data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . Only blood products returned because of nonconformities were taken into consideration. Data about the reasons for returns are presented and discussed.

The top 4 reasons for returns were positive DAT, labelling errors, blood bag defects and visual appearance of blood units (see Table 1 ). In all cases positive DAT was confirmed in our Institute, and blood donors managed accordingly. Nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. In case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). Conclusion: Management of returned blood products is important tool in improving the quality of blood products. 

Introduction: Hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. Attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. Registration of the impact of all participants is a tremendous job as such. Information management may enhance the chances for a successful hemovigilance. Aim: The aim is, to establish a basis for all the information related to the chain, such as Standard Operating Procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. Additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. By clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times.

The primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. Secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. By creating distinct databases for these 6 'W's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. All the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format.

Collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. It establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. It allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. In addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. Conclusion: By outlining the process, followed by defining, analysing, securing, on the 'W6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components.

Background: Transfusion Medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in Bulgaria.

In order to improve the post-graduate training of medical doctors in Transfusion Medicine, a new curriculum was elaborated in 2004. Purpose of the new program: independent work of transfusion medicine specialists on all levels of the Blood Transfusion Service (BTS) -organization of BTS, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching Transfusion Medicine to BTS and clinical personnel. Admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. The overall duration of training is 4 years, of which at least 2 are obligatory for training in the National or Regional Blood Transfusion Centers. Main sections of the curriculum:

1. Organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. Collection, processing, storage and transport of blood and blood components 3. Laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, NAT techniques, flowcytometry) 4. Clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion).

The new curriculum of Transfusion Medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use.

Introduction: Transfusion medicine is integrated medical discipline based on clinical and laboratory practice. It is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. The aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the Medical Faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. Material and methods: There will be presented how transfusion medicine is implemented in educational and health sector in R.

Results: There is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). We have postgraduate studies in TM started 15 years ago. Transfusiology, as subject at the Medical Faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. Also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in TM. Transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in TM and get certificate. Conclusion: Although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines.

Introduction: In hospitals of Saint Petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. The service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. It demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. Clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). Clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. Employment are carried out in departments of city blood bank, hospitals blood departments. Final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). The described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. Conclusion: The existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff.

Introduction: Nowadays in hospitals of Saint-Petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. They are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. For this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. Therefore an actual problem is organization of postgraduate special education for them. Because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. Aim: To give basic knowledge and practice to those general practitioners MoH has established a special training programme with the collaboration of medical schools for 5 years.

Methods: This is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. The trainees are evaluated by a final examination. Each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and MoH training hospitals. Conclusion: Almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals.

Background: Even the history of public blood banking in Turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in Turkey until the last few years. Aim: The main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice.

Methods: There were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in Turkey until the last few years. Blood Banks and Transfusion Society of Turkey (BBTST) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. BBTS has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. Due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. In most of the university hospitals and training hospitals this is around 19%. Conclusion: Like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in Turkey by the direct affect of well organized educational activities.

Training of general practitioners in blood banking and transfusion medicine N SOLAZ*, B KESKINKILIC † and C ORUC † *Ankara University, Ankara, † Ministry of Health, Ankara, Turkey

Background: Turkish Ministry of Health runs majority of the hospital blood banks in Turkey. Most of the MoH hospital blood banks give service under the medical supervision of general practitioners. Because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. Aim: To give basic knowledge and practice to those general practitioners MoH has established a special training programme with the collaboration of medical schools for 5 years.

Methods: This is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. The trainees are evaluated by a final examination. Each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and MoH training hospitals. Conclusion: Almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals.

Background: It has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. The lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (MBL). The level of MBL in serum shows a wide variation. A low level of MBL can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. The effect of transfusions of erythrocyteconcentrates and plasma on the MBL levels and thereby on complications after cardiac surgery are not known. Aim of the study: The role of pre-and postoperative MBL levels and the effect of transfusions on postoperative complications after cardiac surgery.

In a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. MBL measurements were performed by ELISA assays. The data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-MODS and with peri-operative erythrocyte and plasma transfusions. Results: The mean pre-operative MBL-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. There were no differences in preand post-operative MBL levels between patients with infections or MODS compared with non-infected and non-MODS patients. The difference in pre-operative MBL between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (P = 0.20). Postoperative MBL levels between survived and non-survived patients was smaller. Conclusions: pre-and postoperative MBL levels in cardiac surgery are not related to postoperative infections, MODS and hospitalmortality. Whether large amount of blood transfusions are affecting the MBL-levels and thereby the patients outcome is not known.

Introduction: Patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and Multiple-Organ-Dysfunction-Syndrome (MODS), these complications are influencing the survival of the patients. During cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. We found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to MODS by transfusion of leukocytedepleted erythrocyte concentrates (LD) compared to leukocyte-containing, buffy-coat depleted erythrocytes (PC). Aim of the study: The effect of LD on the concentration of proinflammatory cytokine Il-12 and anti-inflammatory cytokine IL-10 in relation to clinical outcome and complications after cardiac surgery. Methods: In 474 participating patients blood samples were taken before and after surgery. Using ELISA IL-10 and IL-12 were measured in these samples. The results were linked with the endpoints of the randomized trial: postoperative infections, MODS and in-hospital mortality. Results: All pre-operative concentrations of IL-10 and IL-12 were low. Mean postoperative level for IL-10 was 70 ± 121 and for IL-12 57 ± 74. Compared with patients without MODS and without infections and survived patients only the IL-10 was significant higher in patients who died and in patients with MODS. There were no differences in mean levels related to LD and PC. The levels of IL-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. Conclusion: There is an association between MODS and mortality and IL-10, not with IL-12. LD has no influence on mean levels of IL-10 and IL-12. There is a correlation between transfusion of more then 10 units and IL-10, not with IL-12. These cytokine-profiles are not associated with the beneficial effect of LD on the postoperative outcome in cardiac surgery patients.

Anemia and blood transfusion practice in critically ill patients E GROUZI*, E TSIGOU*, P EVAGELOPOULOU † , G BALTOPOULOS † and I SPILIOTOPOULOU* *KAT General Hospital, Transfusion Service, Athens, † Athens University School of Nursing ICU, Athens, Greece Background: Anemia is a common problem in critically ill patients admitted to intensive Care Unit (ICU), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. Aim: To define the incidence of anemia and red blood cell (RBC) transfusion practice in critically ill patients in the ICU) of our hospital, and to examine the relationship of anemia and RBC transfusion to clinical outcome. Patients and methods: The period study was from July 2003 to December 2004. Patients were enrolled within 48 h of ICU admission. Follow-up time was 30 days, hospital discharge or death, whichever occurred first. Results: A total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. The mean hemoglobin (Hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. Overall 63.9% (108/169) of the patients received one or more RBC units while in the ICU stay (mean 3.1 ± 3.8 units per patients). The mean pretransfusion Hb was 8.7 ± 1.8 g/dl and the mean time to first ICU transfusion was 2.6 ± 4.1 days. More RBC transfusions were given in the first week of the ICU stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). The number of RBC units which a patient received during the study was positive associated with longer ICU length of stay and an increase in mortality (r = 0.19, P < 0.05 and r = 0.26, P < 0.05 respectively). Baseline Hb level was significantly related to the number of RBC transfusion (r = 0.55, P < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, P > 0.05 and r = 0.13, P > 0.05 respectively). The mean baseline APACHE II and SAPS scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. Furthermore both baseline APACHE II and SAPS scores were significantly higher for patients with a baseline Hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the APACHE II values were positive associated with a significantly increased likelihood of RBC transfusion (Pearson Correlation P < 0.005). Conclusions: Anemia is common in the critically ill patients, it appears early in the ICU course and persists throughout the duration of the ICU stay. RBC transfusion seems to be associated with worse clinical outcome. Despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. The results of our study suggest that approaches to reduce RBC transfusion would be desirable. However, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion Hb threshold and the risk and benefit of RBC transfusion in the critically ill.

Consumption of blood products in a large, general hospital HE HEIER*, J PILLGRAM-LARSEN † , M HESTNES ‡ , B GRAN*, A KROG* and NO SKAGA ‡ *Ullevaal University Hospital, Oslo, † Ullevaal University Hospital, Dept. of Thoracic Surgery, Oslo, ‡ Ullevaal University Hospital, Dept. of Anestesiology, Oslo, Norway UUH is the largest general hospital in Norway, and trauma referral centre for half of the Norwegian population (2.5 mill) The trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. The aim of our study was to analyze transfusion practice at UUH with specific focus on trauma. Materials and methods: Blood consumption during this period was recorded. Clinical data of trauma patients were collected from our Trauma Registry and anonymized before analysis. Results: 7012 units of erythrocytes (ER), 914 of thrombocytes (THR) (buffy coat preparations from 4 donors) and 903 of S/D-treated whole plasma (OP) (OctaplasÒ) were transfused in UUH during this period. 56.6% of ER were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of ER were given to patients above 45 years. ER units were given to 1475 patients (mean 4.75 units/patient; range 1-63). Mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. For transfusion of this group local guidelines state that haemodynamically unstable patients should receive ER if hgb <9 g/dl, irrespective of age and sex. Eighty-eight patients (27.8%) received ER, 15 of them as massive transfusion ( 3 10 ER units in <24 hours) (17.0%), 6 of these died (40%). Altogether trauma patients received 851 units of ER (12.1% of total UUH consumption), 51.5 THR (6.1% of total) and 150 units of OP (16.5% of total). Massive transfusion consumed 340 ER (40%), 29.5 THR (57.3%) and 110 OP (73.3%) units. Fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 ER units only. Lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. Five patients received ER transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 ER were given because of a hyperacute clinical situation. Fifty-five% of issued ER units had been stored for >15 days, while only 10% were issued before 10 days of storage. Discussion: Life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. Transfusion practice in trauma at UUH seems fairly well in accordance with internationally accepted guidelines. The trauma unit consumed a surprisingly small part of total UUH transfusion resources. Trauma patients deviate from the general UUH patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. Further analysis is needed on transfusion indications and results to optimize the total use of blood products in UUH. Special focus should be on transfusion to non-bleeding patients without haematological disease. It seems that only a small part of transfusion efforts results in the saving of life.

In Croatia national guidelines for the use of RHD gamma globulin were laid down in the year 2000. In accordance with the guidelines, RHD gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to RHD negative women after delivery of RH positive children, after abortions, in Week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. As to the latter, detection and measurement of fetomaternal hemorrhage are recommended. Three years after the issuance of the guidelines a survey was conducted regarding the use of RHD gamma globulin that involved 33 health institutions across the country. As many as 38 601 deliveries and 10 974 abortions were reported in 2003. The institutions reported the usage of 5103 doses of 250 mg RHD gamma globulin or 110 doses/1000 deliveries + abortions. We compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. In that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of RHD gamma globulin or 100 doses/1000 deliveries + abortions were administered. Institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. The range of the consumption of RHD gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. The number of pregnancies which should have been protected with RHD immunization in 2003 year was obtained by the following formula: (frequency of RHD negative subjects) ¥ (frequency of the R gene) = 0.17 ¥ 0.59 = 0.10. If a complete antenatal and postnatal preventive measures involving RHD immunization had been taken in 2003, 12 287 doses of RHD gamma globulin or 250 mg/1000 deliveries + abortions should have been given. Our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the RHD immunization in pregnant women is still not adequate in Croatia. In fact, it has not improved since a year prior to the adoption of the guidelines. It has also been established that the category of the institution is a factor influencing the implementation of the protective measures. We consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the RHD immunization.

Consumption of plasma derivates in Croatia G JAKLIN*, B GOLUBIC-CEPULIC † and M DONDUR* *General Hospital, Varazdin, † Clinical Hospital Center Zagreb, Zagreb, Croatia A increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that Croatia is faced with nowadays, as well as many other countries. In 2003 a study was carried out regarding the usage of plasma derivates. As many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. The data regarding the use of plasma derivates were collected as follows: albumin, I.V. gamma globulin, and concentrate of coagulation factor VIII in two periods of time. We divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. Institutional practice patterns regarding the use plasma derivates were compared among them. The parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. Data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. We compared our data with similar study carried out in 1997. The consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. In 1997 in Croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. The consumption of I.V. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. In 1997 in Croatia the consumption of I.V. gamma globulin was 22.20 kg or 4.93/million inhabitants. The consumption of the concentrate of factor VIII was 8807.000 IU or 1.96 IU per inhabitant in 2003. 1539.500 IU, i.e. 17.48%, was a recombinant factor VIII. In 1997 the consumption of factor VIII was 4876.350 IU or 1.08 IU per inhabitant. Discussion: The use of albumin showed stagnation. However, the use of I.V. gamma globulin increased 2.6 times and the use of the concentrate of factor VIII increased 1.8 times if compared with the results in 1997. Self-sufficiency has not been reached in plasma derivates in Croatia We needed 20 000 L plasma for gamma globulin and 58 000 L plasma for concentrate of factor VIII for the level of usage of these plasma derivates in 2003. Significant differences in the level of usage among hospitals have been observed. These reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. Therefore, we would recommend that the National Society sets out guidelines for the use of the products.

Background: Blood bank good practice requires avoidance of Rh alloimmunization. Several studies report a probability of immunization of more than 80% following transfusion with RhD+ RBC's and as many as 19% in the case of platelets. Aims: The purpose of this study was to investigate the development of anti-D and the causes of alloimmunization in a University Hospital (1300 beds), reviewing our practice on the use of RhD+ blood components in RhD-recipients. Method: A retrospective study was performed whereby 15.872 RhD-patients, from 1990 to 2004, were evaluated for the development of anti-D analysing the data on the blood bank information system. Results: From the 15.872 RhD-patients we found out 274 identified anti-D, 77% (211) detected before any transfusion in our hospital. Of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to RhD+ blood components: 12 were transfused only with RhD+ platelets (including two women of childbearing age); 24 with RhD + RBC's. The remaining 13 had been exposed exclusively to RhD -RBC's suggesting that some units could be mistyped. This idea was corroborated in three patients where there was a common donor (he was called for investigation). Conclusions: Transfusion services avoid as far as possible administration of RhD+ blood components to RhD-patients, although there are situations in which such transfusions are necessary. The retrospective review of records revealed the need for specific recommendations regarding the use of Rh immunoglobulin to prevent anti-D immunization. The possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks.

The purpose of the work: To present the usage of whole blood (WB) and blood products (BP) in the treatment of patients who are hospitalized in the internal disease department in Gevgelija. Material and methods: Retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . Statistical methods which were taken from the history of the hospitalized patients in the Internal Department were used for analysis and the data of transfused WB units and units of BP were taken from the Department of transfusion medicine. Results: From the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received WB and BP, and there have been transfused 1411 units WB and BP. Every patient, on an average, has been transfused with 1.86 units WB and BP. At the same time 326 (23.1%) units have been transfused as a WB, 928 (65.77%) have been transfused as red blood cells (RBCs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (FFP). The data for every year particularly will be presented in our paper to the Congress. 

The collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. Our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of WB and RBCs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. The necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the Department of transfusion medicine.

Femoral neck fracture repair is one of the commonest orthopedic procedures. It mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. The aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at Katerini General Hospital during 2002-2003 and to compare them with other studies. One hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. Seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. The mean Hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean Hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). A total of 297 units of RBC were transfused (a mean of 1.8 units per patient). Blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). Patients with preoperative Hb values <10 g/dl were transfused more often than those with Hb values >10 g/dl (100% versus 73%) P: 0.017. Women were transfused more often men (80.2% versus 65.3%) P: 0.042. Patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) P: 0.008. Finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) P: 0.001. Conclusions: In our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. Blood transfusion frequency is greater in patients with Hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair.

Fresh frozen plasma guidelines and practices AS SALTAMAVROS*, G TALAMPOUKA*, C KOUMOUNDOUROU*, S DIMITRAKOPOULOS † and P TSELIOU* *St. Andrews Hospital Patras Greece, Patras, † Pyrgos General Hospital, Pyrgos, Greece Introduction: Fresh frozen plasma transfusions should be done according to specific standards and guidelines. Aims: We have reviewed the FFP transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of FFP transfusions. Methods: Retrospective review of FFP transfusions practices during the entire year of 2004. We classified transfusion practices according to the diagnosis of the patient and also of the Clinical depart-ment that demanded transfusion. Then we analyzed our results by comparing the requests with the internationally approved guidelines for FFP transfusion and counted the percentage of FFP to RBC units. Finally we discussed with our fellow physicians the proper use of FFP and informed them about the accepted guidelines. As we can see from the underneath table, diagnoses and departments with high consumption were: Internal Medicine 22%, Plasmapheresis to patients suffering from Guillain Barre and TTP (Thrombotic Thrombopenic purpura) 21.04% and Trauma/Surgery 20.68%, Cancer 13.60%. Summary/conclusions: The use of FFP corresponds to 27.88% of the RBC units transfused in our hospital. Plasma units should be used properly and according to internationally accepted standards. Plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care.

To show gradual discontinuation of using whole blood and increased use of red blood cells (RBC) in the management of patients in the Transfusion Department of the Clinical Hospital 'Zvezdara' . Methods: Data of WB and RBC use from 1982 to 2004 in our hospital. Results: Our data are presented in Table 1 . Conclusion: After 1982, WB usage rates systematically decreased from 51% to 1.3%. However, in the period of 1991-2004, the WB usage rates increased slightly, and we arrived at a generally very low rate of WB use, namely only in about 3% of the cases. Education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. Therefore, we started an education program and we established a Hospital Transfusion Committee (HTC) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. Members of the HTC are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. We have been organizing courses for the medical staff, doctors and medical technicians since 1995. Background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. Currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. By free oscillating rheometry (FOR), using a ReoRox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. Aim of the study: The aim of this study was to find out if FOR using the ReoRox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. Methods: The change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the ReoRox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). The effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with FOR (n = 8). The change in elasticity per minute (G'max slope) and maximum elasticity (G'max) were evaluated from the elasticity curves. Results: The G'max, G'max slope and platelet count increased after transfusion for all patients. The thrombocytopenic patients had significantly lower G'max and G'max slope values both pre and post transfusion compared with healthy control subjects (P < 0.01). The measurement in platelet rich plasma showed that G'max and G'max slope increased with increasing platelet concentration. However patients with similar platelet count developed different clot elasticity.

The ReoRox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. The fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. The FOR method seems promising to evaluate platelet function.

Quality control of pre-storage leukodepleted red cell concentrates VU URLEP SALINOVIC, K PERBIL LAZIC and L LOKAR Teaching Hospital Maribor, Maribor, Slovenia Background:

The system of quality assurance including quality control (QC) in transfusion medicine is most important for safe blood supply. The safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (WB). Aim: The QC of leukodepleted red cell concentrates (RCC), prepared from pre-storage filtration of WB (quadruple blood bag systems IMUFLEX WB-RP Terumo) is performed with the aim to be sure that the quality of leukodepleted RCC is in accordance with the Guidelines of Council of Europe.

In three years (2002) (2003) (2004) , 35 559 units of WB were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these QC was done. Within 4 hours after collection, WB was filtered at room temperature. Filtered WB was centrifuged at 4000 g for 20 minutes and separated in RCC and plasma. The samples for QC were collected before and after filtration. For each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (WBC) count were measured. The number of residual WBC after filtration was determined in the Nageotte chambre. For all parameters the mean value and standard deviation were calculated.

The results of QC for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the Guidelines of Council of Europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. During filtration of WB, WBC were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%.

The pre-storage filtration of WB is highly efficient, 99.99% of WBC were removed and the loss of hemoglobin was small. Background: The patients of cardiosurgery use big amount of blood components. There is no uniform approach by treatment with blood components in these patients. The safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. Aim: The aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004.

The data about use of red cell concentrates (RCC), platelet concentrates (PC) and fresh frozen plasma (FFP) in the period from 1998 to 2004 were collected from information system Datec. The average use of all three blood components per patient is presented. We were interested, if the average use per patient was diminished in the analysed period. Results: The use of three blood components and average use of all blood components are presented in the table. The data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. Conclusion: During seven years period the use of blood components per patient in cardiosurgery was not diminished. For more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. Background: The presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as NHFPTRs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (CMV, HTLV). The removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. This is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. Aim: To show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced Er. Concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our Daily transfusion hospital at Medical center -Stip. Methods: 123 patients with malignant diseases have been treated in our Daily transfusion hospital in the last four years. Most of these patients suffer from Ca pulmonum, Ca collonis, Ca uteri, Ca mammae. Also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced Er. concentrates. The blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. The blood was collected in Baxter and Terumo bags, and it was filtrated with Baxter -Sepacell RS -2000 and Pall -purecell RN filters. The analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. The analyses samples were taken from the tubing system before and after the filter. Haemathologic parameters were automatically made in the Central Clinic Laboratory. Results: Er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. With the routine monitoring of all patients none posttransfusion reactions were registered. The therapeutic effect is also important because of the fact that the number of Er and the level of Hg and Hct remain almost unchanged.

The aim of the Blood banks is to help and to increase the safety of the blood components. The leucoreduction through of Er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. The time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. We have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration.

Background: The effect of leukocyte reduction of RBCs by filtration has been the topic of several RCTs. These RCTs investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. Some RCTs came up with answers that conflicted with the results of other RCTs. Aim of the study: With including the individual patient datasets of several RCTs in one database, combined analyses are possible that may explain the differences in the reported results. Using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these RCTs. Methods: We coordinated several RCTs comparing the perioperative use of buffy-coat depleted RBCs with the use of filtered RBCs. Cardiac surgery patients were included in three RCTs (1¥ CABG and/or valve; 1¥ re-CABG and/or valve; 1¥ valve with or without CABG). Oncologic surgery patients were also included in three RCTs (1¥ colorectal cancer; 1¥ GI-oncology or vascular surgery; 1¥ GI-oncology, vascular surgery or orthopedic surgery).

The electronic data files of these RCTs were uniformly recoded and entered in a single database. As different primary endpoints were investigated in the RCTs, we focused on common endpoints, recorded in 4 or more of the RCTs. Multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on ICU, postoperative infections, and MODS. Results: In the RCTs, individual datasets from 3875 surgery patients were collected (1630 Onco; 1272 Cardiac; 569 Vascular; 352 Orthopedic, 52 other). In the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (P = 0.01) and at 30 days post-surgery (P = 0.03). No association between randomization and stay in hospital (P = 0.10) or stay on ICU (P = 0.56) was seen in the combined study population. Also, no association of randomization with the incidence or duration of MODS was seen. The analyses of post-operative infections in the total population showed the trial arm to be associated (P = 0.016), and 'hospital' to be far stronger associated (P < 0.001). However, when the oldest RCT, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (P = 0.34) and the trial arm became more strongly associated with infections (P = 0.002).

In the analyses of 3875 surgery patients, the use of filtered RBCs, compared to the use of buffy-coat depleted RBCs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. The association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. However, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost.

Background/aims: UK NEQAS for blood transfusion laboratory practice (BTLP) operates an EQA service for 472 UK laboratories (including Eire) and participants throughout Europe, including major groups in Denmark (n = 52) and Portugal (n = 32). In November 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (PMFs). Results were analysed by country to establish any variation in practice relating to the selection of K negative (K-) and Rhc negative (c-) blood, since antibodies to these antigens are now the major cause of HDN. Results: Overall return rate was 84% (UK) and 64% (non-UK), although only centres treating PMFs were included in this analysis. K-blood was selected for PMFs by 81% in Wales (n = 16) and 71% in England (n = 291), but only 6% in Scotland (n = 32), 8% in Northern Ireland (n = 12) and 10% in Eire (n = 31). In mainland Europe, variation was also observed: 12% in Denmark (n = 17), 75% in Portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). Fewer laboratories selected c-blood for PMFs: 8% in England and Northern Ireland, 0% in Scotland and Eire, rising to 81% in Wales. Mainland Europe showed similar variation: 12% in Denmark, 75% in Portugal (all CcEe matched) and 51% in other countries. There are no guidelines requiring selection of K-and c-blood for PMFs, but in the UK D negative blood is required for D negative females aged <60 years. Trend analysis of questionnaire data in the UK, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select K + blood for a 30 year old female (with pre-existing antibodies other than anti-K), from 58% (1996), 42% (1999) to 26% in 2004. In this survey, K + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a K + unit for a female aged 54 (no antibodies), despite this patient being treated as a PMF for provision of D negative blood. A similar distinction was made between the 30 and 54 year old females in Portugal and mainland Europe. Conclusions: Variation in practice may at least in part be due to the availability of blood routinely labelled for Rh and K. In the UK all donations are labelled, except for those from new donors, as are most units in Portugal. UK questionnaire data (1999) suggested that >50% laboratories would change to selecting K-blood for PMFs if all units were labelled. However, labelling alone does not account for the differences seen within the UK, and perhaps the trend towards providing K-(and to a lesser extent c-) blood for PMFs is influenced by advice from transfusion services. It would be of great interest to monitor and compare the incidence of HDN due to anti-K and anti-c in different countries to measure the outcome of differing practices for provision of blood to PMFs and to thereby inform future policy. There is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. Current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. Objective: We hypothesized that the quantitative measurement of GPIb expression by FACS can be used to predict the outcome of platelet survival post transfusion.

In our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (Blood Dec-2004) . We isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with PMA-matured THP-1 cells (37°C). Binding was measured by FACS analysis of CD42b/CD14 positive particles, and phagocytosis by counting mepacrine/CD14 positive particles. We measured GPIb expression before and after plt-macrophages interaction by FACS flowcytometry. Results: GPIb expression at surface of C22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. The GPIb expression at surface of plt decreased and showed three populations with different densities high (GPIb-1), low (GPIb-2) and in-between (GPIb-3). The binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to GPIb-1, and direct relation to GPIb-2 expression. Anti-human Pselectin (CD62p) delayed 45 ± 10% the binding and annexing V 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. These results show that GPIb expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion.

Transfusion and lung injury JD DODIG*, D SOVIC † , M TOMICIC † and H SAGER* *University hospital 'Sestre milosrdnice', Zagreb, † Institute for Transfusion Med, Zagreb, Croatia

Background: The respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. In recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. Case report: A 59-year-old men was admitted due to chronic macrohematuria. He had no history or current evidence of cardiac failure. The Hb level was measured 36 g/L. He received 500 ml 0.9% NaCl and 500 ml Ringer solutions. After that, he was transfused with 2 units of RBCs. During transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. Massive pulmonary edema was noted. He was treated with mechanical ventilation, oxygen, diuretics, Aminophillin, antihistaminic and corticosteroides. The patient recovered after being on ventilation for 6 hours. Two days after the patient was operated. After surgery he was transfused with 4 units of RBCs. All transfusions were regularly performed. Results: Chest X-ray confirmed bilaterally pulmonary edema. The samples (patient and donors of first two units RBCs) tested were negative for the presence of HLA specific and granulocyte antibodies. Granulocyte agglutination and lymphocytotoxicity test were negative. TNF-a in recipient serum was slightly increased. Conclusion: Our patient is the first reported case suspected of TRALI, but all of the investigations didn't give us the answer.

against neutrophils using flowcytometric detection of cell surface CD11b expression and L-selectin shedding. Methods: A hundred microL of volunteers' heparinized whole blood were incubated for 15 minutes at 37°C with fMLP, LPS or PMA. Monoclonal antibodies against HNA1a and HLA-I, or patient serum were incubated with whole blood for 30 min. Surface CD11b and L-selectin were detected using FACSCalibur. Results: Neutrophil activation was detected after fMLP, LPS or PMA stimulation. P38 MAP kinase inhibitor reduced activation induced by fMLP and LPS. Anti-HNA1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 MAP kinase inhibitor. Ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. Five sera out of 8 samples having anti-neutriphil and/or HLA antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. Conclusion: These findings indicate that neutrophils activation is regulated through MAK kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. Results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. Post transfusion purpura or suspected TRALI was not seen. Fatal complication was not seen. The reactions to plasma were predominately anaphylactic. We detected no case of bacterial contamination among the cultures of transfusion bags. Conclusions: The incidence of reactions among patients malignancy was high, while among surgical patients was lower. The incidence of transfusion reactions during the months had no statistically significant difference. We suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications.

Measurements of IGE immunoglobulin in thalassemic patients, before and after blood transfusion Background: Many studies have reported the implications of proinflammatory cytokines including interleukin (IL)-1 beta, IL-8 and tumor necrosis factor alpha (TNF-alpha) in febrile nonhemolytic transfusion reactions. IL-8 has been shown to accumulate in packed RBCs even after the procedure of filtration, which is explained by the release of IL-8 from RBC receptors into the packed RBC super-natant. Stress induced elevation in TNF-alpha levels was demonstrated in healthy individuals. Aim: To assess the level of proinflammatory cytokines in peripheral blood of blood donors. Material and methods: Immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°C. Upon thawing, the level of the IL-1 beta, IL-8 and TNF-alpha cytokines was determined in plasma samples by ELISA method using commercial kits for cytokine determination (Roche Molecular Biochemicals).

The level of IL-1 beta was at the test detection limit in all donor plasma samples. In 1 (1.3%) BD, the level of IL-8 was 36.8 pg/mL, exceeding the test sensitivity limit of 6.2 pg/mL. In 12 (16.6%) BD, the level of TNF-alpha was within the range of 14-60 pg/mL, with a test sensitivity limit of 12 pg/mL. TNF-alpha levels >800 pg/mL were measured in plasma samples of 3 (4.1%) blood donors.

The increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines IL-8 and TNF-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. This hypothesis will be thoroughly investigated in our future studies.

Background: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. Aim: We report here a 'probable' case of TRALI syndrome in an elderly female patient. Case presentation: An eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (POD) following the abrupt onset of acute pulmonary insufficiency (paO 2 = 49 mmHg, O 2 saturation = 79%), hypotension (BP = 90/50 mmHg) and fever (38°C). This occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (PRC). The patient had no history of any cardiac or pulmonary disease. She was intubated and placed on mechanical respiration and supported hemodynamically. The CVP (12 cm H 2 O) ruled out fluid overload. The chest X-ray revealed bilateral pulmonary oedema, the ECHO cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. After treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th POD. Reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of FFP (fresh frozen plasma) five hours prior to the incident. The donor review revealed that the unit of FFP was from a 29-year old female with a history of multiple abortions whereas the PRC unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. There are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. Methods: Traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. Cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. Homogeneity of reporting is achieved by the introduction of unique reporting form. A reporting route was defined: patients physician sends notification of ATR to the local blood transfusion service. ATR reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the Centre for haemovigilance, which is going to be established very soon. Data analysis will be responsibility of the Centre for haemovigilance at the governmental level. Type of adverse transfusion reactions and events is defined. Education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. Results: In the years 2002-2004 there were 410.000 blood components issued in Slovenia and 333 ATRs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). In the year 2004 there were considerable differences between the number of ATR reports compared to the number of blood components issued among Slovenian hospitals, ranging from 1 in 3375 to 1 ATR in 490 blood components issued. 9.6% of all ATR were not classified. Conclusions: Although the number of reported ATRs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. Feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of ATRs and events in the future. The Slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . Considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in Iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. Six healthy Iran's native cow, 2.5 years old, with average weight of 210 kg were used. The animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. Three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. After that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. Cross matching was done at each transfusion. After each transfusion the research parameters do determined. Results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. This is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( Van der Valt, et al. (1969) Background and objectives: Blood transfusion may lead to the manifestation of anti-HLA and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. It appears that the study of antibodies against HLA-Class I and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. The aim of this study was to detect anti-HLA and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including Acute Leukemia, Aplastic Anemia) and patients with ITP.

In this descriptive study, anti-HLA and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with ITP. The results of anti-HLA antibodies were then compared by Panel Reactive Antibodies (PRA). Results: Our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-HLA Class-I antibodies in their sera. The frequency of each antibody isotype was found to be as follows: IgM (51.2%), IgG (32.9%) and IgA (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: IgM (40.2%), IgG (30.5%) and IgA (12.2%). 27 (31.7%) out of 82 patients had both antibodies. No difference was found between the two groups in platelet specific antibodies. Despite significant correlation between flowcytometry and PRA methods, PRA can only detect antibodies which react with complement.

Conclusions: With increase in the number of platelet transfusion, immunization to HLA antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. The presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. Conducting similar studies with higher number of samples, platelet cross-match, and the use of HLAmatched platelets for these patients are recommended.

Post transfusion purpura (PTP) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. In this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. The disease is rare occurring mainly in multiparous women. The majority of reported cases involved antibodies against the platelet specific alloantigen HPA-1a in a homozygous HPA-1b patient. In a minority of cases the offending antibodies were directed against HPA-1b, -3a, -3b, 4a, -5a, -5b. Although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. PTP is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. We present three patients with severe thrombocytopenia initially misdiagnosed. The first patient, a 54-year old women, had a past history of systemic lupus erythematosus and Coombs positive autoimmune hemolytic anemia. At the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. When severe thrombocytopenia appeared, she was wrongly diagnosed as Evans syndrome. The second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of DIC. The third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. History of recent blood transfusion rose the suspicion of PTP in all this cases, and appropriate therapy with high dose IV immunoglobulin was started. Adequate laboratory work-up confirmed the diagnosis. Three different anti HPA-antibodies were identified: anti HPA-3a, anti HPA-1b and anti HPA-3b, respectively. The platelets genotype of the first patient was HPA-3b/3b, of the second HPA-1a/1a and of the third patient, HPA-3a/3a. The reported cases emphasized the importance of keeping in mind the possibility of PTP. Incorrect diagnosis may lead to wrong treatment and fatal outcome.

Health Sciences Authority, Singapore, Singapore

Introduction: The Haemovigilance Programme in Singapore was started by the Centre for Transfusion Medicine (CTM) in 2002. The system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (ATR). The programme runs on a voluntary, non-punitive and confidential basis. Aims of the study: (1) To gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components.

(2) To use the information acquired to determine the morbidity of transfusion. (3) To provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) To improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. Methods: (1) A common report form is used and made available to all participating hospitals. Within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital.

(2) Reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, TRALI), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute GVHD), transfusion-transmitted infections, incorrect components transfused, and near misses.

(3) Within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to CTM for collation. Within the CTM, the Haemovigilance Coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats.

Results:

(1) The total number of reported cases has steadily increased since the introduction of the programme.

(2) The number of participating healthcare institutions has also increased to 100% (n = 12). Please refer to table entitled 'Summary of the Haemovigilance Report 2002-2004' .

(1) The implementation of the Haemovigilance Programme in Singapore is feasible with respect to the Asian setting, and can significantly contribute to blood safety.

(2) There has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) The results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. Of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. Of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). Overall relatively few errors were reported in comparison to other systems. A small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. Autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 

The rate of post transfusion hepatitis (PTH) in Israel in unknown. This information is important in order to learn about the residual infection risk in blood recipients. Aim: To summarize the data on reported cases of PTH. Methods: Suspected cases of PTH are reported to MDA Blood Services. The investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. Donors involved in suspected PTH-B are tested for HBsAg and anti-HBc. Anti-HBc+ donors are tested for anti-HBs. HBV-DNA testing is done if anti-HBs is less than 10 mIU/ml. Donors involved in suspected PTH-C, are tested for anti-HCV and ALT. Since 2000 HCV-Ag or HCV-RNA are performed, when appropriate. Investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. Results: Between 1993 Between -2004 suspected PTH cases were reported: 74 (57%) were PTH-B, 54 were PTH-C (42%) and in 1 patient both HBV and HCV infections were reported. Investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. HBsAg was not detected in any of the 773 retested donors. Anti-HBc was detected in 26 donors involved in 7 PTH-B cases of which 21 were also positive for Anti-HBs. PCR for the detection of HBV-DNA was performed on the 5 'anti-HBc+ only' donors, and none was found positive. Only in 1/449 donors suspected to be involved in PT-HCV, Anti-HCV antibodies were subsequently detected. This donation was collected and transfused before the introduction of anti-HCV testing in Israel, which was implemented in 1991. In another PTH-C case, the implicated donor is still negative for anti-HCV, in follow up samples of up to 28 months, but was found positive for HCV-Ag and HCV-RNA. PTH investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. Conclusion: In the past 12 years an average of 11 cases/year of suspected PTH were reported to the Israeli national blood services. Investigation was completed in 41% of the reported cases. So far, there was no clear evidence of HBV transmission. In 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) HCV seemed to be associated with blood transfusion: One caseprior to the implementation of anti-HCV testing and the otherprior to the implementation of HCV-Ag testing in a donor that did not develop anti-HCV antibodies. These findings suggest that other modes of HBV and HCV transmission should be sought in blood recipients in Israel. 3-2.9, P < 0.05). The seroprevalence for a-HIV in the BD population was 0.096% (SE: 0.028; CI: 0.034-0.153; P < 0.01) whereas in the a-HBc positive BD was 1.08% (P < 0.01) and in the a-HBc negative BD was 0.07%. From 113 a-HIV positive donations, 33 were also positive for a-HBc, that means that the sensitivity of the a-HBc in the detection of a donation a-HIV positive was 29.2%. The relative prevalence (percentaje of positive donations for a-HIV in the a-HBc positive population divided the percentage of this marker in the donations a-HBc negative: RP) was 15.43, which indicates that the number of BD a-HIV positive is 15.43 times higher in the a-HBc positive that in the a-HBc negative population of BD. As regards a-HTLV, the seroprevalence in the BD population was 0.042% (SE: 0.019; CI: 0.01-0.084, P < 0.01), in the a-HBc positive BD was 0.42% (P < 0.01) and in the a-HBc negative BD was 0.031%. From 49 a-HTLV positive donations, 13 were also positive for a-HBc, that means that the sensitivity of the a-HBc in the detection of a donation a-HTLV positive was 26.5%. The RP for a-HBc as regards a-HTLV was 13.54, which indicates that the number of BD a-HTLV positive is 13.54 times higher in the a-HBc positive that in the a-HBc negative population of BD.

Our results suggest that, in our BD population the screening with a-HBc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 

1. Despite the high specificity of currently available ELISAs, the positive predictive value is lower in blood donors. Therefore, Immunoblot tests and polymerase chain reaction (PCR) have been adopted for routine testing in ELISA +ve blood donors, by our service. 2. Our data show that the real anti-HCV prevalence of our donor population is very low (0.04%). 3. The selection and evaluation of appropriate assays of all donated blood for HCV infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. Given that donors who are ELISA positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. Donor re-entry in the pool of donors is an issue for further discussion. 6. The introduction of NAT technology may elicit more accurate responses and improve the screening process.

