Abstracts of the 33rd International Congress of the International Society of Blood Transfusion, in conjunction with the 33rd Congress of the KSBT and the 2014 Congress of the Korean Hematology Societies, May 31-June 5, 2014, Seoul, Korea.

Those Pesky Red Cell Antibodies 1

ISBT Academy Day Those Pesky Red Cell Antibodies 1A-A01-01

RED CELL ANTIBODY DETECTION BY SEROLOGY Nance SJ American Red Cross, Philadelphia, PA, United States of America Red Cell Antibody Detection in pregnancy and before red cell transfusion is important to appropriately manage transfusion requirements for alloimmunized patients. Alloimmunization in pregnancy can lead to clinical consequences with varying severity up to and including fetal demise. Red cell alloimmunization in the transfusion setting is equally variable from no consequence with serologic detection only to clinically severe hemolytic transfusion reaction with destruction of transfused red cells. A more severe consequence has been reported where not only the transfused red cells, but also the patient’s own red cells are destroyed resulting in a lower hemoglobin post- transfusion. It is important to detect alloantibodies as described above, but it can also be important to detect alloimmunization when the allo antibody is present concomitantly with an autoantibody. This can be achieved with autoadsorption of the patient’s serum with autologous red cells treated to remove some of the autoantibody allowing adsorption of the autoantibody. When the autoantibody is removed from the serum, testing can commence to detect if alloantibodies are present. Patients with auto immune hemolytic anemia have been reported to be the highest contributor to cost of all alloimmunized patient groups according to a recent study (Transfusion 2014;54:271–277). Obstetric patients, the lowest. Methods of detection of red cell antibodies are diverse, but all are intended to detect primarily clinically relevant antibodies. Generally clinically relevant antibodies are reactive at 37°C and detected by macroscopically visible agglutination at 37 or after addition of anti-IgG which acts as a crosslinking agent to visualize sensitization on red cells. Manual methods (tests are prepared by hand, test results read, interpreted, and recorded by a trained person). The popularity of the methods varies between countries. The following methods have been named as antibody detection methods in a recent survey of international methods: saline, albumin, PEG, micro-column, and solid phase. Some of the methods have been automated (microcolumn, solid phase) which results in better process control and ability to electronically manage data. Methods have varying sensitivity, thus clinically irrelevant antibodies may also be detected at a higher rate in some methods. Antibodies are known to change in their detectability over time, losing reactivity and becoming undetectable in some cases. In the highly mobile patient base, record keeping and record sharing are also important in minimizing patient harm due to delayed transfusion reactions. With increasing concentration on health care costs, it is important to choose antibody detection methods carefully to maximize detection of clinically relevant antibodies and minimize detection of clinically irrelevant antibodies. It is also important to minimize errors, and that is usually accomplished with automation, which inherently has better process controls. Selection of the appropriate patient groups for antibody detection studies can also assist in cost control. Red cell antibody detection is important in pre-transfusion testing and in prenatal testing and the methods selected should optimize the process.

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RED CELL ANTIGEN TESTING Scharberg EA, Richter E and Bugert P Institute for Transfusion Medicine and Immunohematology, DRK-BSD Ba-Wue-He, Baden-Baden, Germany Background: The testing of red cell antigens in blood donors and blood recipients is the basic and most important part of the pre-transfusion diagnostics. Depending on the national guidelines the testing includes different mandatory typing ranging from ABO only up to ABO, RhD, RhCcEe, K and RhD genotyping of serologically RhD negative donors. Testing of additional blood group antigens in blood donors and recipients is needed to provide compatible blood products for patients with clinically significant red cell antibodies.

Aims: The potential and limitations of different methods and reagent types for the red cell antigen testing will be evaluated. The differences between the critical issues of antigen typing in blood donors and recipients will be analyzed. The risk and the advantages of different techniques are to be described. The choice of the best methods depending on the indication for the testing will be discussed. Methods: The following techniques are frequently used for antigen testing by serology: tube test, gel technique, slide/plate test, micro titer, solid phase and the lateral flow method. Most techniques are applicable for manual and automated systems. Especially for donor testing automated high throughput systems have become essential. In terms of reagents monoclonal test sera (mostly IgM) are widely used for routine typing of A, B, RhDCcEe, K, M, N, S antigens. However for most antigens only polyclonal antibodies for the indirect antiglobulin test are available. For some serological testing the red cells are treated by enzymes like papain, ficine or bromelain to enhance the sensitivity of the reagents. In the last years molecular methods for DNA typing of corresponding blood group genes have been implemented. Results: The monoclonal test sera (mostly IgM) allow quick and reliable red cell antigen testing even in individuals with a positive direct antiglobulin test. However for some complex/partial antigens, like the RhDCcEe, they may show false negative (e.g. RhD, category VI) or false positive (e. g. RhceRT allele) results. Polyclonal sera mostly detect weak and partial antigens but cannot be used for samples with a positive direct antiglobulin test. For some specificities (e. g. anti-Do(a/b)) there are no available licensed test sera because of source limitations. Serological antigen testing is also not possible in shortly multi-transfused patients. When serological methods cannot be used DNA typing for blood group alleles can be very useful. It has become essential in the non-invasive pre-natal blood group diagnostics and for screening of blood donors for rare blood groups. Genotyping predicts the phenotype but in few cases it may show false results, mostly because of not detecting null alleles or unknown mutations. Conclusions: The testing of red cell antigens in blood donors and blood recipients can be performed in different serological techniques using monoclonal and polyclonal reagents. Automation and molecular DNA typing of blood group alleles rise in significance and get widely available for routine. It is most important to choose or combine the right methods for different indications and to be aware of the advantages, potential and limitations of the used reagents and techniques.

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HANDLING A TRANSFUSION HEMOLYTIC REACTION Yahalom V1 and Zelig O2 Magen David Adom – National Blood Services, Ramat Gan, Israel 2Hadassah Medical Center, Jerusalem, Israel

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Advances in blood collection, component preparation, testing, transfusion protocols and quality assurance systems have improved blood safety dramatically. Nevertheless, even in 2012 Hemolytic Transfusion Reactions (HTRs) remained a major cause of morbidity and mortality as reported to the SHOT and FDA with a calculated risk of major morbidity and death of: 46.5 and 3.1 per 1,000,000 components issued respectively according to SHOT. Hemolytic Transfusion reactions can be immediate/ acute (AHTR) usually due to immune ABO incompatibility or delayed (DHTR) seen days to weeks following transfusion and caused by an anamnestic immune response to red cell antigens (Kidd, Rh, Duffy and others) that might have been missed by antibody screening and/or cross match procedures or due to clerical and communication errors. In rare occasions HTRs may occur with large volume incompatible plasma transfusion for example following apheresis platelet transfusions. HTRs are more frequent in patient populations with Sickle Cell Disease (SCD), Thalassemia and hematological conditions such as autoimmune hemolytic anemia (AIHA) and myelodysplastic syndromes (MDS). In these patients distinction between hemolysis caused by alloantibodies or auto antibodies is challenging, especially if hyperhemolysis is present. Signs and symptoms of AHTR range from mild symptoms as fever, abdominal or flank pain and chills to overt hemolysis with hypotension, dyspnea, hemoglobinuria, disseminated intravascular coagulopathy (DIC), acute renal failure (ARF) and shock. Transfusion of more than 50 ml is associated with increased risk of severe morbidity and death (Janatpour KA et al, AM J Pathol; 2008). DHTR may be asymptomatic, present as weakness, fever with anemia or as overt hemolysis similar to that occurring in AHTR. Factors influencing the outcome of HTRs include antibody and antigen characteristics as well as patient age and comorbidities. Awareness, close observation of patients receiving transfusion, early recognition of a transfusion reaction, prompt cessation of transfusion and appropriate supportive care are essential to minimize morbidity and mortality. Investigation of HTRs should include all aspects of the transfusion chain – administrative, clinical and laboratory. Laboratory testing of hemolysis markers, direct anti-globulin test (DAT), repeat examination of pre and post transfusion samples (ABO, Rh, antibody screen, cross match) and further work

© 2014 The Author Vox Sanguinis © 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107 (Suppl. 1), 1–56

2 ISBT Academy Day

up according to the investigation results. In patients in whom the blood bank laboratory cannot identify a cause for hemolysis, other reasons for immune and non immune hemolysis should be sought such as polyaaglutination, infections, microangiopathic hemolytic anemia as well as causes relating to improper storage and administration of RBC. Recent insights into the pathogenesis of hemolysis including the major role of the complement system (Stowell SR et al: Clin Dev Immunol, 2012) the deleterious effects of cell free hemoglobin (Hyen Baek L et al, JCI 2012) and non transferrin bound iron (Hod EA et al, Blood 2011) might aid in developing new strategies for amelioration and treatment of HTRs. Prevention of HTRs should be a major goal. This necessitates multidisciplinary cooperation, root cause analysis of HTRs and appropriate education as well asimplementation of corrective actions.

Six Degrees of Blood Donation 1A-A02-01

YOUNG DONOR HEALTH AND SAFETY: ARE WE DOING ENOUGH? Steele WR American Red Cross, Rockville, MD, United States of America Background: In some countries, blood collection is permitted from donors as young as 16 years of age. Young donors (donors aged ≤22) represent a unique challenge for blood collection organizations as they must balance their need for donors against protecting the health and safety of this potentially vulnerable donor population. Aims: Explain the importance of young donors in ensuring the adequacy of the current and future blood supply. Discuss rates of adverse reactions in young blood donors and the different interventions being utilized by blood collection organizations to reduce them. Review what is known about the effect of iron deficiency on young blood donors and possible mitigation strategies. Briefly review the role of donor hemovigilance in monitoring the health and safety of young blood donors. Methods: Relevant research studies and review articles from the literature were reviewed to summarize what is currently known about the aims outlined above. Results: Young donors compose varying proportions of the blood supply in countries worldwide. In some countries young donors provide a significant portion of units collected. However, research has shown that young donors suffer disproportionally from adverse donor reactions. After this observation, many blood collection organizations launched interventions to mitigate these adverse reactions. The primary intervention has been to restrict blood donation in those donors who are likely to lose a significant portion of their total estimated blood volume (EBV). These EBV criteria have been applied to young donors up to age 22 and have been successful in reducing both presyncope and syncope. Other blood collectors have experimented with additional approaches to reducing reactions such as reducing the volume of blood taken from donors with lower EBV, administration of water prior to donation, applied muscle tension, increased donor education, reorganization of high school drive environments and changes in staff training. While no intervention has completely eliminated adverse donor reactions, progress has been made in reducing their occurrence. Recent research has demonstrated that many donors are iron deficient. This has led to an increased concern about how iron deficiency may be affecting young donors. The effects of iron depletion on young blood donors are not well understood, but could be significant as these donors are still undergoing neurological development and may suffer from cognitive impairment and fatigue that impacts their functional status. The paucity of data on the effects of iron deficiency in young donors leaves us with more questions than answers, but clearly indicates an area where more research is needed and interventions may eventually need to be applied. Conclusions: With increased reliance on young donors to provide a significant part of the blood supply, it is of the utmost importance that these donors have a good experience when they donate blood and do not suffer any adverse health and safety effects from their altruistic act. Adverse donor reactions and iron deficiency still pose threats to the health and safety of young donors. Continued research is needed in both of these areas to adequately protect young donors.


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USING INFORMATION TECHNOLOGY TO IMPROVE DONOR RETENTION AND MANAGEMENT Kim JN1, Kwon SY2, Kim CY1, Lee MJ1, Kim MH1 and Cho NS1 1

Korean Red Cross Blood Service Headquarters, Seoul, South-Korea 2Blood Transfusion Research Institute, Korean Red Cross, Seoul, South-Korea Background: Korean Red Cross Blood Services (KRCBS) has developed ‘Blood Information Management System (BIMS)’ in 2003. BIMS is the core of all processes of the blood service, managing all data generated from donor recruitment to blood issue at a nationwide level. Since its implementation, it is continuously upgraded to meet current needs. Besides BIMS, we have also implemented and modified various information technology (IT) tools for our blood service. With a well established infrastructure of digital network in South Korea, the application of IT has played an important role in improving donor management and blood safety. Aim: To introduce IT tools of KRCBS applied for donor management and blood safety. Methods and results: BIMS consists of several sections covering donor recruitment, donor management and collection, laboratory testing, blood processing, blood issue, and statistics. In the donor recruitment section, recruitment plans for each group donation can be stored to be used for the next time. In the donor management and collection part, data about donor’s donation history, adverse reactions, and self-deferral can be retrieved and used for donor management on-site. For example, information of every reported adverse reaction is collected in BIMS. By monitoring and analyzing these data including the location, time, type and severity, counseling history, and progress of medical care, it is possible to manage each donor more carefully thereby improving the donor safety management at a more comprehensive level. For better donor experience which directly affects donor retention, features of the IT tool, quality of information provided and accessibility are considered most important. To make use of the high smartphone penetration and wireless internet access rate, we have developed ‘Smart Blood Donation’, a mobile device application, for donors. Using this application, donors can conveniently access the services that had been provided only on KRCBS web site including the electronic donor history questionnaire and the search for the nearest fixed donation site from present location. This application also supplies information about nationwide blood inventory status and urgent donation request for rare blood types. Several IT tools are synced for the communication with donors and provide donorspecific information for each donor. On the KRCBS web site, a donor can find his/ her donation history such as type of donation, screening test results and the date for the next donation. One of the distinctive features of our IT system is the notification service for mobile donations scheduled near a donor’s predefined area. We also use SMS/LMS to reassure donor safety, by repeatedly sending post-donation instructions, that had been also provided before the donation, to donors after the donation session. Conclusions: Expanding the use of IT improves donor satisfaction by providing information to donors in a more convenient and accessible way, which is expected to have a positive effect on donor retention. By managing donor data in real-time at a nationwide level, it also contributes to safety of both donor and the blood product.

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OPTIMISING THE APHERESIS DONATION Vrielink H Sanquin Blood Supply, Amsterdam, The Netherlands In blood banking processes, we would like to achieve an effective and efficient way to provide quality products. In general, quality can be defined as ‘how good and how bad something is’. Translating this to the blood bank world, you would like to have a methodology to achieve quality blood components for patient care from healthy and content donors within an economically feasible way. Blood components can be collected by whole blood and by apheresis donations. In apheresis techniques, the whole blood is separated in plasma and various blood components during the collection procedure. The requested blood component is stored in a collection bag and the remaining blood components are returned to the donor. When solely plasma is collected, this is named plasmapheresis, and if a cellular blood component is collected, cytapheresis, more specifically when platelets are collected, plateletapheresis. If two or more different blood components, for instance red blood cells and platelets, in one apheresis procedure are collected, this is named a multicomponent apheresis procedure. So in one apheresis procedure, one or more complete units of blood components complying with the (inter)national specifications and ready for transfusion can be collected from one donor.

What’s in Your Immunohaematology Toolbox? 3

Basically every donor who is suitable for whole blood donation can also be suitable for plasmapheresis or cytapheresis. However by careful selection of donors, the blood bank can achieve components with a better ‘quality’ for patient, donor and organization. By selecting for instance never transfused male donors for plasma and platelet donations to avoid HLA and/or HNA antibodies, the frequency of TRALI in transfused patients is significantly reduced. This collection process can be simplified by using apheresis techniques. For logistic reasons, a shorter procedure time can be needed. To achieve this, the draw and return speed of the apheresis machine can be increased, but this can be at the expense of the quality for the donor because of an increased chance of adverse events, and possibly also of the quality of the collected blood component. Easier is selection of donors based on ABO blood type (preferably compatible with the patient’s blood type) and platelet count of the donor. When the donor’s platelet count is ≥250,000/ll, within a reasonable time even two or more complete units of platelets can be achieved. This also makes the use of the rather expensive apheresis disposables more feasible for economical reasons. In case not that much cellular blood components from donors with AB0 blood type AB or B are needed, these donors can be invited for plasmapheresis. As known, plasma with blood type AB is considered as universal plasma, and therefore very wanted. Also, male donors weighing >70 kg with blood group 0 RhD negative and hemoglobin values >140 g/l can be invited for an apheresis procedure to collect two units of red blood cells. In summary, by careful selection of donors applying (multi)component apheresis, the process can be adjusted to the needs of the stock of blood transfusion services, to the donor’s and patient’s needs for achieving quality products.

What’s in Your Immunohaematology Toolbox? 1B-A03-01

SEROLOGICAL TOOLS FOR INVESTIGATING IMMUNOHAEMATOLOGY PROBLEMS Thornton NM IBGRL, Bristol, United Kingdom Antibody identification and the resolution of red blood cell phenotype discrepancies are fundamental to the practice of immunohaematology. Although the molecular era is undoubtedly upon us, serological techniques remain the essential tools required for problem-solving in the transfusion laboratory. Reading and interpreting haemagglutination reactions is the primary technique which underpins most serological testing, both manual and automated. Direct agglutination tests, the indirect antiglobulin test (IAT) and the use of cells treated with proteases such as papain or ficin make up the standard battery of tests employed in the routine immunohaematology laboratory and in most cases these tests alone are sufficient to give the information needed to make clinical decisions regarding suitable blood for transfusion. When the use of standard serological techniques are insufficient to resolve an immunohaematology problem we must turn to the additional serological tools at our disposal, which include: testing over a wide thermal range, adsorption and elution techniques, the use of cells treated with a range of enzymes and chemicals, inhibitory substances such as plasma (e.g. Ch/Rg) or soluble recombinant proteins, artificially C4 coated cells, rare cells and serum and the MAIEA assay. Whilst these supplementary serological tools are undeniably very useful, knowledge of blood groups, antibody characteristics and fundamental basic serological methods is essential for choosing the appropriate tests in a stepwise and efficient manner. In addition, there are now a variety of molecular based techniques used in the immunohaematology laboratory and it is important that serologists also grasp the basics of blood group genetics in order to utilise the molecular testing available to complement the serological testing repertoire. The overall goal is to minimise delay in patient care, therefore a systematic approach is required when investigating immunohaematology problems to ensure the necessary tools are used effectively and efficiently, to achieve a timely resolution.

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MOLECULAR TOOLS FOR INVESTIGATING IMMUNOHAEMATOLOGY PROBLEMS Peyrard T Institut National de la Transfusion Sanguine (INTS), Paris, France The characterization of the genes encoding the 33 officially recognized blood group systems by the International Society of Blood Transfusion (ISBT) and knowledge of most molecular bases of RBC antigens and phenotypes have made possible the use of molecular testing to predict blood types. Many molecular mechanisms are involved in the genetic basis of blood group diversity and the so-called single nucleotide polymorphism (SNP) is the most common. Blood types are classically determined by haemagglutination methods, which have proven to be the gold standard for over one century. However, several backgrounds represent limitations for serological techniques, which may be overcome by molecular testing: blood typing in recently transfused patients; distinction between auto and alloantibodies; short supply, poor quality or unavailable antisera; patients with a positive IgG direct antiglobulin test (when indirect antiglobulin testing is required for antisera); distinction of partial and weak phenotypes; phenotype discrepancies, etc. Several molecular tools are now available and can be separated in five groups: lowthroughput tests, medium-throughput assays, standard genomic DNA sequencing, high-throughput DNA sequencing devices and other specialized techniques. The low-throughput tests include PCR-restriction fragment length polymorphism (RFLP), allele-specific PCR (single or multiplex assay) and real-time PCR (hybridization or hydrolysis probes). They can be used for RBC genotyping, study of RHD zygosity and cell-free fetal DNA genotyping in maternal plasma. The medium-throughput systems, especially microarray DNA chips (bead-array, glass-array, liquid-array, nanofluidic-array, dynamic-array) and mass spectrometry (MALDI-TOF/MS technology), allow for several dozen different SNPs to be simultaneously and rapidly tested. Standard genomic DNA sequencing is based on the classical Sanger technique, after exon-specific primer amplification (usually including flanking intron regions). It may be used when genotyping assays are unavailable or when screening for common mutations by medium-throughput devices is inconclusive (e.g. RHD and RHCE, with hundreds of reported alleles). The high-throughput systems, so-called ‘next generation’ or ‘massive parallel’ sequencing technologies are able to analyze 10 Mb to 1000 Gb per run, with a turnaround time between 4 h and about 10 days. Their implementation in transfusion medicine is currently developed by a few teams worldwide. Assuming a significant decrease of cost per test, these platforms may become in the next future a major investigating tool in immunohaematology reference laboratories. Specialized techniques, reserved for complex cases or research, are performed after messenger RNA extraction, followed by reverse transcription into complementary DNA (cDNA). cDNA may be directly sequenced or cloned with plasmid vectors. Cloning is a valuable technique for single-allele sequencing and to know whether mutations are located in cis or trans. Finally, electronic databases are helpful tools in molecular immunohaematology, for example the resources from the National Center for Biotechnology Information (NCBI) (Blood Group Antigen Gene Mutation Database/dBRC, GenBank, RefSeqGene, Single Nucleotide Polymorphism Database/dbSNP), Online Mendelian Inheritance in Man (OMIM), ISBT Website of the Working Party on Red Cell Immunogenetics and Blood Group Terminology and Rhesus Base. Despite the few limitations of DNA analysis, molecular testing has gradually become an essential adjunct tool to serology and can now be considered full part of the immunohaematology field.

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DETERMINING THE CLINICAL SIGNIFICANCE OF ALLOANTIBODIES Meny GM University of Texas Health Science Center at San Antonio, San Antonio, TX, United States of America The detection and identification of red cell alloantibodies are important processes familiar to all laboratory professionals. A term frequently utilized to describe an alloantibody likely to cause an adverse event (such as a hemolytic transfusion reaction or hemolytic disease of the fetus and newborn) is ‘clinically significant’. The


4 ISBT Academy Day

laboratory usually assesses the clinical significance of alloantibodies based upon specificity, serologic phases of reactivity and crossmatch results and make transfusion recommendations accordingly. However, using specificity, reactivity patterns and crossmatch results alone cannot always distinguish between clinically significant and nonsignificant alloantibodies. Costly, time-consuming searches for ‘compatible’ blood can occur when alloantibodies are identified and ‘incompatible’ crossmatches occur, leading to inappropriate delays in transfusion and potentially causing patient harm. Bioassays, such as the monocyte monolayer assay (MMA), can be used as an adjunct to predict transfusion outcome. Current routine serology assays will not predict with absolute certainty whether ‘incompatible’ red cells transfused to a recipient with an alloantibody will be destroyed or whether that recipient will suffer the devastating clinical consequences of a hemolytic transfusion reaction. The MMA, a cell based in vitro assay, can be of particular assistance when the antibody specificity is of ‘variable’ clinical significance (e.g. anti-Yta, -Ge) or the antibody specificity is to a high incidence antigen (e.g. anti-Joa, -Vel, -Lub) and rare blood is requested. A positive MMA indicates the recipient should receive antigen negative blood. A negative MMA indicates that serologically ‘incompatible’ blood (not rare antigen negative blood) can be administered without clinical or laboratory evidence of a hemolytic transfusion reaction. A positive MMA does not need to be repeated. However, when a negative MMA is obtained, it is recommended that a repeat MMA be performed as close as possible to each subsequent transfusion due to potential change in antibody composition. Finally, it is important to remember that laboratory professionals will be called upon for advice and information by clinicians and patients regarding transfusion recommendations. Communication gaps exist due to differing degrees of specialization. We are all partners working together to determine to the optimal transfusion option. And we must reconfirm to all that transfusion should never be withheld from a patient whose life depends on urgent transfusion.

or safety. These are called critical control points and for the donation process, include: Selection of blood donors. Collection of the donation. Handling and storage of the donation. Transport of the donation to the processing centre. A number of the developments are new technologies that automate the critical control points as far as possible, such as decision support systems to improve the consistency of interpretation of donor selection guidelines, vein viewer systems that improve the venepuncture process and transport containers that can control temperatures. There have also been developments of internationally accepted component standards and principles of good manufacturing practices that provide the framework for quality in blood establishments and have been able to drive significant improvements in processes and component quality. In combination, these recent developments can provide a high degree of assurance that the starting point for the manufacture of safe and efficacious blood components will be a quality donation.

1B-A04-02 Abstract not available.

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MAKING THE RIGHT CLINICAL DECISIONS IN COMPONENT THERAPY

Getting the Most Out of Blood Components 1B-A04-01

QUALITY IN BLOOD COMPONENTS – IT ALL STARTS WITH THE DONATION Armstrong VA Australian Red Cross Blood Service, Perth, WA, Australia Blood transfusion is not risk free, so should only be prescribed when it is in the patient’s best interest to do so. Blood establishments have a recognised obligation to promote optimal use of blood and blood components while minimising risk to the health of the recipient. Processes are therefore focused on producing quality components that are efficacious and safe. In manufacturing processes, the quality of final products is heavily dependent on the quality and reliability of the starting materials, and therefore control of these materials to reduce variation is a critical quality assurance activity. The starting material or primary product for the manufacture of blood components is the blood donation. If the quality of that donation is not assured, the efficacy and safety of the blood components manufactured from it will be potentially compromised no matter how good the rest of the manufacturing process may be. The challenge, however, is that blood is a biological material and collection is subject to a number of variations: Donations are collected from donors of various ages with different demographics, health profiles and behaviours. The donations have different characteristics, for example volume of donation, numbers of platelets, levels of immunoglobulin, blood group antibodies etc. The collection process depends largely on manual procedures with the exception of apheresis collections. Donations may be collected, stored and transported under varying environmental conditions. Because of these variations, controlling the quality and consistency of the primary blood donation can be more challenging than controlling starting material for an industry such as pharmaceutical manufacture. In recent years, there have been a number of developments aimed at improving the quality of the blood donation. These have largely focused on controlling and reducing variations in the process steps that are most likely to have an impact on quality


Allard S NHS Blood & Transplant, London, United Kingdom Blood transfusion may be essential in the management of various diseases where it can be lifesaving. However, donated blood is a limited resource and clinical transfusion practice must focus on ensuring safe and appropriate use underpinned by evidence based guidelines and supported by education and training with regular audit of practice. The decision to transfuse requires a careful assessment of the patient’s clinical status and is not just dependent on laboratory parameters such as the haemoglobin level, platelet count or clotting screen. The risks and benefits of component therapy must be assessed together with the availability of transfusion alternatives. The patient should be informed with appropriate documentation in the clinical records. Prospective randomised controlled studies in key clinical areas such as intensive care, hip fracture surgery, cardiac surgery and acute gastrointestinal haemorrhage have demonstrated that a restrictive red cell transfusion strategy is associated with at least equivalent or possibly better clinical outcomes then more liberal transfusion. Accordingly a threshold for red cell transfusion of 70–80 g/l haemoglobin is appropriate in the majority of patients but pending further studies, a higher threshold of 80–90 g/l is indicated in patients with acute coronary syndromes. Platelet transfusions are indicated for the prevention and treatment of haemorrhage in patients with thrombocytopenia or platelet function defects. The decision to transfuse platelets should take into account clinical risk factors for bleeding and the extent and site of bleeding together with the platelet count. Fresh frozen plasma (FFP) is used to replace labile plasma coagulation factors in the presence of bleeding and abnormal coagulation. Wherever possible prothrombin complex concentrates rather than FFP should be used in patients on oral anticoagulants where urgent reversal is indicated. The management of major haemorrhage in any setting provides particular challenges and requires a multidisciplinary approach. There have been significant advances in techniques for resuscitation as well as surgical, radiological and endoscopy interventions to control bleeding. The timely provision of blood component therapy can improve outcomes but requires good communication and effective team work between clinical disciplines and the laboratory staff. The CRASH-2 study has demonstrated the benefits of early use of antifibrinolytics. There is an emphasis on early use of fresh frozen plasma and an increasing focus on prompt correction of hypofibrinigenaemia but further prospective studies are needed. The prompt availability of laboratory results or near patient coagulation testing can help with appropriate clinical decision making around component therapy but are often lacking in this setting. There continues to be considerable inappropriate use of blood and components as shown in various audits despite the increasing evidence base and the development of numerous guidelines for transfusion. This warrants an increased focus on strate-

Facing Up To Transfusion Transmitted Infectious Diseases 5

gies to improve practice including the use of information technology to support the transfusion process. There is a collective need to implement the principles of Patient Blood Management (PBM) with an evidence based multidisciplinary approach to optimising the care of all patients who might need transfusion with the timely use of alternatives wherever available.

Facing Up To Transfusion Transmitted Infectious Diseases 1C-A05-01

SELECTING LOW RISK DONORS – GETTING THE RIGHT BALANCE BETWEEN SAFETY AND SUFFICIENCY Lin CK Hong Kong Red Cross Blood Transfusion Service, Hong Kong, Hong Kong The primary responsibility of a blood transfusion service (BTS) is to provide a safe, sufficient and timely supply of blood and blood products. In fulfilling this responsibility, the BTS should ensure that the act of blood donation is safe and causes no harm to the donors. It should build and maintain a pool of safe, voluntary nonremunerated blood donors and take all necessary steps to ensure that the products derived from donated blood are efficacious for the recipient, with a minimal risk of any infection that could be transmitted through transfusion. All prospective blood donors should therefore be assessed for their suitability to donate blood, on each occasion of donation. Although the chief purpose of blood donor selection is to protect both donor and patient safety, we should all be reminded that no donors should be unnecessarily deferred if sufficiency of blood supply is to be maintained. It is therefore important for each country to establish its own national donor selection system to ensure the collection of donations from the lowest risk donors possible, with consideration given to the issue of sufficiency, balancing any risk of infection against the risk of blood shortages resulting from too stringent selection. Donors should be in good health at the time of donation and free of infections transmissible by blood. The BTS should provide clear and unambiguous guidance for staff involved in donor selection. Rigorous donor selection should be consistently applied to all blood donors either donating whole blood or through apheresis, whether first-time or repeat donors. The process should be planned to make best use of staff and donor time, and make blood donation as convenient as possible for blood donors, without long waiting periods. Key principles of blood donor selection are as follows: Health and safety of the donor as well as the recipient must be safeguarded. Only individuals in good health should be accepted as donors. Selection of blood donors should be based on regularly reviewed selection criteria. A prospective donor’s health status and medical history should be evaluated for each donation, on the day of donation prior to blood collection. Appropriate information should be provided to blood donors. Staff should be suitably qualified and trained in the donor selection process. Counselling of deferred donors. In summary, an adequate and safe national blood supply depends on the availability of a suitable pool of healthy volunteer blood donors who are well informed of the purpose and process of donor selection and are able to meet the criteria. A rigorous and balanced process to assess the suitability of prospective donors is essential to protect the safety and sufficiency of the blood supply, and safeguard the health of recipients of transfusion and blood donors themselves, while ensuring that suitable donors are not deferred unnecessarily.