Background: The introduction of HCV antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated HCV hepatitis and in identification of infected donors. The study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. Evaluating risk factors in HCV infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs Objectives:

1. To recognize the epidemiology of hepatitis C and how it differs geographically; 2. To investigate the risk factors for presence of anti-HCV antibody in blood donors; 3. To evaluate the effectiveness of our donor selection program in local level.

Methods: Serological testing for HCV was performed according to standard procedures. Initial screening was performed using secondgeneration EIA and, after January 1996, using third-generation EIA.

Our study included a confirmation test (RIBA) A questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. The study also included testing of sexual partners and family members. Changes in rates of HCV infections were evaluated by comparing yearly prevalence estimates.

The overall prevalence of anti-HCV (EIA) was 0.11% out of 168 374 blood donations. The average prevalence of HCV infection by RIBA was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. The cumulative number of HCV infected donors was 68, with 54 cases in males and 14 cases in females. Most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). The seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). The annual prevalence decreased throughout years. The relative importance of risk factors for Hepatitis C was: Transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, Sexual Transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. According to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. The importance of sexual activity in the transmission of HCV has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. Conclusions: Our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. Introduction: Apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. The latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. One measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. Normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (PPC). In case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. Aim of the study: In this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (SDP) instead of pooled platelet concentrates (PPC) on contamination risks will be assessed. These risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. HCV, HBV, HIV) resulting from window period donations, and 3) the risk of contamination with TSE or emerging infections for which no screening test exists.

The contamination probability of SDP versus PPC is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). Reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. As patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through SDP is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. The platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the University Medical Center Utrecht (UMCU) in the year 2001. Results: In the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. Our analysis indicate that in platelet recipients, general application of SDP instead of PPC will reduce the risk for transfusion acquired TSE infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. The confidence intervals surrounding the results were obtained by bootstrapping. The large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. Conclusion: Our analysis indicates that general application of SDP instead of PPC will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. Whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. Introduction: Hepatitis B is serious health problem world wide. Its prevention, particularly in the population of blood donors is essential for providing good health care and protection. Aim: The aim of this study is to present the distribution of HbsAg(+) and HBsAg(-) blood donors according to their profession. Methods: Specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. Results: Table 1 shows the distribution of the blood donors in different professions. Administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of HBsAg(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the HBsAg(-) group of blood donors are dominantly more frequent than the other categories of professions. Health care professionals, housewives and farmers in both groups of blood donors are least frequent. Taking in consideration the distribution of blood donors by the given professions in both groups, for U = 21 and P > 0.05 there is no significant difference found. The analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for D = 0.436 and P < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. In relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on Table 2 . In the group of HbsAg(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). In the group of HBsAg(-) blood donors there is no significant difference between the types of professions. Conclusion: Having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and P < 0.01 there is a significant difference in the presented distribution. According to our study, which shows the horizontal transmission of HBV infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. Introduction: Blood donors, as part of the healthy population are tested for HBsAg with each blood unit they give. Therefore, they can be an epidemiological model for exploring the appearance of HBV infection in general population. Aim: This study aims to show the distribution of HBV infection in blood donors in relation with their living conditions, space and facilities.

The material needed for the study consists of the data obtained from 126 confirmed HBsAg(+) blood donors and 100 confirmed HBsAg(-) blood donors as control group.

Results: The Table 1 shows almost equal number of HBsAg(+) blood donors that live in houses or flats. In the HBsAg(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. Having in consideration the presented distribution (Table 10) for c 2 = 3.96 and P < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. As for the distribution of blood donors according the living space they use by member of the family (Table 2) , we can show that 24 (19.04%) of the HBsAg(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the HBsAg(-) group. The bigger number of HBsAg(+) blood donors with small living space gives bigger possibility for transmission of HBV infection in the family. The differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. For U = 19 and P > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. Introduction: When one of the sexual partners has HBV infection the other is also infected in 1 from 3 cases. Aim: The aim of this study is to outline that the risk from transmission of HBV infection between sexual partners is smaller if they use condom as protection. Methods: Two groups of blood donors-HBsAg(+) and HBsAg(-) have been interviewed whether they are using condoms as protection, or not. Results: Table1 shows the distribution of blood donors concerning the use of condoms. Table1: In the group of HBsAg(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. These data are in favor of eventually possible sexual transmission of HBV infection in HBsAg(+) blood donors. In the group of HBsAg(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. The given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and P < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of Er -concentrates are leukodepleted. The choice of bacteriological control of empty bags for blood, bags with Er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. The control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. The pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. Three months later the iso group and the universal plasma kept on the temperature of -30°C is bacteriologically controlled again. Bacteriological control is performed with standard procedures in the Institute for health protection in Stip. The transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. The aim of this study was to determine the prevalence of transmissible infections in blood donor population in Kashan, Iran. A total of 600 consecutive sera were tested for CMV-IgM antibody, HBsAg, Hepatitis B core (HBc) antibody, Hepatitis C (HCV) antibody, and HIV antibody with standard methods. Of the sera tested, 14 specimens (2.3%) were CMV-IgM positive. The frequency of seropositive revealed no significant differences between male and female donors. The frequency rates of CMV-IgM seropositive tests tend to decline with increasing the age. There was no relation between the frequency rates of CMV-IgM seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. The prevalence of HBV, HCV, and HIV antibody were 0.5%, 0.5%, and 0%, respectively. These findings implied important clinical applications because detection of CMV positive sera may reduce the risk for transmission of CMV in blood transfusion and thereby decrease the risk on CMV-induced complications. Introduction: The worlds problem, AIDS, steel can't be said that is a problem in these three centers in R. Macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (HIV-1, HCV, HBV). With the ELISA/AXSYM assay 29 of the 992 blood units which were negative by the Procleix Ultrio assay were positive for anti-HbcAg and negative for anti-HbsAg and HbsAg. From those 29 blood units 23 units were given for transfusion following our Blood Centre protocol and the remaining 6 units were discarded. The protocol consists of a good medical history, liver enzymes (AST, ALT, gGT). We must take into consideration that from those 5 that were found positive by the Procleix Ultrio assay 1 was positive for anti-HCV and 4 were positive for anti-HbsAg. Summary/conclusions: Despite the fact of the short period of time we perform this method, the ability of the NAT technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in Blood donation as a screening method. In spite of the high cost of the method, it is clear that this assay is a valuable tool in our Blood Centre to provide fast and safer blood.

Bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. In Greece it is recommended that platelets (PLT) must be used or discarded within five days post-collection. Recent reports from Europe have advocated the use of bacterial culturing of platelets on Day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. Aim of the study: To assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. Materials and methods: Eligible blood donors were bled according to standard operating procedures used in Greece. PLT were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. PLTs were stored for up to 7 days at 20 to 24°C with end-over-end agitation. Other 111 PLTs prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. PLTs were sampled in the bacteriology laboratory. PLTs were sampled on day 2, 5 and 7. Both aerobic and anaerobic culture bottles were inoculated with a 7-mL platelet sample. Culture bottles were incubated at 35°C in an automated microbe -detection system (BacT/Alert system) until a positive reaction was detected or for 10 days. All samples that were reactive were confirmed by routine culture. Each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. Results: A total of 1889 PLT concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. On of storage day 2 two out of 1889 (0.1%) PLT units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. Out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. After further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 PLT concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. From the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the PLT concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of Coagulase -negative staphylococcus.

Despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. Bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice.

Bacterial screening of platelet concentrates using BacT Background: Bacterial screening of blood components is a routine measure in the evaluation of blood product quality. At our Institute bacterial screening is performed using BacT/Alert system. Sampling and culturing of blood products is performed according to Paul-Erlich Institute recommendations. Methods: Quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. An initially positive (IP) and true positive (CP) rate, organisms isolated and time of detection are presented.

Results: A total of 5359 platelet products were tested during the 6year period. Thirty (0.56%) were found initially positive by BacT/Alert. The cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. Bacterial contamination was confirmed in 14 (0.26%) PLT concentrates. Positive cultures were confirmed and identified in an independent laboratory ( HbsAg, Anti-core, Anti-Hbs, Anti-HCV, Anti-HIV I/II, Anti-HTLV I/II, RPR. These patients were transfused with 5-131 units of concentrated red cells, depending on their problem. A total 530 units were delivered from the beginning of their problem until December 2003. The control were done using last generation enzyme-linked immunoassay (DADE BEHRING, ORTHO, BIOMEURIEUX), as also using automated enzyme-linked immunoassay (AXGYM). The same patients were checked by their physicians before the initiation of transfusions for the same diseases. Results: We found: 12 patients Anti-Hbc (+) and Anti-Hbs (+). 5 patients Anti-Hbc (-) and Ánti-Hbs (-) 3 patients Anti-Hbc (-) and Anti-Hbs (+) 1 patient Anti-Hbc (+) and Anti-Hbs (-) 1 patient HbsAg (+), Anti-Hbc (+) and Anti-Hbs (-). All patients were negative for HCV, HIV, HTLV, and RPR. The same results were found also from patients' physicians. Conclusions: We conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. These results are in accordance with current international literature. This is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. All these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. Neither in the pipetor nor in the extractor runs was found contamination. No false positive were detected and all the positive samples confirmed. (see Tables 1 and 2) . For HIV and HCV the specificity was 99.99%.

The validation criterion were met, so the system was implemented routinely in our laboratory.

The purpose of our study was to analyse the applicability of the Pall enhanced Bacterial Detection System (eBDS) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. Methods: Apheresis PCs, obtained by Trima (Cobe) and Amicus (Baxter) separators and re-suspended in 30% plasma and 70% SSP solution (Macopharma), were submitted to microbiologic control using Pall eBDS System which uses oxygen percentage decrease as a surrogate marker of bacterial growth. The working steps were the following: 16-24 hour after donation, about 3 ml of PC were sampled into the eBDS collection pouch and then incubated at 35°C under continuous agitation (incubator Helmer 900) for 24 hours. After this period, oxygen percentage was measured using an oxygen analyser (Pall BDSO2). The test is based on the 'Pass/Fail' principle.

In case of 'Fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. The incidence of post transfusion hepatitis has been reduced by blood donor screening for HbsAg, but the HBV infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. In this study the HbsAg negative blood units were evaluated for anti-HBc and HBV DNA by PCR method. An extra sample was collected from 2000 HbsAg, Anti-HCV, Anti-HIV and RPR-Negative blood donors. All of samples were examined by approved anti-HBc assay. All of Anti-HBc positive samples were tested by HbsAb assay and evaluated for HBV DNA (PCR). The sensitivity of the HBV DNA (PCR) assay was estimated 50 geq/ml according to VQC proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for Anti-HBc.166 (78.3%) out of 212 Anti-HBc positive samples were HbsAb positive, and 46 (21.7%) were HbsAb negative. All of 212 samples were assayed for HBV DNA (PCR) single and all of them were negative for HBV DNA (PCR). Further study for evaluation of Anti-HBc test as a screening assay for blood unites in high HBV infection prevalence area strongly recommended. 

Early detection of hepatitis B surface antigen: a comparison of ten assays

HBsAg detection is the corner stone of detection of hepatitis B virus infection in blood donors and patients with HBV infection. One of the most challenges is sensitivity of the technique and kit. In this study ten different assays evaluated by seroconversion and performance panels. Some of them can not be used as screening assay due to low sensitivity. The sensitivity of ten HBsAg assays from BIORAD, Dade Behring, Biomeriux, Diasorn, Radim, Diesse, Thermo. Biokit, GB and Shanghai companies were evaluated by two or three seroconversion and two performance panels from Boston Biomedica Ink. Seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. For evaluation of the assay sensitivity WHO and other notified body in the world-wide recommended the seroconversion and performance panels. The HBsAg assays are two groups. Group one with high sensitivity included six assays. They can detect ad and ay subtypes from 0.1 ng/ml BBI to 0.4-0.5 ng/ml BBI respectively. Low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml BBI respectively. For blood safety, the high sensitivity HBsAg assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels.

Diagnosis of chronic HDV infection is usually by antibody testing and HBV DNA detected by PCR method. It is rare to find patients with two replicating hepatotropic viruses and if the accompanying HBV is replicating, prognosis will be very poor. To clarify the correlation between hepatitis delta virus infection and hepatitis B virus DNA positivity, sensitive HBV DNA (PCR) assay was used. The presence of HBV DNA was investigated in 274 patients referred during the 21 Aug. to 28 Dec. 2003. All of them were HBsAg positive. All samples were evaluated for HBV DNA (PCR). The sensitivity of the HBV DNA (PCR) assay was estimated 50 geq/ml according to VQC proficiency panel and run control. Anti-HDV was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were HBV DNA positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of HBV DNA (PCR) negative patients were positive for delta agent. The serum alanine aminotransferase (ALT) levels in 5 out of 8 HBV DNA (PCR) and anti-HDV positive patients were higher than reference interval, but only in 2 out of 13 HBV DNA (PCR) negative and anti-HDV positive samples were higher than reference interval. The present data indicate that 7.7% of patients with chronic hepatitis B have hepatitisdelta infection. Patients with HBV DNA (PCR) negativity had a significantly higher prevalence of delta marker (13.6%) than those with HBV DNA (PCR) positivity (4.4%). Delta RNA testing in positive HBV DNA and anti-HDV patients is recommended.

Introduction: Reduction of the window period of Hepatitis C Virus (HCV) infection represents an important goal in the transfusional and diagnostic setting and Nucleic Acid Technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. A prototype assay designed to simultaneously detect circulating HCV antigen and anti-HCV has been developed by Biorad (Biorad Laboratories Limited, Marnes La Coquette, France). Aim of the present study: To define the cut-off (CO) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. Methods: In order to establish the CO value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n Conclusion: The new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or HCV-RNA detection, are not currently recommended, but also in the screening of blood donations, when Nucleic Acid Technologies is not feasible due to costs, organization, emergency and/or logistic difficulties.

Introduction: In recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. This safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. More recently, the implementation of the nucleic acid amplification technologies for the detection of HIV-1, HCV and HBV, has increase this aim by reducing the 'window period' of the infections. Other viruses, such as Parvovirus B19 (PB19) and Hepatitis A Virus (HAV), can raise problems to the blood safety. These infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. Material and methods: An observational study was performed to determine the prevalence of PB19 and HAV in Portuguese blood donors. We gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-HCV prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. We observed that the anti-HCV prevalence has a decline tendency during years in blood donors. According to sex the anti-HCV prevalence in men is 0.7% and women 0.6% (P = 0.004). Over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; P = 0.033) and increasing tendency in women (0.4% to 0.9%; P = 0.007). According to age group the anti-HCV is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (P = 0.001).

The prevalence of anti-HCV is higher in FDs than VDs, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . A total 14627 FTD and 684 AD have been tested for HBsAg. A sample was considered as HBsAg positive when found repeatedly reactive by 3rd generation. immunoassay method (ELISA). The Chi-square test was used for statistical analysis. Results: The 5-year overall HBsAg prevalence among first time blood donors was 8.9%. and AD 6.7%. Among autologous blood donors was observed a decreasing HBV prevalence from 6.3% to 3.7% in 2003. According to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (P < 00.5). Among AD, a decreased HBsAg prevalence according to age was observed in men and women. The same trends by sex and age were observed in FTD.

The prevalence of HBsAg in AD was lower than in FTD. However, from 1999-2003 HBsAg prevalence has decreased in the same proportion in both population. This decreased can explain by to main factor: the improvement HBsAg screening method in blood donors and decreased the HBsAg prevalence in general population. (1 : 30 250) . The HBV DNA-emia in HBsAg negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. In two donors anti-HBc total was positive and in one anti-HBe was also detected. In one donor the Glycin 145 Alanin mutation in the S region was identified.

The frequency of HBV DNA pos/HBsAg neg donors in Poland is high (0.002%) therefore the decision to introduce routine HBV NAT screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. Of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the eBDS pouch and the platelet mother bag by culture) representing 1/5133. The bacteria detected were Staphylococcus or Streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the eBDS pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, eBDS pouch not tested) representing 1/6559. There was one reported case of a missed detection with confirmed presence of bacteria (Staphylococcus epidermidis) in the mother bag by culture. Subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 CFU/mL. In all cases the Pall eBDS was able to detect. This supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure.

The results from blood centers routinely using Pall eBDS demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). This is comparable to a recent survey result with other culture based systems. Summary/Conclusions: The minority group of pregnant women who come to labor without prenatal testing of hepatitis B and C revealed essentially similar prevalence of anti-HCV with healthy BD even if definitive confirmation is probably increased in this minority group. There is however markedly higher prevalence of HBV infection in the PW so that screening for HBV is essential for the prevention of vertical transmission. The systematic screening of BD with anti-HBc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. Methods: Blood samples were screened for the presence of HBsAg, HCV and HIV antibodies using enzyme immune assay and for syphilis using the TPHA test. The results were analysed retrospectively. All samples with results at or above the minimum positive value were considered reactive. The tests for HBsAg, anti-HIV and anti-HCV were repeated in duplicate in all reactive donations. Blood units that were reactive in the primary or secondary assays were discarded. HIV positivity was confirmed by Western blot analysis using HIV Blot 2.2 (Genelab Diagnostics) Results: Results from a total of 77 903 screened donors were analysed. Hepatitis B surface antigen rates was 1.99%; anti-HCV seropositivity was 0.54%; anti-HIV seropositivity was 0.01% and TPHA seropositivity was 0.06%.

One study calculated this risk to be one in 63 000 for HBV, one in 493 000 for HIV and one in 103 000 for HCV. It is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. In view of the high infectivity of HIV positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. A careful history should identify those who should not give blood. In Turkey, among blood donors the average HBsAg prevalence in 1985-1998 was 5.1%. But it had decreased to approximately 2.1% in 2003. Anti-HCV positivity has been reported to be 0.6% between 1992 and 1999. But it was approximately 0.5% in 2003. RPR positivity in blood donors in Turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. In 2003, the RPR rates was 0.09%. In our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. Anti-HIV seropositivity was found around 0. Introduction: The serological detection of specific antibodies to Treponema pallidum (TP) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. Aims of the study: To develop a qualitative Syphilis assay for the detection of TP immunoglobulin M (IgM) and G (IgG) antibodies. The assay will be used for the serological diagnosis of Syphilis using the Architect platform, which has the capacity to test 200 specimens/hour.

The two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant TP antigens (TpN15, TpN17 and TpN47) and acridinium labelled anti-human IgG and IgM monoclonal antibodies as conjugates. In the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, Pre-Trigger and Trigger are added to produce chemiluminescence, which is measured as relative light units (RLU). Specimens yielding RLUs less than the cut-off are considered negative, while those yielding RLUs greater than the cut-off are considered positive.

The sensitivity of ARCHITECT syphilis TP was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in Fujirebio TPPA; no prozoning was observed with high positive specimens (over 2 16 titer by TPPA). The specificity generated from testing 564 hospitalised patients previously screened as TPPA negative, was 99.6%. Testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. The precision (CV%) with a positive control was 5.8% (95% Confidence Interval: 4.9-7.0%) by the standard 20-Day NCCLS analysis (EP5A2). In a study conducted at Asahikawa Medical College Hospital, in which, 81 positive and 82 negative specimens were tested, concordance with Fujirebio TPPA was determined to be 100%. No significant interference to the assay was observed from Bilirubin (conjugated type and free type), haemoglobin or lipid.

The Architect Syphilis TP assay is an automated, specific and sensitive test for the detection of antibodies to T. pallidum.

Background: HCV exposure of blood donors is serologically determined by the detection of anti-HCV antibodies in serum or plasma. However a 'window' period of 30-70 days after exposure exists during which specific antibodies to HCV antigens cannot be detected. HCV RNA detection and/or HCV Core protein testing result in dramatic reductions in the preseroconversion window period. The new Bio-Rad test, based on the simultaneous detection of HCV core antigen and anti-HCV (core, NS3, NS4) antibodies, improves the detection of HCV infection in the early phase. Aims: The aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, Monolisa HCV Ag-Ab Ultra, by using the Bio-Rad Evolis automated microplate processor system. Methods: This two-step ELISA assay is based on the combination of an indirect test for the detection of antibodies (core, NS3, NS4) and a sandwich test for core antigen detection. Results are available within 2.5 hours, with sample addition monitoring and color coded reagents. No specimen pretreatment is required. Evolis is a self-contained microplate processor designed for full automation of microplate-based EIA techniques. The walkaway system can process four microplates at a time with continuous loading of samples and reagents. Positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. The Monolisa HCV Ag-Ab Ultra/Evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. The results are compared to viral RNA detection and conventional HCV Ab screening assays. Specificity is evaluated by using random blood donor samples. Results: Among the seroconversion panels that begining with samples negative for HCV RNA and anti-HCV antibodies, the Monolisa HCV Ag-Ab Ultra assay detects exposure to HCV an average of 25 days earlier than the Monolisa HCV Plus V2 test. The mean delay of the Monolisa HCV Ag-Ab assay in detecting HCV infection compared to HCV RNA testing is around 2.5 days.

The Monolisa HCV Ag-Ab Ultra/Evolis system allows simultaneous detection of HCV core antigen and anti-HCV (core, NS3, NS4) antibodies, thus significantly reducing the time gap between the initial detection of HCV RNA and the first appearance of detectable anti-HCV antibodies. The fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management.

Operational evaluation of pall eBDS bacterial detection system L LARREA GONZALEZ, MA SOLER and RJ ROIG Centro de Transfusion, Valencia, Spain

Introduction: Regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed Standards for Blood Banks and Transfusion Services). One system currently available is the PALL eBDS Bacterial Detection System which utilises percentage oxygen as a surrogate marker for bacterial growth. Aims: To evaluate the PALL eBDS in routine use in our blood centre. In particular, to assess feasibility and adaptability to daily labour routines.

The Orbisac (Gambro BCT) system was used to produce leucocyte depleted buffy coat (BC) platelet pools (4 BC/pool) stored in platelet additive solution (ssp Macopharma). Mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. eBDS installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. eBDS pouches were sterile connected onto platelet pools 22 hours after blood donation. Platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°C. After this time, percentage oxygen was measured.

No positive results were found in this study. This was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. Minimal training was required to use the eBDS. The system was easy to use and did not require the use of a laminar flow cabinet to take samples. It was quick and simple to take samples and perform oxygen measurement. After pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. The data management system allowed full traceability of product and work flow. Results were very easy to interpret. Conclusion: The PALL eBDS was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. The percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. The age of donors ranged from 18 to 65 years old. The assay used for the detection of HbsAg, HbeAg, Anti-HBc IgG/total, Anti-HBc IgM, anti-HBe and anti-HBs was the automated microparticle enzyme immunoassay (AXSYM) of the ABBOT company. All the units were tested for HbsAg, and Anti-HBc IgG. If the anti-HBc IgG was detected, the specimens were automatically tested for anti-HBs. The units were wasted if the anti-HBs was negative, and the specimens were manually programmed for the testing of the Anti-HBc IgM, HbeAg, and anti-HBe.

From the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of HBV infection, that means the 14.11% of the health adult population was infected in the past by the HBV. The 13.69% (n = 1160) was previously infected and now immunized with HbsAg(-) and Anti-core IgG(+) and 0.41% (n = 35) were chronic carriers of the HBV with HbsAg(+). The 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. In other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. The combinations of the serologic markers for HBV are illustrated in the table attached.

These results indicate that the incidence of HBV infection in the Northeastern department of Greece is equivalent to the incidence of HBV in other Greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. As a consequence, the best source for safe blood collection is the population of volunteers.

Earlier detection of human immunodeficiency type 1, hepatitis C and hepatitis B viruses using the Procleix® Ultrio TM assay on the Procleix® system and 

The study objective was to assess the ability of the Ultrio Assay and associated discriminatory assays to reduce the detection windows for HIV-1, HCV, and HBV. Commercially available seroconversion panels were used for testing. Methods: HIV-1 (n = 13), HCV (n = 12), and HBV (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the Ultrio Assay. Panels were tested neat in the appropriate discriminatory assay. Times to detection of HIV-1, HCV, and HBV nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. 

P-210 Effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method?

The successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. Bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. Therefore, sample drawing for bacteria screening must not be done immediately after blood donation. The established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood.

Here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. The Paul Ehrlich Institute (PEI) developed PEI Bacteria Standards, which are characterized concerning their behavior in blood components. They contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. There are two strategies to improve bacteria safety of blood: Screening and pathogen reduction. Neither of them is perfect, but screening methods are successfully established since several years in routine (Belgium, The Netherlands), and represent the current state of the art. Further development and collecting of experience will produce the basis for assessments in the future. It is of high importance to apply the screening methods in dependence on their properties. Methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . For example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. Both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. Selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion).

Results from the Procleix HIV-1/HCV and HIV-1/HCV/HBV (Procleix Ultrio) assays for the detection of HIV-1 RNA, HCV RNA and HBV DNA in blood donors of two blood transfusion centers of SW Greece In discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for HCV RNA only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for HIV-1 RNA only. None were positive for both HIV-1 and HCV. The standard serological assays gave the same results for the above positive samples. Two samples that tested positive by the standard serological assays tested negative in the Procleix HIV-1/HCV assay. Of the 4151 samples tested by the Ultrio assay, 21 (0.5%) tested reactive for HIV-1/HCV/HBV. In discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for HIV-1 RNA, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for HCV RNA, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for HBV DNA. All were single positive i.e. none tested positive for more than 1 virus. Three out of 16 positive samples for HBV DNA tested negative by the standard serological tests. The opposite was not observed.

The Procleix Ultrio assay is a definite improvement over the Procleix assay in a region with a high incidence of HBV carriers. Up until its use, it is obvious that HBV positive blood with very low antibody titers was transfused into patients. More results will show whether Procleix Ultrio can eventually replace the standard serological tests.

The Introduction: Patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. In Albania, the frequency of hepatitis is also linked with HBsAg testing with ELISA (introduced in1993), and HCV testing (introduced in 1997). Aim of the study: Evaluation of the prevalence of the markers of hepatitis B, C, and D in patients with hemophilia. Methods: Our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. Blood testing for anti-HCV, anti-HDV, and HBsAg was performed with ELISA -Gen. III.

Results: Of 145 patients tested, 26 cases (17%) were HBsAg positive, 87 cases (60%) were anti-HCV positive, and 7 cases (5%) were anti-HDV positive. Co-infection of HBsAg and HCV was found in 16 cases (11%), whereas co-infection of HCV, HDV, and HBV was found in 4 persons (3%). The highest rates of infections and coinfections were found in patients above 30 years of age. Conclusion: Mandatory blood testing has decreased the levels of post-transfusion hepatitis. In Albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . Results: 2/16 760 (0.012%) samples from RBD were anti HIV 1 + 2 nonreactive and RR for p24 Ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from FBD was RR for p24Ag/anti HIV 1 + 2 nonreactive and it was confirmed positive by neutralization. This BD had been autoexcluded himself after blood donation. He showed seroconvertion 21 days later: p24Ag nonreactive, anti HIV 1 + 2 reactive and Western Blot positive.

The only BD p24Ag positive/anti HIV 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. Although a larger populations of BD is necessary to be studied and in spite of the low prevalence we have found, we consider p24Ag screening is an alternative up to implementation of Nucleic Acid Testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. Owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. To these, one has to add the (now unknown) proportion of potentially HBsAg negative + HBV DNA positive FTBDs. HCV: Since the introduction of the screening in 1995, the general incidence in RBD has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. The prevalence in FTBD has stabilized at 12 ± 2‰. Based on reasons similar to these employed for HBV, the residual incidence in RBD suggests that 1 potentially infectious donation in 10 000 RBD escapes the screening (= to a total of aprox. 40, annually). A limited investigation using HCV-Antigen EIA evidenced a 1‰ escape rate in FTBDs (= to a total of aprox. 50, annually Table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. Conclusion: These system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. 

Background: The HBsAg, anti-HCV, anti-HIV1/2, p24 antigen, ALT and syphilis tests are performed for blood donations in Czech republic. No NAT tests are mandatory in Czech republic. The aim of this pilot study was: 1. HCV RNA PCR testing in anti-HCV negative blood donations; 2. correlation between HCV NAT and anti-HCV testing results. Methods: Blood samples (anti-HCV serologically negative, ALT not elevated) were pooled using the Guardian Plus SPII into pools of 24 samples. Pools of 1 ml were tested using the Cobas Ampliscreen HCV Test v. 2.0 (Roche). Results: 891 pools of 24 samples from 21 384 a-HCV serologically negative donations were tested from October 2002 to July 2004. No one pool was initially reactive. Invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). Invalid tests were repeated. In none of 891 pools a positive HCV NAT result was observed. Conclusions: No discrepancy between HCV NAT and a-HCV results was observed in our study. All of the NAT tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-HCV serologically negative. The HCV incidence in the Czech republic blood donor population is low but it is slightly growing up in general population. HCV NAT testing could improve the safety of blood supply by reducing the window period for HCV.

Introduction: Parvovirus B19 is the only parvovirus known to be a human pathogen. Most commonly, it causes a mild childhood rash, Erythema infectiosum, but in some cases more serious symptoms can be linked to B19, such as acute or persistent arthropathies, critical failures of red cell production, Hydrops fetalis, fetal loss, myocarditis or hepatitis. Inactivation of the non-enveloped virus has proven difficult. As a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus B19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (NAT). In our institute all blood donations were screened for human parvovirus B19 by NAT since April 2000. Methods: Over the last 5 years 5.5 million donations were screened for B19 by NAT. Samples with a virus load over 105 IU/ml were defined as positive, whereas samples with a virus load between the detection limit (103 IU/ml) and 105 IU/ml were defined as weak positive. Weak positive products were released, whereas positive products were discarded. In addition infection markers of 50 B19 positive donors (case group) were determined over a time period of one year. Virus load and B19 antibody status was compared with 100 B19 negative donors (randomised control group). B19 antibodies (IgG VP2, IgM VP2, NS1) were analysed by two commercial antibody tests. Results: Overall 496 B19 NAT-positive donors were identified with a virus load over 105 IU/ml out of 5.5 million tested. There was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. VP2 IgG was detected in 90.9% and 74% of the case and control group, respectively (P = 0.01). These data demonstrated statistically significance (P = 0.01). All donor samples which were B19 NAT positive for more than three months developed neutralizing VP2 antibodies. In contrast, NS1 antibodies were observed in 83% of the case group and in 15% of the control group (P < 0.01). NS1 antibodies were detected more frequently in samples, which were B19 NAT positive for more than six months. Conclusion: B19 NAT could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. All B19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 IU/mL. These data support our testing algorithm all components of high positive donations (virus load over 105 IU/ml) were discarded. Donors with NS1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus B19 longer than six months.

Background: On recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. On account of the importance of this screening the tests used are becoming more and more sensitive. Aims: To evaluate an ELISA screening test (the TmpA test Recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-Treponema pallidum in serum or plasma). Methods: In this study 1696 samples from blood donors were tested by the rotine 'Cardiolipidic Reagent For Syphilis Screening on Microplates' -Diagast Laboratories as well as with 'HDTmpA Recombinant' -Hoslab Diagnostics. Positive samples were then confirmed with FTA abs/TPHA. Results: Using the mentioned tests we obtained the following results:

1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using TmpA Recombinant [of which 23 (1.36%) were confirmed positive by TPHA/FTA abs.

As seen we found 23 samples (1.36%) that were only positives by TmpA Recombinant Test and that were confirmed by TPHA/FTA abs. We concluded that TmpA Recombinant seems to be a suitable test for a quick and automated Syphilis screening of blood donors and provides maximum safety for the recipients.

Background: In recent years, there has been substantial evidence indicating that typing and subtyping for HCV is clinically important in understanding HCV disease and its therapeutycal options. 'Naive' viral load also seems to influence disease severity and responsiveness to therapy. Therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. Aims: The University Hospital of Coimbra studies and tests his own patients and patients from 11 other hospitals in the central Portugal. We also collect and test blood donor candidates from this region. We proposed to analyse the distribution of HCV genotypes in this region, among 1578 patients with cronic HCV infection. We have simultaneously analysed the viremia and correlated it with age and severity of liver disease. Methods: Nucleic acid extraction was done using the semiautomatic 'Xstractor' from Biomerieux Laboratories (boom method). The genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (INNULIPA Introduction: Bacterial contamination of blood products remains a persistent problem. Various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. Bacterial detection systems could be divided into culture systems and rapid technologies. Hemosystem has developed a rapid and sensitive technology for bacteria detection named Scansystem TM . Bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. Therefore Hemosystem developed a positive control in order to validate the Scansystem TM Platelet Kit before use. Aim of the study: The current study was designed to evaluate the performance of the Scansystem TM positive control.

The Scansystem TM positive control is a capsule containing lyophilised Lactobacillus casei subsp rhamnosus. The bacteria concentration per capsule is at least 8 ¥ 10 8 CFU. The positive control has to be stored at room temperature and is stable for 2 years. After dilution in PBS, the preparation has to be used within 1 hour. Two capsules were tested for ten consecutive days with Scansystem TM platelet Kit as well as with optimised Scansystem TM platelet Kit. In an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with Scansystem TM platelet kit and optimised Scansystem TM platelet kit. Results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. The ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both Scansystem TM platelet kit and optimized Scansystem TM platelet kit. Therefore by definition all tested capsules were positive.

The lyophilized positive control capsules enable the user to validate the Scansystem TM Platelet Kit before use. Because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. Scansystem TM is currently the only method that provides this safety measure.

Introduction: Whereas implementation of NAT for blood donor screening reduced the risk for transfusion transmitted HIV and HCV infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. Especially platelet products, which are stored at room temperature, are prone to bacterial contamination. Aim of the study: Several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. The study investigates a new rapid bacterial detection method. Material/Methods: Pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 CFU/ml to 106 CFU/ml. Bacterial concentration was verified on blood agar plates immediately after spiking. Five millilitres of spiked platelet concentrates were centrifuged, stained with Thiazole Orange dye and analysed directly by FACS within five minutes after staining. Aliquots of pool platelets spiked with concentration of 102 CFU/mL and 101 CFU/mL of each bacteria strain were incubated for two to eight hours in special bouillon at 37°C and were analysed by FACS immediately after incubation. Results: Sensitivity of FACS analysis differed between 103 CFU/mL for E. coli and 105 CFU/mL for Klebsiella pneumoniae without preincubation and was enhanced to 101 CFU/ml when a pre-incubation step of two to four hours was included. Conclusion: Bacteria detection by FACS analysis combined with a short pre-incubation (2-4 h) at 37°C is a quick and simple method with sensitivity comparable to other commercially available detection systems. The advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. Introduction: Detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. For blood transfusion services the method must have a high sensitivity, an easy performance and a low price. Aim of the study: In this spiking study we evaluated the new optimised Scansystem TM Platelet Kit detection method for use in apheresis platelets. Methods: Apheresis platelet concentrates (APCs) were spiked with strains of ten different bacteria species. After different incubation periods, APCs spiked with 10 CFU/mL were analysed by the optimised Scansystem TM Platelet Kit. The number of bacteria was monitored by plating on blood agar. Results: All bacteria strains were detected with the optimised Scansystem TM Platelet Kit when the sample was collected 24 h after spiking. Identity of the spiked bacteria was confirmed by Gram staining and DNA fingerprints. Conclusion: In summary, the optimised Scansystem TM Platelet Kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking.

Background: Since year 2000 our laboratory started routine screening of HCV-RNA in plasma minipools for all plasma intended for fractionation. Although NAT testing is not yet mandatory all blood products are released depending upon NAT results. Aim: To test and compare two different methods of RNA extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. Methods: Plasma minipools of 24 donations are prepared either on a Tecan Genesis robot or on a Hamilton AT plus. HCV-RNA is isolated from 2 ml plasma by using either the Qiagen Biorobot 9604 and Qiamp Virus Biorobot 960 kit or the manual extraction with Cobas Ampliscreen HCV PCR Kit v 2.0. Results: Between March 2000 and December 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of HCV-RNA. Four pools were found to be positive for HCV-RNA. Of the four NAT-positive pools with no EIA-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. All the positive donations were detected independently of the extraction method used (manual or automated). Our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of Biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for HCV less than 50 IU/ml. The advantage of the manual method is that it has better recovery of nucleic acids than the Qiagen extraction. Concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour.

The automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. Anti-HCV similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). Anti-HIV reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. RPR test for syphilis was around 0.1%. Directed donors were 95% of all and volunteer donors consisted nearly 5%. Donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (DQF) are filled out by the donor candidates. Using DQFs have been mandatory at all blood banks in Turkey by law since 1996. From that time infectious disease marker rates were dramatically reduced at all centers. Donor Information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. The interviewing staff was trained specifically for this topic. This steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. Conclusion: Education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. Rigorous donor selection will contribute this ultimate success. We should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood.

Background: Human T cell Lymphotropic Virus type I is endemic in Japan, the Caribbean, southeastern United States and parts of South America and Africa. In non-endemic areas such as Europe, HTLV-I is less common and most infections are identified in immigrants. The epidemiology of HTLV-II is different, being predominantly found among indigenous American-Indian populations and among IVDUs, but the routes of transmission are the same. Aim: Our study's aim was to ascertain the prevalence of HTLV I/II in blood donors in order to understand the epidemiology of HTLV in Greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. Overall, anti-HTLV seroprevalence levels among blood donors, are low. Although the number of annual donations in this study is relatively small, the data for HTLV indicate that rates of this infection are low and that infected donors will be seen infrequently. As all blood donations are screened for HTLV I/II during the last six years, a national survey is necessary in order to define the epidemiology of HTLV in Greece.

Introduction: Toxoplasma gondii is the causative organism of toxoplasmosis. The disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). The parasite can also be acquired transplacentally by organ transplantation or from blood transfusion.

The purpose of this study was survey of Toxoplasma antibodies in some Iranian blood donors at Tehran blood center. Blood samples were randomly collected for detecting of IgG and IgM antibodies (by Elisa Technique).The total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. Results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma IgG antibodies and 6 (2.8%) were IgM antibodies positive and 2 of them (0.9%) were borderline for IgM antibodies. Among males the frequency of positivity was higher than woman but this different was not significant. The most frequency of seropositivity was found in age group 41 to 50 years. Conclusions: Diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. The acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised.

In spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. The possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. Post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. Objective: To show the prevalence of Hepatitis B (HBsAg) and Hepatitis C (anti HCV antibodies) in multitransfused thalassemic patients.

In our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. They receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. The patients are tested periodically for the presence of viral markers (HBsAg, anti HCV antibodies), using tests for HBsAg (Abbott Auxyme Monoclonal EIA) and for anti HCV (Abbott HCV EIA 3.0).

The presence of markers for Hepatitis B and Hepatitis C has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. The tests in all 5 patients were negative.

The blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. 

Introduction: We observe yearly the prevalence of transfusion transmitted diseases following instructions of SKAE (National Coordination Haemovigilance Centre). Aims: To investigate the prevalence of the most important blood borne infections in our Blood Transfusion Centre in the state Achaia during the last five years (2000) (2001) (2002) (2003) (2004) . Materials and methods: The detection of HbsAg, anti-HCV, anti-HIV 1/2 was made by automated microparticle enzyme immunoassay (AXSYM, Abbott) and by enzyme-linked immunoassay methods (Dade Behring, Ortho, Biomerieux) Syphilis tests were made by using RPR kits. Confirmation for anti-HCV positive samples was made RIBA or INNO-LIA, while the confirmation of anti-HIV1/2 positive samples was made by 'St. Andrews' General Hospital of Patras Reference Centre. Results: All the seropositive donors were first time donors. Conclusion: (1) We observe that there is a decrease in all four infections. (2) The absence of anti-HIV seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our Centre during the last four years. Methods: A prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 Italian regions. Each centre was required to enrol all first-time donors born before December 31st, 1978, and thus not included in the HBV mass vaccination campaign. The selected donors were tested for HBsAg (mandatory by law) and for anti-HBc by commercial assays. All HBsAg and/or anti-HBc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-HBs, anti-HBe, anti-HBc/IgM and anti-HBc avidity index by an experimental procedure) and for the determination of HBV-DNA (both qualitative and quantitative) by real-time PCR.

Results: In the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. Among eligible donors, 0.4% were both HBsAg and anti-HBc positive, and 10.3% were HBsAg negative/anti-HBc positive. HBV positivity rates were higher in Southern than in Northern regions, although a high variability in HBV prevalence was observed between neighbouring areas in the North. HBsAg positives were mostly males, 93% were positive for anti-HBe, 6% had raised ALT and 8% were concurrently positive for anti-HBs. Among HBsAg negative/anti-HBc positive donors, 18% were negative and 82% were positive for anti-HBs. Among anti-HBs positives, 35% showed values <100 mIU/ml and 65% > 100 mIU/ml. The avidity index results suggested that approximately 30% of anti-HBc positive individuals were recently infected. Conclusions: Our preliminary data indicate that approximately 70% of the Italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against HBV, and that 10.7% of them have serological markers of ongoing or past HBV infection. Anti-HBc alone was detected in nearly 2% of the study population. HBV-DNA testing is underway at the time of this writing.

In our country mandatory tests for each blood donations are: HBsAg, anti-HCV, anti-HIV1/2 and TP Ab. to C + NS3 + NS4, 9 (18.4%) to C + NS3, 4 (8.2%) to C + NS3 + NS5, 2 (4%) to NS3 + NS4 + NS5, 2 (4%) to C + NS4 + NS5, 2 (4%) to NS3 + NS5.

The use of the HCV core Ag Elisa Test System may provide substantially earlier identification of HCV infection than it is possible with current serological assays. Although all of six anti-HCV assays are very sensitive and specific screening assays, they didn't detect HCV infection in one patient. Majority of anti-HCV positive patients (77.5%) had anti-HCV Ab for 3 or more different epitopes of HCV.

International comparison of performance of Abbott PRISM assays used for blood donor screening

Background: The National Serology Reference Laboratory, Australia (NRL), coordinates a Quality Control (QC) programme for laboratories that screen for anti-HIV 1&2, anti-HCV, HBsAg and anti-HTLV I/II using the Abbott PRISM assays. Nineteen laboratories from Australia, Belgium, Canada, Ireland, Israel, The Netherlands, New Zealand, Norway, Singapore, South Africa and Thailand have submitted data for this programme. Aims: To determine the accuracy and precision of results from laboratories, individual PRISM instruments and different reagent lots by analysing data accumulated between October 2001 and January 2005.

The multi-marker QC sample 'PeliSpy s2058 Type 7' (s2058), produced by Viral Quality Control (now AcroMetrix-Viral Quality Control), was provided to participants. Laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. PeliSpy was used as a 'Go/NoGo Control' and results were required to be reactive (S/Co > 1) for a test run to be deemed valid. Data were collected and analysed using the NRL's internet-based application EDCNet (https://www.nrlqa.net). After submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. Data for five different s2058 lots were exported from EDCNet and analysed. Results: Nearly 95 000 results were submitted: all results were reactive (S/Co > 1). Fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. A further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. A total of 94 189 results, reported using 332 different PRISM reagent lots (145 for anti-HIV, 79 for anti-HCV, 55 for anti-HTLV and 53 for HBsAg), were analysed. Results from PRISM HBsAg and anti-HIV showed the least variation with coefficient of variations (CV) of <10% for all s2058 lots. Results from PRISM anti-HCV and anti-HTLV produced CVs between 9.17% and 15.38% for all s2058 lots. Data reported for s2058 lot PS030618 (n = 38 662, range 8080 for anti-HTLV to 10 225 for HBsAg) were analysed further to review performance of PRISM reagent lots. HBsAg showed the least variability between PRISM reagent lots with <8% Bias for the 14 PRISM HBsAg reagent lots used (Bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). PRISM anti-HTLV showed greater variability between reagent lots with a single reagent lot generating a +63% Bias. PRISM anti-HCV showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a CV between 10% and 13%. Conclusion: In 94 255 results in a QC sample distributed to 19 laboratories the Abbott PRISM performance was found to be consistent over four assays. EDCNet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time.

Introduction: Since HCV RNA testing of all blood donors started in Finland in 2000, the NAT screening process has continuously been improved. Investments in process automation have made the work more efficient and blood safety has further increased since HIV-1 RNA screening of all blood donors started late 2004. Aim of the study: To implement HIV-1 RNA testing in the NAT screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. To study if the sensitivity for both HIV-1 RNA and HCV-RNA will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. Methods: The nucleic acids were isolated from plasma samples of 1 mL with the MagNA Pure LC instrument using the MagNa Pure LC Total Nucleic Acid Isolation Large Volume Kit. The internal controls from the Cobas AmpliScreen Multiprep Specimen Preparation and Control Kit and the Cobas Amplicor HCV Specimen Preparation Kit were added to the Lysis/Binding Buffer (5 mL/mL of each). From the final volume of the nucleic acid eluate (100 mL) 50 mL was used for the detection of HIV-1 RNA (Roche Cobas AmpliScreen) and 30 mL for the detection of HCV RNA (Roche Cobas Amplicor). A Run Control containing both HIV-1 RNA (200 IU/ml) and HCV RNA (50 IU/mL) was included in all extraction runs. Sensitivity of the both assays was assessed by testing dilution series of the WHO standards for HIV-1 RNA and HCV RNA. Specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). Results: Detection limits of the HIV-1 and HCV assays (95% hit rate) were calculated to be 100.1 IU/mL and 18.7 IU/mL respectively. Specificity for both assays was 100% and during the validation phase also the robustness was good.

The sensitivity of both assays with a pool size of 96 was below the recommendations by the Council of Europe for blood donor screening (for HIV-1 RNA 10 000 IU/mL and for HCV 5000 IU/mL per individual donation). Specificity of the assays was excellent, false reactive results were not observed. Implementation of the HIV-1 NAT assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 mL sample volume and a single extraction for two assays were used.

The The very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. In many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. These ties to industry create a tension that actually is or can be perceived as

• work internationally;

• send young volunteers to international youth forums; • employ young people in your organisation;

Why use volunteers in blood donor recruitment?

• They have networks to scout-groups, sports-organizations, tradeunions, Rotary, staff of large companies etc. • They bring in fellow volunteers -with different prospects of society; • Often paid recruiters are underpaid (!) and tend not to remain • You can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • So you need direct personal contact = need many people (e.g. young ambassadors); • A large number of volunteer recruiters is a gift from heaven! Paid donation gives the act of blood donation LOW status. The act of blood donation should be respected, and praised by role models, queens and presidents. Efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! With such a prospect, it was important to evaluate the practices of blood donor selection in the EU. Material and methods: A questionnaire was designed and sent in 2003 to the relevant institutions of the 15 EU countries plus Switzerland. The questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. The questionnaire also inquired about each country's exclusion period for each contraindication (CI) to donation. Results: Predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. They were supported by written questionnaires in nearly all countries. In half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). The 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. In other countries the age limit could be brought forward to 17 and extended to 70 years old. The time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. The questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/EC directive. This particularly concerned How to meet the expectations of European donors, so that they come back to your blood center! Know your donors: Make regular donor surveys. Age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. Use local donor organisations Let volunteers help! They work for free, but donor recruitment and -retention costs money. Each blood center should have a local donor organisation, run by volunteers. The donor organisation should receive a payment for each bag collected. The reputation of the blood system Tell the donors, what the blood is used for. That all blood is tested.

And that blood provides safe medical treatment Safety of the donor. Insurance is a must Good quality and efficiency in blood services Decentralized blood collection No waste, and minimal outdating Efficient service:

• a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc.

Donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. Donors should be recognized continuously. Use directed press-coverage to higher the self-esteem of the donors. Donors should be well informed:

• leaflets, posters and questionnaires should be 100% correct;

• Use E-mail and web-sites for quick up-date of donor information.

Be visible!

• Have an offensive and comprehensive media approach;

• Have a yearly national campaign 14 June up to World Blood Donor Day; • Have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses.

Directed campaign towards young people:

• young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. Analyzing ineligibility times, on the other hand, revealed wide differences. For example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. Previous transfusion history could not be a CI or could be one varying from 6 months to indefinite CI (this point recently changed in several countries).

The results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. However, the conditions under which donor selection interviews are conducted were similar in all countries. The enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout EU. A next step will be to use it to appreciate their evolutions and especially the impact of the 2004/EC/33 directive on these practices in the 25 EU countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety.

Background: In Europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. Whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. Like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. Aim: The aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. Results: Most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. Complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (Newman BH: Blood donor complications after whole-blood donation. Curr Opin Hematol 2004; 11: 339-345.) Serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). Fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. A recent study of a consecutive sample of 528 Swedish blood donors (Nilsson Sojka B, Sojka P: The blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. Vox Sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: How does blood donation affect you? Physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). The duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. Investigations on the long-term effects of blood donation are scarce. Yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (Nilsson Sojka B, Sojka P: The blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. Vox Sang 2003; 84: 120-128). Conclusion: Both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). Serious reactions to whole-blood donations are extremely rare. More studies are needed in particular with respect to the long-term effects of regular wholeblood donations.

T-PA-056 Establishing a national adverse event reporting system for blood donors -a prospective study of 1. There was no national system and significant regional variation showed that the data was scientifically unsound. A coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. Aims: 1. To develop and implement a high quality, reliable national Donor Adverse Events Reporting (DAER) system; 2. To define and categorise adverse events; 3. To record data systematically and prospectively using the existing computerised donor database. Methods: From summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. Detailed training material was written in May 2003 and the new protocol was validated in one region. From July to December a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 Blood Centres. DAER was fully implemented by January 2004. Adverse events are assessed by Health Care Professionals on session and the relevant code entered onto the donor's computer record by clerical staff. Information received after the session is entered by Centre based doctors using the same system. The database is interrogated monthly for statistics. Results: In the first 9 months 1.86 million donors attended sessions throughout England and North Wales. Results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (Donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. Nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). Bruising was reported after the session by 1 : 1300 donors. Summary: A robust system has been developed and successfully implemented across a large, national Blood Service. Based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored.

The community role in enhancement of voluntary, Non-remunerated blood donation in the new millennium

Introduction: In the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. Only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. And around 80% of the global blood population has access to only 20% of a safe blood supply. Conclusion: Blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it.

Background: In response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. Aim: To identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. Methods: A national telephone-survey was conducted among a cross-sectional sample of the adult Norwegian population (1000 participants). The survey was performed in November 2004. Results: Five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. In the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. However, 36% of the young non-donors reported having intentions of becoming a blood donor. Fifty-five percent of young non-donors had a negative attitude towards ever donating blood. All non-donors were asked why they did not donate. Thirty-six percent of all non-donors reported health related reasons for not donating blood. Thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. In comparison only 28% of young non-donors reported medical reasons for not donating. Thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. Summary/conclusion: Although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. The most important reason why young people do not donate was the lack of a personal request. Indifference regarding donation was not very widespread. A relatively high proportion of young people considered themselves as not medically disqualified to donate. In light of these findings, efforts to recruit young people as blood donors are strongly recommended.

Background: The ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the Blood banking infrastructure. Aim: Employing new technology as an instrument for building relationships of trust between Blood bank and Blood donors. Material: 1. A new software module supporting the management of magnetic cards was added to e-aima blood bank management application. The magnetic card supports the following information: • Front-side: Donor's photograph, surname, name and blood group (including Rhesus phenotype & Kell) and sign of Blood collection centre. • Magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. Specific hardware allowing reading from and writing to magnetic cards is integrated to the software module:

• Magnetic card scanners were added to PCs serving to Donation collection, including laptop for mobile team collection. • Web cameras to capture the photograph of the donor to be printed on the card. • Card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. Consumables: blank plastic magnetic cards, ink cartridges for printer. Methods: In order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction.

1. First-time Donors increased 7.8% in the 6 months of application.

Among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. The questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card.

• 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. The turnover of repeat Donors also increased 1.3% after replacing their plain old paper cards with new ones. Further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity.

• 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. Conclusions: In 2004, Greek health policy provided the legal basis for establishing the electronic national health card. The introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. Apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure.

T-PA-060 Emerging technologies in transfusion. DNA based assays

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. Recent emergence of Nucleic Acid Technologies (NAT) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. However, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems such as DNA Arrays permit a conceptually new approach. These miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. However, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the DNA array. Future technologies such as 'Lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. Another major challenge in the area of DNA detection is the development of methods that do not rely on target-amplification systems. Almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (SNPs).

To facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by DNA microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. Methods: A multiplex PCR was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. Each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. Results: Two blinded panels encompassing 94 donors were genotyped for HPA-1 through -5 and 15; no discrepancies were found. Currently, arrays are prepared for the red cell systems. The Fya/Fyb, Fy-GATA1 mutation, Jka/Jkb, K/k, Kpa/Kpb, M/N, RhC/c, RhE/e, RhDpseudogen, RhDVI negative, rs, Doa/Dob, genotypes can be determined. The set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of DNA. The latter can be overcome by Phi29 DNA polymerase-mediated isothermal genomic DNA amplification, from minute amounts of DNA present in stored red cell fractions or antiserum.

The results show that the blood group typing DNA microarray will provide a reliable and fast genotyping procedure. The method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak D types should be regarded as potential anti-D immunizers. For correct determination of weak D both serological typing (polyclonal and monoclonal), as RHD DNA typing are mandatory. When serology indicates weak D, more anti-D antibodies are tested (9 epitope model) to distinguish partial D from weak D. In addition, an RHD MPX PCR is performed to detect the presence of RHD exons 3, 4, 5, 6, 7 and 9. In all known weak D types, all six RHD specific exons are amplified (except for weak D type 4 which lacks RHD exons 4 and 5), whereas partial D phenotypes usually show aberrant patterns. Aim: The aim of this study was to evaluate the diagnostic scheme for weak D typing. Methods: Between 2002 and 2004, 24 samples were investigated for weak D characteristics. Four PCR-SSP assays were developed for identification of weak D types 1 (809T > G), 2 (1154G > C), 3 (8C > G) and 5 (446C > A). Weak D type 4 was identified by the combination of serology and absence of exons 4 and 5 by RHD MPX PCR. RHD-specific exon sequencing was performed when serology and molecular typing were inconsistent. Results: All 24 samples were subjected to the RHD MPX PCR and 1 sample showed absence of RHD exons 4 and 5, indicative of a weak D type 4 when combined with serology. The remaining 23 samples were analyzed by the weak D PCR-SSPs, resulting in 12 weak D type 1 samples, 4 weak D type 2 samples, 4 weak D type 3 samples and 1 weak D type 5 sample. Two samples remained undetermined and were sequenced for all 10 RHD exons and the RHD promotor region. One sample showed the mutations corresponding to the DAU 2 partial D phenotype (209G>A, 998G>A and 1136C>T). The other sample had only one, not previously known mutation (1193A>T), which is located intracellularly at the COOHtail. Extensive serology using the 37 epitope model showed a pattern matching weak D. This new weak D variant was registered as weak D type 41. Conclusions: Based on these results it may be concluded that weak D phenotypes should be confirmed on molecular level to avoid misinterpretation of partial D that cannot be detected by RHD MPX PCR analyses. Patients with weak D phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-D immunization after transfusion of RhD-positive blood products and should therefore be treated with RhD-negative bloodproducts. In this evaluation, 4 out of 24 patients carried such alleles. Introduction: Although Kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-KEL1 is found in obstetric patients, this is a relatively rare cause of HDN. Anemia is produced by immune destruction of fetal RBCs and suppression of erythropoiesis. Maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. PCR techniques for the determination of blood groups using fetal DNA isolated from maternal plasma, allows the application of noninvasive methods. Clinical cases: We describe two cases of pregnancies in women with anti-KEL1 acquired by transfusion/previous pregnancies: 1st case: In July 2000, a 30-year-old woman (gravida 2, para 0), RhDnegative, KEL1-negative was referred at 20 weeks gestation. The father's phenotype was RhD-positive, KEL1-positive. A maternal antibody screen revealed D and KEL1 alloantibodies. DNA was extracted from amniotic liquid. The KEL genotype was determined by PCR-RFLP using the Bsm I. PCR-SSP was used to studied intron 4 and exon 7 of the RHD gene. The results showed that the fetus was positive for RHD sequences and showed KEL2 homozygosity; 2nd case: In August 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. She had a history of transfusion with 2 RBCs units in 1996 -one of the donors was KEL1positive. The woman typed RhD-negative and her husband typed RhD-positive. RBCs from both were KEL1-negative. The maternal antibody screen revealed anti-KEL1. Doubts existed about the putative father of this child. DNA was extracted from maternal plasma using the MagNA Pure LC (Roche). Real-time PCR was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the RHD gene and the SRY gene by SYBR Green; and the alleles KEL1/KEL2 by hybridization probes. All RHD sequences were detected (with the exception of the pseudogene) and the KEL genotype gave a KEL2/KEL2 result. In both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a HDN due to anti-Kell antibodies, and the results of the molecular analysis were confirmed by testing the cord RBCs after birth. Discussion: These cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. A determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. Evidence-based medicine (EBM) is defined as: 'The conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. The practice of EBM requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' Otherwise healthy individuals without cardiopulmonary dysfunction (CDF) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. However, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. Symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. Acute, normovolemic anemia seems to be progressively less tolerated with increasing age and CDF. Controversy has existed on whether or not to correct hypoalbuminemia in ASB or ICP by infusion of albumin. Recently a large trial showed no outcome differences between ICP patients treated with albumin or saline. Thus in general there is no indication for albumin in ASB or ICP. However, albumin may yet be advantageous in e.g. patients with head injuries. Furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. Thus more refined albumin preparations may carry advantages still to be investigated. Erythrocytes are given to increase the total oxygen transportation capacity of the organism. The effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. In the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in ICP, except possibly in ICP with unstable angina pectoris or heart infarction. However, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. On the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. The effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. Grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. The transfusion trigger for thrombocytes in ASB or ICP remains to be established by clinical trials, however. The same applies for fresh frozen plasma, which is infused as a source of coagulation factors. On the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in ASB and ICP, but many clinicians appear hesitant to use them. Another interesting haemostatic agent is recombinant FVIIa, the use of which to control ASB in blunt trauma is supported by one well controlled clinical trial. Evidence by systematic research is insufficient to decide what is optimal transfusion practice. The procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . Concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. Aims: The goal of this study is to evaluate if a restrictive transfusion policy (Hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (Hb < 6.0 mmol/l) without a decrease in HRQoL. Because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. Material and methods: After a run in period of 3 months (Hb transfusion trigger of Hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. Patients are followed then 12 months. HRQoL is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. Also anaemia related complications and red cell antibodies are scored. Hb values were blinded for the patients during the study period.

Results: From July 2002 till June 2004 15 patients were included (4 RA, 5 RARS, 4 RCMD, 1 RAEB, 1 CMMoL) in 2 general hospitals and 1 university hospital. Two patients died in the run in period. Eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. The mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. Two patients in the liberal group died after randomisation. One patient received growth factors. In the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. The mean Hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). No anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. Conclusion: There were some concerns after introduction of the restrictive transfusion policy. This preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. This study will be continued to compare HRQoL scores in both groups. Introduction: Strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (ABD) and erythropoietin (EPO) for reducing exposure to allogeneic blood. However, use of these interventions is highly variable among institutions and frequently sub-optimal. The Program to Reduce Orthopedic Blood Exposure (PROBE) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (THJA) at Ontario hospitals. Aim of the study: The objective of PROBE was to determine whether a comprehensive blood conservation algorithm (BCA) was more effective than usual care (UC) for reducing exposure to allogeneic blood in patients undergoing THJA. Methods: We randomized 30 hospitals that perform high volume elective primary THJA to implement either a BCA or to continue with UC. The BCA consisted of three components: physician and patient education, blood conservation interventions (use of ABD or EPO), and transfusion guidelines. T-PA-071 Table) . Mortality for non-transfused patients was significantly lower than for patients receiving either LR-or S-PRBC at all time points (P < 0.0001). 

T-PA-074 Thalassaemia: the impact on blood transfusion services

Thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. It can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. Although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. This presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. The basic principle in the modern management of thalassaemia patients is that of a global approach to care. Within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. If thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. In countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. The acquisition and preparation of blood, genotyping the patients' blood group (including at least Rh, Kell, Kidd and Duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. Blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. The availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. Sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. High technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. Additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. The patients should be transfused with red cell concentrates, (RCCs) preferably not more than one week's old and leucodepleted. Other processes i.e washing of RCCs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood.

Should patients with thalassemia intermedia be regularly transfused?

Thalassemia major (TM) and thalassemia intermedia (TI) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 The consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. What differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. The consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. In untreated TM cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. Over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. This rate is even better in well-treated patients, almost 80% of whom survive at age 40. Regular therapy extends survival mainly by preventing early development of cardiac complications. In addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. Bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. Patients with TI remain as a rule without regular therapy until a number of severe complications arise. The consequences of chronic anemia develop slowly compared to untreated TM cases and dominate patients' clinical picture usually by the third decade of life. At this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. Bone marrow expansion results to bone deformities and fractures often occur. Extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. Hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. Pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in TI patients. Nowadays, the beneficial effects of regular transfusion and chelation therapy in TM are beyond any doubt. The occasional application of transfusions in TI has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. Intensive and regular transfusion and chelation therapy in TI has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. Given the 30-year experience on intensive therapy in TM and the first encouraging data in TI, the earlier application of such treatment seems to be crucial in TI. The timing however of therapeutic intervention in TI in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. 

The impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major

Backround: Regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. Febrile non haemolytic transfusion reactions (FNHTR) are common complications due to alloimmunization of recipients against HLA and/or specific antigens on donor's WBCs or to the accumulation during storage of biologic response modifiers (BMRs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. Post storage leucoreduction (LR) has reduced the FNHTR in these patients from 13% to 0.5% per unit. It is unknown whether introduction of prestorage leucodepletion (LD) has reduced the incidence of NHFTR further.

We analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 RBC units from January 1998 to December 2003. All units were fresh, stored less than 7 days. 11.950 units were LR-RBC, 8.370 units were LD-RBC and 12.670 were washed LR-RBC. Results: The incidence of FNHTR and allergic reactions in patients receiving LR-RBC was 0.16% and 0.35%/per unit respectively, in those receiving LD-RBC was 0.10% and 0.36%/per unit respectively, while in those receiving washed LR-RBCs was 0.10% and 0.25%/per unit respectively. The relative risk (RR) of FNHRT and allergic reactions following transfusion of LD-RBC and washed LR-RBC compared to LR-RBC is shown in table.

Conclusion: Prestorage leucodepletion and washing of RBCs reduced the risk of FNHRT in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. These findings show that FNHTR after RBC transfusions are due not only to alloimmunization but also to accumulation of BMRs even in patients transfused with fresh RBCs. Washing is as effective as prestorage leucodepletion in reducing FNHTR. Prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. 

T-PA-078 Viral inactivation/elimination of plasma derived medicinal products

The safety of Medicinal Plasma Products (MPPs) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cGMP conditions. Viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. MPP manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. It is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. Very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (HIV, HCV and HBV). Additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (HAV and Parvovirus B19). Pasteurisation (liquid heat treatment at 60°C) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. Solvent-detergent (SD) treatment which is specific to enveloped viruses is used primarily for coagulation factors. Since the introduction of SD-treated products, no HIV, HCV or HBV transmission has been reported. Viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. Acidic treatment is also an efficient means of inactivating viruses in IgG products. Nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. This technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. This property could be helpful in cases of new emerging pathogenic agents. New inactivation tech-niques are currently under development such as UV treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. These new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. The efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. Their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. In this context, a recent European Guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of MPPs, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. Whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. Developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given MPPs an excellent level of safety never previously achieved.

To date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. Safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and NAT viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. Over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and NAT testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. The excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as West Nile virus (WNV). However, multiple viral reduction treatments have generally decreased product recovery. In addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. As time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of R&D objectives. These developments in the plasma fractionation scene of the Western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. In this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. Safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered.

The INTERCEPT Blood System for Plasma uses a synthetic psoralen, amotosalen HCl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (INTERCEPT Plasma, I-FFP). Phase 3 clinical trials have shown that I-FFP retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for TTP. A prototype plasma processing set was used for the clinical trials. For commercialization, a new processing set has been developed to improve productivity. The prototype set accommodated approximately 250 mL of plasma, whereas the improved set accommodates up to 635 mL of plasma, resulting in up to three I-FFP doses per treatment. Aims: This study was designed to characterize pro-and anti-thrombotic proteins in I-FFP prepared using the improved processing set. Proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonWillebrand complex, endogenous inhibitors, and markers of thrombin generation. Methods: Six fresh jumbo (600 mL) apheresis plasma units, collected using the Haemonetics PCS device (Gambro), were photochemically treated. Sodium citrate was used as the anticoagulant. Plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°C until batch analysis. Standardized clinical assays were used for all analyses.

Results: (Results in the table below are expressed as the percent activity in I-FFP in proportion to the activity in plasma before treatment [mean ± SD]). Retention of procoagulant factors in I-FFP plasma ranged from 77% to 92%. Factor VIII and vonWillebrand factor activity, antigen, cleaving protease activity (vWf : CP, ADAMTS-13), and multimeric composition remained within normal ranges after treatment. Endogenous inhibitors of coagulation were retained 93% to 100%. Plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. Retention of contact factors was variable; some factors were below the reference range prior to PCT. With the exception of TAT, all markers of coagulation activation were well within normal ranges. The TAT level in one I-FFP unit was slightly above the normal range; all other units had TAT levels that were well within the normal range. The significance of this is unclear.

CEPT Plasma is similar to conventional plasma. The improved processing set, intended for commercialization, allows up to 3 doses of I-FFP to be produced from a single photochemical treatment.

Background: Guidelines of the European Directive 2004/33/EC require that fresh plasma prior to freezing contains <6109 residual RBCs per litre. This RBCs content is below the sensitivity limit of the automated cell counters used in routine laboratories. Aim of the study: It was therefore essential to make available an alternative method to detect and quantify RBCs in plasma. We implemented a method by flow cytometry using a PE conjugated anti-glycophorin A (GPA) monoclonal antibody that recognises RBCs and erythroid precursors.

To quantify residual RBCs in fresh plasma, the method uses the same TruCount test tubes (Becton Dickinson) as those used to quantify WBCs and that contain a known number of fluorescent beads. After addition of plasma and PE-GPA antibody, cell counting is performed on flow cytometer (BD FACSCalibur). Validation of the method: Assessment of accuracy, linearity, and reproducibility with 2 different PE-GPA antibodies (Immunotech and Pharmingen) Application of the method: Quantification of RBCS in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. Results: Validation of the method: for both anti GPA antibodies, detection threshold is 0.05 ¥ 10 9 residual RBCs/L; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 RBCs/L showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed C.V of respectively 5.7% and 6%. For values >2 ¥ 10 9 RBCs/L, it appeared necessary to introduce a correction factor of 1.3 for the anti GPA Pharmingen. Quantification of RBCs in plasma: Group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 RBCs/L; Group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 RBCs/L in all the cases; Group 3 (apheresis plasma): <0.05°¥ 10 9 RBCs/L in all the cases. Conclusion: Quantification of RBCs in plasma by flow cytometry is a precise, quick and reproducible test. In addition this study shows that even if there are differences in residual RBCs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. Improving basic transfusion knowledge amongst health workers; 2. Improving pre-operative preparation for surgery 3. Strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood.

My presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. Well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. The various training methods employed to improve knowledge in this area will be described.

The increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. In addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. In contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. The first two cases of transfusion transmitted vCJD provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. Preoperative autologous blood donation (PABD) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. Adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. This implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. In this respect, double red cell apheresis may play a significant role. With this approach, donation schedules assumed to enhance erythropoiesis can be adopted. Moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. In conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy Preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. Acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. Intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. Finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. The goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. However, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed.

Compensatory fluid replacement of surgical blood losses: The transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. To reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. As a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. When normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial O2 extraction. However, once the Hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (RBC) is initiated to increase arterial oxygen content (CaO2) and to preserve a margin of safety for tissue oxygenation and organ function. As an alternative to immediate RBC transfusion, ventilation with pure O2 (hyperoxic ventilation) can be employed to rapidly raise CaO2 by increasing the amount of physically dissolved O2 in plasma (hyperoxia).

However, molecular O2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. As a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and O2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary Hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. The 3 patients undergoing TKR and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. According to our patients data, the 55.5% of unused autologous blood units belongs to patients with TKR and implant removal. Therefore a better schedule is needed for these type of surgery. All the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. Fourteen of our cases were supported by erythropoietin S.C. in a dose of 300 IU/Kg every other day. The majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. All of them underwent THR except one woman who also had a TKR 6 months later. The autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. Furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on PABD program.

Our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. However, every effort should be made to render the practice of PABD more efficient and to minimize its costs.

Colloid solutions and their establishment in clinical practice

Background: Various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. This disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. The final result will most likely be an increased mortality and morbidity. Aim: The important issue from clinical aspect is to define the optimal volume and type of fluid therapy. The debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. Method: We searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. Results: Dextrans reduce blood viscosity and von Willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. In clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. After the initiation of Dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. Dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. Gelatins also cause a decrease in circulating levels of vWF : ag, vWF R : Co, thrombin-antithrombin complexes and F1+2. In clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on RBCs protection from mechanical stress. In respect to anaphylactoid reactions, they have the greatest relative risk. HES has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. In addition, it may improve splanchnic blood flow and tissue oxygenation. HES130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. Clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after HES administration, especially. Caution must be held when administering HES during renal transplantation. Albumin became the scapegoat of transfusion strategy during the past 20 years. Resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. A positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. Conclusions: Although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. Instead, we try to extract conclusions through met analysis. Results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. Also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other.

Recombinant human erythropoietin therapy in critically ill patients -a dose response study* Objective: The aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rHuEPO) in increasing hemoglobin (Hb) level and reducing the exposure to red blood cells (RBC) transfusion in critically ill patients. Design: A prospective, randomized, multicenter trial. Patients: A total of 148 patients who met eligibility criteria were enrolled. Intervention: Patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (Control group), i.v. iron saccharate and subcutaneous rHuEPO 40 000 units once per week (Group A) and i.v. iron saccharate and subcutaneous rHuEPO 40 000 units three times per week (Group B). rHuEPO was given for a minimum of 2 weeks or until ICU discharge or death. The maximum duration of therapy was 3 weeks.

The requirement for RBC transfusions was significantly higher in control group than that in group A and B. No significant difference was observed between group A and B. The mean increase in hematocrit (DHct) and Hb (DHb) from baseline to final measurement were significantly higher in group B than these in control group. DHct was significantly higher in group B than that in group A. DHct in group A was significantly higher than that in controls, whereas DHb did not differ significantly between control and group A. Conclusion: Administration of rHuEPO in critically ill patients significantly reduced the need for RBC transfusions. The magnitude of the reduction did not differ between the low and high dose of rHuEPO, whereas there was a dose response of Hct and Hb to rHuEPO in these patients. In transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. There is an increasing awareness of antibodies towards platelet antigens. Detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. Also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. The real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. In our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. If crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified MAIPA procedure and with HLA class I beads (FlowPra from One Lambda, USA) in flow cytometry. In some cases both HLA class I and human platelet antigen (HPA) specific antibodies are detected and HLA class I, HPA matched donors are chosen for crossmatch. If the crossmatch is negative, there is >90% chance of successful transfusions. In some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. In the case of suspected Heparin induced antibodies, a beads assay is performed (DiaMed, Switzerland). Two percent of Caucasian women have the platelet type HPA 1bb. Ten percent of these women make anti-HPA 1a antibodies in their first HPA 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, NAITP). Results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ICH) and there was no still-born babies in the study. Pregnant women with a-HPA 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of HPA compatible platelets if the new-born had platelet count <35 ¥ 10 E 9 /l. In previous studies, it is reported the ICH appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ICH, die. Our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. NAITP represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. Methods: Apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, PO-80 and control (PL2410, Baxter), which have 2660 and 2024 mL/m 2 *day*atm of oxygen permeability, respectively. On days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, pO2, pCO2, pH, glucose, lactate, aggregation, and P-selectin expression were evaluated. Six experiments were performed. Results: The swirling pattern was preserved better for up to 9 days in PO-80 (6/6) than in control (3/6) bags. Dropped pH less than 6.2 on day 9 was observed 1/6 in PO-80 whereas 3/6 in the control. Aggressive drop of glucose (0 mmol/L) with prominent lactate accumulation (327 mg/L) was also observed on day 9 in 1 of control bags. The pO2 level in the control dropped more significantly by 45% (46.6 mmHg) on day 3 than in PO-80 (65.8 mmHg) compared with the initial level (84.9 mmHg) (P < 0.001). These results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. And less lactate generation with slower glucose consumption is also suggested in PO-80 bags than in control bags. The %HSR and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. P-selectin expression was higher in control bags than in PO-80 on days 7 and 9 with no statistical difference. In two control bags P-selectin expression reached >84% and was accompanied by a loss of swirling.

These functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers.

T-PA-096

Background: Maintenance of a neutral pH in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. Furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. However, a platelet additive solution (PAS) containing glucose having a pH of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. In order to have optimal pH, the currently available PAS such as T-Sol does not contain any glucose. This study describes a novel twostep approach to provide a glucose containing additive solution (PAS-G) by using an acid, glucose containing electrolyte solution (pH 5.5) for resuspension and processing of the pooled buffy coats (BC), followed by transfer of the processed platelet concentrate (PC) into a storage bag containing bicarbonate for pH neutralization and maintenance during extended storage. Aim: Compare the platelet quality of pooled BC PC stored in PAS-G with pooled BC PC stored in T-Sol. Methods: A paired study design was used, where a pool of 8 BCs obtained from standard day 1-old CPD-WB units, was divided into two equal parts: One part was resuspended and processed with a Pall leukoreduction system (ATSBC) using T-Sol, the other part was processed in a similar manner with the acid part of PAS-G. Percentage plasma carryover ranged from 20-35%. Both processed PC products were transferred and stored in ELX TM bags with the PAS-G PC ELX bag containing a bicarbonate tablet. Ten replicate studies were performed. Results: The yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (PAGS vs T-Sol). Statistically significant (P < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in PAS-G as compared to T-Sol. The table below shows results at 7 and 9 days of storage for pH, extent of shape change (ESC) and hypotonic shock response (HSR). The results for T-Sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for ESC, and r = 0.68 for HSR at 7 days storage), while no significant correlations were found for PAS-G stored platelets. Conclusion: This study demonstrated the practicality of using a two step procedure to store BC PC in a glucose containing additive solution with neutral pH during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. 

The effect of irradiation on white cell reduced platelet concentrates, stored for 7 days

Background: The storage of white cell (WBC)-reduced platelet concentrates (PCs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. Irradiation up to 50 Gray (Gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. Method: Two WBC reduced PCs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'A' and study group 'B' . PCs in group 'B' were irradiated immediately after preparation with 25 Gy. PCs in both groups 'A' and 'B' were then stored on a continuous flat bed shaker at 20-24°C. Swirl, pH and CD62P expression were determined on day 1, 6 and 8. Twelve experiments were performed and compared with a paired t-test, P < 0.05 was considered significant. Results: See table (day 8 values; mean ± SD; n = 12). Pooling and dividing of the PCs was successful with respect to volume and platelet number. On day 8, the pH in group 'B' was slightly lower than in group 'A', but the difference is not significant. In group 'A', pH on day 8 was <6.8 in 3/12 PCs, versus 5/12 in group 'B' (not significant). The CD62P expression in irradiated PCs is not significantly higher than in non-irradiated PCs. Conclusion: Irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, PRA 63%, 12 donors. In patient 3 -three transfu-sions were effective (two crossmatches neg by LCT and PIFT, one pos LCT, neg PIFT) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. It is very likely that for the same reason patient 4 and 5 were refractory to two and one HPA compatible platelet units respectively. In patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed.

Conclusions: 1. The frequency of occurrence of anti-HPA antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. In 2 patients who developed anti-HPA alone, transfusions of platelets without relevant HPA antigens were successful. 3.The effectiveness of compatible platelets in patients with both anti-HPA and -HLA was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. In one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective.