1C-A05-02

PROGRESS IN INFECTIOUS DISEASE TESTING – NAT AND BEYOND Stramer SL and Dodd RY American Red Cross, Gaithersburg, MD, United States of America Aims: To update membership on state-of-the-art testing technologies for infectious disease blood donation screening including new methods such as expanding NAT

platforms, and nucleic acid and protein microarrays. Changes to testing algorithms with the availability of inactivation by pathogen reduction will also be highlighted. Background: Testing donated blood for markers of infectious disease plays a major role in establishing and maintaining blood safety. Minimal expectations worldwide are that blood is tested for syphilis, HIV, HCV and HBV. The norm for viral testing has been serology, but increasingly, nucleic acid testing is being implemented where resources allow, even for emerging infectious disease (EID) agents such as the chikungunya virus outbreak that started in Dec 2013 in the Caribbean, marking the first introduction of this virus in the Americas. The appropriate mix of tests and algorithms will depend on local epidemiology, infrastructure and economic considerations. Although NAT will detect acute infections, representing the greatest infectious threat, not all pathogens have adequate concentrations of nucleic acid for detection in the early acute or later chronic phases of infection (if chronicity is a feature of infection). Thus, for many agents, either serology only or a combination of serology and NAT exist. In developing test strategies, it is important to understand the actual risk of transfusion transmission and the impact of the test systems on such risk, especially where resources are limited. Methods: NAT has been implemented in over 30 nations worldwide as documented by an International Forum organized by the ISBT Transfusion-Transmitted Infectious Disease Working Party that captured implementation from 1999 to 2009 for HCV, HIV and HBV (Roth et al., Vox Sang 2012;102:82–90). Testing has progressed from manual technologies using single markers to highly multiplexed automated assays. Routine NAT has been added, either as a required marker or under investigational studies for additional agents including, parvovirus B19, HAV, HEV, arboviruses including WNV, dengue, chikungunya and Zika virus, the red-cell parasite, Babesia, and likely others. In addition to NAT, new technologies including pathogen reduction, and nucleic acid and protein microarrays will have an important impact on testing. Recently, the AABB EID working group has updated a listing of pathogen reduction technologies available including over 40 clinical trials in support of their safety and efficacy (http://www.aabb.org/resources/bct/eid/Pages/eidpostpub.aspx). The impact is either to prevent the need to introduce new tests, assuming availability of the technology to inactivate the component in which the agent is transmitted, and that the technology has sufficient robustness to produce an effective ‘kill’. As an additional benefit, changes to existing testing algorithms may be feasible such that redundant tests may be eliminated. With respect to nucleic acid and protein microarrays, technological advances will allow the simultaneous detection and differentiation of hundreds of pathogens, but many hurdles prior to routine adoption exist. Conclusions: Blood systems worldwide must be ready to adapt to changes in their infectious disease epidemiology, emergence of new agents, and changes in their economic conditions and public expectations to accommodate the changing landscape of infectious disease testing. 1C-A05-03

ASSESSING INFECTIOUS THREATS – TRICK OR THREAT? Seed CR and Kiely P Australian Red Cross Blood Service, Perth, WA, Australia Background: Effective surveillance for, and response to, emerging infectious disease (EID) agents is a pivotal component of blood safety risk management policy for blood services. The residual risk of transmission associated with classical infectious disease agents for which donor screening has been implemented, including HIV, has never been lower. However, the rate of emergence of new potential agents is increasing which demands a robust risk management strategy for rapidly assessing and ranking potential threats. Aim: To discuss the general principles of assessing the threat of EID agents and define possible ranking criteria. Methods: Various schema for the management of the risk posed by EID agents have been proposed, usually based on the ‘precautionary principle’ which prescribes action for ‘serious’ threats even in the absence of an adequate understanding of the pathogen. Examples are the AABB ‘toolkit’ for EID which includes a framework for recognition, assessment and management of EID agents and the Australian Red Cross Blood Service EID management strategy developed in collaboration with the industry regulator and national fractionator. Both systems incorporate EID agent ‘templates’ which consolidate key information about the agent, particularly from a blood safety perspective, to assist with ranking the threat level and formulating an appropriate response. Results: Threats are primarily assessed based on two main criteria; first, transmissibility by blood, and second, the severity of clinical disease in recipients. Where the threat is deemed significant, quantitative risk assessment and cost-effectiveness analysis for potential interventions can assist in defining the most appropriate mitigation strategy. Importantly, threat assessment is a dynamic process as the emergence of EIDs is unpredictable. Hepatitis E virus (HEV) is an excellent contemporary


6 ISBT Academy Day

case study with transmissibility now proven but further studies required to define the extent of clinically significant disease in immunocompetent recipients. Conclusions: The emergence of vCJD in the UK and subsequently West Nile virus in North America highlighted the unpredictable nature of EIDs and underscored the importance of an effective EID risk management framework. However, not all EIDs necessarily pose a significant threat to blood safety and therefore an effective ranking system is required which we suggest should primarily assess prevalence, transmissibility and recipient disease outcome.

ommendation and quality of published evidence. Those evidence based guidelines present this information in a standardized format – one page fact sheet – which allow for concise but yet comprehensive data to be presented. The references used to make those guidelines are also included. The sixth edition of those guidelines was recently published. This session will also describe how the guidelines for the most recent edition were created and how can they be used in making the decision to treat or not with Therapeutic Apheresis and if to treat- how to treat the patient right while protecting the patient’s safety and ensure the quality of Therapeutic Apheresis delivered.

Treating the Patient Right

1C-A06-03

1C-A06-01

APPROACH TO MANAGING PLATELET REFRACTORY PATIENTS

IMPROVING CLINICAL PRACTICE THROUGH THE USE OF RED CELL TRANSFUSION GUIDELINES Carson JL and Czaja M Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ, United States of America Guidelines are being developed widely in all fields of medicine. The purpose of guidelines is to provide recommendations designed to optimize patient care. The Guidelines International Network database includes over 3700 guidelines. Thus, it is not surprising that it is hard to know which guideline is reliable and high quality. The US Institute of Medicine has proposed standards to produce high quality guidelines. These principles were used to develop AABB guidelines that were recently published. The AABB guideline committee had representatives from the transfusion community, surgery, hematology, anesthesia, cardiology, and others. The guidelines were based on a systematic review of the literature, which focused on clinical trials. The quality of literature was evaluated using the GRADE methodology. Four questions were posed. (i) In hospitalized, hemodynamically stable patients, at what hemoglobin concentration should a decision to transfuse RBCs be considered? (ii) In hospitalized, hemodynamically stable patients with preexisting cardiovascular disease, at what hemoglobin concentration should a decision to transfuse RBCs be considered? (iii) In hospitalized, hemodynamically stable patients with the acute coronary syndrome, at what hemoglobin concentration should an RBC transfusion be considered? (iv) In hospitalized, hemodynamically stable patients, should transfusion be guided by symptoms rather than hemoglobin concentration? A restrictive transfusion strategy was recommended in most patients except those with acute coronary syndrome where adequate is not available. The guidelines also emphasized the other limitations of our knowledge especially in high risk patients which have not be evaluated in clinical trials such as traumatic brain injury, and transfusion dependent patients. It is important that guidelines identify gaps in knowledge and not extend recommendations when the optimal treatment is unknown. Guidelines have the potential to improve patient care if they are carefully developed based on high quality evidence and applied to the appropriate patients.

1C-A06-02

IMPROVING THE QUALITY AND SAFETY OF THERAPEUTIC APHERESIS Schwartz J Columbia University Medical Center, New York, NY, United States of America To treat or not to treat – this question arise many times when a request for Therapeutic Apheresis is received at the Apheresis service. This is due to the fact that the decision of treating patient with Therapeutic Apheresis depends upon multiple factors that need to be taken into account. Some of those include: is the disease or the condition of the patient amenable to treatment with Therapeutic Apheresis and if so, what are the risks vs the benefits of treating the patient with this modality. In this session some of these factors will be discussed using clinical case scenarios. In addition, the American Society for Apheresis (ASFA) has started to publish in 1986 systematic reviews of Therapeutic Apheresis applications which were revised every 7 years. Those reviews, also termed and known as the Special Issue, are also addressing those dilemmas and helps in clinical decision making. Since 2007, the Special Issue has been restructured to use an evidence-based approach, as well as staying current by being updated every 3 years. In this guidelines structure, each disease, including specific clinical and condition presentations within the disease, are categorized for the role of Therapeutic Apheresis and graded for strength of rec-


Ayob Y National Blood Centre, Kuala Lumpur, Malaysia Platelet transfusion can prevent and treat hemorrhage in patients with thrombocytopenia or platelet dysfunction. However in some patients the expected result is not achieved, leading to an increased risk of morbidity and mortality. When there is repeated failure to obtain satisfactory response to platelet transfusion, platelet refractoriness is said to have occurred. Schiffer et al recommended that the diagnosis of refractoriness should be made when at least on two sequential occasions, a 10-min to 1-h post-transfusion, the CCI is A (p.65 Gly>Asp). The fifth sample carried the JK*02N.09 allele with c.191G>A (p.64 Arg>Gln). Of the 2 samples with Jka typing discrepancies, one carried the JK*01W.01 allele with c.130G>A (p.44 Glu>Lys) that is associated with weakened expression of Jka. The other carried a novel JK*01 allele with c.814C>T (p.272 Leu>Phe). The SNP in exon 9 likely caused allele dropout due to failure to amplify the PCR product or hybridize the probe that is necessary to detect the c.838G/A polymorphism responsible for Jka and Jkb. Discussion: HEA genotyping panels are used routinely in many blood centers and in large hospitals to predict the blood group antigen status of donors and patients. This study describes the investigation of discrepancies between genotype-predicted phenotype and serologic phenotype in the Kidd system. Though such discrepancies are rare (7 JK discrepancies reported in >60,000 samples), they demonstrate the limitations of SNP genotyping. This work also highlights the ability of additional molecular testing including DNA sequence analysis to resolve the discrepancies.

4A-S25-03

DUFFY BLOOD GROUP TYPING: IS GENOTYPE MORE ACCURATE THAN PHENOTYPE FOR DUFFY BLOOD GROUPING? Hyland CA, Lopez G, McBean R, Liew YW, Condon J and Flower RL Australian Red Cross Blood Service, Brisbane, Qld, Australia Background: Duffy blood group genotype/phenotype discrepancies are frequently observed, exemplified by reports for reagent red cell donor panels. These discrepancies frequently arise from the FY*02W.01 allele that encodes a weak Fyb antigen, Fybw. Genetic diversity in the Duffy system has generated both the erythroid null FY*02N.01 type, observed in 68% of Africans and less frequent ‘true null’ types. In this study we report a review of typing procedures for specimens requiring complex analysis, and have identified protocols for correct assignment in all of these cases. Aims: To study samples with Duffy genotype/phenotype discrepancies to evaluate Duffy testing algorithms. Methods: Red cells were phenotyped in a Reference Laboratory and genomic DNA analysed for Duffy blood group polymorphisms by high-resolution melt PCR assays and a SNP array platform (BLOODchip Reference v4.0, Progenika). Discrepancies between the predicted and the observed phenotype were followed up by Sanger sequencing. The following groups of samples were tested: 1. Samples (n = 155) referred and phenotyped as: a. Fy(a + b) (n = 21); Fy(ab+) (n = 31); and Fy(a + b+) (n = 27). b. Fyb+w (n = 13) or Fyb reactivity unresolved (n = 16). c. Fy(ab) (n = 47), including one with anti-Fy3. 2. Random donor samples (n = 100) including Fy(a+b) (n = 19). 3. A sample exhibiting Fy(a + b+) phenotype with reduced Fya expression (n = 1). Results: In this study seven cases with complex genotype/phenotype discrepancies were further analysed. Three predictions for Fybw were discordant with an Fyb negative serotype: group 1a (1/21), 1c (1/47) and group 2 (1/19). In group 1b, genotyping clarified one additional case, as Fy(a + b)(1/16). Three predictions for Fy(a + b) were discordant with observed Fy(ab) in group 1c. Sequencing showed that one patient, who had formed an anti-Fy3, was homozygous for FY*01N.02 with a 14 bp deletion, a ‘true null’ phenotype. Two were heterozygous for the FY*A and FY*02N.01: One carried the FY*A null FY*01N.04 (287G>A) and one carried a novel FY*A allele with c.719delG (GenBank accession number KC924823). This deletion is predicted to produce a termination codon after Gly242. One sample, Group 3, exhibited a variance between predicted Fy(a + b+w) phenotype and observed Fy(a + wb+). Sequencing defined a novel FY*A allele carrying both the 265T and 298A variants (GenBank accession number KF784871; provisional allelic name FY*01W.02).


38 Orals

Conclusion: In this study, serology failed for 5% of Fy(a + b) samples in which Fyb+w was encoded by the FY*02W.01 allele. For three cases additional genotyping was required because SNP analysis for the FY*02N.01 GATA box mutation did not provide a correct type, and sequencing was required to identify the silent FY*A alleles. In the final case, variation between genotype and phenotype revealed a FY*A allele with the 265T and 298A variants, anticipated by early researchers to be a weak form of FY*A as ‘FY*X’ is a weak form of FY*B. Both this allele and the silent FY*A alleles should be considered in refinement of genotyping algorithms for extended blood group typing and a range of genotyping procedures are required to complement serology in Duffy typing.

4A-S25-04

CAN CHIMERISM CAUSE FALSE POSITIVE RESULTS IN NONINVASIVE BLOOD GROUP GENOTYPING? van der Schoot CE1, Thurik FF1, Ait Soussan A1, Woortmeijer H1, Page-Christiaens GCML2 and de Haas M1 1

Sanquin, Amsterdam, The Netherlands 2UMCU, Utrecht, The Netherlands

Noninvasive prenatal blood group typing is increasingly used, but the extent of interference of placental microchimerism is presently unknown. Objectives: To analyze the frequency of placental chimerism causing false-positive fetal RHD typing results. Method: Fetal RHD screening was performed in the 27th week of gestation by multiplex real-time-PCR in triplicate for RHD exon 5 respectively 7 using cell-free DNA isolated from maternal plasma. PCR results were compared to cord blood (CB) serology and false-positive samples were analyzed. The presence of RHD variant genes was analyzed on DNA from maternal leukocytes and CB. In false-positive samples with ≥5 out of 6 positive Ct-values multiplex STR-PCR on 17 highly polymorphic systems was performed on maternal blood and CB to verify that the cord blood was obtained from the same pregnancy as the maternal blood. When back-up plasma was available RHD-PCR was repeated alongside a DYS14 (Y-chromosome specific)-PCR. Results: CB serology was available in 25,789 women (80.04%) in whom fetal RHD typing had been performed. Comparison of RHD typing and CB-serology revealed 225 false-positive fetal RHD typing results (0.87%). In 100 cases a variant RHD gene explained the discrepancy, in 4 cases there was maternal blood contamination in the CB sample, 71 cases were reported positive with 3–4 positive replicates, because the scoring-algorithm aims to prevent false-negativity. In the remaining 50 samples ≥5 amplifications in the RHD-PCR were seen and these samples were further investigated for possible causes of these false-positive results. STR-PCR could be done in 23/50 cases, and showed that in five cases maternal blood instead of CB had been sent in and in 1 case no mother-child relationship was found. In 28/44 remaining cases back-up plasma was available and tested with RHD and DYS14 PCRs. RHD was again positive in only 14 samples. In eight of these cases also the DYS14 PCR was positive. In seven of those cases CB was available and in four no Y-chromosome sequences were present in CB. Contamination with material from RhD-positive males is unlikely, since RHD-PCR was performed on two separate DNA isolations from samples derived from series of only pregnant women. Conclusions: These data are strongly indicative for placental chimerism as a cause of false positive results of non invasive fetal D testing. An unrecognized or unreported early demise of a co-twin is the most likely explanation for such chimerism. Although unlikely, we did not rule out the possibility of contamination yet. Furthermore, maternal chimerism either caused by organ transplantation or naturally occurring cannot be excluded. Naturally occurring causes of maternal chimerism can be either of previous pregnancies or due to mother’s own vanishing twin or circulating maternal cells of her own mother. These latter causes are less likely, because of the extent of this phenomenon and we do not encounter any false-positive results in non-pregnant women. If all reproducibly false-positive RHD cases are caused by a vanishing twin, and RHD allele frequencies are taken into account, the estimated frequency of placental chimerism due to a vanishing twin would be about 0.5%.