T-PA-099

The successful implementation of nucleic acid testing (NAT) for HIV, HBV, HCV and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. Today, other risks get into the focus of haemovigilance. Bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. Particularly platelet concentrates (PC) are vulnerable to bacterial growth due to their storage conditions. Patients receiving such products have a potential risk of severe complications or even death. Modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. However, a small risk remains. Therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) Testing and/or (b) Inactivation. Testing for bacterial contamination is possible by different methods: Direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. Bacteria might rapidly grow in a contaminated PC, so testing should be performed as close as possible to transfusion to the recipient. Biochemical methods like oxygen consumption might not detect anaerobic germs. Automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). Novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. Three different principles of pathogen inactivation can be distinguished: Photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. Examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin B2). Photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. Chemicals of this group are psoralens like amotosalen as well as PEN-110 or S-303. The third method, the solvent detergent (SD) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. Methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. These two qualities have to be fulfilled up to the end of the storage period. Toxic or mutagenic compounds must not remain in the final product. The technology must be easily integrated into the existing work cycle of a blood bank. Finally, costs per product must be acceptable. In summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. Pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed.

Bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. There are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. The problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. Reactions are more commonly noted and more severe with platelets stored for greater lengths of time. Previous studies at Johns Hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). These reactions caused fatalities in 4 of 23 cases. Although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, Many potential solutions have been proposed to prevent these reactions. Improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. The same limitation applies to methods that divert potential skin plugs from the collection bag. Antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. Although cold storage of platelets is currently being revisited, it is not yet a practical solution. Pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. As a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. In March 2004, the AABB Standards required testing of platelets for bacterial contamination. Testing programs have been implemented in the US widely as a result of the AABB action. Licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. Although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. These data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. Whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. The use of pH monitoring, Gram stain, glucose measurements, and inspection for swirling have all been attempted. These methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. It is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. It is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the US. Results: Twenty four HCV RNA (+)/anti-HCV(-) repeat donors were previously tested in routine HCV RNA in mini-pools and were negative. Twenty available look back samples were individually tested for HCV RNA and in one the virus was detected. To make sure that the failure of HCV RNA detection in routine NAT was not due to the pooling procedure, the HCV RNA was tested in undiluted look back sample and dilutions of this sample by HCV negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were HCV RNA negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. The results of Cobas Amplicor Monitor (sensitivity 600 IU/ml) were negative, which means that viremia in HCV RNA mini-pool negative donation was below 600 IU/ml. In the recipient of red blood cell concentrate from this donation hepatitis C was diagnosed. However, the possibility of pretransfusion HCV infection cannot be excluded as no HCV marker tests were performed before transfusion. The patient and the donor were infected with genotype 3a.

The low HCV viremia (below 600 IU/ml) in the preseroconversion window period was responsible for no HCV RNA detection in routine mini-pool HCV RNA testing.

Introduction and aim of the study: Bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. Bacterial culturing (BC) of platelets as well as pathogen reduction (PR) reduce the likelihood of such contamination. Where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. Therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. This question we will answer by cost-effectiveness and sensitivity analyses. Methods: The balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of PR. Model parameters and valuations of health states were obtained from literature and information from Dutch Sanquin blood banks. . While the estimates in comparison to the situation without BC or PR are surrounded with large uncertainties, the conclusion that PR is not cost-effective in comparison to BC is very robust. The cost-effectiveness of BC and PR are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of PR relative to BC is not. This conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with HIV, HCV and HBV.

The estimates indicate that culturing in the Netherlands is cost-effective, even with the deviation bag in place. The estimates however appear to be very sensitivity to the probability of sepsis. A decision to use PR will, after the introduction of BC and the use of a deviation bag, never meet cost-effectiveness criteria. Even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to BC is very robust and does not alter when varying underlying parameters within their margins of uncertainty. Table) . 

of CB collection. Two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). This compares favourably with the numbers transplanted from unrelated CB banks. DCB collection is therefore at least as efficient a method as unrelated CB collection for transplantation albeit in the limited number of cases where a DCB collection is possible. DCB collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. It is therefore a useful service to provide and complements the work of unrelated cord blood banks.

Increased yield of mature platelets in cultures of CD34-enriched cord blood cells maintained at 39°C

Introduction: The future use in transplantation of ex vivo expanded hematopoietic stem (HSC) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. Also ex vivo cultures of HSCs may eventually permit to produce donor-free blood components such as platelets for transfusion. Culture of animal cells is routinely done at 37°C. However there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients.

We have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived HSC in megakaryocytes (MK) and mature platelets.

The cord blood-derived CD34 cells were cultured continuously at 37°C or 39°C for 14 days in cytokine conditions optimized for MK development and maturation. The cultures were regularly monitored for various parameters. Results: Compared to 37°C, the cultures maintained at 39°C produced significantly more total cells (4.9 fold) and total MKs (7 fold), and showed accelerated and enhanced MK maturation with increased yield of proplatelets and mature platelets (16.6 fold). Accordingly, the cells cultured at 39°C contained an increased frequency of CFC-MK (8 fold) at day 14. Cultures done at 38°C and 40°C were also more efficient than at 37°C but less than at 39°C. Platelets produced in 39°C cultures could be normally activated by thrombin. As expected, the cells cultured at 39°C contained an increased amount of the heat shock protein HSP70. Control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°C temperature.

The unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°C temperatures on HSC proliferation and differentiation indicate that the routine culture of normal human cells at 37°C is a paradigm that needs to be revised. The responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the MK and possibly other lineages. The synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. It remains to be seen if the stimulatory effects of higher than 37°C temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system.

Several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. Indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. Tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. As well, arguments in favor of integrating tissue banking within a Blood System will be discussed, one of the more important aspect of which being the expertise of the Blood Centre staff with cGMPs. The experience of a blood establishment (Héma-Québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. Finally, Tissue Banking is an activity which brings more expertise to a Blood Centre, expands its knowledge of its customers and gives more opportunities to its personnel.

Umbilical cord blood (CB) is an important source of stem cells for clinical transplantation and may cause less GvH disease than non-T-depleted bone marrow (BM). The relatively low numerical cell dose available from CB has usually restricted its use for transplantation in adults. Only 25-30% of patients have an HLA matched sibling and for others an unrelated BM or a stored unrelated CB donation may also not be available. For some children the collection of CB following the birth of a sibling may be the only opportunity for a transplant. Directed CB (DCB) donations from matched siblings have been shown to give better long-term overall results than matched unrelated CB or BM. DCB collection is however not as easy to control as CB for banking where dedicated hospitals and trained staff are used. Here we review DCB banking in Oxford over a 2.5-year period. Requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. Failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. The remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority SCID). Three collections tested positive for anti-HCV antibody but negative for HCV by PCR. Collections were not excluded on the basis of volume or cell number. Mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean TNC count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). The mean CD34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). All collections were cryopreserved within 24 hours using DMSO/Dextran/Saline without volume reduction. The mean TNC viability prior to freezing was 98% (range 86-100%) and the mean CD34+ve viability post freezing was 82% (range 71-96%). The reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . RHD and RHCE typing was performed by multiplex-PCR with 24 fluorescent primer pairs. Positive results were obtained for RHD-exons 2-7, 9, 10 and polymorphisms associated with antigens C, c, Cw, E and e. Sequencing of RHCE-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an ABI310 (Applied Biosystems). Background: A number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. Among these reactions, transfusionassociated graft-versus-host disease (TA-GVHD) has a mortality of greater than 90%. Mirasol ® Pathogen Reduction Technology (PRT) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. The technology is based on light and riboflavin photochemistry. This study was performed in order to evaluate the effectiveness of the Mirasol ® PRT process for inactivation of human PBMNCs. Methods: Human PBMNCs were collected from Trima platelet apheresis disposable sets, purified by Ficoll-Hypaque discontinuous gradient centrifugation and divided into test and control samples. The test cells were treated with Mirasol ® PRT in autologous plasma on day 0. Both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, T-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to LPS stimulation was measured using a CBA assay kit. Results: Although Mirasol ® PRT treatment did not significantly change the distribution of CD3+, CD3+CD4+, CD3+CD8+, CD19+ and CD16+CD56+ human lymphocyte subpopulations there were significant functional change. The expression of the activation marker, CD69, was observed in 65.4% (SD = 15.6%) of control T cells upon activation with PMA, while only a 1.7% (SD = 1.3%) of the test T cells increased CD69 expression. Proliferation assays showed that 3H-thymidine incorporation did not increase in the test cells in response to either PHA or allogeneic stimulator PBMNC compared to the significant increase in thymidine incorporation levels observed with control cells. The test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder PBMNC proliferation. The release of IL-10, IL-6, IL-1b and IL-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (SD = 73), >5000 pg/ml, 404 pg/ml (SD = 301) and >5000 pg/ml for control cells, respectively. Under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (SD = 0.7) for IL-10, 2.4 pg/ml (SD = 1.2) for IL-6, 11 pg/ml (SD = 3) for IL-1b and 1022 pg/ml (SD = 286) for IL-8. Addition of LPS further stimulated the release of TNF-a, IL-10, IL-6, IL-1b and IL-8 in the control samples, but not in the test cell samples.

In vitro studies demonstrate that Mirasol ® PRT treatment does not change lymphocyte immunophenotype, inhibits Tcell activation by PMA, abolishes PBMNC proliferative activity, eliminates PBMNC stimulatory activity for responder cell proliferation and suppresses the production of cytokines by PBMNC in both the absence or presence of LPS.

Introduction: It has been discovered that vaccination of dendritic cells (DCs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. Aim of the study: The purpose of the study was to investigate whether human monocyte-derived dendritic cells (DCs) were able to present p210Bcr-Abl protein and induce antigen-specific CTL responses in vitro after transfected with total RNA of K562 cells (K562-RNA). Methods: DCs were derived from human PBMNCs, which were incubated for 5 days in the presence of GM-CSF and IL-4, and then were transfected with K562-RNA using electroporation or DOTAP lipofection. To verify the successful transfection of DCs with K562-RNA, Bcr-Abl fusion genes expression of DCs was detected by RT-PCR and Western blot. The immune phenotypes of the DCs were analyzed by flow cytometry. The cytotoxicity of CTL was assayed by propidium iodide (PI) staining and flow cytometry. Results: It was shown that the Bcr-Abl fusion gene was detected in the DCs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210Bcr-Abl protein and expressing increased CD80, CD83, CD86, HLA-DR. Moreover, the transfected DCs could significantly promote the T lymphocytes to kill the target K562 cells. Conclusion: Human dendritic cells transfected with total RNA of K562 cells in vitro could induce effective p210Bcr-Abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors.

appear to be relevant to the clinical response. IVIg has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. Judicial use of IVIg based on results from controlled studies is recommended.

T-PL3-09 Donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation

The notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. Subsequently, pooled leukocytes from patients with CML were found to effect responses in patients with advanced leukemia. Response correlated with cell dose and with severity of GVHD. In the 1970's, the graft-versus-leukemia (GVL) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. Such studies suggested that GVL could be enhanced without causing severe GVHD. The era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (DLI) for relapsed acute and chronic leukemias after bone marrow transplant.

It is now clear that chronic myelocytic leukemia (CML) in chronic phase is highly susceptible to GVL effects mediated by DLI which induce durable remission in 70-80% of relapsed patients. The success rate is 30% or less in patients with accelerated phase or blast crisis. Since DLI cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance GVL without exacerbating GVHD. Unfortunately, the response to DLI in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. DLI have been used successfully to treat viral infections and virus-associated malignancies following transplant. Both unfractionated DLI and ex vivo-generated Tcell clones have suppressed reactivated cytomegalovirus and eradicated Epstein-Barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin B cells that occurs after transplant. Where tumor-specific antigens have been defined, efforts to target DLI have been undertaken and donor and patient immunization has been investigated. Acute or chronic GVHD develops in approximately 60% of patients receiving DLI for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor T-cell dose. DLI-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. Complications of aplasia include infection, bleeding, increased transfusion requirements. Efforts to limit the adverse effects of DLI while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens.

Available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (IVIg), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive T cells and inflammatory disorders e.g. an imbalance in cytokine networks. 

TrimaR-collected apheresis platelet concentrates (PCs) were exposed to 17.2 J/mL UV light in the presence of 50 uM riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mM for 5 days. The control platelets were not stressed by UV light exposure and were stored under the same conditions without 2-DOG presence. All test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. Results: Lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (SD = 0.0097) and 0.0268 mmol/1012 cells/h (SD = 0.0114) for control samples to 0.1371 (SD = 0.0281) and 0.0724 (SD = 0.0151) for UV-treated platelets, respectively. UV treatment also caused a decrease in pH from 7.50 (SD = 0.07) for controls to 6.55 (SD = 0.26) for treated platelets at day 5, HSR from 75% (SD = 12.4) to 33% (SD = 25.1), ESC from 24.3% (SD = 3.9) to 3.8% (SD = 3.6), swirl from 2.8 (SD = 0.7) to 1.0 (SD = 1.0), and increased P-selectin expression from 21.2% (SD = 7.3) to 85.2% (SD = 9.4). Addition of 2-DOG up to 20 mM significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (SD = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (SD = 0.0060), and maintained pH above 7.35 (SD = 0.09) for 5 days of storage. The effect of 2-DOG exhibited a dose-dependent response. However, the addition of 2-DOG had no effects on HSR (28.1 + 11.4% at day 5), ESC (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and P-selectin expression (69.9 + 6.4% at day 5) during platelet storage. ATP contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. Furthermore, an exaggeration of UV-stressed platelet aggregation by addition of 2-DOG was also observed. Conclusions: Increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. The results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. Aim of the study: Was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of HIV, HCV and HBV, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. Methods: Secondary Standard traceable to WHO International Standard for HIV-1 (97/ 656), International Standards for HCV (96/798) and HBV (97/746) were used to determine the analytical sensitivity. Sensitivity and inclusivity for HIV-1 subtypes other than HIV-1B, for HIV-2 and for Hepatitis B and C genotypes as well as specificity was evaluated with >1000 specimens. Results: Results from this study indicate that high analytical sensitivities (39 IU/mL HIV-1M, 114 cp/mL HIV-1O and 1.1 cp/mL HIV-2, 7 IU/mL HCV and 3 IU/mL HBV) and a specificity of >99.4% are accomplishable for the MPX Test. The 95% detection rate for HIV-1M subtype isolates (A through H) was between 10 to 50 IU/mL, for HCV genotype isolates (1a through 6) between 2 to 10 IU/mL and for HBV genotype isolates (A through G and Precore mutant) between 2 to 5 IU/mL. Investigating seroconversion panels, HIV-1 RNA was detected an average of 6 and 5 days earlier than HIV-1 antigen with Abbott HIVAG-1 Monoclonal and Coulter p24 Antigen tests, respectively, HCV RNA an average of 31 or 21 days earlier than HCV antibody with the Abbott HCV EIA 2.0 or Ortho EIA 3.0 tests, HBV DNA an average of 18 days earlier than HBsAg with the Abbott HBsAg EIA IMx test. For all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human DNA or bilirubin. Conclusion: Automated pooling, sample preparation, and real time PCR using the Blood Screening System 200 TaqScreen MPX Test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. The MPX Test is another evolution step in the development of PCR automation by Roche Molecular Diagnostics, and further represents Roche's commitment to increasing the safety of the global blood supply. Aim of the study: A prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of IP that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment.

Methods: This plan is proposed to blood establishments and transfusion prescribers who have already decided to implement IP. This is an observational, non randomized, non controlled plan. No patient selection, inclusion or exclusion criteria are required. All IP transfusions are documented using an Internet form, whether or not a reaction is observed. Patient population data are collected anonymously, for epidemiological purposes. Results: Between October 2003 and September 2004, 2512 apheresis IP units have been transfused in 2 sites and registered in the database. IP platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). The population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). The most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). The patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). The number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). Half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. Transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% CI 0.84-1.75), and 8.5% of patients. Only 2 (0.1%) were considered serious. After further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% CI 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. The most often reported symptoms were chills (0.9%) and fever (0.6%). Itching, skin rash or urticaria was observed in 0.5% of transfusions. Of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. None of the reactions occurred in cardiovascular surgery patients. Patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, P = 0.15).

The active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. The risk profile of IP transfusions appears favorable.

Quality of Theraflex MB-plasma during storage and treatment S REICHENBERG* and N MÜLLER † *Maco Pharma International GmbH, Langen, † Inst. for Transfusion Medicine, Essen, Germany

Background: Although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. Additionally new viruses like West Nile Virus enter the transfusion chain. Therefore, the treatment of therapeutic plasma with methylene blue (MB) is a technique used in several European countries for pathogen inactivation. MacoPharma has developed the proprietary Theraflex MB-Plasma bag system including a MB pill and a final MB filtration step. Aims: Aim of the study is to show the quality of the MB plasma during the preparation procedure and during storage using the Theraflex system. Methods: For the preparation process every single step was evaluated using 18 single donor plasma units. For the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with PLAS4, after dissolution of the MB pill, after illumination, after treatment). Because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. Six samples of each stage were pooled at three days. A whole panel of plasma factors was measured for the resulting three pools. Global tests: Quick, INR, aPTT, thrombin time Coagulation factors: Fibrinogen, factor II, factor V, factor VIII:c, factor IX, factor X, factor XI Inhibitors: AT III, protein C, protein S Fibrinolysis: plasmin inhibitor, alpha1-antitrypsin Complement: CH50 Activation: TAT, factor XIIa, D-dimer Stability data were generated using three plasma pools. Six plasmas were pooled and afterwards divided into six aliquots. Each was treated as single unit and then each was divided into six storage samples. The same plasma factors as for the manufacturing process were evaluated. Results: A moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. This was mainly fibrinogen (17.5%), factor VIII (22.2%), and factor X (13.4%). Despite this reduction the values were within the ranges found in non-treated plasma. All investigated plasma factors remained stable during the investigated storage time. Summary/conclusions: The investigation showed that plasma treated with the Theraflex procedure showed slight reduction during treatment and no reduction during storage. All plasma factors remained within the threshold values. The treatment of therapeutic plasma with MB is a valid technique of pathogen inactivation.

Validation of intercept treatment of pooled platelets G SANTOS, C SILVA, F PEREIRA and G SOUSA Lisbon Regional Blood Centre, Lisbon, Portugal Background: INTERCEPT BLOOD SYSTEM for platelets uses amotosalen HCl and UVA light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. Aims: The purpose of the study was to assess the feasibility of introducing this technology in the routine of Lisbon Regional Blood Center (CRSL) and validate the procedure in our center. Material and Methods: Whole Blood units of 450 mL were collected from volunteer blood donors in quadruple top and bottom blood bags (Optipure RC soft T& B Baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 mL were obtained in the Opipress II and kept overnight at room temperature before pooling. Five buffy coats were pooled with 280 mL InterSol using the octopus system INTERCEPT Buffy coat pooling set with an integrated filter. The pools were treated using the INTERCEPT. Samples were taken before treatment, after CAD remotion, on days 2, 5 and 7. The following tests were performed: platelet count, mean platelet volume, pH, swirling.

Results: All pools met the Intercept guardbands. Platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). Platelet pool volume was 362.3 mL (sd-10.7). Plasma % was within 32.9% and 36.5%. All pools had leucocytes within Council of Europe specifications. After photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). The average platelet loss was 0.35 ¥ 10 11 . The pH was within specifications during all the storage period. Conclusions: Intercept treatment of pooled buffy coat platelets is feasible in the routine of CRSL and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown IP and conventional platelets (CP), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. Extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. Clinical efficacy and safety of IP stored for 7 days were investigated. Methods: A randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat IP vs buffy coat CP, each stored for 7 days. Patients were randomized to receive one 7-day IP transfusion and one 7-day CP transfusion in random order. After each study transfusion, the 1hour platelet count, CI, and CCI; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (SAEs) were assessed. The primary endpoint, 1-hour CCI, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation).

The per protocol population included 20 patients, 9 randomized to the IP-CP sequence and 11 to the CP-IP sequence. More patients received allogeneic stem cell transplant in the CP-IP sequence than the IP-CP sequence (64% vs 33%; P = 0.37). Mean platelet dose (¥10E11) was 2.8 for IP and 3.0 for CP (P = 0.23). There was a significant period by treatment interaction (P = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. Including both treatment periods, mean (±SD) 1-hour CCI (¥10E3) was 6.6 ± 4.5 for IP vs 8.9 ± 5.5 for CP. The mean paired difference for both sequences was 2.4 ¥ 10E3 (P = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). For the first period only, mean 1-hour CCI (¥10E3) was 8.7 ± 3.8 for IP vs 7.4 ± 5.4 for CP) The mean paired difference for the first period sequences was 2.4 ¥ 10E3 (P = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). The non-inferiority margin for the study was 2.2 ¥ 10E3 for mean treatment difference in CCI (CP-IP). Median time to next transfusion was 25 h for IP vs 24 h for CP following the first transfusion (P = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h IP vs 34 h CP after the second transfusion (P = 0.02). Bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and Grade 2 or lower, and responded similarly to IP and CP. No significant transfusion reactions or SAEs were reported.

In this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour CCI was not met. However, transfusion with 7-day-old platelets treated by the INTERCEPT Blood System showed only a marginally and probably clinically insignificantly lower 1-hour CCI compared to 7-day-old conventional platelets. Methods: New Zealand white rabbits were transfused with syngeneic blood (10 mL/kg), across a major antigen (HgD) mismatch. High anti-S-303 Ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with KLH-(S-303) hapten (KLHhapten) in complete Freund's adjuvant. Ab titers against SRBC were determined by gel card agglutination, or by FACScan with FITC-Goat_anti-rabbit_IgG. Survival of infused RBC (4 mL/kg) treated with different methods was assessed by RBC biotinylation. Blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using Streptavidin_PE and FACScan to determine the proportion of circulating biotinylated RBC. Results: Groups (G) of rabbits were transfused with control rabbit RBC (CRBC; n = 4, G1), or O-SRBC (n = 6, G2). No Ab against O-SRBC developed after biweekly transfusions over 24 weeks in G2 rabbits. High Ab titers to O-SRBC could however be induced by KLH-hapten immunization in a different group of animals (n = 2; G3). Transfused rabbits (G1 & G2) exhibited no change in hematocrit or body weight and maintained good vital signs. High titer anti-S-303 Abs were then induced by KLH-hapten immunization in rabbits from G1 (n = 2) and G2 (n = 4), and in a group (n = 6, G4) of naïve rabbits. Non-immunized rabbits (G1, n = 2), and (G2, n = 2) were maintained on the biweekly transfusion schedule of CRBC and O-SRBC, respectively. All rabbits treated with KLH-hapten developed comparably high Ab titers. KLH-hapten immunization did not affect the viability of CRBC in G1 rabbits. Transfusion of O-SRBC demonstrated reduced viability in hyper-immune G4 rabbits, but not any of the G2 rabbits. G2 rabbits exposed to 12 O-SRBC transfusions prior to hyper-immunization with KLH-hapten, had viability of O-SRBC comparable to CRBC, suggesting induction of immune tolerance by repeated exposure to O-SRBC. After depletion of labeled O-SRBC from circulation, G2 and G4 were transfused with M-SRBC. Viability of M-SRBC in all G2 rabbits (hyper-immune or not) and the hyperimmune G4 rabbits was equivalent to CRBC circulation in G1 rabbits.

In pre-immunized rabbits with high titer anti-S-303 Ab, O-SRBC are cleared faster than control. In contrast, M-SRBC survive normally in rabbits with high anti-S-303 Ab titers. Repeated transfusion of O-SRBC does not result in alloimmunization of naive rabbits. The modified S-303 RBC process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of RBC viability.

Introduction: The Bombay phenotype is extremely rare and characterized by complete absence of ABH activity both on erythrocytes and in secretions. Those individuals can produce anti-H, which is active over a wide thermal range. Method: Using LISS indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. A cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. Introduction: Immunohematology Reference Laboratory in Kuwait Central Blood Bank receives samples from all hospitals in Kuwait both governmental and private sector. The laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. Material and method: A tube and Gel cards are two methods in the reference laboratory for antibody identification. The laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive Direct Antiglobulin Test that occur mostly in autoimmune hemolytic anemia. There are facilities to phenotype most of rare red blood cell antigens. Results:

Records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records.

Central Blood Bank has the potential to identify rare phenotypes such as Kpb-, Jsb-, Lan-, Bombay, RzR2, RzR1, r¢r¢, r¢r≤, r≤r≤, Ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-Ge2, anti-H, anti-Lan, anti-Kpb, anti-Jsb, anti-Wrb, anti-Ena, anti-Csa and low frequency antibodies anti-Kpa-, anti-Jsa-, anti-Dia, anti-Lua, anti-Cob as well as hightiter-low-avidity antibodies such as anti-Chido. Samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program.

Purpose of the work: To present the substitution of the blood groups O, A, B, AB in ABO blood group system and Rh (D) blood group in Rh (D) blood group system in the population in the Gevgelija-Valandovo region. Material and methods: A retrospective analysis was done on the data of following the blood groups O, A, B, AB and D in the blood group system ABO and Rh in the Gevgelija-Valandovo region. The asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. The period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. The examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems ABO and Rh in the population. Tests were done with different series of commercial anti serum tests from domestic and foreign origin. Results: Totally 43.395 examinees were typified. With O blood group were 15.761 (36.31%); with blood group A were 17.675 (40.73%); B blood group 7.119 (16.4%) and AB blood group were 3.019 (7.52%) examinees. Totally 37.365 (86.11%) were D positive and 6.029 (13.89%) were D negative. Discussion: The given results from our examinations for the frequency of O, A, B, AB and Rh (D) blood groups from ABO and Rh (D) blood group systems in the region Gevgelija-Valandovo show that the most present is the blood group A from ABO blood group system 17.675 (40.73%) and D blood group in Rh system with 37.365 (86.11) form examined population. The results are in correlation with data from the literature for other European nations.

Introduction: The policy of our center is to transfuse all Heamatologic multitransfused patients with their own Rhesus and Kell phenotype. In addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important Duffy and Kidd systems while all the other patients receive blood compatible only with ABO and Rhesus system. Nevertheless, it is observed a significant positivity of the Indirect Antiglobulin Test, due to alloimmunization. Aim of the study: In this study we tried to evaluate the prevalence of alloimmunization in patients of our region. The most frequent detectable alloantibody remains the anti-D, with high prevalence 88.9% of anti-D in females versus 11.1% in males, due to alloimmunization during the pregnancy. As a consequence, the high incidence of anti-D is not transfusion related and anti-E is evidenced to be the most frequent transfusion related alloantibody, followed by anti-Kell. Care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-E and anti-Kell.

Severe hemolytic reaction due to anti-J K3

Background: Red blood cell alloantibodies directed against antigens of the Kidd system are notorious for causing delayed hemolytic transfusion reactions. The antibodies are formed because of pregnancy or transfusion. Blood donors with the red blood cell (RBC) phenotype Jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. However, the rare phenotype Jk(a-b-) is more common in Polynesians (1.4%). Individuals with Jk(a-b-) phenotypes typically form anti-Jk3 with inseparable anti-Jka and anti-Jkb activity. Some Jk(a-b-) patients' sera may show an additional distinct anti-Jka or anti-Jkb component when examined with adsorption studies. Case report: A 63 years-old Caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. It is reported four pregnancies with no history of haemolytic disease of the newborn (HDN). On admission, her haemoglobin was 5.5 g/dL. She was given 4 units of crossmatchcompatible RBC. On day 6 her haemoglobin was 12.1 g/dL, with a total bilirubin of 0.72 mg/dL and lactate dehydrogenase of 141 U/L. On 10th day an unexpected fall in Hb (5.6 g/dL) occurred with an increase of bilirubin to 1.9 mg/dL and of lactate dehydrogenase to 1042 U/L. A new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. Anti-Jk3 antibody high titer was detected in the plasma by gel-test using Liss/Coombs cards (ID-Diamed). The DAT was negative and the antibody reacted equally with Jk(a-b+), and Jk(a-b+) panel cells (Jka:1/16 384 and Jkb:1/16 384). Other alloantibodies could not excluded, because Jk(a-b-) cells are not available. She was started with erythropoietin-a (EPO), folic acid, Fe IV and high dose intravenous immunoglobulin (IVIG). The EPO was discontinued after four week of therapy when the haemoglobin was 12 g/dL. Two months later her haemoglobin was 13.5 g/dL and anti-Jk3 was present in the same titer. A year later her blood cell count was normal and the anti-JK3 was detected in a lessened titer (1/16). No additional distinct anti-Jka or anti-Jkb component was shown after two adsorptions at 37°C using carefully selected phenotyped red cell compatible with patient's Rh, Fy, MNSs, Lu, Le system and Jka(+) and Jkb(-), but two additional alloantibodies anti-c and anti-E of low titer (1/4) were revealed.

The rare anti-Jk3 alloantibody found in this case displayed the erratic nature of many Kidd system antibodies. Although anti-Jk3 may cause mild hemolytic disease of newborn, she did not have a history of HDN. Our patient was sensitized to a Kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. The use of EPO and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible RBC unit.

Background: Differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, Kpb, Lub and Inb. This technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion.

Purpose: Differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. In these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. Methods and result: Case 1. A 63 years-old Caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. Anti-k was detected in the plasma. It is reported two pregnancies and no history of transfusion. The DAT was negative and the plasma did not react with one k-cell present on the red cell panel in use. The anti-k specificity was confirmed using additional k-cells. The patient's red cell were group A, D+, K+, k-, c-, E-, Fy(a-), S-, Le(a-), Kp(a-), Cw-. She was transfused with two units RBC k-. Ten days after the first transfusion the DAT became positive and the one k-cell present on the red cell panel reacted with her plasma. Two adsorptions were carried out at 37°C using carefully selected phenotyped red cell (compatible with patient's Rh, Fy, Jk, MNSs, Lu, Le system and k positive). An anti-Fya was identified in the presence of ant-k. . An antik (titer 1/16) was suspected. Because k-red cell was not present in the panel in use, adsorptions were carried out at 37°C using carefully selected phenotyped red cell (compatible with patient's Rh, Fy, Jk, MNSs, Lu, Le system and k positive). After the anti-k antibody was totally removed, no additional alloantibodies were revealed. Conclusion: Differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion.

Evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using Nacl cards P CHALKIA, S INTZEPELI, V AVGOLOUPI, A TSOUKALA, E NTINOPOULOU and P DIDOUDI AHEPA Hospital, Thssaloniki, Greece

Background: Expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. Accurate results depend on the integrity of the antigens. Purpose: To validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using Nacl cards.

The serum of nineteen patients with common specificities antibodies in Rhesus, Kell, Duffy, Kidd, and MNSs systems tested with commercially prepared 0.8% enzyme treated cells RBC panel (ID-Diamed). Gel tests were performed according to the manufacturer's instructions on in-date RBC and simultaneously on RBC 1 month to 5 months past expiration. Reactivity of the expired antigen positive and antigen cells was compared to in-date cells.

Results: Twenty antibodies detected with enzyme treated red cells in neutral gel cards [D(4), c(3), E(2), K(7), Cw(4)] and were tested with enzyme treated red cells in use and RBC 1 to 5 months post expiration. Seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-K antibodies (negative with K positive cells 2-4 months post expiration). Conclusion: Most RBC antigens studied were detectable 5 months after RBCs expiration date. Tests with 0.8% cells were valid in gel test (Nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. Studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. Background: Wr(a) is a low-incidence blood group antigen (1 : 1000) in the Caucasian population. Despite that anti-Wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. Anti-Wr(a) may occur as an autoantibody or arise without immune stimulus. We report a case of a naturally occurring anti-Wr(a) antibody. Case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. The serological status identified by their Blood Bank was: B RhD positive, with anti-Wr(a) antibody in the serum (Cellbind card method).

Our results: The patient's cells were group B RhD positive (microplate method), DAT negative (tube and gel test method). The antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in LISS IAT (tube test) and gel IAT (ScanGel and DiaMed). Discussion: Our routine tests for antibody detection didn't detect any specific antibody in patient's serum. He was transfused several units of blood, that was Wr(a) negative and showed negative crossmatch reactions. The patient had no transfusion reactions. 10 days after the transfusion anti-Wr(a) specificity was confirmed in the serum with Cellbind test. Only few test cell panels contain Wr (a) positive cells, which are usually not present in commercial screening cells. In our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. The risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. Background: According to requirements of the French Committee for Accreditation (Comité Français pour l'Accréditation COFRAC, ISO 17 025 Standards), it is essential to use validated and standardised methods in Immunohematology. This imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. Aim: A multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (IAT) in filtration technique.

The antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: ID Diamed, Biovue Ortho and Scangel Biorad, the same tests cells, a standard 20 ng/mL anti RH1 (provided by CNRGS), a positive control anti KEL1 and a negative control. All equipment used (oven, chronometer, pipettes) were calibrated according to COFRAC standards. Each antibody sample was tested under the following combined conditions (243 tests/sample): Results: All the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. Conclusion: This study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in IAT using commercial filtration systems maryvonne.

The authors present a retrospective study involving 12 091 blood donors from the University Hospital of Coimbra during the year 2004. The incidence of weak D and Rh (D) phenotype was determined in 1729 individuals who were Rh (D) negative. The AB0 and Rh(D) blood grouping was performed using a column gel agglutination card (Diamed). The Rh (D) typing was done using a anti-D polyclonal and a anti-D monoclonal antibodies. All donors that gave negative or poor agglutination results were tested for weak D with an indirect antiglobulin test, with anti-IgG (gel matrix card) plus anti-D serum (Diamed). Within our target group of blood donors the Rh (D) negative represented 14.3% of the total sample. We also describe AB0, Rh(D) phenotype (C, c, D, E, e) group frequencies and establish reliable estimates frequency for weak D and Rhesus haplotypes.

The Background: In 80s and 90s several authors tried to assess the relationship between the number od IgG molecules per RBC and in vivo haemolysis, but determinations usually concerned small groups of tested patients. Some of the investigators suggested that the number of IgG autoantibody molecules per RBC was a major determinant of the severity of the haemolysis, whereas others found AIHA patients with severe haemolysis and undetectable autoantibodies. Aim: Presentation of our experience with the quantitative ELAT performed on a large group of AIHA patients during long-term observation. Material and methods: Six hundred fifty eight blood samples from 268 warm-type AIHA patients were randomly tested for the number of IgG molecules per RBC. Eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. Autoantibodies on RBCs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (ELAT).

Results: In about 1/3 of tested samples the number of IgG molecules per RBC was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 IgG/RBC). The large number of IgG molecules per RBC (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple IgG subclasses on RBCs and C3d. In 79% of patients tested periodically, the number of IgG molecules per RBC decreased and it significantly correlated with improvement of haemolysis parameters. In 21% of AIHA patients the number of IgG fluctuated and it was a poor prognostic factor. Conclusion: In AIHA patients the dynamics of the changing number of IgG autoantibody molecules per RBC is a more helpful diagnostic and prognostic parameter than the number of IgG molecules per RBC evaluated in one test. -b+) , -H-negative (using the Anti-H lectin). Antibody work-up showed a positive antibody screen (LISS and PEG-tube methods) reacting 4+ with O rbcs at all phases and 2+ with A1 rbcs. The direct antiglobulin test (DAT) was positive with Polyspecific AHG as well as Anti-C3b,-C3d (Table 1) . Prewarming of test system did not change the reactivity (4+ at Antiglobulin phase). A treatment with Dithiothreitol (DTT) was performed and abolished all reactivity of the serum ( Table 2 ). Autoabsorption of the patient's plasma was performed. The absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with O red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (Table 3 ). The patient remained crossmatch incompatible with O and A rbcs, but was compatible with Oh rbcs. Summary: We report an unusually strong IgM anti-H antibody in this patient, who may require Oh phenotype units. The patient is not a para-Bombay since her red cells type strongly as group A.

The cause for the auto-anti-H remains unknown at this time. If a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive H-units if transfusion is required. Introduction: Fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. For this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. However the indirect antiglobulin test (IAT) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. Aim of the study. In this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. Methods: We have studied 1.560 women, 19-43 years old, resulted IAT positive at the first screening and successively assisted by our two Hospitals. All women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and IAT. Results: A positive IAT was confirmed only in 64 cases; moreover a RBC autoimmunization was found in 7 women. Anti-D (16 cases), E (10), C (8), K (8), c (6), S (5), D + Jka (1), D + S + E (2), D + C + K (2), M (3), c + E (1), D + C + G (2) were the identified alloantibody specificities. Anti-S, -E, -K, -C and -Jka were the specificities in autoimmunized women. Conclusion: In conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases IAT resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center.

Background: One of the problems of the RBC transfusion is the alloimmunisation and the delayed haemolytic reactions (DHTR). Besides Rhesus and Kell systems the antibodies against Kidd antigens cause both DHTR and difficulties in their detection. Aim and methods: The exact recording of all blood units according to ABO Rhesus Kell and Kidd systems. The ABO-Rh-Kell Systems are identified through automated microcolumn method (AutoVue, Ortho), while Kidd antigens are identified manually using microcolumn gel (DiaMed). Results: The percentage of Jka+ and Jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the Caucasian population. Conclusions: Given the fact that there is lack of available freezing RBC system in Greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. In the latter case suitable donors can be called and cover the shortage. The identification of all antigentic systems of the donated RBC units is underway.

Background: In many countries transfusion recipients are currently typed and transfused D-positive, if their red cells are agglutinated by 2 IgM monoclonal anti-D that do not react with DVI. The transfusion strategy in weak D patients is not clear defined and it depends on the chosen monoclonal reagents and methods. Patients who are carrying Dw types 1, 2 and 15 were prone to develop anti-D. Aim: The aim of this pilot study was to estimate capability of commercially available monoclonal anti-D reagents to recognize this weak D types as RhD positive. Material and methods: EDTA anticoagulant blood samples were collected from 50 blood donors, previously typed as weak D positive by indirect antiglobulin test. Molecular genotyping of RhD gene and weak D alleles by CDE-SSP and D weak-SSP kits (Inno-train, Germany) were performed. Direct agglutination was tested in a tubes and microplates using the following antibodies: RUM-1, TH-28, MS-201 (Bioscot/Serologicals) and D415 1E401/175-2 (Immucor). Results: Out of 50 samples molecular typing results were as follows: in 5 samples Dw were not determined, in 45 samples, 20 weak D type 1; 12 weak D type 2; 8 weak D type 3, 1 weak D type 11; 3 weak D type 14 and 1 weak D type 15 were determined. By all monoclonal reagents 95% weak D type 1, 100% weak D type 2, 10% weak D type 3 and 100% weak D type 15 negative results were given. By all monoclonal reagents weak D type 11 and weak D type 14 positive results were given. Conclusion: According to weak D types, which were known to be at risk for anti-D immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak D type 1, type 2 and type 15. Such improved typing strategies with novel reagents would enhance the transfusion safety.