4A-S25-05

TECHNICAL PERFORMANCE OF THE FULLY AUTOMATED FETAL RHD SCREENING PROGRAM IN THE NETHERLANDS Thurik FF1, Ait Soussan A1, Woortmeijer H2, Veldhuisen B1, van der Schoot CE1 and de Haas M2 1

Sanquin Research, Amsterdam, The Netherlands 2Sanquin Diagnostics, Amsterdam, The Netherlands Background: A nation-wide fetal RHD screening program (circa 27,000 RhD-negative women/year) has been implemented in the Netherlands since July 2011 to restrict prophylactic anti-D immunoglobulin administration to RhD-negative pregnant women carrying an RhD-positive child. Aims: To evaluate the technical performance of the fully automated fetal RHD screening program performed in the 27th gestational week Methods: Tests are performed at Sanquin Amsterdam. Multiplex real-time PCR is performed for RHD exon 5 and exon 7 (Applied Biosystems) in triplicate using cellfree fetal DNA isolated from 1 ml of maternal plasma (48 samples tested simultaneously in a run). Plasma separation (Xiril AG, Hombrechtikon, Switzerland), DNA isolation (MagNa Pure 96 Instrument, Roche Holding AG, Basel, Switzerland), and PCR set-up (Xiril AG) were robotized. Pooled plasma from RhD-negative pregnant women served as internal standard to judge test sensitivity. Predictions generated by a computer algorithm (i.e. ‘positive’, ‘negative’ or ‘no result, repeat’) were judged daily and could be overruled. Here we report on the technical performance during the first 15 months of the program. PCR results were compared to serological cord blood (CB) typing. Results: Fetal RHD typing was performed on 32,222 plasma samples. These were analyzed in 883 DNA isolation runs; 21 runs (2.4%) had to be repeated because of technical failures of the pipetting robots (n = 7) or because internal standards were out of range (n = 14). We received CB samples in 25,789 pregnancies (Table 1). In one case a negative test result was overruled at discretion of the supervisor, and reported as positive. However, CB serology typing was RhD-negative. In one case a positive test result was overruled at discretion of the supervisor, and reported as negative. CB serology confirmed an RhD-negative phenotype. In 248 (0.96%) cases results were issued after the assay was repeated (in five cases after a new blood sample was requested), because the initial result did not allow a conclusion by the computed algorithm (n = 143) or because the supervisor decided to repeat (n = 105). In 24 of the latter 105 cases this led to a different PCR result which prevented one false-negative result at the expense of 20 more false-positive results. Overall, there was clear discrimination between positive and negative results in 99.27% of the negative results no or a single amplification signal was obtained and in 98.37% of the positive results five or six positive signals were observed. There was a 99.09% concurrence (25,555/25,789; 95% CI 98.97%-99.20%) between reported fetal RHD screening results and CB serology results. In this period nine false-negative results and 225 false-positive results were obtained. Summary/Conclusions: This report shows that high-throughput fetal RHD typing with a fully automated process is an efficient and reliable method to predict fetal RhD phenotype. We report a low rate of technical failures and a low repeat rate. If we would have strictly followed the computed algorithm, the repeat rate would have been almost halved, with the expense of one false-negative and 20 more false-positive results.

Donor Hot Topics 39

Donor Hot Topics

4A-S27-03

4A-S27-01

Brown RW, Dickeson G, Wilson JT and Chesneau S

BLOOD DONATION BY AFRICAN MIGRANTS AND REFUGEES IN AUSTRALIA: THE ROLE OF DEMOGRAPHIC AND SOCIOECONOMIC FACTORS

Australian Red Cross Blood Service, Melbourne, Vic., Australia

McQuilten Z Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic., Australia Changing populationdemographics have important implications for the ability of transfusionservices to meet the transfusion requirements of minority groups. In westerncountries, differences in red blood cell (RBC) antigens between predominantlyCaucasian blood donors and recipients from different backgrounds may result inhigher RBC alloimmunization rates, particularly in those who are chronicallytransfused, and can make it more difficult to source compatible blood. Ensuringblood donors are representative of the general population is particularlyrelevant for patients of African descent, who have a number of important bloodgroup differences from Caucasians. However, there are challenges in ensuring a representative donorpopulation, especially when the national population is verymulti-cultural. This presentation willdiscuss results from a recent Australian cross-sectional survey of African migrants and refugees to establishblood donation rates and identify demographic and socio-economic factors associatedwith blood donation. The presentation willalso review experience from other published studies. To increase the number ofblood donors among African migrants, promoting knowledge and awareness ofissues associated with blood donation should be emphasised.

4A-S27-02

THE IMPACT OF HAVING LESS STRINGENT DEFERRAL POLICIES FOR MEN WHO HAD SEX WITH MEN: THE PREDICTED VS THE OBSERVED Germain M and Delage G Hema-Quebec, Quebec, QC, Canada Most jurisdictions still do not accept blood donations from men who had sex with men (MSM), even if this behavior happened in the remote past. This longstanding policy is regularly attacked by many stakeholders, who view this lifetime deferral as being overly cautious and unjustly discriminatory. Those who defend the status quo argue that a less stringent policy would unduly increase the risk to recipients. In an effort to address this dilemma, investigators in various jurisdictions have tried to project the impact of having less stringent deferral policies on HIV transmission risk, using mathematical models that rely on empirical data, with certain assumptions about the parameters included in these models. Such models can be used to evaluate various scenarios, for example those in which donations would be allowed from MSM who recently abstained from this behavior. In most models that have been published, there are essentially two main components that enter the equation: the number of HIV infected donors who would be added to the donor pool, and the proportion of these donations that would not be properly identified and discarded prior to becoming available to recipients. Not surprisingly, under certain assumptions, these models predicted very small but definite increases in risk. However, in these scenarios, the predicted number of transfusion-transmitted infections resulting from less stringent, temporary deferral policies would be so small as to remain imperceptible in reality, even in very large populations. Some countries have actually changed from a permanent to a temporary deferral policy for MSM, allowing us to verify the accuracy of these predictions. The data from three countries, Australia, the United Kingdom and Canada, indicate that there is no observable increase in HIV transmission by transfusion after the policy changes, consistent with model predictions. At least some models also predicted that there would be a sizeable and measurable increase in the number of HIV positive donors who would present to donate, an outcome that has not been observed in any of the three countries. These data suggest that certain models were overly pessimistic, at least in some of their assumptions. In particular, it seems likely that one or both of the following parameters were overestimated: the proportion of newly eligible MSM who would actually donate, and/or; the proportion of these newly eligible MSM who would unknowingly be infected. The models and the actual experience of countries that implemented temporary MSM deferrals, all support the notion that permanently excluding MSM from blood donation is an overly cautious policy, at least from the perspective of HIV transmission risk.

MOBILE AND STATIC COLLECTION OPERATIONS, A REVIEW OF POPULATION ACCESS AND COST

Background: In many countries blood collection using mobile operations is seen as a requirement to meet clinical demand. Typically, it is thought that mobile operations allow greater access to population than static sites – both in terms of the percentage of population willing to donate (population participation) and the distance those donors are willing to travel. This study seeks to challenge that hypothesis by reviewing experiences in Australia – a country with 21 million people spread over 7 million square kilometres. Currently, Australia collects 25% of all collections on mobile operations. A review was undertaken to determine strategically what this proportion should be in the future, taking into consideration both population access and cost. Aims: To determine the relative efficacy of population access afforded by static and mobile operations; and the relative cost of each modality. Methods: A review of historical donor behaviour and population behaviour was undertaken. This included a 3 year analysis of: donor movement between mobile and static collection sites; donor behaviour post mobile site closure; population participation relative to collection modality; and donor acquisition methods relative to collection modality. A detailed financial model was also built exploring each collection modality. Results: In Australia’s major cities, population participation ranges from 5.2% to 3.2% of citywide age eligible population (average 4.4%, 11 cities, population range 95,000–3,117,000). There was no observed correlation between: population participation and the number of collection locations within the city (including both mobile and static operations); nor between population participation and the mix of static and mobile operations; or areas of high population participation and distance to a collection location. Citywide, and at a suburb level, there was an observed relationship between population participation and various socio-economic factors (e.g. household income averages and country of birth). Donors tended to donate at the same location over a 3 year period (92.5%), with a small proportion moving permanently from one site to the next (5.4%) and smaller proportion actively switching between donation locations (2.1%). Of those donating at the same location, the majority of donors ceased to donate after a site was closed, even if an alternate donation facility was within the same locality. This was in contrast to observed donor travel patterns – generally donors were willing to travel further than expected. Financial modelling showed mobile and static collection modes to be total cost comparable. Summary/Conclusions: Donors appear willing to travel a reasonable distance to donate at a static donation centre, even in a metropolitan area. This means that static sites, and cities serviced by static only networks, are able to achieve population participation rates that were much higher than expected (upwards of 4.5%). While mobiles do provide access to a proportion of the population for whom distance to a donation facility is the key barrier to donation, this proportion is perhaps not as large as previously thought. These findings do not support a requirement for mobile collections in Australia in order to meet clinical demand.

4A-S27-04

DEVELOPMENT OF A DONOR HEALTH CARE CURRICULUM van den Burg PJM, Wagenmans E and de Kort WLAM Sanquin Blood Supply, Amsterdam, The Netherlands Background: Although transfusion medicine is a recognized specialty in a small number of countries, Donor Health Care (DoHeCa) is not recognized as a separate profession. In some countries, DoHeCa education is scantly included in transfusion medicine but more often DoHeCa education hardly gets attention. While independent donor care is getting more and more important, recognized in JACIE legislation, the need for DoHeCa education increases. Moreover, Donor Health Care does not only concern blood donors, but also donors of cells, tissues and organs. Although differences in these areas do exist, these separate fields of DoHeCa have much in common. Aims: The DoHeCa project has had its kick-off meeting in October 2013. The aim is to develop a broadly accessible distance learning curriculum for all professionals working in the fields of blood, cells, tissue and organ donation. The curriculum will be a high quality education program, stressing the similarities in the DoHeCa fields. Methods: The DoHeCa project was established by the initiative of different European organizations. This initiative has been based on an educational plan that was already developed in The Netherlands. The common factor of these organizations


40 Orals

has been recognized and supports the need for an innovative collaboration in the areas of blood, cells, tissues and organs. This initiative was based on an educational plan that was already developed in the Netherlands. Convinced by the international need for DoHeCa education, the international consortium has been established to explore collaboration, commitment and funding opportunities for developing an international curriculum. Results: The Dutch professional medical association for Donor Health Care applied for recognition of Donor Health Care as a new medical specialty in the Netherlands. As a result, Donor Health Care has been formally recognized in the Netherlands by the medical authorities since 2014 and the national association has started with implementation of the educational plan. The international DoHeCa consortium applied for European funding which was granted in 2013 in the framework of the Life Long Learning Programme. The international DoHeCa consortium already has set up an educational frame work for a distance learning curriculum on master’s level. In 2016 the curriculum learning will be available to fulfill the educational need for professionals working in DoHeCa. Summary and conclusions: The need for education for professionals working in DoHeCa is internationally recognized and with commitment of the implicated working fields and professional organizations a curriculum is being developed. The integration of the areas of donors of blood, cells, tissues and organs is unique and stimulates the improvement of quality of the professionals. However, the highest goal is improving the quality of donor care.

Hepatitis 4A-S28-01

THE CURRENT STATUS OF HEV INFECTION AMONG BLOOD DONORS AND TRANSFUSION-TRANSMITTED HEV INFECTION IN JAPAN Satake M1, Matsubayashi K2 and Tadokoro K1 1

Japanese Red Cross, Tokyo, Japan 2JRC Hokkaido Block Blood Center, Sapporo, Japan

Background: Hepatitis E, a zoonotic or food-borne illness in developed countries, is a potential cause of transfusion-transmitted infection (TT-HEV). Despite the high prevalence of HEV-specific antibodies in the general population (at 2–20%), the number of reported cases of TT-HEV is relatively low. The decision to implement nucleic acid test (NAT) screening for HEV in donated blood depends on several critical parameters, namely the following: 1) the frequency of blood donations with HEV-viremia; 2) the transmissibility of HEV by blood transfusion; and 3) the seriousness of hepatitis caused by transfusion with HEV-contaminated blood products. Aims: Both to evaluate the prevalence of HEV-viremia among blood donors and to explore the frequency of TT-HEV and the severity of hepatitis resulting from TTHEV. Methods: The frequency of HEV-viremic blood donation was deduced from the results of NAT screening for HEV that has been conducted in the Hokkaido region of Japan since 2005 and in the Tokyo metropolitan area in 2006. The transmissibility of HEV was evaluated using the following two look back studies: 1) the occurrence of TT-HEV in Hokkaido among patients transfused with HEV-contaminated blood components that had been issued before the establishment of real time HEV-NAT; and 2) a patient look back study performed using the information from a plasma manufacturer that has been screening for HEV using NAT for source plasma. Maximum ALT levels were evaluated after index transfusion in patients with established TT-HEV, and after index donation in followed-up viremic donors. Results: Among more than 2 million donors screened in Hokkaido, an area with the highest HEV endemic rate in Japan, 0.012% were found to be viremic by 20-pool HEV NAT. In Tokyo, 0.007% were found to be viremic among 14,800 donors. Thus, 340–600 donations per year in Japan are expected to contain HEV at levels detectable by the current NAT system. In the Hokkaido study, eight blood components containing HEV were transfused into patients before an NAT result was obtained. Three of these patients had already died, with the deaths attributed to their presenting diseases. Among the remaining five recipients, TT-HEV was excluded in three patients and established in two. Follow-up studies were conducted for 21 patients who were, based on information from the plasma manufacturer, identified as having been transfused with HEV-containing blood products. Among these patients, four had died, seven were confirmed to have acquired infection, and 10 had ambiguous data for TT-HEV. In total, HEV transmissibility was estimated to be between 40 and 80%. Median ALT levels exhibited by patients with established TT-HEV was 673 IU/ l, with higher levels recorded among high-risk patients. Among blood donors who


were followed after viremic donation, one fifth showed subsequent ALT levels of >1000 IU/l. Summary/Conclusions: Considering the high frequency of HEV-viremic donation, high transmissibility of HEV via transfusion, and moderate severity of resultant hepatitis, the number of TT-HEV cases is likely underreported. The issue of the validity of introduction of HEV-NAT screening deserves further consideration.

4A-S28-02

ALTERATION OF A NEW POSTTRANSCRIPTIONAL REGULATORY MECHANISM ASSOCIATED WITH REDUCED HBSAG EXPRESSION IN BLOOD DONORS WITH OCCULT HBV INFECTION Candotti D1, Enjalbert F2, Yuen MF3 and Allain JP2 1

National Health Service Blood & Transplant, Cambridge, United Kingdom 2Dept of Haematology, University of Cambridge, Cambridge, United Kingdom 3Dept of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China

Background and aims: Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the pre-seroconversion window period. Previous work suggested that some OBI might be associated with various mutations in genomic regulatory elements negatively affecting viral replication. Pre-S2/S mRNA splicing might be essential for viral protein expression but must be strongly controlled to prevent the accumulation of nonfunctional spliced mRNA. The functional relationship between S mRNA splicing and S protein expression and the potential negative effect of specific mutations was investigated in OBI donors. Methods: 176 OBI and 381 HBsAg+ strains (genotype A–E) were collected worldwide through the ISBT HBV safety network and their Pre-S2/S mRNA 50 splice donor site sequences were analyzed. The influence of mutations on local RNA folding was evaluated using MFOLD program. The effect of selected mutations on splicing and S protein expression were tested in Huh7 cells transiently transfected with cloned S gene of OBIs and controls with or without the distant HBV posttranscriptional regulatory element (PRE), mutated whole replicating HBV genome, and genome extracted from liver biopsies. Results: In vitro Pre-S2/S mRNA splicing was confirmed irrespective of HBV genotype and was functioning whether an autologous or heterologous promoter controlled S gene transcription and in the natural context of an entire replicating HBV genome. Similar splicing was evidenced in liver biopsies from both HBsAg+ and OBI patients. The 50 splice donor site (nt458) was conserved among HBV strains but new alternative 30 splice acceptor sites were identified in vitro and confirmed in vivo in the vicinity of PRE. Irrespective of HBV genotype, 51% of OBI sequences presented substitutions adjacent to the 50 splicing donor site compared to 31% of controls (P < 0.05). These substitutions were predicted to disrupt a putative stem-loop structure in 54% of OBI variants. OBI strains appeared to be competent in S protein expression. However, the amount of HBsAg in culture supernatant was significantly lower in most cells transfected with sequences from OBI strains when compared to HBsAg+ strains. Mutating the 50 donor site prevented splicing and resulted in significant reduction of HBsAg production. Similarly, the double mutation A453G/G463A observed in two OBI strains interfered with splicing and HBsAg production. In contrast, blocking all alternative 30 acceptor sites abolished S mRNA splicing but did not affect HBsAg production. These mutations affected expression of the Medium surface protein and to a lesser extend of the Large surface protein. No effect was observed on the expression of the pgRNA and the production of HBcAg and HBeAg. Conclusions: Preliminary data indicate that the sequence and context surrounding the 50 splicing site of the S gene is important for efficient HBV S mRNA expression but splicing itself does not appear essential for HBsAg production. This region of the S gene appears to function as a new post-transcriptional regulatory element added to PRE essential for the efficient expression of functional unspliced S mRNA of HBV strains. Involvement in S mRNA nuclear export is suspected.

Hepatitis 41

4A-S28-03

4A-S28-04

HLA-B*07:05: A NOVEL ALLELE ASSOCIATED WITH CHRONIC HCV INFECTION IN CHINESE BLOOD DONORS

HBV DNA LOAD AND SEROLOGIC MARKERS OF HBV NAT YIELD CASES IN KOREAN BLOOD DONORS

Rong X, Huang J, Xiong HP and Fu YS

Kwon SY1, Kang JW1, Youn KW1, Kim BH1, Lee SA1, Kim MH2, Cho NS2, Shin JY3, Choi YS3 and Lee DH3

Guangzhou Blood Center, Guangzhou, China

1

Background: Studies have showed the interactions between the human leukocyte antigens (HLA) and Hepatitis C Virus (HCV) infection are critical to the clearance or persistence of HCV. However, different associations between the HLA allelic distribution and HCV infection have been indicated in varied ethnic groups. There have been few studies focused on the Chinese population, which is the largest HCV infected population worldwide. To fill this knowledge gap, we studied the HLA genetic polymorphism among HCV chronically infected blood donors who were asymptomatic, treatment naive, and are therefore ideal to display the natural outcomes of HCV infection among the general population in China. Aims: To investigate the potential association between HLA alleles and chronic HCV infection in the Chinese voluntary blood donors. Methods: A high resolution HLA typing method was used to determine the polymorphism at the HLA-A, B, and DRB1 loci among 426 HCV-infected (HCV Ab+/ RNA+) and 709 control (HCV Ab/RNA) blood donors. HCV antibodies and nucleic acids were detected by enzyme-linked immunosorbent assay and nucleic acid testing, respectively. The allelic distribution at the HLA-A, B, and DRB1 loci and their association with chronic HCV infection were analyzed using Chi-square test with the SPSS 16.0 software. The strength of the associations was inferred by odds ratio (OR) with 95% confidence interval (95% CI). Bonferroni correction was performed for multiple testing. Results: Of the 426 HCV-infected donors, robust typing results were obtained for 413, 415, and 416 at the HLA-A, B, and DRB1 loci, respectively. Of the 709 control donors, robust results were produced for 658, 650, and 683. In total, 31 HLA-A, 64 HLA-B, and 54 HLA-DRB1 alleles were identified. Alleles with >1% frequency among the studied cohorts were analyzed. A novel allele B*07:05 (OR = 0.299, P = 0.001, Pc = 0.018) at the HLA-B locus were more frequently in the controls than in the HCV-infected donors (Table 1). The allele distributions at HLA-A and DRB1 loci had no statistical difference between the two cohorts after Bonferroni corrections for multiple comparisons. Our study suggests the effects of HLA genetic variations on the host immune responses against HCV are ethnically restrictive. Conclusions: We conducted a high resolution typing of HLA class I (A and B) and II (DRB1) alleles among the Chinese voluntary blood donors that included 426 HCV chronically infected donors and 709 healthy controls. For the first time, a novel allele, HLA-B*0705, was found to significantly associated with chronic HCV infection among voluntary blood donors who represent the general population in China, which suggested that the patterns of HLA polymorphism are ethnically specific. Our study fills the knowledge gap of association between HLA polymorphisms and chronic HCV infection in the Chinese population, which would be insightful in designing regional HCV vaccines.

Blood Transfusion Research Institute, Korean Red Cross, Seoul, South-Korea 2Blood Service Headquarters, Korean Red Cross, Seoul, South-Korea 3Korea Centers for Disease Control and Prevention, Osong, South-Korea

Background: To prevent transfusion-transmitted HBV infection by shortening the window period, individual HBV nucleic acid technology (NAT) testing using Procleix Ultrio Plus assay (Novartis Diagnostics) has been implemented in Korea since July 2012. Aim: To assess HBV DNA load and serologic characteristics of HBV NAT yield cases. Methods: Characteristics of HBV DNA positive donors from July 2012 to July 2013 were analyzed using the Blood Information Management System (BIMS) of the Korean Red Cross. NAT yield cases were tested for HBV DNA load (COBAS AmpliPrep/ COBAS TaqMan HBV Test, v2.0, Roche Diagnostics) and anti-HBc total, anti-HBc IgM, anti-HBe, HBeAg and anti-HBs was evaluated (Architect, Abbott). For comparison, HBV DNA load was also tested in 17 HBsAg positive/HBV DNA positive donors. Results: Among 2,572,513 donations 1523 HBV DNA positives (0.06%) were identified, including 374 NAT yield cases (0.02%). Among 346 donors tested for HBV DNA load, HBV DNA could not be quantitated in 151 donors (43.6%). Among 195 donors (56.4%), all but one donor had a DNA load of 6.4. Hemolysis throughout storage was 6.4 for 10 of the 12 units tested. Average hemolysis by end of storage hemolysis was 1.12% for DINCH and 1.53% for DOPT. Other metabolic parameters suggest comparable red cell quality between DEHP, DINCH, and DOTP.

Sanquin Blood Supply, Amsterdam, The Netherlands Background: The plasticizer di(2-ethylhexyl)-phthalate (DEHP) is a common component in medical plastics and also used in blood collection systems. DEHP is noncovalently bound to the PVC polymer and can leach from PVC devices when the surface comes into contact with fluids. There are concerns that exposure to phthalates might induce developmental and reproductive toxicity. Due to its lipophilic nature, plasma is prone to extract large quantities of plasticizer from the storage container. Omniplasma (Octapharma GmbH), a pooled, SD-treated and prion reduced plasma, is recently introduced as replacement of Fresh Frozen Plasma (FFP). Omniplasma and FFP are derived from the same starting material, i.e. plasmapheresis units. Aims: Determination of the leakage of DEHP and its conversion to mono-ethylhexyl phthalate (MEHP) in thawed Omniplasma and FFP during storage at 4–8°C and at room temperature (20–25°C). Methods: 6 Omniplasma and 6 FFP units were thawed in a water bath at 37°C, according to recommendations. After thawing, 3 units were stored at 4–8°C and 3 at room temperature (20–25°C). At different time points the plasma units were sampled for DEHP and MEHP extraction. Quantification of phthalates was performed using HPLC analysis. Results: The results are shown in the Table. Immediately after thawing, the concentration of total phthalates in Omniplasma was significantly lower than in Q plasma. During subsequent storage, the increase in phthalate concentration was comparable for both plasma types, being more pronounced at higher storage temperature. About 40% of the released phthalates was in the form of MEHP, independent of plasma type. Summary/Conclusions: Immediately after thawing the phthalate concentration in Omniplasma was lower as compared to FFP. This might be due to removal of phtalates during the removal of SD-chemicals. During subsequent storage, the phthalate concentration increases in a temperature-dependent fashion. At all storage times, phthalate concentrations were lower in Omniplasma.

Table 1: Total phthalate (DEHP + MEHP) concentrations in Omniplasma and FFP during storage (mean SD).

Table 1: Hemolysis and metabolic data of red cells in different PVC bags with CPD/SAG-M solution. © 2014 The Author Vox Sanguinis © 2014 International Society of Blood Transfusion Vox Sanguinis (2014) 107 (Suppl. 1), 1–56

44 Orals

4C-S30-04

IMPLEMENTATION OF A COLD CHAIN MANAGEMENT PROCESS TO FACILITATE A SUCCESSFUL REGIONAL BLOOD INVENTORY ROTATION PROGRAM WITH MINIMAL WASTAGE Tocchetti R1, Clarke M2, Sinha R3 and Ireland S1 1

Blood, Organ and Tissue Programs, Department for Health and Ageing, Adelaide, SA, Australia 2Country Health South Australia LHN, Adelaide, SA, Australia 3 Blood, Organ and Tissues Programs, Department for Health and Ageing, Adelaide, SA, Australia Background: Regional South Australia (SA) presents significant geographical and logistical challenges to the supply and maintenance of emergency standby blood stocks. These include the cold chain assurance of regionally held blood stocks and the logistics in the rotation of those stocks back to the supplying metropolitan centres prior to expiry thereby minimising wastage. Aim: To implement a novel blood wastage minimisation program across regional SA covering multiple transfusion laboratory networks and regional SA hospitals without on-site laboratories. The program enabled hospital stored emergency standby blood and unused crossmatched blood to be accepted back into transfusion laboratory inventory. Methods: The program title ‘BloodMove’ was implemented with the support of a central Transfusion Committee and a dedicated team comprised of a medical scientist, nurse management facilitator and a network of transfusion portfolio nurses and site blood contact nurses. Blood cold chain security was assured by replacement of ageing blood refrigerators and revalidation of blood shipment systems. Regional hospital onsite education programs were conducted that included blood refrigerator oversight and blood shipper packing. Regional and remote hospitals were partnered with a primary supplying transfusion laboratory to oversee blood refrigerator temperature and testing records as well as blood storage confirmation documentation. Local ongoing management, audit and communication was established using the network of transfusion portfolio and blood contact nurses. Hospital documentation was provided to receiving laboratories allowing previously discard units to be returned to inventory. Results: The following graph details the blood wastage minimisation reduction following implementation of the BloodMove program in regional SA. Wastage has decreased from a high of 15% of transfusion laboratory inventory to a recent low level of 0.7% (Dec 2013). This decrease was been sustained during 2013. Conclusion: The BloodMove program assisted regional hospitals and supplying transfusion laboratories to comply with the national safety and quality standards and guidelines. Blood wastage due to regional site product expiry and product cold chain failures were reduced. This effective regional blood rotation process has allowed the provision of emergency standby blood to remote sites which were not previously provided with holdings due to transport and wastage concerns. Importantly, BloodMove has engaged regional hospitals and laboratories in stewardship activities and established wastage avoidance as normal practice.

the development of storage lesions in platelet concentrates which is in part caused by collection/production. Perturbation of the platelet proteome and PRT-induced increases in platelet activation raised the question whether delayed treatment could reduce the negative impact on platelet quality. Aims: To understand the impact of varying PRT timing, PRT was employed at different days after apheresis collection and platelet quality was monitored throughout storage. Methods: In a paired pool-and split study, apheresis platelet concentrates were either treated on the day of production, on days 1, 3 and 4 of storage or kept untreated as control. A panel of routine in vitro parameters was used to monitor quality throughout storage. PC units were sampled on days 1, 2, 5, 7 and metabolic parameters (pH, glucose and lactate) were measured. CD62P expression, phosphatidyl-serine exposure and microparticle count were determined by flow cytometry. Extent of shape change was measured after exposure to ADP. Changes in the protein profiles of lysate and releasate were analyzed by immunoblot. Results: On day 7 of storage, units treated on day 4 displayed the best platelet quality compared to the units treated on day of production or on days 1, 3 and 4 indicating a reduced damage to the platelets. Significantly reduced glucose consumption and consequently reduced lactate production resulted in similar pH in units treated at day 4 and the untreated control. Platelet activation was significantly reduced on day 4 and onwards when treated on day 3 and 4 compared to units treated earlier. Platelet response to ADP measured by the extend-of-shape change assay was significantly improved with the delayed time of PRT treatment. On day 7, units treated on day 4 displayed 2.3-fold reductions in MPs compared to treatment on day of production or after 24 h. Phosphorylation levels of proteins p38MAPK or HSP27 known to increase upon riboflavin/UV light treatment displayed no timing-dependent change compared to early treatment. Releasate analyses showed a 1.7 and 2.1-fold decrease in platelet factor 4 and thrombospondin on day 7 when treated at day 4 compared to day 0 or day 1, respectively; this is consistent with the reduced degranulation measured by CD62P expression. Conclusions: The timing of PRT treatment after platelet collection is a strong influence on the quality of apheresis platelets. Based on these results, timing recommendations for PRT should be reexamined providing there is no negative effect on pathogen inactivation. Increased flexibility in timing of PRT application may improve inventory management of PRT-treated platelets.