Background: Vel is a high-incidence antigen found in >99% of the population. Anti-Vel can be IgM or IgG and reacts optimally at IAT, although it can also react at immediate spin and 37C. Anti-Vel may or may not cause severe hemolytic transfusion reactions and mild to severe HDN. Autoanti-Vel has also been reported. The AABB Technical Manual 14th edition states that the Vel antigen is unaffected by protease and sulfhydryl treatment. We have reason to believe that sulfhydryl treatment may have an effect on the Vel antigen as evidenced by a recently referred case. Case report: A 67 year-old Caucasian female presented with symptoms of anemia and renal vascular hypotension. Transfusion history indicated multiple red cell transfusions in 1959. According to the patient, previous attempts to locate compatible units were unsuccessful. The case was referred to our laboratory. The patient's red cells (rbcs) typed as group O, D+ with a negative DAT. The serological picture revealed an antibody reacting 2 + -3 + s at 37 C/LISS, as well as at the antiglobulin phase. The antibody reacted with all rbcs tested and the autocontrol was negative. Further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 M dithiothreitol-(DTT)treated rbcs. A high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by DTT. The antibody reacted with all rbcs tested. Additional rbcs were tested that lacked high-incidence antigens, including Vel. The antibody did not react with four Vel-rbcs tested using LISS and PEG methods. The patient's rbcs typed as Vel-negative. All other clinically significant antibodies were ruled out using Vel-or DTT-treated rbcs. Based on the unusual reactivity demonstrated by the anti-Vel, we tested 12 different examples of anti-Vel (frozen in our rare sera inventory) against two sets of known Vel+ and Vel-rbcs. One rbc set was DTT-treated; the other set was tested neat. LISS enhancement was used to test both sets. One of the 12 antisera failed to react with the positive control and one reacted with the negative control. Both were disqualified from the study. Four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the DTT-treated rbcs. The remaining six antisera showed no change in reactivity. Conclusion: Contrary to the statement in the AABB Technical Manual, we discovered that sulfhydryl treatment (0.2 M DTTtreatment) can have an effect on the Vel antigen. Our experience has demonstrated that in some cases anti-Vel may not react or may show reduced reactivity when tested with DTT-treated rbcs. Therefore, the presence of anti-Vel should not be ruled out if negative reactivity with DTT-treated rbcs is encountered. Additionally, DTT treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-Vel. Additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, HLA antibody screening by lymphocytotoxicity test (LCT) and ELISA in some cases. Patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and LCT were noted. Antibody tests were performed: at pretransfusion testing (PT) by LISS-Coombs (DiaMed) and at blood grouping (BG) by BioVue polyspecific (Ortho) microcolumns -manually in urgency and routinely by Sampler IIF (DiaMed) and MITIS (Ortho) systems. Results: Investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. These findings comprised 15.1% of 1598 unexpected results found at PT and BG. Incidences were 0.18% at routine and 0.21% at urgent BG (37 339 and 16 003 BGs, respectively), and 0.35% both at routine and urgent PT (15 803 and 23 705 PTs, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). Frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. Subsequently positive antibody test during the study had 27.6% tested patients. At PT positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. Majority of reactions were '1+' (50% at PT and 38.6% at BG); reactions '3+' or '4+' were found in only 5.1% cases at PT, compared to 17% at BG. One crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. AHG identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. Lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. Finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, HLA lymphocytotoxic antibodies in 9.7%, 'IgG antibodies of unknown specificity' in 7.5% (HLA noncytotoxic antibodies in 2/2 ELISA tested samples!), contaminated sample in 5.9%, anti-Bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 Lua, 2 M, 1 Kpa, 1 Yka), non-specific autoantibodies in 3.2%, carry-over of DAT-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. Discussion: After introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. Such reactivity was frequently caused by laboratory mistake, but often HLA and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. Relationships that may be helpful are further discussed in abstract part II. Results: Significant differences (P < 0.05) and relationships of interest were noted. Sex. In female vs male patients frequent features were: crossmatch as only reactivity at PT (45.8% vs 28.2%), positive LCT (59.4% vs 26.1%), lymphocytotoxic HLA antibodies (LyTxAb) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. Age. In patients > 60 vs < 30 reactivity was often found at PT (65.1% vs 35.8%), caused by LyTxAb (21.7% vs 5.4%), anti-Bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). Subsequent antibody tests: Tests were subsequently positive more often if reactivity was found at PT (79.3% vs 20.7% at BG), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). Subsequent tests were positive in only 43.8% patients with LyTxAb, 50% with anti-Bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . Techniques. LCT was positive in 60% and 45.5% tested samples found at urgent and routine PT (DiaMed), vs 0 at BG. All 18 cases due to LyTxAb, 8 of 9 anti-Bga and 6 of 8 'positive antibody screening, negative panels' were found by DiaMed. At BG (Ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. Positive antibody test: Identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. LyTxAb were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by LyTxAb were crossmatch-only. Identification. AHG panel was positive in 60% cases with LyTxAb (in 2 with papainized panel) and often due to 'IgG antibodies of unknown specificity' (29.7%), anti-Bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). Strength of reaction: Laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). Diagnosis. In patients with solid and hematologic malignancy reactivity was often found at PT (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by LyTxAb (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). In patients with liver and cardiac diseases reactivity was often found at BG (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. Discussion: Features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. This analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations.

Background: Worldwide screen and type is a very usual method for pre-transfusional testing. The ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. Aim: The aim of this study was to determine the frequency of red blood cell (RBC) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. Materials and methods: Blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. The mean age of the patients was 57 years. Pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (Liss -enzyme). In case of a positive result, identification was performed (panel with autologous control). In addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. When the result was marginal (1/32) a new test was carried out after a seven days period. In the presence of a positive autologous control or an autoantibody, samples were examined with Direct Antiglobulin Test (DAT). Results: Alloantibodies were detected in 580 patients with the incidence of 3.56%. Antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-Kell being the most frequent. The incidence and the specificity of the detected antibodies are summarized in the following table (Table 1 ). In 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-D and anti-C combination. 35 patients (6.03%) were DAT positive. Autoantibodies were found in 10 patients (1.72%), all of which had specificity to Rhesus system. Cold agglutinins were positive in 53 patients (0.32%). No specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated RBCs, were observed. One patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-Jka. The antibody, however, was not detected in the pretransfusion sample re-testing.

The frequency of the pre-transfusion detection of red blood cell alloantibodies in our Center, was 3.56%. The most frequently identified were the anti-Kell and anti-Rh. The high rates of unidentifiable antibodies and non specific reactions in enzyme treated RBCs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. The high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. The routine pre-transfusion screening for alloantibodies probably assures the prevention of DHTRs and provides sufficient time for blood selection for transfusion. 

Introduction: In the United Kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. Before the introduction of prophylactic anti-D, it was reported that 3% of the total Haemolytic Disease of the Newborn (HDN) cases were due to anti-c. Currently, cases of HDN due to anti-c are half as frequent as anti-D and 14% of the UK population are Rhc negative. We present two unusual cases of pregnant women who are D and c negative and have anti-c and anti-D detected in their serum. Case studies and results: Case 1: A 38-year-old Asian woman had three previous uneventful pregnancies. In her 4th pregnancy she presented with miscarriage at 19 weeks gestation. She was group B, D and c negative. Her serum contained anti-c and anti-D. Anti-D was detected by LISS tube IAT and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using R2R2 cells). In a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. Anti-c was then detected by two-stage papain technique only as well as anti-D. Case 2: A 33-year-old Asian woman had a positive antibody screen post caesarean section in Jan 2005. No antibody had been detected during the pregnancy. Standard prophylactic anti-D was given at 28 and 34 weeks gestation as this patient was D Neg. Anti-c and anti-D were confirmed in her serum. The anti-c level was 0.7 iu/ml using rr cells and the anti-D level was <0.1 iu/ml using R1R1 cells (prophylactic anti-D Ig). She was phenotyped as O r¢r¢. At delivery the baby was found to have a negative DAT and was phenotyped as r¢r. Discussion: Cde/Cde (r¢r¢) is an uncommon phenotype in the UK population with a frequency of 1/10 000. In routine antenatal testing, ABO/D grouping is only performed for pregnant women at booking and 28 weeks gestation according to BCSH guidelines. Full Rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. In routine testing of the above cases, this extremely rare phenotype is missed. Prophylactic anti-D was given to both patients and immunisation due to anti-D was prevented. There is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. Antenatal management of patients with anti-c and anti-D during pregnancy can be problematic: (i), problem in antibody identification;

(ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-D) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn.

Study on the frequency of red cell phenotypes (e.g. Duffy, Kidd and MNS blood group system) in our local population L LEOU, YF WONG, MBC KOH and D TEO Health Sciences Authority, Singapore, Singapore

Background: The frequency of various red cell antigens in the Caucasian population has been well studied. To date, the frequency of these antigens in the local population composed of a multi-racial mixture of Chinese, Malay, Indian and Others is still unclear, especially in the Malays with paucity of data in the literature. Aims: To investigate the frequency of clinically significant red cell antigens Duffy, Kidd and MNS across the local ethnic groups. To investigate the occurrence of rare phenotypes. To be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies.

Typing for the Duffy, Kidd and Ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. A total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. Agglutination is demonstrated by the indirect antiglobulin technique. Result and discussion: Table 1 -A higher frequency of the Fy(a+b-) phenotype is seen in Chinese, Malay and others in contrast to the Indian and Caucasian population. The clinically significant allo-antibody Anti-Fya is rarer in our local population. It occurs predominantly in Malays and Indians and usually in combination with other antibodies. It means that provision of antigen negative blood may be difficult. Table 2 -All groups show similarity of distribution of the Kidd phenotype with the Caucasians and distinct from the American Blacks. The Jk(a+b-) and Jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. Table 3 -The S-s+ phenotype is most common amongst all races. The Indians are more similar to the Caucasians with a relatively high frequency of S+s+ phenotype. The Chinese and Malay distribution are unique with > 99% being s+. Conclusion: There is a unique distribution of red cell antigen groups in the 4 races and the data on Malays is especially useful. This data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies.

Miltenberger phenotypes among Taiwanese Table 1 . Conjointly, in order to obtain the frequency of Mi.V phenotype, we screened 543 samples among 1230 with anti-Hil and four additional cases of Mi.V were found. Conclusion: In this study significant Miltenberger polymorphism was seen among the Taiwanese population. Besides previously described Mi.III phenotype (1.9%), there were also Mi.I/II phenotype (0.4%), Mi.V phenotype (0.7%), Mi.VI phenotype (0.7%), Mi.X phenotype (0.1%), and most interestingly two Miltenberger related not yet classified variants (1%). Related variant A (0.5%) was phenotypes as Mia+, Anek+ and Hil+. Related variant B (0.5%) was phenotyped as Mia+, Anek+. Interestingly, both variants were Mur-(negative). The total estimated frequency of Miltenberger variants in Taiwanese population (including related variants A and B) is therefore 4.8% (table1). The discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study.

Introduction: The use of column technologies for the detection of RBC antibodies improved significantly the screen test sensitivity. Each column-based method has its advantages and disadvantages. Aim: To compare antibody detection by two column agglutination tests; the fully automated Ortho Auto Vue TM method and the manual DiaMedᮀ ID-Micro Typing system. Material and methods: During the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the Ortho Auto Vue TM method (AV) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the DiaMedᮀ System (DM), were reciprocally re-screened, respectively. Positive samples were tested by DiaMed panels for antibody identification. Sera samples were divided into 6 categories according to antibody specificity; 1. Rh system antibodies (n = 150), 2. Clinically significant non Rh system antibodies (n = 85), 3. Clinically non significant antibodies (n = 28), 4. Auto antibodies (n = 13), 5. Not identified (NI) antibodies (n = 48), 6. Negative screening results by the DiaMed technique (n = 90). The 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the Auto Vue (AV > DM), those exhibiting equal strength reactions (AV = DM) and those with weaker reactions by the Auto Vue (AV < DM). Statistical analysis was carried out using the Wilcoxon Signed Ranks Matched-Pairs Test.

Results: A total of 414 samples were compared, 31.2% of them gave stronger reactions by AV, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by AV. Positive screen tests by AV only were detected in 90 samples, no specific antibodies were identified. In contrast to that, positive screen tests by DM only were detected in 9 samples, 5 were Rh system related, 3 Kell system related and 1 not identified, results are summarized in the table.

Discussion and summary: The antibodies detected by DM only, are anti-D and antibodies directed to low frequency antigens. The failure of AV to detect Rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by AV technique. Recently Ortho-Clinical Diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). The possible explanation for the failure to detect low frequency antibodies is that Ortho screen cells do not consistently carry the low frequency antigens Cw and Kpa. Positive screen tests detected by AV only, can be explained either by the fact that antibody identification was carried out on DM panels, or these are false positive reactions. In order to clarify this question we recently repeated these AV only positive samples by the manual Ortho Bio Vue technique. Preliminary results indicate that no specific antibodies were detected. It can be assumed that these results are false positive. Further study of this issue is required. Aim of the study: We have detected blood donor with RoHar variant which was mistaken as D-and his donations used for D-recipients. We tested these patients in order to evaluate possible anti-D or anti-LFA immunization.

Methods: RoHar variant was tested serologically (commercial and workshop MoAbs) and on DNA level (PCR-SSP). Involved recipients were tested by DiaMed column agglutination (GLIAT with normal and enzyme treated rbcs) with commercial rbcs and with RH33 and RH50 rbcs. Results: RoHar variant was confirmed on phenotype and genotype levels. In four D-and two D+ recipients of RoHar positive units no anti-D not anti-RH33 or -RH50 antibodies were detected. In one case anti-Le(a) antibody was found. Conclusion: In our cases massive exposition of recipients (whole transfusion unit) by RoHar red cells did not lead to production of detectable anti-D or anti-LFA. The immunogenic potential of this variant seems to be low.

Unusual AB0 grouping discrepancy -inhibition of anti-B reagent by isolated increase of plasmatic b substance in a patient with group AB and pancreatic cancer M PISACKA*, K PETRTYLOVA † , M KRALOVA* and H FLIDROVA* *UHKT, Prague 2, † Blood Bank, Faculty Hospital M, Prague 5, Czech Republic Introduction: In rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. Changes in ABH and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. Aim of the study: We describe a case of isolated increase of B group substance in plasma of a group AB patient with pancreatic cancer. METHODS: AB0 grouping was performed with registered Immucor reagents (Immuclone: anti-A Birma 1, anti-B LB2) by slide and tube tests. Neutralizing effect was quantified by (i) inhibition od anti-B reaction by titrated patient's serum; and (ii) inhibition of titrated anti-A and anti-B reagents by patient's serum, compared to AB serum of a healthy donor. Results: Slide test: unwashed rbcs: group A, washed rbcs: AB. Tube test (washed rbcs): AB. Titrated patient's serum when added in aliquot to anti-B reagent inhibited agglutination up to titre 64. Titration of reagents /+ aliquot of serum added/: anti-A: titre 4096 (both patient's serum and control); anti-B + patient's serum: titre 4, anti-B + control: titre 4096. Conclusion: Pancreas is known as rich source of blood group specific substances. Malignant transformation is known to be associated with either loss or re-expression of cell-bound ABH antigens. Reported excessive increase of B substance in group AB patient could be either due to loss of A-transferase activity in malignant pancreas cells or isolated increase of B-transferase activity. Further studies on larger groups of pancreatic cancer patients will help to understand this observation. Weakened ABH reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation.

Implementation of the AutoVue Innova, an upgraded column agglutination technology (CAT) for pre-transfusion testing in a large blood establishment C POLITIS, K ARMYROS, A ANTYPAS, V MALAMOU and P KATSEA G. Gennimatas General Hospital, Athens, Greece Objective: Automated CAT testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. A new technology is evaluated in comparison to standard methods. Materials and methods: We used column agglutination technology with the Innova AutoVue System, Ortho, Ratrian N.J. According to the manufacturers this system provides priority to the management of the STAT samples while the random access feature of the system enhances the system throughput. It provides an extended test menu as well as automated antibody identification with the red cells panels. The AutoVue Innova system supports the bi-directional communication with the LIS interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. In this study we performed 1423 tests including forward and reverse ABO group, Rh type and phenotype and Kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (IAT). The results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic ID-Diamed gel agglutination microtyping system and by standard manual methods. Results: Test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the AutoVue Innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. The technical execution was easy with the AutoVue Innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. Computerization of the test results with the AutoVue Innova provides an important advantage in record keeping in the blood establishment. Conclusion: Standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the AutoVue Innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. A heavy workload is expected to significantly decrease time performance.

Clearance of senescent erythrocytes in young and old individuals AL RACCA, A ENSINCK, C COTORRUELO, S GARCÍA BORRÁS, L RACCA and CS BIONDI Universidad Nacional de Rosario, Rosario, Argentina Introduction: after a lifespan of 120 days, human Red Blood Cells (RBC) are captured and phagocytized by monocytes/macrophages. The accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of Senescent (Se) RBC. An alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. The physiological elimination of SeRBC might be modified by individual's age. Aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: SeRBC, RBC stored with or without serum and desialinized RBC. Methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. Different suspensions from each sample were obtained: (i) Se and Young (Y) RBC by differential centrifugation; (ii) RBC stored with its own serum (RBCS) and without serum (RBCwS); and (iii) RBC desialinized with neuraminidase (Ne) and tripsine (T). The suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°C. Two hundred cells were analyzed to determine the percentage of active monocytes (AM) with phagocytosed and adherent red cells. Non sensitized RBC (NRBC) and ex vivo sensitized RBC (SRBC) were used as negative and positive controls respectively. Results: the% of AM obtained with old individuals were: SeRBC: 21.9 + 1.0, YRBC: 2.9 + 1.2, RBCS: 16.2 + 1.4, RBCwS: 3.1 + 0.8, NeRBC: 11.1 + 1.3, TRBC: 3.2 + 1.1, NRBC: 3.0 + 1.2; SRBC: 31.2 + 1.8. The values of AM obtained with young individuals were: SeRBC: 17.1 + 1.5, YRBC: 3.1 + 0.9, RBCS: 11.1 + 1.1, RBCwS: 2.8 + 1.2, NeRBC: 10.8 + 1.4, TRBC: 3.5 + 1.0. NRBC: 2.9 + 1.3; SRBC: 30.5 + 1.7. Conclusions: No differences in the% of AM were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. The values of AM with SeRBC were higher (P < 0.001) than those obtained with YRBC in both populations analysed. The rate of erythrophagocytosis with SeRBC in old individuals was significantly higher (P < 0.001) than that obtained in young donors. The increase observed may be due to agedependent changes of RBC that occur with human ageing. The% of AM with RBCS were higher (P < 0.001) in old individuals. No modifications were observed with RBCwS. The significant increase in the rate of erythrophagocytosis with SeRBC and RBCS show the involvement of autologous IgG in the selective removal of erythrocytes. These values were higher in old individuals indicating that this process would increase in aged donors. The tripsine activity was not enough to modify the% AM. The values of AM obtained with neuraminidase treated RBC were higher than those observed with YRBC (P < 0.001). However these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. Cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. Therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. Erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. The study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. The main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-A antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. Agglutinates of two A erythrocytes (doublets) induced by specific monoclonal antibodies anti-A were used. Adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. The chamber was installed on the stage of an optical inverted microscope (Union Optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. A CCD (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a Digital Image Processor (IPPLUS System) to digitize, record and analyze microscopic images.

The doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. In this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. The sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. Digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. These results show that, while the shear stress applied is raised, the contact area between both cells diminishes. The obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. As consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. Glycophorins (GP) A, B and C are abundant transmembrane integral proteins in red blood cell (RBC). Their highly glycosylated nature and high sialic acid content account for the net negative charge of mature RBC membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. In this study, we have tested anti-GP specific mouse monoclonal antibodies (MoAb) as primary antibody to directly agglutinate RBC. FRET technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (Alexa488TM, DiO and DiI) located in the RBC membrane or combined to secondary antibody directed against the primary agglutining MoAb. Combined intensity (spectral) and Lifetime (FLIM) Imaging was used to discriminate the FRET signal of molecules on their different lifetimes whereas their emission spectra overlap (Alexa488TM or DIO/DiI) as independent phenomena of the fluorophore concentration and photobleaching. Furthermore, GP-cytoskeleton interactions were analyzed by 3D-fluorescence microscopy after lipid extraction with Triton X-100 in red cell. All the antibody used was found to directly agglutinate the human RBC. In view of the fluorescence properties depicted in RBC for the pair of fluorophore Alexa-488TM (donor) and DiI (acceptor), it may be expected that upon agglutination of RBC, effective FRET should be observed. FLIM in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. The results demonstrate that's upon excitation at 780 nm the FLIM-FRET from Alexa488TM to DiI for the anti-GPB MoAbs only with a lifetime distribution in the picosecond range. Similarly, effective FRET was not observed for the anti-GPA MoAbs. The contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3D-fluorescence microscopy images showed interactions between GPC or GPB and cytoskeleton and did not show interactions between GPA and cytoskeleton. All together, these results only revealed by FRET-FLIM and 3D-fluorescence microscopy strongly support the importance of the specific reactivity with Glycophorin A or B of agglutining MoAb.

Introduction: The frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. Aim of the study: In order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (DAT) as well as the antibody screening results in 612 transfused sickle cell patients. Methods: All the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the C, c, E, e, K antigens. The transfusion range was 1-94 units. The DAT performed was a polyspecific gel test. In cases of positive DAT, was performed the monospecific gel test (IgG, IgA, IgM, C3c, C3d) . In almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (Liss/Coombs and papain). An antibody screening by gel test (Liss/Coombs and papain) was performed in all the 612 patients.

The DAT was positive in 98 (16%) of the 612 patients. In 96 cases (98%), the DAT was IgG type, in 1 case (1%) IgG + C3d and in 1 case (1%) IgG + C3c + C3d. The acid elution was performed in 97 cases. The eluate was positive in 38 cases (39%). In 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (Rh5), 1 anti-C (Rh2), 1 anti-Ce (Rh7). In 6 cases (16%) we found alloantibodies whose specificity were 2 anti-E(Rh3), 2 anti-K(K1), 1 anti-C (Rh2) and 1 anti-Jka (JK1). In the 59 cases (61%) no antibody was identified on the eluate and the cause of positive DAT is unclear. We could correlate the positive DAT with a presence of autoantibodies in 32 (5.2%) of the 612 patients. Of these, 13 (40%) had a blood transfusion association. The global frequency of red cell alloimmunization in this set of 612 patients was 15%. Seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. Conclusion: The mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. The loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation.

Forward and reverse blood grouping with lateral flow based assays P SCHWIND*, I AEBISCHER*, K LOESTER † and P MONOD* *Medion Diagnostics GmbH, Duedingen, Switzerland, † Prisma Diagnostika GmbH, Berlin, Germany Background: Recently, a lateral flow assay for simultaneous typing of ABOD, Rhesus subgroups and Kell with stable end-point and without a centrifugation step was presented (Loester K, Fleischhauer S, Schwind P: Lateral flow assay for simultaneous typing of of ABO, Rhesus subgroups and Kell. Vox Sang 2004, 87 (Suppl. 3), 40). In many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for ABO grouping. Aims: To develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay.

A lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (Reverse-Cyte A1, A2, B, 0, Medion Diagnostics, Switzerland) are mixed in each incubation well with 100 microliters of plasma. The resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. Results can be read after 3 min in the detection areas. A positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. Results: The plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. The results for both methods were in full agreement. Conclusions: A simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. Both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments.

The performance of the AutoVueTM system for red cell antibody screening of blood donors during

Background: In Israel every donation is tested for the presence of red cell antibodies (RBC Abs). Screening is performed on the AutoVueTM system, using Ortho 2 screening RBC since the year 2000. Aim: (i) To summarize the performance of the AutoVueTM system for RBC Abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) To evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. Methods and results: RBC Abs screening was performed using IgG cassettes. Positive results were confirmed by DiaMed gel cards, with 3 screening RBC, followed by DiaMed panels for Abs identification. Results: Summary of the results is presented in the table. (i) In 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the AutoVueTM system, a positive initial result for RBC Abs was detected, which was confirmed in 40.2% (0.22% of the donations). An increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by Ortho. (ii) During the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the AutoVueTM. 57/83 (69%) gave negative results in both repeats. Only 1/57 was confirmed positive and anti-M was identified by DiaMed gel test. The remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. Summary: AutoVueTM can be used as a competent method for RBC Abs screening, in Blood Services which require high thoughput automated systems. Samples with a low positive agglutination, which were negative in two repeats, can be considered negative. Retesting these samples on the AutoVueTM, can reduce the number of tests sent for confirmation and allow early release of blood components.

RK TAGI-ZADEH*, AA KARIMOV*, AR HASANOV † , LY NOVRUZOVA* and SI DONSKOV ‡ *Hematology and Transfusiology, Baku, † Blood Transfusion Centre, Ganja, Azerbaijan, † Centre for Hematology, Moscow, Russian Federation Background: The main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular RBC transfusions. The use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. However, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to RBC antigens. In order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. Objective: Examine the distribution of RBC antigens among thalassemic patients and blood donors. Methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of Scientific Research Institute of Hematology and Transfusiology Baku, Azerbaijan) and 1690 blood donors of Azeri nationality were examined. 108 patients' blood samples were typed on RBC Rh (C, c, D, E, e) and Kell (K, k) antigens. Gel test Bio-Rad (France) was used to detect RBC antigens. Results: Phenotyping of the patients' RBC Rh antigens (D, C, c, E, e, Cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. Studying of Rh phenotype distribution showed that the patients' CcDee (44.1%) •• CcDEe (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). Phenotype CCDee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). The similar picture was observed in relation to ccDee and ccDEe phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). Additionally, the results demonstrated that K antigen of the Kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. The same picture was observed with two other antigens of this system. Thus among the patients typed on RBC antigens Kpa antigen was detected in just one case whereas Kpb was detected in the rest the patients.

The results of the analysis showed that for thalassemic patients with phenotypes CcDee and CcDEe it is easier to find compatible blood than for patients with phenotypes CCDee, ccDee and ccDEe. Furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the McLeod syndrome is genetically associated with the sharp suppression of the Kell alloantigen expression (absence of gene Kx). The fact of the discovered absence of antigen K in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the Kell system, like the scarcity in McLeod syndrome. All the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of RBC Rh and Kell antigens must be carried out.

Hyperhaemolysis in sickle cell disease due to complement activation. Tessa thorp RCI national blood service Manchester UK TM THORP National Blood Service, Manchester, UK Introduction: Sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. When critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. Clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. Mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. Aim: The objective of this study was to measure the amount of C3c and C3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. These levels were then compared to 'normal' sickle negative blood donors. Haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. The hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. Method: Flow cytometric analysis using rabbit anti-C3c or anti-C3d and FITC labelled goat anti-rabbit was developed using a Beckman EPICS XLMCL flow cytometer. Control samples were prepared using a fruitstone buffer technique of complement coating. Results: A total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. Of the 11 homozygous patients analysed only one was undergoing a sickle crisis. A positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. The results obtained in this research project indicate an increase in C3c levels bound to red cells of homozygous sickle patients in vivo. Statistical analysis of results obtained suggest that there was no increase in C3d levels in either sickle trait or homozygous sickle patients. Conclusion: These findings support research carried out by Mold et al. (1995) and are present in more than 90% of Caucasian population. It has been reported that anti-Chido and anti-Rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. Case report: positive IAT and DAT were detected in a 34 years old Swedish woman, A Rh negative, at 34th week of pregnancy. Previous IAT and DAT determinations were negative. At time of detection DAT wsa weakly positive and IgG subclasses identified. Antibody identification revealed the presence of high titre (1 : 64) anti-Chido and anti-Rogers antibodies. A slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. This pattern showed no variation until the end of the pregnancy. At 39th week a healthy baby was delivered, DAT negative. The mother DAT and IAT remained positive for four months after delivery. The platelets count raised again to 200.000/ul. The same laboratory findings were detected in a Previuos pregnancy in Sweden. The patient reported the presence of antibodies at 35th week that disappeared few months after delivery. A healthy baby, DAT negative was delivered in this case too. Conclusions: this is the first report of the presence of Chido and Rogers as autoantibodies during the last months of pregnancy. The association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. P-289 RBC alloantibody frequency and their prevalence within Chinese, Malay and Indian community in Singapore E WIDJAJA, MBC KOH and D TEO Health Science Authority, Singapore, Singapore Background and aim: There is a recognised existence of different alloantibodies in different ethnic groups. While this has been studied in the Caucasian population, their frequencies remain less well documented in the Asian population. The frequency of different alloantibodies and their distribution in terms of age, sex in Singapore population is studied, as were their prevalence within the Chinese, Malay and Indian races in Singapore. Design and method: We conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. These blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. They were sent to the Centre for Transfusion Medicine for antibody identification. Small number of these samples came from hospitals where preliminary antibody testing was not done. The antibody distribution across different ethnic groups (Chinese, Malays, Indians) age and sex were studied. Results: The most frequent alloantibodies in the population is Mia (40.7%), E (20%), Le a (17.5%), Le b (14.8%), P1 (6.1%), M (5.4%), D (4.5%), c (2.7%), Jka (1.9%) and C (1.4%). Within the Chinese community, the most frequent alloantibodies were similar: Mia (51.9%), E (24.1%), Le a (11%), Le b (8.8%) and P1 (6.1%). In the Malays, the most frequent alloantibodies were Le a (35%), Le b (26.4%), Mia (24.2%), E(13.4%) and P1 (8.2%); while in the Indians, these were Mia (28.9%), Le b (28.9%), Le a (27.8%), D (17.5%), and E (12.4%), with the anti-D reflecting the higher incidence of Rh D negativity in the Indians race. For the lower incidence antibodies, anti-c was more common in the Malays and Indians (4%) compared to Chinese (1.9%). Anti Jka tended to occur mainly in the Malay race and anti-C was rare in all (<2%) reflecting the high prevalence of C in the Singapore population (R1R1 phenotype). The ratio of alloimmunised male to female (M : F) is 1 : 3. Most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for Mia where M : F ratio is almost equal at 1 : 1.9. Alloimmunisation increased with age for Mia, E, K, P1, Jka and Fyb while the frequency of alloimmunisation to Lea, Leb, D, M and C decresed with age. The prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. Conclusion: Anti Mia is very common within the Asian population especially in the Chinese. Anti D is common in the Indians. Most antibodies show increased frequency with age except for anti Lea + b, D and M. The majority of alloimmunised patients are females.

Study aim: To identify the present antibodies in newborns, after a positive direct antiglobulin test (DAT). Material and method: During a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. Each newborn was examined for ABO group, Rhesus with phenotype, Kell and DAT. Each mother was also tested for ABO group, Rhesus with phenotype, Kell and indirect antiglobulin test (IAT). After each positive DAT, the study was continued with the elution test, in order to identify the present antibody. DAT test was performed with DC Screening I-DIAMED SA, Switzerland. For the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. Results: The DAT test was positive in 132 (5%) of newborns and the antibodies found were IgG type immunoglobulin. Anti-A antibody was detected in 89 (67.4%) (newborns of group A with mothers of group O), anti-B antibody in 25 (19%) (newborns of group B with mothers of group O), anti-D antibody in 2 (1.5%) (newborns D+ from D-mothers), anti-E in one case and anti-Jka in another one. The eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. The results are presented in the following table. Conclusion: The majority of antibodies in newborns with a positive DAT test, is due to ABO incompatibility (the mother belongs to group O and the newborn to group A or B). anti-D (2), anti-E (6), anti-K (7), anti-Fya (5), anti-s (2), anti-Jkb (1), anti-N (1), anti-Kpb (1). In two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). It is remarkable that only in 10 out of 30 patients with both DAT and IAT positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. In 19 patients with only IAT positive, 15 had an irregular antibody and 4 had unspecific antibodies. In 3 out of 31 patients with both DAT and IAT negative the cause of incompatibility was the positive DAT in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure.

The results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-K, anti-E, anti-Fya, while anti-D is important for D negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. Finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure.

Multiple isoforms excluding normal RHD mRNA detected in RH blood group del phenotype with RHD 1227a allele

Introduction: Del phenotype is very common in Rh-negative Chinese. The rates in Hans (more than 91% in China) were reported from 26% to 30%. Moreover all Del individuals in this population were found mainly carrying a same allele, RHD 1227A, through genomic DNA analysis. Those individuals always possess one or two of this allele with Ccee or CCee phenotypes. Aim and the study: We focused on the mRNA investigations of Del individuals carrying RHD 1227A alleles, in Chinese, to expect it could be explained that why a silent mutation is associated with Del phenotype.

The full-length RhD mRNA was analyzed in 2 Rh-positive donors with CDe/CDe and CDe/cDE genotypes, respectively, and 4 Del phenotype individuals carrying RHD 1227A allele with CDe/cDe, CDe/CDe, CDe/cde and CDe/Cde genotypes, respectively, through reversed-trancriptase PCRs and cDNA direct or cloning sequencing. Results: Five transcripts and 6 isoforms were detected in Rh-positive and Del, respectively. Among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. The normal RhD mRNA was only observed in Rh-positive, but not in Del individuals. In stead, two additional transcripts were found in Del individuals. Its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of RHD. Through 2 additional reversedtrancriptase PCRs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in Del anyway. Conclusion: (i) A normal RhD protein does not exist in a Del individual with RHD 1227A allele since the exon 9 was always spliced out in all isoforms. All 6 transcripts in Del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different C-terminals (GenBank AY751491, AY751492, AY751493, AY751494, AY751495, AY751496). Among them, the sequence of Del9 (isoform with exon 9 spliced) transcript was the most similar as normal RhD mRNA. This isoform was first described by Chang et al. in Taiwan in 1998 . It encodes 463 amino acid residues and has 46 amino acids more than normal RhD. It is different from RhD after codon 384. In normal RhD protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. Therefore a further study on if a Del red cell possesses all epitopes of normal D antigen may be significative. (ii) A normal Rh-positive individual has also the transcript of Del9 that was found in Del. (iii) There is only one polymorphism in the region of 170 bp segment between RHD intron 7 and 2 of the Del transcripts, which indicated that other polymorphisms may exist in intron 7 of RHD 1227A allele compared RHD to explain that this situation was not happened in normal Rh-positive individuals.

total WBC were enumerated by flow cytometry and cell counting. WBC subsets were analyzed by flow cytometry with three-color fluorescence. In this study, the third generation bags and filters are used. Results: Before filtration, the total number of WBC, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. Although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of T cells was decreased, whereas the percentage of B cells and monocytes was increased after filtration.

In conclusion, both pre and post storage WBC filtration affect the proportions of WBC in the final product but pre storage WBC filtration of platelet concentrates is superior than post storage WBC filtration.

The effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations

Background and objective: The white blood cells (WBC) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against HLA antigens and cytomegalovirus transmission. In this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (PCs). The total number of white blood cells as well as the distribution of WBC subsets, in units filtered before and after storage were compared. Materials and methods: platelet rich plasma -derived PCs were filtered either fresh (5 pooled We reported earlier that metabolic arrest followed by incubation at 4°C reduces the platelet storage lesion (Badlou et al. Transfusion 2005) . Here we report that this treatment also reduces binding and phagocytosis by macrophages. Metabolic suppressed platelets (MSP) were prepared by incubation in glucose-free, antimycin A containing medium (40 min, 37°C) followed by storage (48 h, 4°C) and recovery with glucose (1 h, 37°C). Controls were (i) platelets in glucose-rich medium stored for 48 h at 22°C and recovery with glucose (C22) and (ii) platelets stored for 48 h at 0°C (C0) with rewarming. Platelets were labelled with mepacrine and incubated with PMA-matured THP-1 cells (37°C). Binding was measured by FACS analysis of CD42b/CD14 positive particles, and phagocytosis by counting mepacrine/CD14 positive particles. Binding of MSP, C22, C0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. Phagocytosis of MSP, C22 and C0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± SEM, n = 4). Before recovery of MSP, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. These data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion.

Platelet compatibility testing and alloimunization in multiply transfused hematologic patients

Purpose: Multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. Refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. Two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. Alloimmunization occurs most frequently against the HLA, and rarely against the HPA system. Nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. We performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused.

We performed 164 crossmatchingcompatibility tests of single donor leucodepleted, ABO compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. We also obtained samples from the patients for platelet antibody detection. We evaluated the CCI (corrected count increment) 18 h after the transfusion. The solid phase RBC adherence assay (Modified Capture P System/Immucor) was used for platelet compatibility and antibody detection.

A total of 164 compatibility tests were performed, of which 84 were compatible. Twenty five compatible platelet concentrates out of 84 were clinically evaluated. Twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. The 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. We found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (P~0.01). Additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple HLA antibodies. Three patients transfused with compatible platelets, during the study, developed alloantibodies.

We found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. The platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. Furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. NAITP is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. It is caused by maternal immunization against paternally inherited antigens present on foetal platelets. Screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of NAITP. We have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (HPA and/or HLA systems) and the pertinent risk of NAITP in neonates using a fully automated system with a solid phase red cell adherence methodology (SPRC-Capture P) and paternal or random donors (indirect test). Screening started in June 2003 and is still in course: in January 2005, 2300 blood samples were examined. Identification of antibodies in maternal serum was carried out using ELISA methodologies: MAIPA and commercial kits (Pak-Plus and Quick screen-GTI). Of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti HPA1a: 8, anti

HPA1b: 2, anti HPA1a + HLA: 2, anti HPA5b. 6, anti HLA: 24, auto HPA-5b: 1. Specificity of HPA antibodies was confirmed by determination of parents' HPA genotype (HPA-1,2,3,4,5,6) using PCR-SSP or PCR-RFLP. The infants with HPA 1 immunization suffered from severe (Plt count 5-103/ml) and symptomatic NAITP (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. We confirm that NAITP due to HPA-5 and HLA immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. It would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. Background: Fetal or neonatal alloimmune thrombocytopenia (FMAIT) results from a maternal alloimmunization against fetal platelet antigens. It is the commonest cause of severe thrombocytopenia in the neonatal period. The diagnosis of FMAIT is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. In Caucasians, HPA-1a is the most frequently implicated antigen. Other antigens such as HPA-3a, or HPA-4a are less often implicated. During the past few years FMAIT has been reported associated with rare or private antigens. The diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. However it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. If the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. Due to the risk of hemorrhage, particularly intracranial hemorrhage (ICH), during the course of severe thrombocytopenia, specific therapy is mandatory. Because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. Study design and methods: Since the first documented case of feto-maternal alloimmune thrombocytopenia (FMAIT) due to anti HPA-9bw (Maxa+), no additional cases have been reported. We present here a retrospective analysis of the cases referred to our laboratories in recent years. Since 1999 we have screened for rare or private antigens in suspected cases of FMAIT when there is no incompatibility for the most frequently implicated antigens. The diagnosis was performed by genotyping and identification of the maternal alloantibody by the MAIPA technique. Results: Parental genotyping showed HPA-9bw (Maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. In the last one, incompatibility was found for HPA-3 without anti HPA-3b maternal alloantibody. As the father was found to be HPA-9bw (Maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. The maternal alloantibody was identified in the MAIPA technique. However, our data strongly suggest that recognition of the HPA-9bw (Maxa+) epitope is not uniform. The neonatal thrombocytopenia was severe in most cases with bleeding. The outcome was good in all the cases but one.