Hemoglobinopathies 4C-S32-01

MANAGEMENT OF ALPHA-THALASSEMIA Viprakasit V Mahidol University, Bangkok, Thailand

4C-S30-05

TIMING OF PATHOGEN INACTIVATION MAY IMPROVE PLATELET QUALITY RETENTION Schubert P, Karwal S, Culibrk B and Devine DV Canadian Blood Services, Vancouver, BC, Canada Background: Treatment of apheresis platelet concentrates with riboflavin/UV light is deemed to be carried out within 22 h after collection. Research studies have demonstrated that the application of pathogen reduction technologies (PRT) accelerate


Background: Heterozygotes for a0-and a+-thalassemias are usually asymptomatic or have microcytic-hypochromic red blood cells. Interactions of a0-and a+-thalassemias result in a non-fatal form of a-thalassemia syndrome; Hemoglobin H (Hb H) disease. Patients with this condition present with a diverse clinical severity, causing from mild to moderate severity included in the broader syndrome of non-transfusion dependent thalassemia (NTDT). Aims: To provide a guideline for clinical management in patients with Hb H disease. Methods: Five hundred Thai patients with confirmed molecular analysis of the aglobin genes and their related clinical data were analysed. Interactions of other genetic modifiers have been taken into account and re-evaluate with other clinical complications including gallstones, infection, iron overload etc. Results: In general, patients with non-deletional (–/aTa) are usually more severe than deletional Hb H (–/-a) types. Moreover, certain non-deletional Hb H patients have the most severe phenotype; referred to as Hb H hydrops fetalis. In these rare cases, intrauterine and neonatal complications develop with hydropic features. These patients require regular blood transfusion for survival similar to patients with b-thalassemia major. Other mechanisms beside imbalanced globin synthesis might influence the Hb H disease pathophysiology resulting in heterogeneous clinical phenotypes. Although stem cell transplantation offers a curative measure for these severe a-thalassemia syndromes, its application has been limited by donor availability. Summary/Conclusion: Clinical phenotype of patients with Hb H disease is more heterogeneous than previous thoughts; ranging from a symptomatic to severe transfusion dependent Hb H and Hb H hydrops. Molecular analysis of the globin gene is useful as a primary tool to predict clinical severity and tailor appropriate clinical management including blood transfusion, splenectomy to stem cell transplantation.

Platelet Biology 45

4C-S32-02

THALASSAEMIC PATIENTS IN HIGH RISK FOR RED BLOOD CELL ALLOIMMUNIZATION Adamidou D, Vlachaki E, Oikonomou M, Lefkou E, Teli K, Farmaki E and Theodoridou S Ippokratio Hospital, Thessaloniki, Greece Background: The development of alloantibodies against red blood cell (RBC) antigens constitutes a major issue when managing transfusion dependent patients causing not only difficulties in finding compatible units but also serious transfusion delays and haemolytic reactions of various clinical significance. Aims: To record the incidence of RBC alloimmunization in our thalassaemic cohort and try to reveal factors associated with increased risk for the development of alloantibodies. Methods: A group of 177 patients (94 female-83 male) diagnosed with major thalassaemia b (133), intermedia b (35) and compound heterozygosity (HbS/b+, HbS/b0) (9) with a median age of 30 (4–74) years was included in the study. Age at first transfusion was 4 (0.1–50) years and patients remain transfusion dependent for 24 (1–59) years. 59 patients underwent splenectomy in the age of 19 (4–67) years, 34 had history of HCV infection whereas in 64 patients transfusions were initiated before 1984 when our centre started to use Rhesus phenotype matched units. With regard to genotypes, 22 were IVS1-110/IVS1-110, 14 IVS1-110/cd39, 11 IVS1-110/ IVS1-6 and 8 cd39/cd39. We recorded the ΑBΟ/Rhesus blood group, ferritin levels and the type of chelation therapy. Indirect Antiglobulin Test-IAT (Diamed) was used for the detection of alloantibodies. We analyzed 52 samples by flow cytometry (FC 500 Flow cytometer) in order to count the lymphocyte subpopulations and determine the percentage of the regulatory T cells (Treg). Results: Over the course of time 21/177 thalassaemics (12%) developed alloantibodies against the following red cell antigens: Rhesus (2D, 2C, 2Cw, 1E), Kell (5K, 2Kpa), Kidd (4Jka, 2Jkb), Lutheran (1Lua), MNS (1S, 1s). Two patients developed multiple antibodies and for one the picture is still undefined. The first detection occurred in 24 (6– 57) years since the initiation of transfusions. In 9/21 patients alloantibodies disappeared within a median period of 4 (0.1–13) years. Non persistent antibodies were against mainly the Kidd, Lutheran jaι MNS blood group and none against Rhesus and Kell. In a univariate analysis we found that female gender was associated with higher incidence of alloimmunization (16/21) (P < 0.020). With regard to diagnosis patients with intermedia thalassaemia b were more prone to the formation of RBC alloantibodies (8/35) (P < 0.030). Older age at first transfusion was significantly related to alloimmunization (8 vs 3.5 years for patients with or without alloantibodies respectively, P < 0.05). The multivariate analysis revealed the female gender as the only independent factor for the development of RBC alloantibodies (P < 0.012). Of notice the fact that alloimmunized chronically transfused patients showed lower Treg compared to non-alloimmunized (2 vs 2,7)%, though the difference was not statistically significant. Conclusions: Based in our centre’s experience, RBC alloimmunization still continues to trouble multitransfused patients despite the use of leukoreduced units that are phenotipically matched for RH/KEL blood groups. Our data suggest that it might be of clinical value to recognize this group of high risk patients and try to implement special transfusion policies based on extended RBC antigen phenotype in order to avoid the development of alloantibodies.

4C-S32-03

IL-1, TNF, IL-4 AND IL-10 POLYMORPHISMS IN PATIENTS WITH SICKLE CELL DISEASE ALLOIMMUNIZED AND NONALLOIMMUNIZED TO RED CELL ANTIGENS

cytokines may be directly involved in antibody-mediated immune response, we investigated the possible association of inflammatory cytokines (IL-1 and TNF-a) polymorphisms and cytokines directly involved in the humoral immune response (IL-4 and IL-10) polymorphisms with RBC alloimmunization. Aim: We aimed to determine the frequency of TNF-a -308G/A, IL1B -511C/T, IL4 intron 3 VNTR, IL10 -592A/C e IL10 -1082G/A polymorphisms in alloimmunized and non-alloimmunized patients with SCD to identify genetic risk factors for RBC alloimmunization. Methods: DNA samples from 67 patients with SCD (27 alloimmunized and 40 not alloimmunized) and 34 blood donors of African origin were genotyped for TNF-a -308G/A, IL1B -511C/T, IL4 intron 3 VNTR, IL10 -592A/C e IL10 -1082G/A polymorphisms. Genotyping for TNF-a-308G/A, IL1B -511C/T, IL10 -592 A/C, IL10 -1082G/A and IL4 intron 3 VNTR was performed by manual PCR using laboratory developed tests (LDT). Clinical data and RBC alloimmunization were obtained from patients’ medical records. The Hardy–Weinberg equilibrium (HWE) and the allelic and genotypic frequencies were obtained by Arlequin version 3.1 software. Comparisons of frequencies were performed at the Open Epi version 3.01 program using the chi -square test with Yates correction or the exact Fisher test when appropriate. Results: The distribution of genotypes and alleles of TNF-a -308G/A, IL1B -511C/T, IL4 intron 3 VNTR, IL10 -592A/C and IL10 -1082G/A did not differ significantly between alloimmunized, non- alloimmunized patients and blood donors of African descent. The association of TNF-a -308G, IL1-511T, IL4*1 (254pb), IL10-592C, IL101082A (GT1CA) alleles showed a higher frequency in alloimmunized patients compared to non-alloimmunized (19.1% vs 0%, P = 0.0084). Summary/Conclusion: Our results showed that the GT1CA allele association may be associated with susceptibility to RBC alloimmunization in patients with sickle cell anemia. The knowledge of genetic factors that predispose to alloimmunization in sickle cell patients can assist in choosing strategies based transfusion units of compatible blood for patients at high risk, in particular those suffering from rare phenotypes or Rh variants.

Platelet Biology 4C-S33-01

PLATELET-DERIVED MICROPARTICLES Boilard E CHU de Quebec, Quebec City, QC, Canada Nearly one trillion platelets sentinel the blood vessels to monitor and preserve the integrity of the vasculature. A normal platelet count in humans is approximately 150–400 9 109/l, and individuals with low counts are more prone to bleeding and bruising. The transfusion of platelets is a life-saving medical procedure for patients undergoing a major surgery, with massive bleedings, for subjects with abnormal platelets (not fully functional) and those with diseases, like cancers or thrombocytopenia. However, the transfusion of platelets can trigger adverse reactions. Beyond their role in hemostasis, platelets are also tiny cells capable of producing inflammatory mediators potentially implicated in unwanted post-transfusion reactions. These inflammatory mediators can be soluble, and can be encapsulated in microparticles; small vesicles with submicron dimensions shed from the platelet’s cytoplasmic membrane. In this presentation, different approaches for the detection and the characterization of platelet-derived microparticles will be presented. Furthermore, recent observations revealed the unexpected heterogeneity of the microparticles produced by platelets. The distinct inflammatory functions played by these platelet microparticle subtypes will be discussed and put in a context of transfusion.

Sippert EA, Gaspardi AC, Gilli SCO, Carvalho MA and Castilho L Hemocentro-Unicamp, Campinas, Brazil Background: One of the most serious consequences of red blood cell (RBC) transfusions in patients with sickle cell disease (SCD) is alloimmunization. In fact, it has been observed that a group of polytransfused patients with SCD develops alo-/autoanticorpos against multiple RBC antigen but an interesting date is that not all patients develop alloantibodies following exposure to transfused RBCs, despite receiving a similar number of transfusions. Many factors may influence the immune system to respond to blood group antigens including dosage, immunogenicity of the antigen, as well as genetic factors related to patients or acquired system. Whereas


46 Orals

4C-S33-02

MOLECULAR MECHANISMS REGULATING PLATELET CLEARANCE AND THROMBOPOIETIN PRODUCTION

aggregate together. Contrarily, there is no PAC-1 on the microparticles, they don’t spontaneously combined with fibrin and result in thrombus. So, it is expected to become a hemostatic agent.

Grozovsky R, Falet H and Hoffmeister KM Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States of America The human body produces and removes 1011 platelets daily tomaintain a normal steady-state platelet count. Platelet production must betightly regulated to avoid spontaneous bleeding if counts are low (thrombocytopenia) or arterial occlusion and organ damage if counts are high (thrombocytosis). Thrombopoietin (TPO) is the primary regulator of plateletproduction, supporting the survival, proliferation and differentiation of theplatelet precursors, bone marrow megakaryocytes. Although it is clear thathepatocytes are the primary source of TPO, the mechanisms regulatingcirculating TPO levels have been subject to discussion for decades. Twomajor models for TPO regulation have been proposed. In one model, hepatic TPOproduction is constitutive and TPO serum levels are maintained solely by itsuptake and metabolism by the TPO receptor c-Mpl on plateletsandmegakaryocytes. This conclusion is based on models of acute immune orchemotherapy induced thrombocytopenia, such as those induced by antibodies or busulfan, in which serum TPO levels increase, while hepatic TPO mRNA levels are not changed. However, discrepancies between circulating TPO levels and platelet counts observed in severalhuman diseases such as Immune Thrombocytopenia (ITP) and EssentialThrombocythemia (ET) give credence to the assertion that platelet-TPOmetabolism is not the sole determinant of plasma TPO levels. In anothermodel of TPO regulation, platelet counts are sensed by an unidentifiedmechanism, resulting in regulated levels of TPO expression in the liver and thebone marrow. This hypothesis is based on the observations that liver TPO mRNAexpression is not constitutive, as it can be stimulated by interleukin 6 (IL-6)during reactive inflammatory thrombocytosis, epithelial ovarian cancer, orafter selective liver damage, providing a regulated pathway to increaseplatelet production during acute inflammatory responses. However, thephysiological ligand-receptor pair capable of regulating steady state TPOproduction remained mysterious for more than two decades. Recent studies havehighlighted the role of glycan modifications on platelet surface proteins inmediating platelet clearance. We and others have found that the hepaticAshwell-Morell receptor binds and removes desialylated platelets. Recently we showed that clearance ofdesialylated senile platelets by the hepatic Ashwell Morell Receptor (AMR) definesa direct feedback pathway for TPO mRNA expression and secretion. Inthis review, we will discuss the past and current knowledge of platelet and TPOhomeostasis regulatory mechanisms.

4C-S33-03

PREPARATION AND PRELIMINARY INVESTIGATION OF ARTIFICIAL PLATELET MICROPARTICLE Liu J Institute of Blood Transfusion, Chinese Academy of Medical Science, Chengdu, China Backgound: Recently, lots of researches have demonstrated the fact platelet concentrates could spontaneously generate platelet microparticles in the storage and the microparticles have a relationship with haemostatic function. We could have good use of the microparticles’ haemostatic function as one haemostatic agent only if preparing the microparticles through simple process. Aims: To prepare artificial platelet microparticles which have haemostatic function. Methods: Outdatedplatelet concentrates was separated from red blood cells, white blood cells and plasma, by differential centrifugation. Then the platelets precipitate were resuspended in physiological saline, and disrupted by repeated freezing and thawing, three times. The frozen and thawed suspension was centrifuged to remove intracellular residue components. And the lysed platelets were then homogenized by sonicating for 200 cycles. The sonicated platelets suspension was centrifuged to get microparticles which remain in the supernatant. The microparticles were used for the following research: the size of the microparticles, surface protein receptor of microparticles, aggregation function and thrombin generation ability. Results: The artificial platelet microparticle size is about 140 nm, and retain glycoprotein (GP)Iba (CD42b), GPIIb-IIIa (CD41/CD61), CD31, CD62p except PAC-1 (activated CD41/CD61 complex). In vitro, Ristocetin and III-collagen could induce particles aggregation. Thrombin generation experiments showed that the artificial platelet microparticles could provide catalytic surface for tenase complex and thrombin generation. Conclusion: Byfreezing-thawing and ultrasonic sonication, we could obtain platelet microparticles with uniform particle size. The microparticle surface still remains GPIba, GPIIb-IIIa, CD31, CD62p, which were associated with haemostatic function. And microparticles were activated by the aggregation reagents and they could


4C-S33-04

C-REACTIVE PROTEIN BOOSTS ANTIBODY-MEDIATED PLATELET DESTRUCTION Vidarsson G1, Kapur R1, Heitink K2, Porcelijn L1, Bentlage AEH1, Bruin M2, Visser R1, Roos D1, Schasfoort RBM3, de Haas M1 and van der Schoot CE1 Sanquin Research, Amsterdam, The Netherlands 2University Medical Centre, Utrecht, The Netherlands 3MIRA Institute, University of Twente, Enschede, The Netherlands

1

Background: Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcc-receptor (FccR)-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating other factors to play a role. Aims: To identify if serum factors besides antibodies contribute to phagocytosis of platelets and platelet clearance. Methods: Platelets opsonized with Foetal/Neonatal Alloimmune Thrombocytopenia (FNAIT) sera or recombinant anti human platelet antigen 1a (HPA1a) IgG1 were subjected to phagocytosis- and respiratory burst assays by human neutrophils and monocytes in the presence or absence of serum and chemical inhibitors. Binding of identified serum factor (the acute-phase protein C-Reactive Protein, CRP) to platelets was further investigated by FACS and label-free cellular Surface Plasmon Resonance Imaging (cSPRi). CRP levels were measured before and after treatment with IVIg in newly diagnosed ITP patients, and the levels of CRP were correlated with platelet counts. The effect of CRP on IgG-mediated platelet destruction was followed in a mouse model of ITP induced with rat-IgG anti-mouse CD41. Results: We identified a Ca2+-dependent cofactor present at various concentrations in normal human sera (NHS) that was required for activating phagocyte respiratory burst against IgG-opsonized platelets. We identified this factor as CRP, because depletion and subsequent supplementation of CRP abrogated or reinstated the observed phagocyte- enhancing effect, respectively. Without anti-platelet antibodies, CRP was inert towards platelets, but it bound to phosphorylcholine exposed after oxidation triggered by anti-platelet antibodies. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared to healthy controls, albeit the values were still in the normal range. Within a week, IVIg-treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, which correlated significantly with increased platelet numbers and clinically decreased bleeding severity. To investigate the causal relationship between increased CRP levels and thrombocytopenia, we studied the effect of CRP in a mouse model of antibody induced ITP. While CRP alone did not cause thrombocytopenia in mice, it deepened the thrombocytopenia significantly in the presence of anti-platelet IgG. Conclusions: These data suggest CRP to amplify platelet destruction by anti-platelet IgG. They may further explain the aggravation of autoimmune thrombocytopenia’s upon infections. Hence, targeting of CRP could offer new therapeutic opportunities for these patients.

Managing Risk of Transfusion Transmitted Infectious Diseases 4D-S34-01

TESTING FOR HIV – THE FIRST 3 DECADES Busch MP Blood Systems Research Institute and University of California, San Francisco, CA, United States of America Background: HIV was discovered in the spring of 1984, with high rates of virus isolation and antibody (Ab) detection in AIDS patients. Early studies demonstrating isolation of similar strains of HIV in transfusion recipients and linked infected donors

Managing Risk of Transfusion Transmitted Infectious Diseases 47

contributed to proof that HIV was the cause of AIDS, and established the critical need to develop tests for blood screening. Aims/Methods: Review evolution of HIV donor screening, and broader applications of HIV tests and infected donor surveillance to research and public health. Results: In mid-1984 scientists in governments and industry collaborated to develop HIV Ab tests that were implemented in blood banks ~1 year following discovery of HIV. Look-back studies triggered by detection of Ab+ donors found that ~90% of recipients transfused with Ab+ blood were infected, indicating high rates of persistent infection/infectivity in seropositive donors. Retrospective analyses estimated that incidence of donor infection and consequent transfusion-transmitted (TT)-HIV in high risk cities like San Francisco rose rapidly from 1978 to a peak in 1982 of ~1%/unit, followed by a decline to 0.1% at implementation of Ab screening. Initial Ab screening did not eliminate TT-HIV, with numerous breakthrough cases detected shortly following introduction of first-generation assays. Analyses of transmission rates by prior donations from seroconverting donors yielded estimates for an infectious window period (WP) of ~56 days prior to detection by first-generation Ab assays. Improvements in sensitivity of second-generation tests in the late-1980s and third-generation tests in the mid-1990s reduced the pre-Ab-seroconversion WP to approximately 3 weeks. Subsequent additions of p24 antigen and then nucleic acid technology (NAT) screening in the late 1990s closed the infectious WP to 5–10 days, resulting in current risk estimates of approximately 1 infectious unit per 1–2 million transfusions in the US and other countries where NAT screening is performed, including high incidence settings like South Africa and South-East Asia where individual-donation NAT screening has been adopted. However, rare transmissions of HIV by NAT-negative blood indicate that even sensitive NAT and serological screening cannot eliminate HIV risk, both due to high infectivity of virus in the early ramp-up phase of acute infection and rare failures of assays to detect genetically diverse strains of HIV. NAT screening assays and algorithms and fourth-generation Ag/Ab serological tests developed for blood bank screening and staging early HIV infections are now in widespread use in clinical diagnostic, research and public health settings. NAT assays developed for donor screening have been applied not only to detect acute infections, but also for studies of patients on HAART and ‘elite controllers’ involved in ‘HIV-eradication’ studies to detect and quantify persistent low-level viral replication. Conclusions: Beyond advancing blood safety, advances in HIV testing have contributed to HIV diagnostics, research and public health. Further opportunities to cross-fertilize transfusion medicine and HIV research and surveillance include establishing a global program to characterize incident HIV infections in donors (risk factors, clades, resistance profiles) to help monitor the pulse of the epidemic, and referral of acutely infected donors to research programs focused on understanding this critical phase of infection.

4D-S34-02

DO BLOOD DONOR TRAVEL ACTIVITIES PLAY A ROLE IN BLOOD TRANSFUSION RISK? A FLAVIVIRIDAE-BASED RISK ASSESSMENT MODEL Caballero M1, Niederhauser C2, Andina N2, Piazza U2, Rotzetter K2, Baumer H2, Stadler A2, Gowland P2 and Fontana S2 Inselspital, University Hospital, Bern, Switzerland 2Blood Transfusion Service of the Swiss Red Cross, Bern, Switzerland

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Background: Frequent blood donor travel activities and environmental changes have increased the risk of mosqito-transmitted human flaviviridae infections in Europe. Increasing flavivirus infection rates in the general population has led to a potential increased risk of contaminated blood. Currently most European countries rely on ECDC risk area definitions for the implementation of safety measures, without taking into account their effect on blood supply and infection risk for patients. Aim: To develop a model to evaluate the effect of deferral policies on the infection risk of donors traveling to risk countries, and to apply this model to West Nile Virus (WNV), the currently most relevant flavivirus infection in European blood transfusion medicine. Methods: A simple excel-based tool was developed. It incorporated disease specific variables, epidemiological data in the risk country, information about donors traveling abroad, and number of blood donations performed in our region during the WNV season 2013 (1 July–30 November). The effect of a 1 month deferral recommendation on the contamination risk of blood with WNV for affected countries publishing epidemiological data was calculated. Results: The WNV calculated risks related to donor travels are summarized in the table. Assumptions taken in the model: mean asymptomatic infectivity 5.6 days, duration of epidemic 150 days, reported cases per country = number of confirmed cases reported by ECDC, proportion of reported symptomatic cases = 30%, length of stay 7 days, blood

donations during the epidemic period in our region 39,293, traveling donors = effective numbers in 2013. Risk of infected donation applies to a donor donating within 30 days after returning from an endemic country. The risk of an infected transfusion applies to a delivered component, considering the number of donors who donate within 30 days after travel and the total number of donations (Tab. 1). Conclusions: The calculated risks of donors and patients acquiring a WNV infection are variable but are very low. The loss of virus infectivity during manufacturing and storage was not taken into account. This model allows comparing the effects of preventive measures on blood supply and on WNV infection risk, from both the perspective of blood banks and hospitals, who are involved in taking risk-adapted decisions. The model can be adapted to other transfusion-transmitted infections associated with travels abroad.

4D-S34-03

RISK OF MALARIA IN BLOOD DONORS WITH MALARIA ANTIBODY REACTIVE RESULT Nobre Fernandes S, Koch C, Abreu C, Sabio F, Guedes E, Almeida C, Abrunhosa S, Sarmento A and Ara ujo F Centro Hospitalar de S. Jo~ao, E.P.E., Oporto, Portugal Background: In May 2012, according to Portuguese legislation and European regulations, a malaria antibody test, was implemented for individuals who had history of travel/residence in a malaria endemic country or had past history of malaria, as a mean of reducing the long term donor deferral. Aims: Evaluate the malaria infectious risk in blood donors whose malaria antibody result was reactive. Methods: All potential blood donors were subjected to stringent screening procedures. Eligible blood donors, who had a history of travel/residence in a malaria endemic country or a past history of malaria, were tested using an enzyme immunoassay (Newmarket Malaria EIA), the unique reagent actually approved for blood donors. The assay detects antibodies (IgG, IgM, IgA) against P. falciparum and P. vivax and shows 80% cross reactivity with P. ovale and 67% with P. malariae. This immunoassay does not distinguish between IgG, IgM and IgA antibodies, or between antibodies to Plasmodium species. Reactive blood donors were notified to receive personal information, collect a new blood sample for retesting and, in the case of result confirmation, to be referred to Infectious Disease Specialist. They were further tested with NOWâ ICT malaria Pf/Pv teste (Binax Inc.), a rapid, in vitro, immunodiagnostic test for the detection of circulatingP. falciparum antigen and a pan-malaria antigen. Malaria smears and Plasmodium PCR in blood were done. Deficiency of glucose 6-phosphate dehydrogenase was excluded in those donors. Then, they were treated with primaquine, considering P. vivax and P. ovale hipnozoits erradication. Results: From May 1st 2012 to December 31st 2013, a total of 134 blood donors were tested for anti-Plasmodium antibodies (EIA). The mean age was 59 years old, ranging from 45 to 64. Overall, 117 (87.3%) were EIA non-reactive and 17 were EIA reactive. 1/17 blood donors was a former resident in endemic country and 16 donors had past malaria infection (over 20 years ago), being malaria acquired and treated in sub-Saharan Africa countries. 13/17 blood donors were referred to infectious diseases appointment. Malaria tests (smear, antigens and PCR) were all negative and no clinical suspicions of present malaria were elicited. 7/13 individuals were treated with primaquine, 15 mg/day for 14 days and they were retested 6 months later for Plasmodium antibodies. Five remained reactive, 1 became non-reactive and 1 is waiting for retest.1/13 blood donors turned into non-reactive status. 5/13 blood donors remain reactive. Before 2007, all 16 donors with past malaria were repeat blood donors of 536 donations, which produced 746 released blood components transfused to 707 patients. No cases of associated transfusionmalaria were elicited. Conclusion: Our findings showed that from 134 blood donors tested for malaria antibodies, 17 were reactive, more than 20 years after leaving malaria endemic regions. Additional malaria diagnostic tests were negative in all donors (13) who presented at infectious diseases appointment. Albeit these individuals are clinical cured, they are not accepted as active blood donors. If our results are confirmed in larger studies regulatory laws for the acceptance of blood products must be reviewed.


48 Orals

4D-S34-04

RISK OF BLOOD TRANSFUSION-TRANSMITTED BRUCELLOSIS IN AN ENDEMIC AREA OF XINJIANG, CHINA Li C1, Wang W1, Liao Q1, Wu X2, Hou S3, Wang Y4, Wu J1, Shen C2, Chen S5 and Allain JP6 1

Southern Medical University, Guangzhou, China 2Kashi Central Blood Station, Kashi, Xinjiang, China 3Center of Disease Control and Prevention (CDC) of Guangzhou, Guangzhou, China 4Shihezi University, Shihezi, Xinjiang, China 5Center of Disease Control and Prevention (CDC), Guangzhou, China 6University of Cambridge, Cambridge, United Kingdom Background: Brucellosis is a severe zoonotic disease that is increasingly prevalent in North China. However, the prevalence of Brucella infection in blood donors has not been investigated in the high epidemic areas of China. Aims: This study was to evaluate potential risk of blood transfusion-transmitted brucellosis in the Kashi area of Xinjiang, China. Methods: Donor blood samples were collected between 18th February and 5th May 2013 at Kashi Central Blood Station, a Brucella-epidemic focus area of China. Bloodsamples were screened for antibody to Brucellae by the Rose Bengal Plate Test (RBPT), and the reactive samples were further tested by the Standard Tube Agglutination Test (SAT), ELISA and Western blot. Brucella DNA was detected by real-time quantitative PCR (qPCR), nested-PCR and DNA sequencing. Results: A total of 3896 blood donor samples were tested. 135 (3.5%) and 120 (3.1%) plasmas were reactive by RBPT or SAT, respectively. Of 135 sero-reactive plasmas, 39 (1.0%) were positive by ELISA with the native membrane protein extract of B. melitensis and 25 were confirmed reactive to either rBP26 or rOMP31 antigens by Western Blot. 14 plasmas were positive for Brucella DNA by qPCR, in which 13 were detected as Brucella BP26 DNA by the nested-PCR. Two donor follow-up blood samples were tested positive for Brucella antibodies and DNA amplifications. Overall 15 (1:300) Kashi blood donorswere detectedas Brucella bacteremia by the nucleic acid testing (NAT) and confirmed specific by DNA sequencing, which suggested high risk oftransfusion-transmitted brucellosis yet to be documented in transfusion recipients. Conclusions: Blood donation screening for Brucella infection may be considered in the high Brucella-endemic areas of China, for which implementation of NAT would be the most efficient.

4D-S34-05

HEPATITIS E VIRUS RNA SCREENING IN BLOOD DONORS IN HOKKAIDO, JAPAN Sakata H1, Matsubayashi K1, Iida J1, Yoshimasa T1, Sato S1, Kato T1, Hino S2, Ikeda H1 and Takamoto S1 Japanese Red Cross Hokkaido Block Blood Center, Sapporo, Japan 2Japanese Red Cross Blood Service Headquarters, Tokyo, Japan

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Background: Autochthonous hepatitis E virus (HEV) in developed countries is far more common than previously recognized. We reported two cases of transfusiontransmitted HEV (TT-HEV) in Hokkaido where hepatitis E is most prevalent in Japan so far. Thereafter, HEV RNA screening (HEV NAT) for research has been conducted in blood donors in Hokkaido, Japan since 2005. Aims: To clarify the current status of HEV infection among blood donors in Hokkaido, Japan. Methods: From January 2005 to December 2013, 2,484,249 blood donors were tested for the presence of HEV RNA by real-time reverse transcription (RT)-PCR using 20-pool samples in Hokkaido. Blood samples that tested positive for HEV RNA were also tested for the presence of IgM and IgG anti-HEV by a commercial ELISA kit, and HEV RNA load by quantitative real-time RT-PCR. A partial sequence of 412 nt of HEV ORF2 region was determined for HEV strains from the HEV-positive donors by direct sequencing. Questionnaires were sent to the HEV RNA-positive donors to collect data on their history of intake of animal meat before the index donation. The HEV positive donors were looked-back and followed-up before and after the index donation. Results: HEV RNA was detected in 279 (209 males and 70 females) donors and the overall prevalence of the HEV-infected blood was 0.011% (0.013% in males and 0.008% in females) from 2005 to 2013 in Hokkaido. The yearly HEV prevalence fluctuated between 0.008% and 0.016% and no clear seasonality was observed. Geographical differences in HEV prevalence was observed in sub-prefectures of Hokkaido, with the prevalence of HEV RNA-positive donors higher in densely populated areas. Of the 279 donors, 229 (82%) had neither IgM nor IgG anti-HEV and they were presumably in the early stage of HEV infection at donation. Mean HEV viral


load was 3.3 1.0 log copies/ml and ranged from 2 points prompts activation of the MTP. Results: One hospital reported an impressive 50% decrease in mean time of first blood product arrival to patient’s bed-side (decrease from 45 to 22 min); the other hospitals experienced similar dramatic improvements. There were 434 activations from October 2011 to October 2013 (average: 18 per month). 39% were for trauma patients, while 30% were for major surgical patients with heavy intra-operative bleeding. 25% and 6% were for patients with gastrointestinal bleeding and peri-partum haemorrhage respectively. Mean blood component use per MTP activation was 9.7 RBC, 7.3 FFP, 1.89 CSP/ PPLT, and 4.26 Cryoprecipitate (in units). Mean transfusion ratio achieved was 1 red cell unit: 0.75 FFP units: 0.78 wholeblood derived platelet units: 0.44 units of cryoprecipitate. Despite initial concerns, the blood service was able to fully stock standby MTP Inventory for all hospitals more than 95% of the time, while keeping hospital MTP standby stock outdate below 3% for platelets & below 0.5% for Red Cells. Summary/Conclusions: The National MTP is a highly successful collaboration between the National Blood Service and all public-funded hospitals in Singapore; this is also the first time a common transfusion protocol has been universally adopted across the hospitals. This augurs well for future introduction of more common clinical transfusion protocols on a national level.

4D-S37-03

MASSIVE TRANSFUSION PROTOCOL IN THE SINGAPORE – OUR EXPERIENCE IN INTRODUCING AND RUNNING A COMMON NATIONAL MT PROTOCOL (2011–2013) Chay JWM1, Koh MBC1, Tan HH1, Ng HJ2, Ng JCF2, Chua CML2, Chia NCH3, Tey XS3, Desouza K4, Toh AH4, Mattar NM4, Ong KH5, Tan J5, Tan SC5, Kuperan P5, Yuen J5, Lew E6, Heng KML6, Tan LK7, Koh PL7, Lim STT7, Khng YL7, Bin Mohd Fathil BMF8, Ng HP8, Lim JL8, Wijaya SI8 and Ang AL1 Health Sciences Authority, Singapore, Singapore 2Singapore General Hospital, Singapore, Singapore 3Khoo Teck Puat Hospital, Singapore, Singapore 4Changi General Hospital, Singapore, Singapore 5Tan Tock Seng Hospital, Singapore, Singapore 6Kandang Kerbau Women’s and Children’s Hospital, Singapore, Singapore 7 National University Hospital, Singapore, Singapore 8Jurong Health Services, Singapore, Singapore

Figure 1: Acknowldegements page.