Conclusion: This analysis confirms that anti HPA-9bw (Maxa+) FMAIT is not uncommon and was found to be around 2% of our confirmed FMAIT cases with parental incompatibilities and presence of maternal alloantibodies. It is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. Laboratory investigation of a suspected FMAIT case should be carried out in a specialist laboratory wellexperienced in optimal testing. Therapy requires strict collaboration between clinicians and blood bank services. Appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. Introduction: The human platelet (plt) antigen (HPA) system is independent of the HLA system. Therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (HSCT) even in HLAmatched donor-recipient pairs. We report the first case on a stem cell recipient developing thrombocytopenia due to host-derived HPA-1a antibodies after non-myeloablative allogeneic HSCT. A 52year-old male patient was diagnosed with multiple myeloma in 6/2003. Treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. Because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgBW CD34 + cells) from his histocompatible (HLA A,B,C,DR identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). He had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic HSCT. Stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. Between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. The corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. Therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. Methods: The patient's serum was tested by antigen capture ELISA assays (PAKPLUS® and PAK1®, GTI) and a solid phase assay (Capture-P®, Immucor). The MAIPA assay was used to confirm the results obtained by the above mentioned assays. In addition, we tested the patient's serum by the MASPAT kit (CLB) against plt from the donor and against homozygous HPA-1a plt obtained from our donor pool. Stored recipient's DNA from the time before HSCT was used for genotyping. Genotyping for HPA-1, -2, -3 and -5 of the donor and the recipient was performed by PCR-SSP (HPA, Protrans).

The patient's serum obtained on d19 after HSCT reacted strongly with the donor's plt due to anti-HPA-1a antibodies and antibodies against HLA class I antigens. The patient's genotype before transplantation was HPA-1bb, -2aa, -3ab, and -5aa; the donor was HPA-1ab, -2aa, -3aa, and -5aa. Thus, the antibodies were host derived and directed against the donor's plt. Serum samples obtained on d38, d45 and d65 after HSCT contained antibodies against HLA class I antigens but HPA-1a antibodies were not anymore detectable. No HLA antibodies were detectable on d149 after HSCT.

The severe thrombocytopenia was caused by hostderived HPA-1a antibodies. Fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. The decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. We conclude that the HPA mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions.

Severe neonatal alloimmune thrombocytopenia with anti-HPA-5B antibodies: case report P MONCHARMONT, M VIGNAL, Y MÉRIEUX and D RIGAL EFS Rhone Alpes Site De Lyon, Lyon Cedex 07, France

Usually, in case of feto-maternal incompatibility, the platelet (PLT) specific anti-HPA-5b antibodies (Ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (NAIT). Contrary to this observation here is reported the case of a severe NAIT. A 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (IUGR) and anamniotic fetus. Five years before, she had had a first pregnancy with IUGR of the fetus but no NAIT. The second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. The APGAR score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. No bleeding, hepatomegaly, splenomegaly or infectious signs were noted. Five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. The PLT count which was 82.0, 79.0 and 73.0 giga/L at day (D) 0, D1 and D2 respectively dropped dramatically at 9.0 giga/L at D5. Simultaneously, an intracranial hemorrhage grade II was diagnosed on ultrasound scan. Because of the clinical signs and of the decreasing PLT count the mother's serum was tested for PLT-specific Ab by immunocapture and the Ab identified by the monoclonal Ab-specific immobilization of PLT antigen (MAIPA) assay. A PLT genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. The mother was HPA-5a/a and anti-HPA-5b Ab were detected in her serum. The baby was heterozygous, HPA-5a/b. PLT were transfused to the baby and the PLT count rose to 80.0, 115.0 and 315.0 giga/L at D6, 10 and 18 respectively. No further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. In conclusion, when a mild thrombocytopenia with IUGR and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal PLT specific Ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. Anti-HPA-5b Ab can be detected.

Partial results of the incidence of heparin induced thrombocytopenia type II OSC OLIVEIRA*, RA RACHED*, C CAVALHEIRO-FILHO*, JC NICOLAU*, SHGL PASQUALUCCI*, DAF CHAMONE † and SP BYDLOWSKI † *Heart Institute, University of São Paulo, † University of São Paulo, São Paulo, Brazil

Heparin induced Thrombocytopenia type II (HIT) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. HIT type II occurs in up to 5% of patients who are exposed to unfractionated heparin (UFH). In our institution patients that are under heparin treatment are mostly cardiac patients. The purpose of this study is to determine the incidence of HIT type II in these patients. Material and methods: 111 patients from the Intensive Care Unit and Cardiac Care Unit treated with UFH or Low Molecular Weight Heparin (LMWH) for 5 or more days were studied. Known causes of thrombocytopenia were excluded. Platelet count was monitored pre and post heparin therapy. All selected patients were tested for detection of anti heparin/PF4 antibody test (DiaMed ID-Card). Results: From the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. Six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/PF4 antibody. Three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/PF4 antibody.

The results demonstrate that 9 (8.1%) patients were positive for anti-heparin/PF4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. Moreover, a higher frequency of patients with heparin/PF4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. Introduction: Neonatal alloimmune thrombocytopenia (NATP) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. Although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). The commonest antibodies are anti-HPA-1a. Treatment consists of IVIG, compatible donor platelet concentrates or washed maternal platelets. The administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. Case report: A baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, Apgar score 8. Immediately after birth, severe thrombocytopenia (7 ¥ 109/L) and signs of haemorrhagic diathesis (generalized petechiae and ICH gr. II-III) were observed. Coagulation tests were abnormal and K-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as HPA-1a/a) were given. Twenty-four hours later platelet count rose to 26 ¥ 109/L and no new petechiae were observed. On third day of life the blood platelet count was 13 ¥ 109/L and the newborn received IVIG 2 g/kg and corticosteroides. Twenty-four hours later the platelet count rose to 107 ¥ 109/L and further clinical course was uneventful. NATP due to HPA-1a was serologically confirmed. Conclusion: Optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. Random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of HPA-1a incompatibility. Accordingly, random donor platelets may be considered appropriate in emergency situations. Background: RhD is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. The weak D phenotype is the most common D variant, with a frequency of 0.2% to 1% in Caucasian individuals. There are several weak D types, with different frequencies in European countries, which may pose serologic problems and have the potential for alloimmunization. The objective of the study was to determine the frequency of the principal weak D types in Portugal. Study design and methods: Lisbon Regional Centre of the Portuguese Blood Institute and Oporto São João University Hospital selected samples from blood donors and patients. RhD was tested by two (Oporto) or three (Lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. When discrepant results were observed, the samples were tested with panels of monoclonal anti-D by LISS-IAT. Samples that reacted weakly with IgM anti-D but positive with IgG anti-D were sent to the Molecular Biology Centre. PCR with sequence-specific primers was performed using two commercially available kits (Inno-Train and BAGene). Real-time PCR, carried out on a Light Cycler, was applied when the interpretation was dubious. Results: 101 samples were referred after being characterized as weak D. In 99 cases we obtained a positive result, with a preponderance of weak D type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). Two samples were not categorized.

The high incidence of weak D type 2 in our population is in marked contrast to studies performed in other European populations where weak D type 1 was the most frequent. This might be due to our sample selection criteria or ethnic variation in the causes of weak D. There are advantages in genotyping serologically depressed D samples: to avoid the waste of D-negative RBC units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization.

Analysis of RHD zygosity in different RH phenotypes cal except for a 4-bp T insertion. The deletion of the RHD gene, found in most RhD-negative Caucasians, was theoretically due to recombination of the upstream and downstream Rhesus boxes resulting in the formation of a hybrid Rhesus box. Thus, the detection of a hybrid Rhesus box in an RhD-positive individual denotes an RHD heterozygous status. Aim of the study: to determine the RHD zygosity in different Rh phenotypes.

Methods: blood samples from 106 white trios (father, mother and child) were studied. The Rh phenotype was performed by hemmaglutination and the RHD zygosity was inferred in each member of the family groups. The RHD deleted allele was determined by a PCR strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid Rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid Rhesus boxes. These primers selectively amplify a 1980-bp segment of the hybrid Rhesus box in RhDnegative and RhD-positive heterozygous samples. Serological and PCR inconsistencies were studied by a PCR-RFLP method to detect another polymorphic site of the hybrid Rhesus box. Frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). Results: 179 (84.4%) RhD-positive and 33 (15.6%) RhD-negative samples were phenotyped. Of the RhD-positive donors, 75 (41.9%) were RHD homozygous and 104 (58.1%) were RHD heterozygous according to PCR. PCR-RFLP analysis confirmed the results of PCR in serological and molecular discrepancies. These results were coherent within each family group and did not differ from those published in the literature for Caucasians based on the most probable genotype method. However, the homozygosity indexes were significantly higher in the DccEe (20.0% vs 6.6%) and Dccee (16.6% vs 3.3%) phenotypes due to an increase of the Dce haplotype. In all samples with the Dce haplotype the RHces allele, frequent in individuals of African descent, was investigated by PCR-SSP. This allele was found in 33.0% of the Dce haplotypes. . RHD and RHCE typing was performed by multiplex-PCR with 24 fluorescent primer pairs. Positive results were obtained for RHD-exons 2-7, 9, 10 and polymorphisms associated with antigens C, c, Cw, E and e. Sequencing of RHCE-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using BigDye-terminators v.1.1 in an ABI 310 (Applied Biosystems).

Results: see Table 1 .

The substitutions of RHCE-specific nucleotides in exons 3, 5 and 6 with their RHD-specific counterparts lead to 3 different new RhC-antigens with weakened expression. Since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization.

Inheritance of a RHD cat VI type II in a twin pregnancy: a case report

Introduction: Determination of HDN-relevant fetal blood groups in amniocentic fluid with PCR is a routinely used method. Misinterpretation of test results -e.g. overlooking RHD category -decreases depending on the number of examinated RHD-specific exons. Case report: A 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an Anti-D. After amniocentesis of both foetuses tests for the occurrence of RHD intron 4, exons 7 and 10 were performed in the hospital's lab. The sample of fetus I showed PCR-products for intron 4, exon 7 and exon 10 while sample of fetus II lacked RHD-specific intron 4. Therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. Methods: Total DNA was amplified in a multiplex PCR with fluorogenic primers for RHD exons 2-7, 9 & 10; polymorphisms for Dweak type 1-5, D-VII, D-HMi and RHCE polymorphisms for C, c, Cw, E, e. Capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an ABI 310. RHD zygosity was determined by quantitative real-time PCR with co-amplification of RHDand RHCE-specific exon 3 and subsequent calculation of D ct value (ct RHCE minus ct RHD). Results: While maternal DNA sample has been genotyped as RHDnegative, amplification of paternal DNA for RHD-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. Determination of RHD-Zygosity revealed a homozygous constellation of the RHD-Gen. Investigation of amniocentic fluid from both foetuses resulted in a RHD-wildtyp for fetus I and a RHD cat. VI type II for fetus II which was the reason for missing amplification in RHD intron 4 PCR. Results: Weak D type 3 was not identified in our research population. Weak D type 1 was identified in 2 CN, weak D type 2 was identified in 1 CN and weak D type 4 was found in 1 CN, 3 BSA, 4 BE, 1 BC and 1 SAA. Conclusions: Although weak D types 1 to 3 represent the majority of all weak D phenotypes in Caucasian populations, none of these weak D alleles were found in Black populations. Since none of the frequent weak D types were identified in non-Caucasians one might not expected to find type 4 in all populations. However, regarding RHD phylogeny, the weak D type 4 mutations (602C > G and 667T > G) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. Therefore, it is not surprising that weak D type 4 was identified in non-Caucasians. Based on these results it may be concluded that weak D phenotypes have evolved relatively recently since they are present in Caucasian and Asian Methods: DNA was extracted from 9 A, 14 B, 11 AB subgroups, and 21 provisional cis-AB after serological ABO typing. Allelespecific PCR, RFLP, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. Results: ABO*A205 allele was observed in an Aint subgroup. Two new A alleles that showed 784G > A base change and 990C > T of intron 6 and a polymorphism of 532C > T in A(pro) intron 5 were discovered. O04 and O20 alleles were also observed. In B subgroups, a silent substitution 255C > T (Leu85Leu) was observed as a new B allele. Another new B allele which showed 547G > A was also found in 4 B subgroups. Conclusion: We discovered new ABO alleles and polymorphisms in Korean populations. Many other polymorphisms and alleles already reported in Japanese were also observed in Koreans.

Evaluation of a multiplex SNaPshot-PCR method for red blood cell marker genotyping C JUNGBAUER*, C HOBEL*, EM DAUBER † , PK RABITSCH*, DWM SCHWARTZ* and WR MAYR* *Austrian Red Cross, † Medical University Vienna, Vienna, Austria

Once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. The approach of multiplexgenotyping using PCR-SSP has apparently technical constraints so we are currently analyzing whether a modification using SNaPshot PCR-technique could provide a routine application for such purposes. Compared to SSP-technique, the SNaPshot PCR only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. Briefly, we perform SNaPshot PCR as follows: In a first step, DNA segments covering the essential polymorphic regions for the ABO, RHD, KEL, JK and FY genes are amplified using a standard PCR step. After purification treatment of the PCR products (Roche High Pure PCR Product Purification Kit or shrimp alkaline phosphatase (SAP)/Exo1 treatment according to the ABI Prism SNaPshot Multiplex Kit protocol) the labeling reaction is performed using the ABI Prism SNaPshot Multiplex Kit. After a second purification step the products are analyzed on a ABI Prism 310 Genetic Analyser in fragment analysis mode. Our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. Generally a better signal quality from controls and samples is obtained compared to the SSP-technique with consecutive gel electrophoresis. We also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). In contrast, the method involves more manual steps and higher costs. We may conclude that the implementation of SNaPshot-PCR techniques for red cell marker genotyping is a promising alternative to PCR-SSP. The obvious quality improvements compared to PCR-SSP might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories.

Quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique The monoclonal antibodies (MoAb) are biological reagents of homogeneous activity. They are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, Ag identification, blood group determination, oncology, organs transplant, etc. MoAb can be characterized by its specificity and affinity. Affinity may be expressed as the equilibrium association constant (K). Several techniques are available to determine the equilibrium constant of the Ag-Ab interaction. In this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. The laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. A silicon wafer was chosen as reflectant surface. Once fixed the principal angle of incidence, an amount of anti-AB is added to the sample. The reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. The mathematical analysis of the results verifies that the antibodies adsorption follows Langmuir's kinetics. From the curve analysis, the parameters related to the anti-AB concentration are extracted. From them, the calibration curve is constructed. This curve allows the desired commercial monoclonal antibody quantification. The developed technique shows to be sensible and precise. The obtained graphics are very well approximated (R > 0.94) verifying that the monoclonal anti-AB associa- 

Study aim: To prevent the sensitization to Rh0(D), to a D-patient who was transfused with D+ blood. Material and method: On September 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. The patient was on an olighemic shock (Ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. She had a blood group: O, D-and her phenotype was ccdee with Du-. After her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. Initially she was transfused with blood of the same group (O, D-) but when we were short out of Dblood, we were asked for 7 more units. The necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with D+ blood. The indirect antiglobulin test (IAT) and papaine were negative, so there would be no problem with the transfusion with D+ blood. The patient received finally 3 units (850 ml) of D+ condensed red cells, out of 7 that were initially asked. Before the 48h from the surgery had passed, it was decided that human anti-D immunoglobulin (Rhesogamma P) should be provided, in order to prevent the sensitization of the patient to Rh0(D). The indicted dose was 500-1250 IU/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time PCR amplification. Based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. We optimized the amplification conditions for all 12 primer pairs using our SYBR green real-time quantitative PCR protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive DNA was added into 100 000 copies of negative DNA followed by allele-specific PCR amplification. We also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of TA-MC using both HLA-DR and InDel panels. Results: For the short-term samples, 10 additional MC cases was identified in Non-LR group using InDel panel; and one additional MC case was detected in LR group (Table 1) . For the long-term follow-up samples, 4 additional MC cases were found. When evaluating analytical sensitivity, we were able to detect a single copy of positive DNA mixed with 100 000 copies of negative DNA in a single amplification tube for all 12 primer pairs. We were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of TA-MC if we consider both HLA-DR and InDel panels. Conclusion: Using our new InDel panel, we were able to detect more instances of MC in this cohort of patients. We conclude that the DR assay underestimates the presence of MC. Moreover, the tandem use of both panels provides a powerful tool for the detection of MC with 99.5% of recipients having at least one informative allele.

Background: We reported severe immunesuppression and longterm transfusion-associated microchimerism (TA-MC) in transfused trauma patients. We have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 Dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. In order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying TA-MC, we established an animal model using immunedeficient knock-out mice. Aim: Our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine TA-MC model. Material and methods: Female RAG-2/Common gamma double knock-out mice were transfused with fresh blood collected from male Balb/C mice, which were either not infected (Non-primed, NP) or infected twice (Primed, P) with 100 million viral particles of murine CMV (mCMV). At different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mCMV intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mCMV viral load in recipient's circulation. Each female knock-out received only one challenge of mCMV. If the subject died, we quantitated mCMV viral load in the brain, spleen, lung and liver. We used real-time quantitative PCR targeting murine Y-chromosome, H2K and mCMV to quantitate male donor cells, transfused recipient cell DNA input, and mCMV vrial load, respectively. The number of recipient cell DNA input served as a denominator to calculate the concentration of male donor cells and the mCMV viral load. Results: Results of overall mortality are summarized in the table.

All female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mCMV infection. All non-transfused control and recipients transfused with non-primed donor blood died after mCMV infection; these two groups also had higher mCMV viral load in blood than the recipients transfused with primed donor blood. When the subject died, we were able to detect mCMV in all four organs we analyzed, with liver having the highest mCMV viral load. There was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood.

The preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine CMV infection. The time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. 

Aims: To compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing PABD. Methods: Seven adult patients scheduled for operations were administered Aranesp (300 mg sc once or q3 weeks if needed) for PABD (Aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (Eprex 300 IU/kg biweekly). The two groups were matched according to the Ht, ferritin levels, number of the predonated units and type of the operation performed. CBC count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. Erythropoiesis-stimulating factor was administered when Ht £ 36% during blood donations and blood donation was not performed if Ht < 34%.

Results: There was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. Five of seven patients from both groups received one or two autologous blood units. Both factors were well tolerated without any side effects. The cost per patient was 609.09€ in the Aranesp group and 414.2€ in the epoetin group. Conclusion: Despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing PABD. Darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. However smaller doses should be examined in order to reduce the cost. Larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in PABD.

Background: Incompatibility with many blood units is a major problem in transfusion therapy. In selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. We present here the experience of our Blood Transfusion Centre from 4 operations in 3 patients with anti-erythroid antibodies. Materials: Three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. Introduction: Because of improvements in surgical techniques, preoperative autologous blood donation (PABD) in patients undergoing radical retropubic prostatectomy (RP) is contested. Aim of the study: We wanted to develop and validate an algorithm to determine the patients who probably do not benefit from PABD. Methods: We calculated the perioperative Hb-loss of 153 consecutive patients (group 1) who donated two red cell units (RBC) of autologous blood 5 and 4 weeks before undergoing RP: Hb-loss (g) = preop-Hb ¥ BV + (n RBC ¥ 65) -(postop-Hb ¥ BV) (BV = blood volume (L) = body weight (kg) ¥ 0.08; postop-Hb = Hb (12-18 h after RP); n RBC = number of transfused autologous and allogeneic RBC). Hb of RBC was taken as 65 g. RBC requirement is probably if initial-Hb -(Hb-loss: BV) -trigger-Hb (taken as 80 g/L) < 0 (initial-Hb = Hb at the first contact with the PABD-unit). This assumption was validated by the next 125 patients who were also assigned for PABD (group 2). PABD was refused if the probability of RBC requirement (PRR) was <25%. Between 25-50% one RBC was taken after considering the patient's individual risk of PABD. If PRR exceeded 50% two donations were planned. Results: Both groups did not significantly differ in age or initial Hb. Preop-and postop-Hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/L). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic RBC. Hb-loss caused by RP was 269 ± 111 g. Mean PRR in group 2 was 8.5%. 5/125 patients donated one RBC, which was later discarded, and no patients donated two RBC. 7/125 of group 2 needed allogeneic RBC. Mean PRR of these patients was 12% (range 0.65-33.3). Conclusion: Postop-Hb were lower in RP-patients with PABD because of the lower preop-Hb and the restrictive indication for transfusion of autologous blood. The individual calculation of PRR, for which only body-weight and initial-Hb of the patient are necessary, shows that PABD in patients undergoing RP is indicated only in rare cases. The algorithm also may be used in other major operations, if Hb-loss is known.

Use of Darbepoetin-alpha in preoperative autologous blood donation: preliminary results

Background: Preoperative autologous blood donation (PABD) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. Darbepoetin-a (Aranesp, Genesis Pharma SA) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (Eprex, Janssen-Cilag) in patients with chronic renal failure and cancer. Darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. This property leads to less frequent administration and may reduce drug cost. So far, no clinical trials with darbepoetin have been published in patients with surgical anemia. Other 44 (15.3%) patients who required autologous and homologous blood, had average predonation Hb level of 13 509 (SD ± 9.01) g/L. We found a significant relationship between the need for postoperative transfusion and the predonation Hb level (P = 0.003), predonation Htc values (P = 0.006), weight (P = 0.003) and gender (P = 0.002): female patients and patients with lower predonation Hb and Htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. No relationship was found between age of patients and the need for transfusion (P = 0.121). 104 (80.6%) patients with pTKA were transfused with autologous blood only, and had average predonation Hb level of 13 867 (SD ± 9.06) g/L. Other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation Hb level of 13 588 (SD ± 8.93) g/L. The significant relationship was found between the need for postoperative transfusion and weight (P = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. No relationships were found between predonation Hb level (P = 0.099) predonation Htc values (P = 0.243), gender (P = 0.241) and age (P = 0.276) of patients and the need for postoperative transfusion. Conclusions: Our results show that over 80% of patients needed only autologous blood. In our patients with pTHA predonation Hb was significant predictive factor for additional transfusion therapy, while in pTKA it was not observed. In both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with pTHA and pTKA. Aim: Prevention results of loosen anastomoses on colon, with fibrin sealent (FS) application and influence on colagen production. Materials and methods: Investigations were done on rats, weight 350-500 g. In control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. FS was applied in examined group. Concentration of colagen was done indirectly, with quantitative L-hydroxyproline determination. Place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed III, V, VII and XIII day postoperatively. Results: Analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with FS application. The highest approximate value of hydroxyproline was registered V day postoperatively. Distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher III day postoperatively in control group but V postoperative day value is intensively growing in group with FS application. Electronic microscopical was done V postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. In group with FS application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. Conclusion: FS application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis.

The study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (PBD) in 100 surgery patients

Introduction: The use of the autologous blood is already under consideration in developed countries. Thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. In this study, pbd as the easiest method to use and the most cost-effective one was selected. Aim of the study: it aims at improving blood safety and raising blood inventory. Methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. The subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. Blood collection procedure was followed at 3-7 day intervals. The blood volume taken from patients in every collection differed from 100-450 ml according to their weight.

Results: This study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. In other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases.

To avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform.

The 

Introduction: The purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111In-Oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. Methods: AGP made. Consent was obtained by patient to conduct HIV and HBV testing. Briefly, 40 cc to 60 cc of blood is withdrawn from the patient. The blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (PRP) , platelet-poor plasma (PPP), and red blood cells (RBC). The red blood cells were discarded. The PRP [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . The procedures of AGP labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming Platelet Gel. Imaging: The scintigraphy was performed 2 h after application of labelled AGP (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a Gamma Camera equipped with medium energy collimator. A later scan was performed at 10 days after graft. The platelet uptake index (PUI) was then calculated by dividing the cpm/pixel in the graft ROI (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background ROI. In vitro sampling: The radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. Results: All patients presented early high concentration of 111In oxine AGP, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in SPET slices reconstructions. All labelled AGP was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. Conclusion: All patients studied well tolerate the implant of AGP; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft.

Serial blood donations in a Ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-Ku biweekly) were administered to the mother to ensure an adequate supply of compatible RBCs for intrauterine transfusions and possible perinatal haemorrhage as well.

Results: Intrauterine transfusions were repeated every 1-3 weeks. By the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-Ku titre was 1/2048 and four intrauterine transfusions had been performed. Cesarian section was decided and the Apgar of the newborn were 8 and 10 at 1 and 5 min. The newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. By the 15th day of life Rh-EPO was administrated to him due to anemia. The maternal red cells completely disappeared from the child's blood by the day 100.

The experience of the use of erythropoietin in pregnancy is minimal. As illustrated by this case treatment with rH-EPO and IV Fe has effectively increased mother's capacity to donate RBCs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. However, further research is necessary to evaluate if rh-EPO crosses the placenta.

Introduction: Blood components should be transported by a system which has been validated. The containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. Whole blood should be stored at different temperatures above 2°C. Aim of the study: Bags with whole blood collected in a collection site are transported in containers without active cooling. We tested temperature of blood in containers put into the extreme weather conditions (+50°C, -20°C) during loading test for transport. Methods: The container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. The bags with blood of the temperature +4°C, +20°C and +37°C were used. Temperature indicators were situated in the bottom, centre and top of the container. Filled container was placed into thermostat (+50°C) or freezer (-20°C). The temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. Results: (see Table 1 ) NT, non tested; tmp, temperature. In the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. Conclusion: Loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. In the light of presented data we corrected transporting system to maintain the recommended temperature during transport. Background: Since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in TSOL platelet additive solution and have stored them in Pall Autostop CLX bags made out of pvc/totm. The residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. For pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. New foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. Aims: Storage bags made out of polyolefin (Pall Autostop ELX) were tested to prove their suitability for prolonged storage of platelets. Methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). The platelet pools were stored on flatbed shakers at 22°C, and sampled at day 1, day 5 and day 7. PH, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods.

Results: Mean pH on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%.

The new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. Prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. The possibility to filter RBC either at 4°C or RT simplifies the preparation process. Filtration at +4°C enables to achieve a better leukoreduction performance.

The NBS has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. The impact of this work on the incidence of TRALI will require detailed, long term analysis of hemovigilance data using existing mechanisms. Active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors.

Quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood Background: Whole blood can be separated into; plasma, buffy coat and red-cell conc (RCC) by differential centrifugation and separation on a separation device. Because of the high hematocrit of the RCC, 60% of the process time is needed for expression of the RCC. By increasing the internal diameter of the tubing at the bottom of a T&B system by 0.7 mm. a decrease of the process time is expected. Methods: 220 Units of whole blood were collected with the new T 3988 'wide boring' blood pack and separated on a routine base. Quality control parameters were checked and the whole process time was monitored. Free hemoglobine was measured up to 42 days. Results: Process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. Average decrease: 86 s. Slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells.

Bloodproducts produced with the new T 3988 meet European guidelines. No significant increase of free hemoglobine due to the faster expression is measured. An significant decrease in process time is measured with the wide boring bloodpack. The new Fresenius HemoCare RCC in-line system: T 3988 can be used for routine production which will speed up the production process considerably. Introduction: Leukoreduction of blood components is required to prevent several transfusion-associated complications. Aim: The aim of this study was the full process validation of the Pall Leukotrap WB system for the preparation of leukoreduced blood components.

We collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an WBF3 in-line filter (Pall) for the removal of leukocytes and platelets. Mixer balances (Baxter) were used and donation occurred within 12 min in all cases. After donation whole blood units were stored at room temperature for 2 h. Subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (Baby Leuko Cart, ITL-Corporation). Filtered units were centrifuged at 5000 g ¥ 10 min by an Heraeus Cryofuge 6000i. An automatic extractor (Bag Press Plus-Bioelettronica) was used to prepare red cell concentrates in SAG-M solution and fresh plasma units. Air in the system was automatically expelled by the extractor. Complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (Pentra Dx120-ABX). We enumerated residual leukocytes in red cell units by flow cytometry (Becton Dickinson-LeucoCount Kit). Results: Pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in Table 1 .

Results are given as mean and standard deviation. Whole blood filtration was completed within 10 min in all cases. Red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). No cases of transfusion reaction were observed.

The Pall Leukotrap WB system was easily introduced in our setting. All blood components prepared by the system fulfilled the Council of Europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. Future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. Background: During an evaluation of the Compodock (Fresenius HemoCare) sterile connection device (SCD), we observed irregularities on the inside of the tubing at the site of the weld. It was our aim to investigate the effect of these observations on the quality of blood products. Methods: Three leukoreduced red cell concentrates (RCCs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a Compodock-weld, and one with a weld made with the Terumo SCD. The RCC was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. The RCCs were stored in PVC containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (Hb) was measured.

The same procedure was also performed using platelet concentrates (PCs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and CD62 expression was measured. Ten experiments were performed per blood component. According to WHO standards processing of blood into labile components are considered an expression of quality of transfusion service. In our practice, modern transfusion principles are successfully applied. They cover blood collection, serological processing of blood units, technological preparation of blood products (GMP, SOP) and rational utilization of blood components and blood derivatives. In the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). NBTI collected X = 62 926 (249 778) blood units into blood bags. In Serbia X = 230 537 (691 611) blood units. Retrospective analysis: LDPC-BC was administered X = 97% with the satisfactory haemostatic effect. Increase of the cyta and plasmapheresis-manual procedures was also noted (100%). Increase of the use of leukocyte poor red blood cells was also registered (95% Introduction: According to the relevant recommendations of the Council of Europe, whole blood is a source material used for the preparation of blood components and blood products. Basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. In that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. Objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. Aim of the study: The purpose of our in vitro study is to compare storage of platelet concentrates at 4°C with platelets stored at 22°C, and to determine the in vitro-effects of pre-incubation at 37°C for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. Methods: Platelets concentrates (PCs) were prepared from pooled buffy coats (BC) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°C; and (iv) at 4°C and pre-incubated for 1 h prior to analysis. This paired in vitro study (n = 8) over 21 days include volume, platelet counts, MPV, volume, pH, pO 2 , pCO 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, HSR, ESC, ATP, LDH and release of a-granule content (RANTES, ß-thromboglobulin and PF4). Results: Platelet count (day 5 and 21; P < 0.05), MPV (day 5; P < 0.01), pH (day 14 and 21; P < 0.01), pCO 2 (day 14 and 21; P < 0.01), bicarbonate (day 21; P < 0.01), glucose (day 5, 7, 10, 14 and 21; P < 0.01), ATP (day 14 and 21; P < 0.01) was significantly higher in platelets stored at 4°C and platelets stored at 4°C with preincubation. LDH (day 21; P < 0.01), bicarbonate (day 5 and 7; P < 0.01), lactate (day 5, 7, 10, 14 and 21; P < 0.01), pH (day 5 and 7; P < 0.01), ESC (day 5, 7, 10, and 14; P < 0.05), HSR (day 5, 7 and 14; P < 0.05) was significantly lower in platelets stored at 4°C and platelets stored at 4°C with pre-incubation. The concentration of RANTES, ß-thromboglobulin and PF4 was significantly higher in platelets stored at 22°C than in platelets stored at 4°C (day 5, 7, 10, 14 and 21; P < 0.01). HSR (day 5 and 10; P < 0.05) and ESC (day 5, 7, 10 and 14; P < 0.01) was significantly higher in preincubated platelets stored at 22°C compared with platelets stored at 22°C. Conclusion: Platelets stored at 4°C maintain metabolic and cellular characteristics to a great extent during 21 days of storage. We confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°C is associated with decreased metabolic rate and decreased release of a-granule content. Aim: As reference centre of the Swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in Switzerland. Method: 20 whole blood donations were collected in a whole blood filtration set with CPDA-1 and stored at room temperature for 1 h before filtration at room temperature. The leucodepleted whole blood was stored for 42 days. Following parameters were analysed on day 0, 35, 42: free haemoglobin in%, K. In addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . Blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. Summary and conclusion: As expected there was a clear increase in K and free haemoglobin after day 35. However the results were within the required specifications from the European and Swiss guidelines up to day 42. We conclude that autologous leucodepleted whole blood can be stored in CPDA-1-for 42 days without loss of stability of the red cells. We will introduce the system to the offi-cial material list of the Swiss blood transfusion service and then implement the procedure to our daily routine. 

Results of FFP production from whole blood, and of FFP and PC produced by use of cell separator over a 5-month period before and after the introduction of measures for TRALI prevention are presented. The following measures were undertaken:

(4) blood of female donors was not used for FFP production, and plasma was only used for fractionation (5) plasma of female donors was not used for KT-BC pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy.

Female donors of whole blood: 16%*; 16%** FFP produced by plasmapheresis: 2%*; 2.5%** Female donor units on cell separator: 9.4%*; 4.5%** FFP from total plasma units: 63%*; 58%** Plasma units used for BC-PC pools: 4%*; 6%** *period before and **after the introduction of measures for TRALI prevention

The exclusion of female donors had no major impact on the production of FFP and BC-PC pools from whole blood because of the very low rate of female subjects in the Croatian blood donor population. The amount of plasma and PC collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total FFP store because of the small rate of plasma and platelets obtained by apheresis. Background: Platelet concentrates (PCs) are currently stored for a maximum of 5 days. Extended storage to 7 days would increase the supply and reduce the waste of PCs. Transfusion-associated graftversus-host-disease (TA-GVHD) is a severe transfusion reaction caused by T-lymphocytes in the transfusion product. The risk of developing TA-GVHD can be prevented by gamma irradiation of the PCs. Various in vitro tests can be used to study the quality of PCs such as inspection of the swirling phenomenon, hypotonic shock response (HSR), detection of platelet surface markers (e.g. CD62P and CD42b), metabolic parameters and blood gases. Free oscillation rheometry (FOR) using the instrument ReoRox® 4 can be used to monitor the coagulation over time in whole blood, PCs and plasma samples, and to obtain information about clotting time and coagulum elasticity. Aim of the study: The purpose of this study was to evaluate the quality of PCs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including FOR. Methods: Platelets were collected from healthy donors (n = 12) using apheresis technique. The PC from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 Gy. The PCs were stored on a flatbed agitator at 22°C for 7 days. Samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. Samples taken on day 1, 5 and 7 were also analysed for HRS, CD62P (P-selectin) and CD42b (GpIb) expression utilising flow cytometry. Evaluation of coagulation by FOR was performed on day 1, 5 and 7. The maximum elasticity (G'max) and the time to G' were evaluated from the FOR elasticity curves. Results: There was no difference between irradiated and nonirradiated PCs regarding any of the tested parameters during the storage period. Swirling, HSR, platelet count and percentage of CD42b expressing cells were well maintained for 7 days of storage. Glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/L to 16.2 mmol/L for lactate and from 16.5 mmol/L to 8.9 mmol/L for glucose. The percent CD62P expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. pO 2 was well maintained but pH increased and pCO 2 decreased significantly between day 0 and 1 whereafter pH decreased and pCO 2 continued to decrease. The FOR parameters G'max and time to G'max increased significantly between day 1 and 5 and the time to G'max continued to increase significantly between day 5 and 7.

The results indicate a well preserved platelet quality after storage for 7 days. Gamma irradiation did not affect the platelet quality.

Cytokine release during storage of buffy coat platelet concentrates produced manually and automatically

Background: Transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. Platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. TGF-b1 (Transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of T-and B-lymphocytes and decreases the secretion of IgG and IgM from B-lymphocytes. Aims: As part of our quality control program, we aimed to quantify the amounts of RANTES and TGF-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (M-PCs) and by an automated method using the Orbisac System (Gambro) (A-PCs). Methods: PCs were produced from 5 buffy coats. Following overnight storage at 20-22°C, buffy coats were pooled with 300 ml T-Sol (Baxter). Forty-two PCs were produced either manually (n = 21) using the Imugard III S-PL set (Terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the Orbisac Validation BC Set (Gambro) equipped with the LRP6 leuko-reduction filter (Pall). Swirling was scored visually, platelet count and MPV were measured on a cell counter (Cobas Argos, Roche), and blood gas analyses, glucose as well as lactate were measured on an ABL700 Series Analyser (Radiometer). Samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°C; supernatants were harvested and frozen at -86°C until analysis. Cytokines were quantified using Quantikine Human RANTES Immunoassay (R&D Systems) and human TGF-b1 ELISA Immunosorbent assay (Bender MedSystems GmbH). All analyses were performed on days 1, 4 and 7.

Results: Platelet concentrate volume (mean): M-PCs: 316 ml, A-PCs: 328 ml. Platelet yield was found to be 3.15 ¥ 10 11 for M-PCs and 3.43 ¥ 10 11 for A-PCs (P < 0.0001). In all PCs pH levels were between 6.9-7.2. Glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. RANTES levels (pg pr 10 9 plts) were significantly higher in A-PCs than in M-PCs (P = 0.04, repeated measures analysis of variance), but no significant difference was found in TGF-b1 levels (pg pr 10 9 plts). Summary and conclusions: Preparation of buffy coat platelet concentrates by the automated Orbisac System improves platelet yield compared to our manual processing procedure, but the levels of the chemokine RANTES were significantly highest in the automatically produced products. The clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process.

The quality of cryopreserved vs liquid stored platelets: a comparative study Table 1 . The mismatches can be divided into the two categories. The first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in HLA-C locus (this locus was not concluded in primary donor search). In the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. The mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. Conclusion: The use of high-resolution DNA methods makes the identification of HLA match/mismatch more accurate and can affect the outcome of unrelated HSCT. This work was supported by the grant IGA MZ, No. NR/7984-3. 

Pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation P BERGAMASCHI, C PEROTTI, G VIARENGO, C DEL FANTE, C PARISI, A MARCHESI, L BELLOTTI and L SALVANESCHI IRCCS Policlinico 'San Matteo', Pavia, Italy Background and aims: Nowadays umbilical cord blood (UCB) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. Thanks to the large-scale banking of unrelated units and the preliminary encouraging results, UCB employ in adults is quickly growing up. In this context, total nucleated cells (TNC) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as CD34+ cell content and short-term culture clonogenic assay, are recommended in accordance to Netcord-FACT stan-dards. In order to guarantee the safety and the prompt availability of a UCB unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. We report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our UCB Bank compared to the pre-freezing values. Methods: Every UCB unit stored in our Bank is accompanied by 3 satellite cryovials available for subsequent controls. For each unit issued for transplantation, one cryotube was thawed in 37°C water bath with gentle agitation without washing out DMSO. TNC and mononucleated cells (MNC) were estimated by an automated cell counter; viability and CD34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin D. CFU assay was performed using commercial reagents (Methocult GF H4434, StemCell Technologies) and colonies were counted after 14 days. The same tests were performed before cryopreservation, taking a sample from each fresh unit. Moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (BacT/ALERT®FA/FN, Biomérieux Inc). Results: The UCB characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. Post-thawing TNC and MNC, as well as CD34+ cells, showed no significant difference in comparison to the pre-freezing values. Despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the CD34+ cells. The colony forming units (CFU) growth after thawing was documented and was always lower as respect to the pre-freezing assay. Finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. Conclusions: In our experience, well standardized evaluation of UCB content could be obtained with regard to TNC, MNC and CD34+ cell. Concerning the results of short-term cultures, the presence of DMSO as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. Finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of UCB to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time.

Bone marrow transplantation or BMT transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. Bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. In fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. This microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. Marrow injury can occur as a consequence of a variety of diseases. Some diseases could be due to a microenvironment that fails to support hematopoiesis. A possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. We can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. Background: Intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. Understanding the genetic bases of this adverse event is still an open goal. There is evidence that motherchild HLA compatibility and HLA-DRB1 foetal genotype are associated with a reduced placental growth and a low birth weight. The recent institution of Cord Blood Banks, with their huge amount of HLA types, offers an unique opportunity to look inside the molecular bases of normal birth weight. Aims: We investigated whether the baby-linked immunogenetic profile, i.e. HLA gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. Methods: 1868 cord blood units (961 from males and 907 from females) were HLA typed with PCR-SSP and/or reverse PCR-SSO techniques and recorded in the Cord Blood Bank database of Pavia-Italy. All were defined at low resolution level for HLA-A and B genes and at high resolution for HLA-DRB1. 686 blood units were also randomly typed for HLA-DQA1 and DQB1 at high resolution. Results: Mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). Comparing the HLA allele distribution in these extreme bands we found that HLA DRB1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, P = 0.032. On the contrary, HLA-DRB1*14 and DQB1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile P = 0.035 and P = 0.019. All infants were analysed as to the effect of the above mentioned alleles. We confirmed the positive association of HLA-DRB1*08 and higher relative birth weight (mean 0.17 vs 0.08; P = 0.033) as well as the association of HLA-DRB1*14 with lower relative birth weight (mean 0.03 vs 0.27, P = 0.04). No significant association was found as far as HLA homozygosity was concerned. Conclusions: The present findings confirm the role of foetal HLA-DRB1 gene in the intrauterine growth. About the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of HLA-DR binding groove. It has been demonstrated that HLA-DRB1*08 and HLA-DRB1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). This implies distinct functional restriction patterns. The sequence motif of HLA-DR14 is characteristic of some autoimmune conditions, such as Hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retarda- which provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. In a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. The extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. After incubation at room temperature with lymphopre solution, the mixture was centrifuged. Two clear and separate rings of mononuclear and PMN leukocytes were obtained. To eliminate any red blood cells, PMNL ring was separated and washed three times with cold ammonium chloride. After a short period of incubation 4°C, mixture was centrifuged and the PMNLs were isolated. The purity and viability of total leukocyte population was counted and the percentage of PMNL obtained was established. The total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. In both cases the PMNs isolated were of high purity and viability. The overall percentage of PMNLs obtained from both groups under study was 98% to 99% when stained with Gimsa or Wright staining method. The viability of isolated PMNLs was also 98% too, which is excellent for numerous immunological or molecular studies. The PMNLs isolated by this method were highly pure and viable in comparison with standard methods used to isolate human PMNLs. Generation a high amount of PMNLs is another advantage of the suggested method. This method to separate PMNLs is recommended for in vitro studies of different subjects. et al. 1950 .) The object of exchange transfusion (ET) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-D, anti-c and anti-K are easily the most important) or remove anti-D positive red cells.

We have studied exchange transfusion in our Hospital in Neonatal Intensive Care Unit, during 1997 to 2004. At delivery cord samples were taken for determination blood group, Rhesus, Hb and Ht have been counted. Also direct antiglobulin test (DAT) has been performed. In cases of positive DAT, Hb and bilirubin levels were monitored. Newborn body-weight were weighted (ranged 710 g to 3100 g). The blood for ET was of group O, D negative and Kell negative and was compatible with the serum of mothers; it was less than 7 days old. The blood was screened for HbS and for anti-CMV as well as being submitted to all usual tests. The method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. Results: 17 exchange transfusions were carried out. Four out of 17 ET due to ABO incompatibility mother-newborn. Five out of 17 due to Rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. The last two years only three ET were carried out due to immaturity. Conclusions: Phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. Phototherapy alone or phototherapy plus high dose IgG therapy has been used to minimize exchange transfusions in this population.

Detection ABO blood group system antibodies of neonatal using fully automated column agglutination technology (Auto-Vue) Background: The ABO blood group system remains the most important in transfusion practice. This is because of the regular occurrence of the antibodies anti-A, anti-B and anti-A, B reactive at 37°C, in persons whose red cells lack the corresponding antigens. The regular presence of anti-A and anti-B is used in the routine determination of ABO blood groups; in addition to testing red cells for A and B antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known ABO groups. Methods: 100 samples were taken from 100 newborns at first 24 h of life for ABO blood group typing during 2001-2004. Simultaneously the presence of anti-A and anti-B antibodies has been studied using fully automated column agglutination technology (Auto Vue Ortho Diagnostic Systems) with Bio-Vue cassettes ABORh/Reverse. The column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. To determine the ABO serum group, test serum and ABO reagent red cells were added to the top of column containing diluents. The ABO grouping columns were centrifuged and examined for agglutination. The presence or absence of agglutination has been recorded.

Results: In 68 out of 100 newborns were found detected antibodies anti-A, anti-B. In 24 out of 100 newborns no antibodies were found. In 8 out of 100 were found antibodies maternal origin. The automated reader detected all positive reactions. Positive results were recorded on a scale from +0.5 to +4. Conclusions: the technical performance of device allows objectivity and precision to detect ABO blood group antibodies of newborn. The origin and the type of antibodies and also factors that influence their presence are to be studied. Introduction: Diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (HDN) has changed from a common to a rare pathology. Aim of the study. In this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. Methods: In the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our Hospitals. AB0 and Rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. Results: Not considering AB0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-D, 9 anti-C, 7 anti-c, 9 anti-E, 3 anti-JKa, 1 anti-D + anti-JKa, 1 anti-D + anti-S, 3 anti-D + anti-C, 1 anti-D + anti-C + anti-K, 5 anti-S, 10 anti-K, 2 anti-M, 1 anti-c + anti-E and 2 anti-D + anti-C + anti-G. The most severe HDN were the 2 D + C + G, the c + E and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/L, bilirubin = 20.5 g/L, reticulocyte count = 10%. In these exchange transfusion needed at the delivery. Other newborns were only treated with phototherapy. Conclusion. Thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. In fact all newborns survived and showed no neurological lesions in the following controls. Conclusion: In order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. This is an interesting case of RhD immunisation in RhD negative woman despite the application of RhD immunoprophylaxis. Case report: A blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our Laboratory for serological analysis. Her blood group was O, ccddee and she had an anti-D antibody reactive only with enzyme treated panel of test erythrocytes. Her husband was A, CCDee, and two children were both A, Ccddee. On the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of RhD immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (FMH). Both of abortions were after the deliveries. Until the end of the pregnancy, detailed serological analysis showed anti-D specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. The fetus was under permanent ultrasound control. She delivered a mature, RhD positive, CcDee male child, without any sign of haemolytic disease.

The proper personal history, measurement of the size of FMH, distinguishing the anti-D and anti-G specificity of the antibody, administration of RhD immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-D antibody. Anti D antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. Introduction: Red blood cell (RBC) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. Clinical indications for RBC transfusion are shock, sepsis and/or anemia with the following laboratory criteria: A) hematocrit (Hct) < 20% or hemoglobin (Hb) < 7 g/dL and reticulocytes < 4%; B) Hct < 30% or Hb < 10 g/dL in these conditions: O2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; C) Hct < 35% or Hb < 12 g/dL with severe respiratory distress. Aim of the study. Aim of this study has been to evaluate the effectiveness of RBC transfusion therapy in premature and at term newborns independently of initial pathological conditions. Methods: Our therapeutic objective has been to achieve an Hct of 50% after the whole cycle of transfusion therapy. For each little patient, the volume of transfused RBC unit has been calculated, according to international guide lines, using the following criteria: weight (Kg) ¥ blood volume (100 mL per Kg if premature or 80 mL per Kg if at term newborn) ¥ (Hct desired -Hct observed)/Hct of unit transfused. Particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. In order to avoid a circulatory overload, the indicated hemocomponent has been always packed RBC with higher possible hematocrit. For the same reason, the rate of infusion has been always 3-10 mL/Kg/hour. Methods: This is a descriptive study. The name of the neonates who received transfusion was obtained from the blood bank of Beheshti Hospital. Information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. Results: Out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. Fifty four percent received one, 35% two and 11% received three types of blood components. The frequency of transfusions were 311 times. The most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). All the blood products except whole blood were used more common in premature and low birth weight infants. Appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (HDN) . In accordance to current regulations, this study is carried out in all pregnant women attending in our Service. According to our protocol, when an alloantibody of any specificity is detected through the LISS-Coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a HDN. There are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. Aims: Demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (Liss-Coombs -Enzymatic) and the prevalence of anti-D associated antibodies, only detected by using enzymatic technique. Analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a LISS-Coombs medium during pregnancy, thus acquiring clinical significance for HDN. Materials and methods: Between January 2000 and December 2004 we studied 1970 D-negative pregnant women. The studies performed were: ABO grouping, D and weak D, Rh phenotype, direct antiglobulin test and irregular antibody detection (IAD) against commercial screen cells with LISS-Coombs (DiaMed) ® gel-medium technique, according to the manufacturer's specifications. When IAD were positive, an antibody identification using two commercial cell panels with gel techniques (LISS-Coombs-Enzymatic) was performed. Along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a LISS-Coombs medium (clinically significant antibodies). Results: Out of 1970 D-negative studied samples, 278 (14.1%) had a positive DAI and it was only showed anti-D specificity in a LISS-Coombs medium. After analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-D associated alloantibodies: 5 anti-K (1.8%), 63 anti-C (22.7%), 10 anti-E (3.6%) and 1 anti-C + E (0.3%). During the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a LISS-Coombs medium and later they were identified in the red cell elution of the newborn. Conclusions: These results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. The detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for HDN. It was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. Background: Fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. This product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor XIII. In contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. Readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. The risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. Many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. Aims: The aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. The influence of different plasma preparation methods and plasma storage temperatures (-30°C and -80°C) on the quality of fibrinogen concentrate was examined. Methods: Whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. Plasma was removed. Four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. Two of these units were stored at -30°C and 2 units of FFP at -80°C. After one month the plasma was thawed at 4°C during 16-18 h. The fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. To compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor XIII were determined. Results and conclusion: The levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°C. There were no significant differences in the level of plasminogen in both tested groups. Double cryoprecipitation of a single unit of plasma (stored -30°C) is an efficient, simple and safe method of obtaining fibrin glue. Background: Talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. In this article I would like to point out another side of the problem-the risk of making preventable medical error. With all its consequences. Aim: How blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. Information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. At the same time it would contribute to avoidance of blaming and shaming of many health care providers. Prevent what preventable could be! Method: Retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). Results: After clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that:

-Great majority of adverse events resulted from medical error -Every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -Any educational level is equally liable to error There is no significant difference about occurrence time:

-Working day/holiday -Emergency/routine request -Routine h/out of routine h -Main error cause were as follows: -Donor sample misidentification -RhD typing error -ABO typing error -Incorrectly performed cross match -Recipient misidentification -Wrong component prepared -Sample confusion during freezing preparation Conclusion: The truth incidence of transfusion medical errors is underestimated. Mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. Those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. But, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. Wastage rate was highest for plasma components. The influence of local practices on such discarding and whether avoidable shall be discussed.

Audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers.

designed technical and economic support. Options include importing finished products and/or procuring products made from locally collected plasma. One approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. An alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. The end products are returned to the country of the plasma supplier. In 1954 the National Center for the production of blood products was established, under the direction of Elias Politis and 9 years later in 1963 begun the production of dried plasma from Greek donors. By the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. In 1992 all the activities of the center settled down due to administrative aspects. At the beginning of 1990s a contract fractionation program was instituted (under the direction of K. Sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. The challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. A new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. A close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. Collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. There is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. The plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. This lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel.

Background: Fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. In 1972, Matras et al. described successful application of fibrin glue for peripheral nerve repair. This encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. The fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. Such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. The second component, a mixture of thrombin and CaCl2, is commercially available. Thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. There are many methods of fibrinogen concentrate preparation but none of them has been described in detail. Aims: The aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in Blood Transfusion Centers. Methods of precipitation of fibrinogen by polyethylene glycol (PEG), ammonium sulphate, ethanol or cryoprecipitation were compared. Methods: Plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. Four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. One of them was stored at -30°C and after one month the plasma was thawed at 4°C during 16-18 h. Fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, PEG and ammonium sulphate. The levels of fibrinogen, fibronectin, plasminogen and factor XIII were determined in each fibrinogen concentrate. Results and conclusion: The level of fibrynogen ratio in fibrinogen concentration, obtained by PEG and ammonium sulphate was significantly higher. Cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system.

Plasma fractionation program in Greece: an unknown history

The provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well-

(Light Cycler, Roche Diagnostic Systems, NJ) was used for the identification of the C282Y, H63D and the S65C point mutations of the hemochromatosis gene, and were based on protocols developed, for C282Y by the Unidad de Medicina Molecular (INGO, Santiago de Compostela, Espanha), and for the other two mutations by Bolhalder M et al. The primers and probes were designed by TIB MOLBIOL (Berlin, Germany). Results: The analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the Table. No differences were found between the patients and the controls. When we compared subgroups of patients based on their Hepatitis C genotypes, a higher value for the C282Y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. Discussion: Hereditary Hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. Follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. Also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution.

Introduction: Portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with Hepatitis B and/or C. Hereditary Hemochromatosis (HFE) is one of the most common causes of known hereditary illnesses with hepatic repercussion. HFE mutations are also found in linkage desiquilibrium with particular HLA haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. In this study we evaluated the prevalence of the main mutations C282Y, H63D and S65C for the HFE in a population with chronic Hepatitis B and/or C and in a cohort control. Background: Haemolytic disease of the newborn (HDN) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. Although postpartum RhIG prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. Material and methods: A total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. All ABO, Rh typing, antibody tests and DAT were carried out in column agglutination tests. Results: From the 918 cases studied it was found 778 cases without incompatibility (85%). From the 140 incompatibilities, 88.6% were ABO, 8.6% were RhD, 1.4% were other Rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were RhD+ and 20% RhD-. Conclusion: Of the 918 pregnant women studied, only 184 were RhD-. From this group 83 (45%) delivered RhD-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. From the 184 pregnant women RhD-, 13% had incompatibility ABO, which decreases to near 2% the risk of development of RhD immunization. Being anti-D immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? The introduction of molecular methods to determine the fetal RhD genotype could rationalize the use of antenatal anti-D immunoglobulin prophylaxis. Introduction: The frequency of HLA A201 haplotype expression has been found about 44-46% in Caucasian and in Greek population particularly, 44.3%. Because of the high frequency, it is used widely in anticancer immunization. The immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. Aim: The probable difference in the frequency of HLA A201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with HLA-combined peptides. Materials and methods: 323 healthy donors who proceeded in the Department of Transfusion Medicine, University Hospital of Heraklion Crete were tested for the HLA A201 expression. In 11 of these donors the expression of CD3, CD4, CD8, CD2, CD19, CD20, HLA DR, CD3+CD4+, CD3+CD8+, CD16-CD56+, CD3+CD16-CD56+, CD3-CD16-CD56+, CD3-CD16-CD56+ was examined. Meanwhile, 132 patients with metastatic cancer who were hospitalized in the Department of Medical Oncology, University Hospital of Heraklion Crete, were tested for their HLA A201 expression, while in 50 of them for their lymphocyte phenotype. The antigens expression was examined in Flow Cytometry.

The HLA A201 expression in healthy donors was 44.8% and in cancer patients 45% (P > 0.05). In Table 1 the Mean, Standard Error, t-test and p of the two groups are included. (see Table 1 ). The two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the CD20 expression, which was higher in cancer patients. Summary and conclusion: The expression of HLA A201 in cancer patients and in healthy donors was comparable. Also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the CD20 (total B-cells). The significance of this result has to be investigated. In the course of original documents research I found out that Dr. Kalic, head of the first organized blood transfusion institution in the Balkan region (at Beograd, Serbia, in 1936), set himself a professional goal: Blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. Dr. Kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. Encouraged by the conclusions of the Congress held in Paris in 1937, Dr. Kalic started preparations for the transport of blood to the inland. He concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. He advised his colleagues to use blood as an intravenous injection. Blood was taken from voluntary female donor in Belgrade (capital), March 1938. After keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from Belgrade. There it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. The patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. Reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. Conclusions: Automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. Using Auto-Vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. The instrument allowed us to leverage current staff to a more productive, less stressful level.

Introduction: Exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (APCs). The finding that exosomes from DC pulsed with tumor-derived peptides elicited potent antitumor Tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. Aim of the study: To establish the method of producing a new kind of tumor vaccine -Exosomes secreted by DC, pulsed with tumor peptides. Methods: Exosomes used in this study were generated from monocyte-derived DC pulsed with peptides from K562 tumor cell lines. Exosomes were purified by the methods of ultrafiltration and ultracentrifugation. The methods of Dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. The function of the exosomes was deter- Objective: To develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (CD44) receptor expression in red blood cells (RBCs) from adults and newborns. Materials and methods: Samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. Several dilutions oh hailuronic acid sodium salt solution 2% (Sigma L-77H0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (PBS) pH 7.4. The RBCs were previously treated with an enzymatic solution of 5% bromeline in PBS pH 7.4 (Sigma L60H0168). Agglutination readings' were been by slow sharking after of 12 h incubation at 4°C.

The results were expressed through the Sensibility Parameter which involves titer and score. This is defined by a mathematical expression a = à6 Si. Di-1. 10-3 (i = 1, 2, 3 . . .) where Si represent the score and Di-1 is dilution inverse. The adult' RBCs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. Our results showed significant differences between both groups. Conclusions: In this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (CD44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in RBC.

A new technology for crossmatching tests adapted to a fully automated system L GAILLARD, V DESVIGNE, A BOULET, L FAUCONNIER and JM PELOSIN Diagast, Loos, France

We have developed a new automated technology for Crossmatching (Compatibility) test suitable for automation and high throughput. The method does not require centrifugation steps thanks to the use of magnetised red blood cells (RBC). All the steps described are performed on the fully automated Qwalys System. This methodology requires washing steps under magnetic field and is based on the fixation of sensitised RBC on the surface of a well coated with monoclonal anti-human globulins. In a first step, the Red blood cells from target blood bags were magnetised during 10 min. Then the patient plasma is distributed on a microplate and incubated with the previously magnetised RBC during 20 min at 37°C. Excess of unbound immunoglobulins is removed by washing steps. In a third step, sensitised magnetised RBC were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. Wells in which antigen-antibody interactions have occurred display a confluent layer of RBC (positive reaction). The negative reaction appeared as a pellet in the middle of the well. The test can be read by an automatic reader or by naked eye. The patterns in the well are stable for at least 24 h at room temperature. The plasma samples are provided by the laboratory of haematology of the CHRU of Lille. The Red Blood Cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the EFS (French Blood Services) Nord de France-Lille. The results are obtained in 40 min. Comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. The mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. One of them PIG3, is induced by p53 through a microsatellite in its promoter region. This microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms.

Microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 TGYCC repeats), at this locus have been hypothesized to provide an increased risk for cancer development.

Aim: In the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, Greek and British. Results: Analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. The last two observations were found in both Greek and British populations. Conclusion: Taken together, these data do not support the notion that this PIG3 polymorphism is associated with an increased risk for cancer susceptibility. Background: Blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. Many weak D and partial D phenotypes which react as D negative or weak D by slide test, are assigned the Rh D + status by gel test. This is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial D phenotype. Case report: We report a female patient (blood group O, C+, c+, E-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-D antibodies in slide tests.

We were asked to type the patient and provide the appropriate blood units. The patient's cells gave a 4+/3+ reaction in the standard screening procedure for the Rh D in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak Ds), a 4+ reaction in a test with monoclonal anti-D and a 4+/3+ reaction in the gel test micro-typing system destined to detect Du and which contains polyclonal anti-D of human origin. However, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-D) we tested the patient's red cells against 6 anti-D reagents in the ID-Partial D Typing System. One of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial D category VII phenotype. The patient's red cells were also tested in the ID-Card 'DiaClon ABO/D' . This card provides the complete profile for ABO/Rh D in one single procedure step, including the confirmation of Rh D. It contains two different anti-D reagents within the gel matrix in two consecutive microtubes. The first anti-D (polyclonal human) is expected to give a positive result with D+ red cells and partial D category VI, while the second (monoclonal rabbit) is expected to give a negative result with DVI+ red cells. Our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-D in this system, indicating a D variant other than DVI. Finally the patient was assigned the Partial D category VII phenotype (according to the pattern of the reactions obtained with the ID-Partial D Typing set) and rhesus D negative blood units were issued.

This case illustrates the diversity of reagents used for Rhesus typing in different laboratories. Failure to disclose some D variants is a disadvantage when typing patients. A combination of techniques is often needed to reveal the real Rh D phenotype. The only single system that could have revealed a D variant in our patient from the beginning, is the ID-Card 'DiaClon ABO/D' with two different anti-D reagents in two consecutive microtubes as described above. A cost-benefit analysis should be undertaken to show whether it should replace other screening tests for ABO and Rh D when typing patients.

Who cares about the quality of life of the chronic patients treated with blood products? D ILCENCO*, E HANGANU-TURTUREANU † , C BURCOVEANU † , C VARTOLOMEI ‡ and D AZOICAI § *Blood Transfusion Center, † Hospital 'Sfantul Spiridon', ‡ Institute of Hygiene, § University of Medicine, Iasi, Romania

Quality of life is one of the methods used to appreciate the quality of the health system. Romania is going to join soon the European Union, so there must be a concern regarding the improvement of the national health system. Blood receiver's life quality never been researched before in Romania. We have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. We used NOTTINGHAM HEALTH PROFILE and BECK'S DEPRESION INDEX. Results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. Evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. In conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support.

Transfusion medicine practice in surgically treated urology patients: our experience IL ILINCIC*, BM BOZOVIC* and TS TADIC † *Clinical Center Dr Dragisa Misovic, † Natio. Blood Transfusion Inst., Belgrade, Serbia

Objective: Multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. Method: Using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (ICU) and at the Urology Center within the CC Dr Dragisa Conclusion: Due to a rather liberal use of primarily FFP in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. Background: The safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. Since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. In the last years a new technology was developed for industrial use, the radio frequency identification (RFID). Aim: The aim of our studies was to check whether RFID can use reasonable in transfusion medicine. Methods: At first we developed a flow chart, where we can use the technology and where are the problems by introduction. So we tested in the Red Cross Donation Centre in Saxony about 1000 passive RFID smart label under real conditions. In cooperation between the AKH Vienna and NovaTech Research a new handheld PC software 'LabelView' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time.

Results: Passive and semiactive (with temperature control) RFID labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). As a result of the contactless identification they are help to make easier the documentation of all processing steps according Good Manufacturing Practice. In clinical practice they are a good supplement to bed side transfusion software. Conclusion: For all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. The lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data.

Our experiences with the immunohaematological analyser OLYMPUS PK 80 applied conventional and no conventional (Hemolytic Medium Time) techniques in 23 sera of patients with ascariasis. Results: The AI and HK tests showed: B epithopes in 4 AE from B patients and in 3 AE from AB patients; A epithopes in 1 AE from AB patient and in 3 AE from A patients; P and P1 epithopes in 18 AE and only P1 epithopes in 3 AE. These 21 patients had both epithopes in their erythrocytes. The hemolytic techniques showed: anti B immune antibodies in 8 sera and anti A immune antibodies in 8 sera.

The presence of ABO and P epithopes in AE and immune antibodies in patients with ascariasis show a relation about Blood Groups and Ascariasis. The fact of to find the same ABO and P antigens in A. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. These epithopes would be involved in the molecular mimicry.

The use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis G POPOSKI*, S KOVACESKI*, B KRSTANOSKI*, S MENA* and N SOLAZ † *Institute of Nephrology, Struga, Macedonia, † Ankara University, Faculty of Medicine, Ankara, Turkey

Introduction: Renal anemia is one of the major chronic complications in end stage renal disease. It is caused by reduced production of erythropoietin (EPO) due to uremic toxin effects, reduced halflife of RBC, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. Allogenic blood transfusion is transplantation of certain or all cell types. However, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. Besides erythrocytes, mononuclear, T and B-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. Leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and HLA antigen and transmission of CMV.

Anti-Le antibodies, forming of immune-complexes, complement activation with pirogenic C3a and C5a immunoinflamatoric citokines cause febrile reactions. Commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of CMV. Aim: The aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at Institute of Nephrology in Struga. Matherial and methods: During 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. The first group 34 pts with febrile non-hemolitic transfusion reaction. The second group-25 pts immunized toward leukocyte and HLA antigen. The third group 57 young candidates for kidney transplantation for prevention of HLA immunization. The fourth group 16 pts with SLE (for immune-complexes and autoantibodies). Total 132 patients (65 males and 67 females) received 319 units of RBC with residual number of leukocytes. Commercial filters of BAXTERÔ (Lekostop 4 LDS) and TERUMOÔ (IMUGARD III RC) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. Erythrocyte concentrates are filtered until 5 days of collection. Result: AABB permits maximum <5 ¥ 106 WBCs/unit for prevention of febrile non-hemolytic reaction. The filters we used reach residual leukocyte number of 2 ¥ 10 5 the Le reduction of 99-99.99%. The number of RBC after filtration is minimum 90% -40 g Hb per unit. In none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. Conclusion: The used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. The use of filters for Le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with SLE, and particularly in young patients candidates for kidney transplantation. Background: FV Leiden, prothrombin G20210A, MTHFR C677T are three most common and important prothrombotic inherited mutations. Aims: The aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. Methods: The study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. Extraction of genomic DNA was followed with genotyping of FVL by PCR-SSP, prothrombin and MTHFR mutation by PCR-RFLP. Results: A statistically significantly higher prevalence of FVL mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), P < 0.0001. The OR for heterozygous carriers was 15.24 (95% CI 6.13-37.94), confirming the association of FVL mutation with the risk of thrombosis. There was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), OR 1.71 (95% CI 0.56-5.21), P = 0.3441. Although the group of thrombotic patients showed a higher prevalence of homozygous carriers of C677T MTHFR than the control group (17.5% vs 10.5%), OR was not significant (1.81, 95% CI 0.91-3.57), P = 0.0859. Analysis of combined effects of mutations showed an additional thrombotic risk for carriers of FVL mutation and both mutated alleles of C677T MTHFR gene (TT and CT) (OR 9.40, 95% CI 3.41-25.80), P < 0.0001. Conclusions: FV Leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. Additional prothrombotic risk have carriers of FVL mutation and C677T MTHFR gene mutation.

A female patient in a high fever due to urinary tract infection does not respond being given antibiotics. On the contrary, leukocytes rose (to 30 ¥ 10 9 /L), anaemia became even deeper, as well as thrombocytopenia. Hemocultures were negative. Hematologist decided to search for hematological disease. The first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). To treat heavy anaemia (Hgb 53 g/L) hematologist asked for red blood cell concentrate. Pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. Antibodies had no apparent specificity. Biochemical parameters (bilirubin, LDH, haptoglobin) suggested mild hemolitic process. Electroforesis revealed polyclonal hypergamaglobulinaemia. The 9th day of hospital treatment, the therapy with corticosteroids was introduced (Solu-Medrol 125 mg per day). Coagulation parameters were tested: PT 1.75 INR, APTT 41s, fibrinogen 0.5 g/L, Trb 66 ¥ 109/L, D-dimer 251 mg/L, ATIII 52%. DIC was suspected. Liver enzymes showed mild liver dysfunction (normal AST, ALT, elevated GGT, low CHE). Substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 IU ATIII, 2 doses of red blood cells and vitamin K 10 mg. Two days after the substitution therapy we saw PT 1.28 INR, APTT 31s, fibrinogen <0.5 g/L, Trb 75 ¥ 10 9 /L, ATIII 71%. During the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. The patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. Till 17th day of therapy, fibrinogen was below 0.5 g/L. There was no hemorrhagic diathesis. After that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. The results of immunological tests, collected later, confirm the diagnosis of Systemic lupus erythematosus. We did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of SLE, without any specific treatment except corticosteroids, fibrinogen recovered. We assume it is quite enough for highly suspected immunological hypofibrinogenaemia. Results: Twenty-four-year-old male patient with severe hemophilia type A suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. Treatment was initiated with 90 mg/Kg/dose of rFVIIa (*) for 7 days, after which there was clinical and endoscopic recovery, and an INH decrease to 1.0 UB/ml (FVIII dosage 19%) . He began to take meprednisone (1 mg/Kg/day) for 45 days, after which the INH titre was 12.0 UB/ml. (Table 1a ,b) The patient underwent surgery the following year (correction of equinus foot). He entered the operating room with an INH of 26.7 UB/ml and was treated with 120 mg/Kg/dose of rFVIIa (*) for 10 days, obtaining an excellent hemostatic response. He had two autologous blood units, but it was not necessary to be administered. The INH titre decreased again (down to 5.7 UB/ml) during the intratreatment stage. Thirty-five days after rFVIIa, the INH titre was 7.5 UB/ml. (Table 2a ,b) The presence of high titre INH against FVIII is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. In this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rFVIIa could reduce the INH titre in sequential dosages.

We have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre INH against FVIII. In this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rFVIIa could reduce the INH titre in sequential dosages. There was also a decrease in the INH titre concomitantly with an increase of the plasma FVIII level during its use. This phenomenon suggests that rFVIIa could produce a modulation in the immune response. 

Evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion L DADIOTIS, A KOLOKYTHA, M DIMOU, A PERDIOU, C ALEPI, P SPYROPOULOU, E IGOUMENIDES, C VELIDOU, V PANAGOPOULOU and S MATSAGOS Tzaneion General Hospital, Pireas, Greece Automated Blood Collection system (ABC) is a device manufactured by Macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (AC) in standard ratio (7 : 1). This is managed by passing the AC, which is stored in a special bag,

Anti-erythrocyte antibodies are immunoglobulins that belong to the IgG, IgM and IgA classes. Their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. They can emerge as auto antibodies and alloantibodies. The blood transfusion in patients may induce a post-transfusion hemolytic reaction (PTHR). In order to avoid or reduce the danger of the PTHR it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. All the irregular anti-erythrocyte antibodies are not clinically relevant. The experience shows that the clinically significant antibodies most often belong to ABO, Rh, Kell, Kidd, Duffy and SSU blood groups. In the period from April, 2003 to November, 2004, we monitored and examined, at the Institute for Blood Transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. We used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect Coombs test, screening test by the commercial test erythrocytes and gel filtration method. The detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. Our results are the following: voluntary blood donors: ANTI D, ANTI C + D and ANTI-Leb antibodies. Patients: ANTI-D, ANTI-K, ANTI-Fya, ANTI-c and ANTI-E antibodies. In nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques.

Improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and G-CSF S PARK*, C KELAIDI † , S GRABAR ‡ , V BARDET ‡ , D VASSILIEFF ‡ , F PICARD ‡ , M GUESNU ‡ , MC QUARRE ‡ , P FENAUX § and F DREYFUS ‡ *Service Hématologie, Hopital Cochin, † Hématologie, Hopital Avicenne, ‡ Service hématologie, Hopital Cochin, § Hématologie, Hopital Avicenne, Paris, France It has previously been shown that serum EPO level and number of previous red blood cell transfusions are predictive factors of response to EPO + G-CSF treatment of myelodysplastic syndromes (MDS). In a subgroup of patients with MDS having sEPO <500 UI/l, known to be good responders to EPO + G-CSCF, the GFM group wanted to refine the model predicting the response to EPO + G-CSF, especially with cytology (WHO classification with dysplasia and percentage of erythroblasts and blasts). In a population of 64 patients (RA, RARS and RAEB <10% blasts) receiving EPO ± GCSF between 2000 and 2004 and having serum EPO <500 UI/l, the response rate at week 12 (IWG criteria) was 60%. Six variables were associated with response to EPO ± G-CSF for MDS: age >70 years (P = 0.02), number of prior red blood cell transfusions <2 packs/months (P = 0.005), serum EPO level <200 UI/l (P = 0.02), percentage of blasts <5% (P = 0.05), percentage of erythroblasts >30% (P = 0.02) and low IPSS score (P = 0.001). We did not found any influence of dysplasia, type of rhEPO (darbopoietin alfa or epoietin alfa) and karyotype on response rate. In multivariate analysis, age through a rotating pump. The ABC can be used with all types of P-417 Pan-European blood safety alliance

The Pan-European Blood Safety Alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in Europe. It was formerly established on 11 February 2005, during the course of the first General meeting of the PBSA, which comprised of 8 founding patient organizations. The objectives of the PBSA are:

1. To promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. To ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -The education of all staff handling blood components, to reduce human error. -The implementation of and access to, proactive blood safety technologies, for each patient across Europe. -Haemovigilance -The adequate access to blood transfusion services, which should be provided free of charge to the patient.

Other objectives are to raise awareness on a local and European level regarding blood safety, to promote EU legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across Europe and to lobby for increased patient influence on EU health policy makers. Very importantly, the Alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the European Commission and other European institutions, when seeking the opinions of patients on blood safety.

cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. Particularly questions also remain as to, whether gene therapy and the production of ectopic factor VIII and IX will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors.

W-PL4-11 Gene therapy for thalassemia: will it become reality?

University of Washington, Seattle, WA, USA Experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. In the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. A major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. These elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. A second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. As a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. Considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. The major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. In vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. Other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. Gene therapy trials on limited number of patients are expected to be initiated relatively soon. If these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice.

W-PL4-10 Gene therapy for haemophilia

Haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. Gene therapy for haemophilia today relies upon addition of normal factor VIII or IX genes. With present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. More than 30 patients with haemophilia have now been treated in Phase 1 gene therapy protocols. All studies have failed to conclusively show that therapeutic levels of factor VIII and IX can be reliably obtained. The first trial reported used IM injection of a factor IX containing recombinant Adeno Associated Virus (rAAV) in adult patients with severe haemophilia B. Only very modest increases in factor IX level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor IX concentrate was needed in 3/6 subjects. A similar study using the same rAAV vector via intrahepatic artery infusion has been conducted. This has been complicated by the observation of AAV vector in the semen of subjects. In six patients enrolled, no durable levels of IX above 1 u/dl were seen. Further development of this rAAV vector is suspended. For haemophilia A, three systems are have been tried. The first study was an ex vivo addition of factor VIII gene to autologous fibroblasts and then laparoscopic reimplantation. Preclinical assessments demonstrated durable expression of factor VIII (>5% of normal) for >1 year in mice following a single treatment. In 4/6 patients treated repeated factor VIII rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. The second protocol used a murine leukemia retrovirus containing factor VIII, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor VIII >1 u/dl. The third study, using a modified, 'gutless', adenovirus containing factor VIII gene has recruited one patient. This patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. Sustained levels of factor VIII of ~1 u/dl have been observed over a number of months. Accrual to the study has been poor. Haemophilia remains a prime target for gene therapy. However, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. A balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. Con-

Reference: Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia: Churchill Livingstone, 2004, pp 375-400. W-PA-114 Autoimmune neutropenia

Introduction: Autoimmune neutropenia (AIN), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic AIN) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary AIN). The condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . In some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . Clinical and laboratory findings: In general, physical examination is negative. Laboratory investigation reveals the existence of isolated neutropenia. Association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . Blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. Bone marrow is hypercellular without maturation arrest of granulocytic series. Hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. Methods for the detection of granulocyte-specific antibodies: Serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. The former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (GAT). In order to overcome these problems, the Second International Granulocyte Serology Workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, GAT and granulocyte immunofluorescence test (GIFT). GAT is mainly mediated by IgM antibodies and is positive in about 2% of cases. GIFT detects IgG antibodies and is positive in about 98% of cases. It is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. A good direct anti-granulocyte test is not available today. This is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing FcgRII and FcgRIIIb to which nonspecific binding of W-PA-113 Practical approach to transfusion in autoimmune hemolytic anemia (AIHA) G GARRATTY American Red Cross Blood Services, Pomona, CA, USA A major problem when transfusing patients with AIHA is that often all units are incompatible. This may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). If the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). Thus, it is essential (as in any other patient) to exclude the presence of allo-ab. It is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. There are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. The preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous RBCs treated with enzymes, or preferably, with ZZAP reagent. The latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and DTT. These two chemicals will destroy significant antigens other than Rh and Kidd (e.g. MNS, Duffy, Kell, Lutheran, Dombrock, Cromer, LW, some Yta and Ge, Inb, JMH, Ch, Rg, Pr antigens), thus will not adsorb alloantibodies to these antigens. If autoadsorption is not possible (e.g. patient has been transfused recently or there are too few RBCs), one has to perform adsorption with enzymes or ZZAP-treated allogeneic RBCs. One does not have to be concerned with covering any antigens destroyed by ZZAP (e.g. Kell and Duffy systems). We use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). If there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. This is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous RBCs are preferred. If time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. Another approach is to select units matching the patient's phenotype as closely as possible. When dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37C. This can be helped by performing adsorptions with enzyme-treated autologous RBCs at 4C, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. It is reported that 30-40% of AIHA have allo-abs; the incidence is even higher in patients who have received multiple transfusions. Thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. One should always negotiate with the attending physician regarding the time it will take to perform adsorptions. A decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant.

serum IgG may occur (NAIg). The presence of immune-comlexes in the serum, such as in patients with Felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to FcgRIIIb molecules expressed on the surface of neutrophils. Elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . Another cause of false-positive results may be the presence of anti-HLA antibodies because of allo-immunization. These allo-antibodies react with HLA molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. These allo-antibodies can be eliminated by platelet absorption. It seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) [12] . With this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. Finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. One negative test does not exclude AIN. Usually, two to three tests have to be run over a period of months [13] . Antigenic specificity: Human neutrophil antigens (HNA) are classified according to an International Granulocyte Antigen Working Party [14] . Three glycoproteins have been found to be involved in the determination of antigenic specificity, FcgRIIIb, GpNB1 (CD177) and Gp70-95. The respective antigens, frequencies and alleles are illustrated in Table 1 . Antigenic specificity can also be studied by using methods applied in Molecular Biology. A promising approach is transfection of mammalian cells by cDNA derived from granulocyte antigen specific mRNA. Cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. Genotyping of HNA antigens can also be stydied with the PCR technique [15] . References are available from the author upon request.

W-PA-115 A rare case of 'Coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of IgA class

Warm autoimmune haemolytic anaemia (WAIHA) is usually associated with red cell auto-antibodies of the IgG class, which can be detected by polyspecific direct antiglobulin test (DAT). Routine polyspecific direct antiglobulin tests contain anti-IgG and anti-C3d components, and are not standardized to react with IgA-or IgMsensitized red blood cells. Haemolytic anaemia caused by warmreacting auto-antibodies solely of the IgA class is exceedingly rare. Those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific Coombs' test, which is a standard in investigation of possible causes of haemolysis. We present a case of severe warm autoimmune haemolytic anaemia caused by IgA class autoantibodies. A 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. He received multiple transfusions but haemoglobin level did not rise above 7 g/dL. The initial polyspecific direct antiglobulin test, containing an anti-IgG and anti-C3d antiserum, was negative. Tests for cold agglutinins and other possible causes of haemolysis were negative. Only by using a monospecific, anti-IgA antiserum could we show that the warm IgA auto-antibodies against red blood cells were present on patient's erythrocytes. We have not detected signs of complement activation by IgA autoantibodies in this patient. The patient received corticosteroids with good initial effect. His haemoglobin level stabilized and he did not require more transfusions. Anti-IgA direct antiglobulin test became negative about to weeks after the therapy was initiated. However, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. It has been shown that human lymphocytes, granulocytes and monocytes contain specific Fc receptors for IgA, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to IgA auto-antibodies has been demonstrated. There is also increasing evidence that IgA auto-antibodies can activate complement, both via the classical and the alternative pathway. A phenomenon of 'reactive haemolysis', which involves C3-independent binding of C5b6 complexes to 'bystander' red blood cells, has also been described. We emphasize the importance of performing additional testing in cases of apparent 'Coombs' negative' haemolytic anaemia due to IgA, IgM or 'low affinity' IgG autoantibodies, and serological aids that are available for that purpose.

described. Both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/mL). The diagnosis of ANNanti HNA-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. In both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. Omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. The first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. This was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. The second neonate received no specific therapy for neutrophil count increase. The children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. In spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. Discussion and conclusion: In our patients, the therapeutic approach to ANN was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. Although in the last few years rh-GCSF is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. The reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-GCSF therapy in patients with neutropenia induced by anti HNA-2a immunization, and (3) the unknown effect of rh-GCSF on developing tissues of the neonate. The choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases.

Male donors for the production of fresh frozen plasma: a special issue for TRALI patients

TRALI is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. There is no good evidence on which to base transfusion support policy for patients who have experienced TRALI. The hypothesis that there may be patient associated factors that contribute to the risk of TRALI is generally accepted. For this reason it seems reasonable to try to avoid further transfusion during the period of illness. If this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially FFP) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. As fresh frozen plasma transfusion accounts for up to half of all TRALI cases and as our center is the only in Greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. Our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion The difficulty of the project consisted on the fact that these donors are assigned to military camps throughout Greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of FFP conform the European Council quality requirements. This was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. The first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for FFP production. The next step was to re-schedule the shifts of the personnel for the on time production of FFP. During 2003, 25% of donations were fulfilling the specifications for the production of FFP and with the flexibility of the schedule the 97% of them were successfully processed to FFP. During 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to FFP. So it is feasible to increase the proportion of male FFP by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts.

Maximising the blood supply chain in times of shortage

Shortages in the blood supply chain may occur for a variety of reasons. They may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. Increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vCJD by transfusion has been the driver for the development of blood shortage contingency plans in the UK. In England and North Wales, hospitals and the National Blood Service (NBS) have worked together to develop an integrated blood shortage plan (IBSP) designed to ensure that hospitals and the NBS work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. An essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. The impetus for hospitals to implement these programmes was a government circular (HSC 2002/009). Hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower Hb triggers, cell salvage and hospital transfusion teams and are participating in the Blood Stocks Management Scheme (BSMS). Audits of compliance with the circular have taken place, and a web based tool kit is available. The demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. The shortage plan introduced in England and North Wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. The plan is structured to provide actions for the NBS and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. Hospitals should have documented emergency blood management arrangements for each of the phases. The national plan is activated when the NBS red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the IBSP and comply with the daily hospital usage budget. The BSMS has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. To monitor progress against the recommendations in HSC 2002/009 hospitals will be benchmarked against a number of performance indicators. These include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood.

There have been no shortages within the NBS for more than six years, it is hoped that the implementation of the IBSP will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion.

W-PA-120 Transfusion during disaster G KLEIN NIH, Bethesda, MD, USA Publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. Blood is rarely needed in excessive quantities at the moment a disaster occurs. The outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. The terrorist attacks on the World Trade Center on September 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. More than a million potential donors contacted blood-collection centers. Hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. Qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. Supplies and storage capabilities were pushed to their limit. Some blood was inadequately processed and stored. Resources were diverted from needed apheresis collections and component preparation to whole blood collection. In the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. Similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. In virtually every civilian disaster in the U.S. during the past century, all the blood that was needed was immediately available from blood inventory. In only four cases were more than 100 units of blood used in the first 24 to 30 h. In 2001 in New York, the five hospitals closest to the disaster site admitted only 139 disaster victims. The New York Blood Center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. The center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. In the area of the Pentagon, the Chesapeake and Potomac Red Cross blood center supplemented hospital blood inventories within h of the disaster. Meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at NIH, and at a building next to the White House. In the week after September 11, America's Blood Centers collected 167 000 more units of blood, and the American National Red Cross collected 287 000 more units than in the same period the previous year. More than 475 000 units were collected for the disaster victims, but only 258 units were used. U.S. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation.

Rehabilitation of blood transfusion service in Azerbaijan CD ASADOV, GA HUSEYNOV and AB HAGIYEV Institute of Hematology and Transfusiolo, Baku, Azerbaijan

At the end of 80th years of the last century in Azerbaijan as well as in other Republics of the former USSR began process of progressive deterioration of Blood Service parameters. In result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. It is connected by that our republic experiences a heavy transition period from scheduled to market economy. After reception by Azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of Blood Service. In 1996 the law about ' the donorship of blood and its components in the Azerbaijan Republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. Now the national program of Blood Service development is developed. At drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. Within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. However in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. The big work on attraction of the international organizations has been carried out. Now the project of the United Nations Development Program (UNDP) 'Rehabilitation of Blood Transfusion Service in Azerbaijan' is carried out at sponsor's support of the Norwegian Government. Realization of this project will lead to reorganization of Blood Transfusion Service in our country according to practice of the European countries. Within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the Europe Council.

Updating of the Russian law 'concerning the donation of blood and blood components'

On June 9, 1993, a law, 'Concerning the donation of blood and blood components, ' was signed by the first Russian President, Boris Eltsin. Now, after more than ten years of market economy and democratic evolution in Russia, this law was significantly changed on August 22, 2004, as shown in the following sections:

1. The development of a voluntary blood donor system. 2. Removal of the upper age limit for blood donors. 3. Funding for blood donations. From January 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals.

costs of these drugs and the need of prolonged growth factor treatment in these disorders.

W-PA-127 Can iron administration reduce peripartum blood transfusion C BREYMANN University of Zurich, Zurich, Switzerland

The prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. The increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. However, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. Infections: It is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. Around 0.03% of are contaminated by bacteria such as Yersinia or Pseudomonas species but are not screened routinely for bacteria. In addition there is no donor screening for Hepatitis A, Herpes species (CMV, EBV, HHV 7, HHV 8), Parvovirus B19, Hepatitis G (3.4%) and TT (transfusion transmitted) virus (1.9%). Numbers for positive testings for 'classic' virus such as HIV, Hep. B and Hep. C vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, PCR sensitivity etc. Recently there is increasing evidence that even Prions which cause the Jakob Creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. Therefore the FDA has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the US (e.g. donors from UK). Beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. Beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in ICU show higher morbidity and mortality compared to patients with restrictive transfusion policy. This might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. For example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. Taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. Prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. In order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. When defining the Hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. It has been found that haemoglobin values <11.0 g/dL in the first and third trimesters, and <10.5 g/dL in the second trimester may point to an anaemic situation which should be further clarified. The first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical W-PA-126 Impact of EPO treatment on transfusion requirements in myelodysplasia C GARDIN and P FENAUX Hopital Avicenne, APHP, University of Paris 13, BOBIGNY, France

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (AML). Despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. Incidence of MDS increases with age and reach 15/100 000 above 70 years of age. Bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to AML. A composite international prognosis scoring system (IPSS) is used in everyday practice to guide the management of these diseases. These disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to AML in approximately 50% of cases. At diagnosis, 80% of MDS patients are anemic, with an hemoglobin level less than 100 g/L, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. Chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. Although transfusion practices and patient's transfusion need are variable, elderly MDS patients require a mean of 10-20 units/year of follow-up, in recent surveys. Therapies able to diminish or abolish the need for RBC transfusion have therefore a major role in the management of MDS, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of MDS patients. High-doses of recombinant erythropoetin (EPO) (150-300 U/kg tiw, or a 40 000-60 000 U as single weekly dose) are typically used in low-risk MDS. The response rate to EPO is 25-40%, including major responses (suppression of RBC dependency or rise of hemoglobin level of more than 20 g/L). Absent or low RBC transfusion needs and a serum epo level less than 500 U/l are strongly predictive of response to EPO, in patients with low-risk MDS. The duration of response is variable (12-20 months) in most studies, with some long-term responders. The use of higher doses of EPO or its prolonged administration may be associated with higher response rates, although no randomized studies are available Combination of EPO and low-dose granulocyte-colony stimulating factor (G-CSF) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with Epo alone. Two randomized trials published in 2004, compared G-CSF-EPO to RBC transfusions and confirmed the efficacy of this combination, and a longer survival of EPO-G-CSF responding patients. Studies are ongoing in MDS, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. Two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk MDS. In both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. Further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on QOL are required, in order to EPO and its derivatives to gain acceptance in MDS, due the high history and a medical examination. This procedure often lays the basis for a correct diagnosis. The current gold standard to detect iron deficiency remains the serum ferritin value. To be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. We recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. These should The Hb level alone is insufficient to guide management. A complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. Since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. A high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. If the C-reactive protein level is also raised, the soluble TfR concentration can be used, since it is unaffected by inflammation. Inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. However, the current type II iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. After correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics.

Today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. Our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia.

W-PA-128 Iron therapy in orthopaedic surgery

Surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 L) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. Because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. Studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. Correction of even slight preoperative anemia is thus mandatory. In the elderly, iron and vitamin deficiency (B12 and/or folic acid) should be looked for as a matter of routine. We recommend the use of iron + Epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/L. With this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). In the post-operative period, anemia worsens because of the existing inflammatory state. This inhibits iron absorption from the intestine and iron release from the macrophages while it affects Epo function and production. There is increasing evidence that i.v. iron combined with Epo induces a rapid correction of post-operative anemia. It is thus recommended to stimulate erythropoiesis by i.v. iron and Epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with Hb as low as 70 g/L. Background: Hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. The effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. Red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session.

We performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ C282Y homozygotes, 1 ¥ C282Y + H63D heterozygote) using Haemonetics MCS 3p cell separator (protocol TAE) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. Collection time, donor convenience, side effects and red cell yield were recorded and analysed. Samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. Background: The collection of 2 units of red blood cells by apheresis (DRBC) has been reported to be safe and effective in increasing the yield of RBC units from a donor population. However several reports demonstrated the risk of inducing iron depletion when the interval between a DRBC donation and a subsequent RBC donation is shorter than 180 days. Aims: to evaluate the recovery from anaemisation and iron stores depletion after DRBC donation. Methods: donors who underwent DRBC donation between December 01, 2002 and February 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (Hb), Htc, serum iron and ferritin values. These parameters have been assessed on the day of donation and, thereafter 7 and 180 days after DRBC procedure. Donors suitable to DRBC apheresis had to have: age between 18 and 60 years, weight > 70 Kg, Hb > 15.5 g/dL and serum ferritin between 50 and 400 ng/mL. A written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. DRBC collection procedures have been performed by using a MCS + (Haemonetics) cell separators. Results: Out of 130 donors who donated DRBC during the study period, only 14 males completed the follow up program and have been analysed. Baseline haematological values and iron metabolism parameters were: mean Hb 16.3 ± 0.5 g/dL, ferritin 85 ± 31 ng/mL, serum iron 121 ± 42 microg/dL. On day 7 mean Hb was 14.3 ± 0.5 g/dL (P < 0.001). On day 180 mean Hb was 15.3 ± 0.8 g/dL (P < 0.001), ferritin 53 ± 22 ng/mL (P < 0.05), serum iron 111 ± 27 (p NS). Only 6 out of 14 donors (43%) had a ferritin value > 50 ng/mL.

In the studied donors the collection of 2 units of RBCs induced an expected reduction of about 2 grams of Hb, however only 50% of this reduction was recovered after 180 days (P < 0.001). Similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. According to these data it appear that the amount of iron 'lost' with the donation of 2 units of RBCs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. Further data are necessary to define the risk of iron depletion after the donation of a DRBC, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a DRBC and a subsequent RBCs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. Background: Increased transferrin saturation and/or serum ferritin have been observed in Italy in approximatively 5% of subjects at first blood donation and, in these subjects, HFE mutations prevalence was 0.11 for C282Y and 0.26 for H63D (Velati et al., 2003) . Aims: The role of the C282Y mutation is well known in the patho-genesis of iron overload, whereas the role of the H63D mutation remains uncertain. The aims of the present study were first to study the main HFE mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the H63D mutation in comparison to a population of blood donors wt/wt for the H63D mutation. Methods: A total of 1165 repeat blood donors were examined in 10 Italian Transfusion Centers (6 in Northern Italy and 4 in Southern) for C282Y and H63D mutations. Out of those, 50 blood donors heterozygous for the H63D mutation and 31 wt/wt for the same HFE mutation, both groups wt/wt for the C282Y, were enrolled to evaluate iron parameters and iron depletion. These two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. Serum ferritin (SF) was the iron index recorded at first and second observation. Results: Table 1 summarizes the allelic frequencies in the 900 blood donors. Table 2 reports the haematological evaluation in the 50 subjects heterozigous for H63D mutation and the 31 wt/wt for the same mutation.

Conclusions: These data suggest that subjects with H63D mutation of the HFE gene have, at first observation, a higher ferritin levels than subjects wt/wt. This seems to be more evident in blood donors of Southern Italy than in Northern. Blood donation induces significant reduction of the iron stores both in H63D heterozygous and in wt/wt subjects. Although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors H63D heteroxygotes or even homozygotes when available, in order to get further insights on the H63D role in iron metabolism. Background: CD 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. Consequently the levels of soluble CD43 as well as the expression on the cell surface is a marker of cell activation. Furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. The myelodysplastic syndromes (MDS) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. The MDS patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. Aims: We studied CD43 expression in transfusion-dependent and non-transfused MDS patient in an effort to investigate mechanisms of regulation of this molecule. We also studied other activationassociated antigens in the absence of manifest infection. Material and methods: Forty-two patients were included in the study suffering from refractory anaemia (RA). Thirty-one were males and 11 females aged 33 to 85 (median 75). Twenty of them had never been transfused (group A) and 22 were regularly transfused (group B). Nineteen age matched healthy individuals were used as controls (group C). Cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. The following mouse monoclonal antibodies were tested: anti-CD11b, anti-CD18, anti-CD43, and anti-CD45. Leukocytes were gated according to CD45. We used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. The R&D ELISA kit was used according to the manufacturer's instructions. Results: The CD43 was found down-regulated in the transfusiondependent MDS patients compared with the non-transfused ones (P < 0.001) and controls (P = 0.012). This downregulation concerned the proportion of CD43 + cells, that was lower in the transfused patients than the non-transfused (P < 0.01) and controls (P = 0.006), and the RFI (relative fluorescence intensity) value that was also lower in the group A compared to the group B (P < 0.01) and group C (P = 0.001). Negative correlation was observed between the CD43 expression and CD11b (P = 0.001) and CD18 (P = 0.001). CD11b was found up-regulated in the transfused patients. The RFI value was significantly elevated in the transfused patient compared with the non-transfused and controls (P = 0.001 and 0.001 respectively) while the percentage of CD11b cells did not differ significantly between the various groups. Increased expression of CD 18 was also found in the group A compared to group B (P < 0.01) and C (P = 0.008). The proportion of CD18 + cells did not differ between the various groups. The levels of immuno-reactive sVCAM-1 as determined by ELISA were found 512.02 + 35.56 in group A, 3.42 + 65 in group B and 194.11 + 45.48 in the control group. Conclusions: Activated hemopoietic and endothelial cells are found in MDS that may be associated to the vascular disorders found in these patients. CD43 downregulation may also be associated to increased susceptibility to infections in these patients.

Despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. Preoperative autologous blood donation (PAD) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. It may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. PAD also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. As any medical intervention, PAD has both advantages and disadvantages. With proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh.

Background: Prestorage pooling of whole blood derived (WBD-PC's) buffy coat platelet concentrates (PC) is common practice in Europe

Event-free survival was significantly better in patients who responded to EPO + G-CSF. We have reviewed data in 2 centers and the GFM has the intention to extend the study to a larger population in at least 10 centers in France

Blood components and preparations. The new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. Different methods are necessary for the quality control of blood components and blood preparations

Privileges for blood donors include: -A paid day off work on the day of blood donation and medical examination for blood donation

Additional paid day off work after blood donation

An extra paid day off work if blood is given during vacation or on a holiday

this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. Before 2005, each 'Honoured Donor of Russia' or 'Honoured Donor of the USSR' had three privileges: Free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities

Previously, municipalities also could have their own blood establishments. This resulted in more than 1500 blood establishments in the Russian Federation. From both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution

Acknowledgements: We thank KSW Microtec AG, Dresden/ Germany for sponsoring the RFID-labels and Novatech Research GMBH, Vienna/Austria for developing the clinic-software.

Background: The national preparation Human Immunoglobulin G 5% for intravenous use (IVIG) that is produced at the Serbian Institute for Blood Transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. Aims: It is our aim to prove the impact of this national medical preparation Human Immunoglobulin G 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. Material and methods: Human Immunoglobulin G 5% for intravenous use (IVIG) has been used in the study. The preparation is liquid, 5% stabilised with glucose of a pH value of 4.25 ± 0.25. It is used in those cases where sepsis developed after surgery. Both an IVIG group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with IVIG. The number of specimens with the IVIG therapy Cholecystitis is (n = 3), and the Control group (n = 2); Pancreatitis (n = 8) Control group (n = 7); Intestinal obstruction (n = 7) Control group (n = 8); Abdominal organ perforation (n = 5) Control group (n = 8); Abdominal perforate injuries (n = 12) Control group (n = 13); Serious abdominal interventions (n = 5) Control group (n = 6).

The period of hospitalisation of the patients in the IVIG group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. The mortality rate in the IVIG group was 40% counter 63.7% in the control group. Summary: Toxic Gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. By using Human Immunoglobulin G 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as Diabetes mellitus, Neoplasma, Cardiac diseases etc.

Background: Manual production PC from buffy coats (BC) is a procedure with some consecutive manipulations. The OrbiSac system (Gambro BCT) automates the steps and we assessed its performance. Material and methods: 160 PC were produced by this device and some parameters were studied. For the preparation of PC, 5 BC were pooled using the OrbiSac set, with an integrated filter (Pall LRP6). BC pool was resuspended in the additive solution T-Sol in order to obtain a final ratio plasma/T-Sol 35/65. The PC was stored in a Gambro ELP bag. Results: The average platelet count per unit was 3.41 ¥ 10E11. The platelet recovery from pooled BC was 85.12% (range 79.75%-93.21%). All products of the tested PC containing <1 ¥ 10E6 WBC (by flow cytometry). The values of pH on day 1 and 5 of storage were 7.45 and 7.43. The swirling phenomenon was good until day 5°. The average loss of haemoglobin per BC was 6.8 g.Conclusions:The OrbiSac system is very suitable for routine PC preparation and it allows increased productivity and better standardization method for PC preparation. Platelet concentrates met the requirements for leucodepleted product.

Increased production of plasma components from male donors Background: We routinely separate whole blood (WB) after hard centrifugation into a red cell concentrate (RCC), a buffy coat (BC) and plasma (PL) by an automated expresser (Compomat, Fresenius). The BCs are subsequently processed into platelet concentrates (PCs) by soft centrifugation and an additional (manual) expression step. The Atreus 3C system (Gambro BCT) eliminates several of those hand-on steps by combining them into one integrated process. A processing 'circular' bag is placed in the device and filled with the WB. While the bag is centrifuged, the system expresses PL, PC and RCC into separate containers. The RCCs are subsequently leukoreduced (manually) with a filter (LR-RCCs). This study was designed to evaluate the storage characteristics of the RCCs obtained with a prototype of the Atreus system in comparison to RCCs obtained by routine procedure. Methods: Whole blood (500mL) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) Atreus 3C. RCCs were leukoreduced with the integrated inline filter: Fresenius (routine group) or Pall RC2D (Atreus). LR-RCCs were stored at 4°C and sampled until day 42. Various in vitro measurements were performed (n = 20 per group).Results: See table (mean ± SD). The LR-RCCs contained significantly more leukocytes in the Atreus group. Despite the RBC loss in the BC, hemoglobin (Hb) content was 6% lower in the Atreus group, but met the requirements. In vitro storage characteristics for the RCCs were similar in both groups. The Atreus PCs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 mL. Although plasma volume was higher in the routine group, subsequent preparation of PCs would have resulted in an additional loss of 50 mL per unit in the control group. Atreus plasma had extremely low levels of residual WBC and RBC. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 Aims: The aim of this study was to examine platelet quality of prestorage pooled PRP-derived PC's for up to 7 days storage. Methods: PC's were manufactured from WBD-PC's using in-line filtration of PRP on day 0. On day 1, either 4, 5, 6, or 8 PC's were pooled into an ELX® container using a sterile connecting device. Studies were performed on days 1, 5 and 7 for the following measure of platelet quality. pH, morphology score (MS), extent of shape change (ESC), hypotonic shock response (HSR), percent in surface expression of P-selectin (P-Sel), phosphatidyl serine (PS), glycoprotein 1b (GP1b) and by thromboelastography of the PRP (Maximum Aplitude, MA). Results: A total of 20 pools were studied, 5 each of 4, 5, 6 and 8 PC's. The mean platelet yield was 4.3 ¥ 10E11 with a range of 2.4-7.0 ¥ 10E11. The five 8 PC's had a mean yield of 6.0 ¥ 10E11 and all maintained a pH > 7.0 on day 7. All products had less than 1 ¥ 10E6 residual WBC. Platelet quality data is presented in the Table. Data are the mean ± 1 SD, n = 20. Conclusion: Platelet pools manufactured from PC's produced by inline filtered PRP and stored in ELX® containers show good quality preservation to day 7 over a range of platelet yields. Introduction: The big progress in treatment of critically ill children significantly increases the need for blood and blood products. Loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. Aim of the study: To present the number of erythrocyte concentrates (EK) that were issued to the Pediatric Clinic in Skopje, as well as to point out how they were distributed. Material and method: This is a retrospective study performed in NITM-Skopje from January till May 2004. The following criteria were followed: Hemoglobin (Hb), Hematocrit (Htc), as well the clinical evaluation, and then final decision for transfusion was made.Results: There were 254 blood units (EK) issued for the mentioned period to Pediatric Clinic for 73 pediatric patients (~3, 5% units/per child). The biggest consumers are children at intensive care unit and at the hematology-oncology unit. One unit of leukodepleted erythrocytes (Er) was split equally to 3-5 bags. For small and prematurely born children and for some other selected patients Er unit was filtered and irradiated. The dosage was 25-100 ml Er (depends on age and body weigh). 173 EK was issued as washed concentrates, 15 EK were filtered and 25 EK were resuspended in AB plasma. Distribution among ABO system was the following: Conclusion: Gynecologic patients consumed RBC more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. The A blood group is the most needed one. We should insist on using the WHO guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and Z. CERMAKOVA University Hospital, Ostrava, Czech Republic Background: Fully automated system OLYMPUS PK 80 is an immunohaematological analyser for detection of red blood cells antigens of AB0, Rh (D, C, c, E, e) and Kell systems without centrifugation by MAM (Microplate Agglutination Method) on unique terraced microplate Olympus. In the Czech Republic Analyser PK 80 is used only in Blood Center Ostrava. Aim: To evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. Methods: 95 880 blood samples of donors were tested between July 2002 and January 2005. All samples were analysed for AB0 blood group. 69 052 samples were tested for RhD antigen and 15 220 ones for Rh (C, c, E, e) and K antigen. The validity of the results was evaluated for AB0 with parallel testing antigens and antibodies, while for Rh (D, C, c, E, e) and K using two diagnostic serums. Sensitivity of MAM i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. Requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control PK during testing and maintenance. Capacity were evaluated as a number of samples tested per day. Reliability determine by occurrence disorders. Results: AB0, Rh (D, C, c, E, e) and K were investigated truly by first testing at 99.5% samples. Two diagnostic serums anti-D Olymp IgM and Totem differentiate directly RhD negative and RhD positive donors. False negative or positive results were not founded out due to MAM or quality of diagnostic serums. About 0.5% samples with abortive tests were analysed next time the same testing or manual technique. Causes of abortive tests were microagglutination several samples except for anticoagulative EDTA, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. In one case analyser evaluated false AB blood group due to hemolysis. Analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. Conclusion: Analyser Olympus PK 80 is an effective alternative full automation for medium serological laboratory and together with MAM easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests.

Introduction and aim of the study: The purpose of this study is to establish Nested-PCR for the detection of hepatitis B virus (HBV) in blood and blood products. Methods: The primer pair set was designed to amplify 513 bp in Sregion of HBV genome in the first PCR and 233 bp of first PCR amplicon with Rubisco (internal control) in the second PCR. To assess the specificity of PCR results, all the samples were tested cross-reactivity or interference in the assay. Results: In case of HBV spiked blood products such as immunoglobulin and coagulation factors, this method could detect HBV DNA up to 62.5 IU/ml. Nested-PCR was compared with PCR-ELISA and hybrid capture II (HC-II), the PCR-ELISA showed a sensitivity of 96% (HC-II; 77%) and a specificity of 98% (HC-II; 100%) (P < 0.005).

The results of the study show that Nested-PCR and PCR-ELISA could be used equally in the management for HBV detection in blood and blood products. P-398 Blood component therapy: slow improvement A MRDJA Health Center Subotica, Subotica, Serbia Background: Transfusion department at general hospital was founded in 1953. Since that time till now it has answered to all demands in blood and blood components. Aim: The aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. Method: Retrospective analysis of blood/component utilization in period from 1. January 1984 to 31. December 2004. Results: In 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (RBC) only 5.94%, washed RBC were used in 9.87% of the cases. In 2004 whole blood participated in the consumption with 31.5%, packed RBC with 61.65%, washed RBC with 0.14% and RBC in additive solution with 6.71%. As far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (FFP) only 2.9%. Since 2003, there has completely been cancelled the production of five day old plasma, only FFP is being used. From 1984 to 2002 for the patients who needed platelets, platelet rich plasma (PRP) was prepared and applied right after preparation. The consumption rate was from 35 units to 103 units per year. In 2002, after the purchase of platelet shaker, began the production of platelet concentrates (PC) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. Conclusions: It is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. Variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. In 2003 the production of plasma old up to five days was cancelled and instead we produced only FFP. PC we prepared for patients only in agreement of treating physician. Although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. Transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy.

Introduction: Blood Groups may act as receptors of parasites, bacteria and viruses. There is evidence that they perform a function and play a biological role. Objective: The aim was to detect ABO and P epithopes in Ascaris lumbricoides extracts (AE) and to study the presence of immune antibodies in patients with ascariasis. Materials and methods: 50 AE were prepared by refrigerated mechanical rupture of adult specimens. Agglutination Inhibition (AI) and Haemogglutination Kinetics (HK) Tests were made with the AE. The patients´ ABO and P blood groups were determined. We 1998  1999  2000  2001  2002  2003  2004  Total   Febrile non haemolitic  2  3  2  3  2  4  2  5  2  4  2  4  2  9  4  34  16  male  transfusion reaction  2  1  1  2  3  2  2  5  1 8  f e m a l e  Alloimunisation on Le/HLA  3  1  2  2  1  3  1  3  3  4  1  5  2  25  6  male  antigens  2  2  1  2  3  3  3  3  1 9  f e m a l e  Kandidates of renal  4  3  4  4  5  4  6  4  6  5  10  8  12  8  10  7  57  43  male  transplantation  1  1  2  1  2  4  3  14  female  Lupus nephritis  3  3  2  3  2  1  1  1  1 6  male  3  3  2  3  2  1  1  1  1 6  f e m a l e  Total  12  12  12  16  16  18  21  25  132  65  female  67 male Introduction: Irradiation of Blood product has been in routine use to prevent Graft-Versus-Host Disease (GVHD) in certain recipients for many yeas. Gamma Irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cGy of gamma radiation reduce lymphocyte response to mitogens by 90%.The aim of the study: 1. To estimate potassium level increment in stored irradiation blood units. 2. To compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. To evaluate the expiratory date of blood units post irradiation.

The study included 40 units of blood collected in CPD-Adsol (AS-1). In twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. All the 40 units were irradiated using Caesium 137 as a source of irradiation, with a dose of 1500-2500 cGy. Baseline samples from the bags were obtained for measuring of extra cellular potassium (K+). Control samples included. Results: There is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. Comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. There is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, P value of more than 0.05.

1. Gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. There is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. There is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. An out date of 28 days post collection (unless they expired before) for irradiated Red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. The percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications.

The use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in Iran. In accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . There is also increasing trend of using fresh frozen plasma (FFP) from 26.4% (1994) to 55. 2% (2003) . Comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. Results: During 1 year period, a total of 5310 units of blood and 867 units of F.F.P were used. More specifically, the results can be shown in the following Table 1 . A high rate of F.F.P usage is observed both in Surgery and Pathology clinics. The main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. Conclusion: The only way for rational usage of F.F.P is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. Acquired factor V inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. It may occur spontaneously or as a result of various clinical conditions. A 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. During the routine preoperative procedure screening coagulation tests showed pathologic values: APTT 41s, PT 35%, fibrinogen 2.8 g/l, FV 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /L. Factor V inhibitor was detected by a modified Bethesda assay. The assay showed a low level of inhibitor of about 1.30 Bethesda units (BU). The patient's medical history showed no major morbidity except appendectomy performed 22 years ago. The patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (FFP) in a dose of 15 mg/kg (~1500 ml). Upon FFP transfusion, repeated determination of the factor V plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. As the measured level of factor V activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. The procedure and postoperative course were uneventful and without major hemorrhage. Laboratory testing for the possible systemic autoimmune disorder produced normal findings. Control examination performed two years later revealed no major clinical or laboratory variation, while a low factor V level persisted (17%) along with the presence of factor V inhibitor at a level of 1.15 BU. 

We have evaluated two groups of RCC's, one we routinely use (Quadruple Leucoflex LCR5 T/T CPD/SAGM) (MACOPHARMA) and one using an automated collection device which gives the ability to collect whole blood in CPD with a rate of 7 : 1 respectively during the whole donation. The mentioned system has been evaluated using the suitable, Quadruple LEU-COFLEX LCR5 T/T CPD/SAGM (MACOPHARMA). Whole blood 450 ml in CPD was collected from random donors. In both groups the whole system was stored at scaled R.T. 20 ± 2°C for 2 to 5 h. After component separation (BECKMAN COULTER J6MI-optipress I-BAXTER), the red cell concentrates were filtered immediately at R.T. 20 ± 2°C. Sampling was done after filtration and WBC measurements were determinated using Nageotte champer (Bright Line-Detection Limit 0.05 WBC/ml) with Leucoplate solution (SOBIODA). The other parameters were measured with (Celldyne 1700 ABBOTT). Conclusion: All products met the accordance of national and European norms for blood components quality. Leucodepletion with Leucoflex LCR5 and ABC Leucoflex LCR5 (5.040 and 5.015) is highly efficient. The use of ABC Leucoflex LCR5 showed better scaled donation in terms of collection (statistical analysis Mann-Whitney, Minitab P-value = 0.0392 < 0.05). Additionally less Hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis Man-Whitney, Minitab, P-value = 0.0382 < 0.05). This new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. Smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. The ABC system gives the ability of fully traceability during donation. Macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the AC. The device can keep records for many parameters and can transfer them to the data base server of the Blood Center. To evaluate the performance of the ABC, we conducted a comparative study between ABC Leucoflex LST1 system and Leucoflex LST1 system we currently use. The later is a well known Macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted CRCs and one unit of leucodepleted plasma. The ABC was handled with the same system modified with the storage bag for the AC. Issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual WB Cells after filtration. We performed 24 donations with the LST1 system and 28 donations with the ABC LST1 System. All the donors were random male volunteers and they were meant to donate 450 mls of whole blood. Our results analysed by Mann-Whitney, MINITAB statistical analysis have as follows:1. No significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the LST1 system compared to the ABC LST1 system. 2. The duration of filtration has been found without statistically significant differences between the two systems. 3. The loss of volume in the filter of LST1 is higher than in the ABC LST1 (P = 0.007 < 0.05, which is statistically significant). 4. There was very good leucodepletion with the LST1 systems (median reduction of the WB Cells 4.7 log) but there was a superiority of the ABC LST1 over the LST1 concerning the leucodepletion per litter and per unit (P: 0.0368 and P: 0.0398 respectively). 5. The personnel after a very short period of training accepted fully the ABC procedure.In conclusion ABC is an easy to handle device which provides with high quality blood products in combination with Leucoflex LST1.

Evaluation of a post-storage filter for WBCs with an incorporated waste bag for washing RCCs

Leucolab LCG4 is a system manufactured by Macopharma for poststorage filtration of a RCC unit (with or without an incorporated waste bag for further washing of the filtered product). To evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of RCCs stored in CPD/SAG-M, aged 2-6 days, filtered and consecutively washed with 200 ml of Normal Saline, span down in the regular way and the supernatant extracted in the waste bag. Issues for evaluation were:1. The duration of priming and filtering the RCCs. 2. The loss of volume in the filter.3. The efficiency of the filtration. 4. The acceptance of the personnel of a new (to them) filtration system.Our results have as follows:1. We counted the duration of priming and filtration. Median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. The median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). This narrow range is apparently due to the non-flexible cell of the filter. 3. The efficiency of the leucodepletion was counted by flow cytometry. The median WBC counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). The median reduction of the WBC count was 4.2 log (range 3.9-4.7). 4. The personnel involved in the procedure found the system easy to handle, even without specific training. In conclusion the LCG4 is a reliable and easy to handle system, for leucodepleting (and washing) RCCs, very efficient in removing WBCs with negligible loss of volume. Objective: Standardization of blood banks and establishing quality assurance are important landmarks in the new era of Transfusion Medicine. As the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. Aim: To investigate the types and capacities of blood bank in the country and evaluate the statistics of them. Material and method: Blood Banks and Transfusion Society of Turkey conducted a nation-wide survey of a comprehensive questionnaire. This is a preliminary report of this investigation and illustrates the capacities of the most blood banks in Turkey. It also guides the nationally planned renewing structure of blood banks. Results: There are nearly 340 blood banks and blood stations in whole country. The overall blood collected at those centers is about 1.800.000 units. Nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). The third major group is the Red Crescent Society Blood Centers-RCSBC (23.5%), followed by the social security hospitals (12.9%). Blood collecting capacities are not appearing in the same order. The major blood banks belong to the RCSBCs, whereas the small ones are mostly government hospitals. The only donor recruitment organization is run by the RCSBCs.

Yearly blood collecting capacity Blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3Conclusion: There are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. These are only possible after centralization of all existing blood banks. In our country, we should first arrange all small capacity blood banks and standardize them.

Controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (PATM)

PATM is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. Its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (TM). They believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. Therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to TM. As such, PATM held its first Medical Opinion Forum where members of the group debated important questions pertaining to TM clinical practice. The forum was held just prior to the AABB annual meeting and attracted 58 physicians. The questions debated were preselected by PATM membership via an email survey. Respondents were asked to select their top four preferences from among 17 topics in five broad categories. The top two topics were selected for the forum from the 80 completed surveys. The subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? The participants were divided into four groups, with each topic assigned to two groups. All the groups were given two h to debate and arrive at consensus on their topic. Each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. There was remarkable consensus between the groups debating the same issue. The conclusions and recommendations on these two topics will be presented in detail. PATM is a new organization that will add an important medical voice and opinion on current topics in TM. At its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field.