1

Background: Singapore introduced a common National Massive Transfusion Protocol (MTP) in 2011. Aims: To describe our experience in establishing and coordinating a National MTP during the first 25 months (October 2011–October 2013). Methods: MTP in Singapore began with successful introduction of a pilot obstetric MTP in 2008 for peri-partum haemorrhage at a local 700-bed obstetric hospital, followed by a well-received pilot trauma MTP at an 800-bed district general hospital in 2009. In 2011, a workgroup of surgeons, anaesthetists, transfusion physicians and haematologists formulated a national MTP for rapid and effective transfusion support during life-threatening bleeding. This MTP supports all adult patients with severe trauma, surgical, medical and obstetric bleeding emergencies in the 7 large publicfunded hospitals and was launched in late 2011. Blood components are transfused in a ratio of 1:1:1 (pRBC: Platelets: FFP), together with anti-fibrinolytic agents (tranexamic acid) and cryoprecipitate replacement. Blood components are issued in ‘MTP PACKS’, each containing 4 red cells units, 4 Fresh Frozen Plasma units and 1 adult dose of platelets (Single-Donor-Platelets or Pooled-Platelets).

4D-S37-04

THE IMAGE MONITORING OF OPERATING ROOMS IMPROVES PRACTICES IN TRANSFUSION MEDICINE; RECENT RESULTS Furumaki H, Yamada C, Watanabe H, Fujihara H, Shibata H, Nagai S, Ishizuka K, Kaneko M, Shimizu D, Adachi M and Takeshita A Hamamatsu University School of Medicine, Hamamatsu, Japan Background: Sufficient information from operating rooms is important for appropriate and speedy transfusions. However, it has been difficult for our transfusion unit to understand real-time information in operating rooms only by previously constructed speech-based interfaces. Recently, we reported the efficacy of screens placed in the transfusion unit, and linked it to each operating room. Aims: We would like to introduce developmental utilization of the system not only by voice but also with images, and discuss its potential in transfusion medicine. Methods: Large liquid crystal displays (Panasonic, Tokyo, Japan) linked to each operating room, as well as to the recovery room, were placed on the board of the transfusion unit. One of the displays can be divided into 16 sections, and each section can be enlarged to a full-size screen. Another shows real time progress notes on transfusion. We studied the amount and frequency of expired blood during perioperative period, and compared them during the 4-year periods before and after adoption of the system. Statistical analyses were performed by using chi-square test and Student’s t-test (SAS, Tokyo, Japan). All statistical analyses were two-tailed with a significance level of 0.05.


50 Orals

Results: In the analyses of all operations, the number of cases with expired red blood concentrates (RCC) significantly decreased from 25 cases to 4 cases (P < 0.001); the amount of expired RCC decreased from 76 to 7 units (P < 0.001); and the frequency of expiration decreased from 0.80% to 0.09% (P < 0.001) after the introduction of the image monitoring system. In the analyses of cardiovascular operations, in which 10 units or more of RCC were used, the amount of RCC, that was requested initially, was 16.8 7.2 (mean SD) units and 15.4 5.2 units before and after introduction of the image monitoring system, respectively (P = 0.03). The number of operations in which more than 30 units of RCC were requested initially, decreased from 43 cases (12.4%) to 3 case (3.5%) (P = 0.001). The number of operations in which more than 10 units of RCC was initially delivered to operations, decreased from 152 cases (44.2%) to 21 cases (24.4%) (P < 0.001). The deficiency of blood in the transfusion unit was alleviated. Conclusions: The system not only by speech-based interfaces but also with images, which was linked to the operating rooms, was very useful. It delivers superior performance with the real time progress notes on transfusion. It increases the safety and swiftness of transfusion, and it decreases the amount of expired blood.

4D-S37-05

PREDICTORS OF BLOOD UTILIZATION IN ORTHOTOPIC LIVER TRANSPLANTATION Eichbaum Q, Woods MC, Domenico HJ and Karp SJ Vanderbilt University School of Medicine, Nashville, TN, United States of America Background: Liver transplantation surgery can result in massive blood loss necessitating the transfusion of substantial amounts of blood products. The amount of blood products utilized varies widely between national transplantation centers. Vanderbilt University Medical Center (VUMC) in 2011 ranked nationally among the highest utilizers or blood products for liver transplantation surgery. At that point, a liver transplant review committee was established at VUMC, tasked with introducing measures to reduce blood utilization. To determine which predictors might be associated with blood utilization, a multivariate analysis of 455 liver transplantation surgeries performed at VUMC between the years 2009 and 2013 was initiated. We report here the preliminary findings of that analysis. Aims: The aim of this study was to identify factors associated with increased utilization of blood products in 455 liver transplantation surgeries performed at VUMC between January 2009 and July 2013. Methods: Four log-linear regression models were built using each surgery as the unit of analysis and intra-operative red blood cells (RBCs), platelets, fresh frozen plasma (FFP), and cryoprecipitate as outcome variables. Predictor variables used in the models included pre-operative laboratory test values, pre-existing patient conditions, patient demographics, the use of cell saver, the operating surgeon and anesthesiologist, as well as the date and time length of the surgical procedure. Results: The analysis demonstrated that the significant predictors of blood product utilization were the following pre-surgery laboratory lab values: the Internationalized Ratio (INR) (P < 0.0111), platelet count (P < 0.0002) and hemoglobin (Hgb) (P < 0.0001). Other predictors were the operating transplant surgeon (P < 0.0015) and anesthesiologist (P < 0.0092). The length of the surgical procedure time was differently associated with blood utilization: for the early period of the study, before any measures aimed at reducing blood utilization were effectively implemented (January 2009–August 2012), surgery times ((5 0.1 h), were associated with increased utilization of blood products (RBC: 12.9 2.1; FFP: 15.9 1.7; platelets 2.1 0.4; cryoprecipitate: 5.5 1.1); but for the later period (September 2012–July 2013), after such measures were implemented, longer surgery times (5.4 0.3 h)were associated with decreased blood utilization (RBC: 6.5 1.8; FFP 9.8 2.2; platelets: 1.0 0.2; cryoprecipitate: 6.1 1.7). Which interventions had the greatest impact on decreased blood utilization has not yet been fully determined but preliminary indicators suggest that improved surgical technique and the introduction of the cell saver (P < 0.0001) may be implicated. By the end of the two-and-a-half year study period, VUMC managed to reduce its RBC utilization from an average 12.9 units per surgery, to an average of 6.5 units. Additional analyses are in progress to pinpoint the reasons for this significant decrease in blood utilization at VUMC. Summary/Conclusions: A number of variables may be predictively associated with blood product utilization in liver transplantation surgery. The pre-surgical INR, hemoglobin and platelet count appear to be significant predictors. In addition, improved surgical technique that lengthens the time of surgery, together with the use of cell saver, may be interventions that result in decreased blood utilization in liver transplant surgery.


Blood Group Studies 4D-S38-01

ABO SUBGROUP STUDIES IN KOREA Cho D and Ryang DW Chonnam National University Medical School, Gwangju, South-Korea ABO subgroups are phenotypes that differ in the amount of A and B antigen carried on red cells. The weak expression of A and B antigens can generally be explained by single nucleotide polymorphisms (SNP) or hybrid formation between common ABO alleles. Until now, numerous examples of A or B subgroup alleles have been described in many geographical and/or ethnic groups. To analyze the phenotypic and genotypic basis of the ABO subgroups including cis-AB, a large-scale study was performed in 169,605 samples from Korean blood donors. The overall incidence of ABO subgroups was 0.13% (227/169,605) in our donor population. Based on genotyping and/or serologic typing, A2B (n = 120), A2B3 (n = 36), A1B3 (n = 22), A2 (n = 13), B3 (n = 13), AintB3 (n = 6), and others (n = 17) have been defined. Among these ABO subgroups, 26.4% (60/227) demonstrated a cis-AB01 allele. Data from other studies show that Korean population has several unique ABO subgroups such as Aw10, Aw14, B306, and B308. To understand why subgroups are manifested by weak or unusual phenotypes, flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing various mutants, three-dimensional (3D) structural analysis of glycosyltransferase (GT), and an assay that predicts the stability of the mutant GT was also used, in addition to ABO genotyping. Our experience with ABO subgroup studies in Korea again suggest that the phenotypic frequencies and genetic basis of ABO subgroups differ between ethnic regions.

4D-S38-02

GENERATION OF MICE EXPRESSING HUMAN RHAG AND RHD BLOOD GROUP GENES Goossens D Institut National de la Transfusion Sanguine; Inserm UMR_S1134, Paris, France Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but since the suppressive mechanism remains uncertain, a mouse model of this pathology would be useful to achieve a better understanding of the processes involved. A number of highly interesting models of mice transgenic for human blood groups, producing alloantibodies through transfusion, and even pregnancy, have been developed in recent years. Expression of RhD has, however, proved difficult in mice, due to its integration in a complex structure, a heterotrimer involving not only Rh, but also its RhAG protein partner. In our experience, RhD could be expressed from a human RHD gene on a BAC or from RHD cDNA under control of b-globin regulatory elements, but RhD erythrocyte membrane expression was obtained only in mice transgenic for both the RHAG and RHD human genes. We here give an overview of our approach to the generation of transgenic mice coexpressing human RhAG and RhD erythrocyte membrane proteins, and discuss further challenges to be resolved in the quest for a model of RHD alloimmunisation and HDFN.

4D-S38-03

PROPHYLACTIC ANTI-D BATCHES DISPLAY VARIABLE DECREASE IN FC-FUCOSYLATION, AFFECTING BINDING TO FCcRIIIA AND CLEARANCE RATE OF RBC Vidarsson G1, Kapur R1, Della Valle L1, Verhagen O1, Hipgrave-Ederveen A2, Ligthart P3, de Haas M1, Kumpel B4, Wuhrer M2 and van der Schoot CE1 Sanquin Research, Amsterdam, The Netherlands 2Leiden University Medical Center, Leiden, The Netherlands 3Sanquin, Amsterdam, The Netherlands 4Bristol Institute for Transfusion Sciences, Bristol, United Kingdom

1

Background: Anti-RhD (anti-D) immunoprophylaxis has greatly reduced the occurrence of hemolytic disease of the fetus or newborn (HDFN). The prophylactic IgG is obtained from hyperimmunized healthy anti-D donors (HID) boosted with D-positive RBCs. Although the exact working-mechanism of anti-D immunoprophylaxis is unknown, one strong hypothesis is that anti-D ensures fast D-positive RBC clearance through FccRIIIa. The level of Fc fucosylation influences the interaction with FccRI-

Organising a National Blood Service with Limited Resources 51

IIa, with a lower degree of Fc-fucosylation resulting in stronger interactions. Previously we demonstrated increased phagocytosis mediated by low Fc-fucosylated IgG. Recently we found a strong reduction in Fc-fucosylation of anti-D in pregnant women, which was found to correlate with decreased neonatal Hb levels. Together these data suggest that inclusion of plasma samples with low Fc-fucosylated anti-D in a pool of anti-D prophylactic products will increase the biological activity. Aims: To investigate if male volunteers also display a lowered core Fc-fucosylation in their anti-D and if the composition of different anti-D products is affected by the donor inclusion criterias. Methods: Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female HID-donors (Dutch anti-D immunoprophylaxis), and commercial international anti-D immunoprophylaxis preparations were analyzed with massspectrometry. Furthermore, the Fc-glycosylation-profiles of HID-donors were evaluated with regard to their immunization history. Results: Both female and male HID-donor serum samples demonstrated a clearly lowered anti-D Fc-fucosylation when compared to normal IgG fucosylation (93%), and this was more pronounced for females than for male HID-donors (46.7% vs 64.7%, P = 0.001). Polyclonal anti-D immunoprophylaxis preparations from 7 different manufacturers varied greatly with respect to the level of Fc-fucosylation (56– 91%). The product in which IgG is mainly derived from female donors immunized by pregnancy showed the lowest level of fucosylation. Although the level of fucosylation was slightly increased upon repeated immunization, the level of fucosylation remained fairly constant over time. Conclusion: Decreased IgG1-Fc-fucosylation seems to be a common response to particulate blood-born antigens. Based on the observed fucosylation patterns of anti-D in combination with the hypothesis that anti-D immunoprophylaxis exerts its effect through FccRIIIa-mediated clearance, anti-D immunoprophylaxis could be further optimized by selection of donors whose anti-D have low amounts of Fc-fucose. Implementing a biological assay in the standardization of anti-D immunoglobulin preparations might be considered.

4D-S38-04

LOW ANTI-RHD IGG-FC-FUCOSYLATION IN PREGNANCY: A NEW VARIABLE PREDICTING SEVERITY IN HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN Sonneveld ME1, Kapur R1, Della Valle L1, Hipgrave Ederveen A2, Visser R1, Ligthart P3, de Haas M1, Wuhrer M2, van der Schoot CE1 and Vidarsson G1 1 Department of Experimental Immunohematology, Sanquin Research, Amsterdam, The Netherlands 2Leiden University Medical Center, Leiden, The Netherlands 3 Erythrocyte Serology, Sanquin, Amsterdam, The Netherlands

Background: Hemolytic disease of the fetus and newborn (HDFN) may occur when maternal anti-RhD (anti-D) IgG antibodies cross the placenta and mediate the destruction of D-positive red blood cells (RBCs) via phagocytic IgG-Fc-receptors (FccR). Clinical severity is not strictly related to titer and is more accurately predicted by functional antibody-dependent cellular cytotoxicity (ADCC), which is a sensitive test but has relatively low specificity. This suggests other factors to be involved in the pathogenesis of HDFN. Binding of IgG to FccR requires the N-linked glycan at position 297 in the IgG Fc-region, consisting of several different glycoforms. However, the exact composition of this glycan is quite heterogeneous. Particularly, the absence of core fucose greatly increases the binding of IgG to FccRIIIa and FccRIIIb, on macrophages/NK cells and granulocytes, respectively. Although approximately all (94%) of IgG-Fc-glycans are fucosylated, indicating it to be of little clinical relevance, this feature is currently being exploited for therapeutic application for example in anti-tumor antibodies. Aim: To investigate if antibody glycosylation may explain clinical features observed in HDFN, and explain the lack of a strong relationship between IgG titer and D-positive RBC count in the neonate. Methods: Anti-D alloantibodies were purified from serum by acid elution after incubation of the serum with D-positive RBC. The amount of IgG1 and IgG3 in the eluate was determined by ELISA and specificity verified by agglutination of RhD+ RBC and lack of agglutination on RhD- RBC. Anti-D specific IgG antibodies, and total IgG antibodies were subjected to tryptic digestion and the resulting IgG1 and IgG3 Fc-derived glycopeptides were analyzed by MALDI-TOF-MS. Cytotoxic lysis of D-positive RBCs was assessed by NK-cell and monocyte based ADCC. Results: Analyzing IgG-derived glyco-peptides from 70 alloimmunized pregnant women, we found a variable decrease in Fc-fucosylation in the majority of anti-D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated like in healthy individuals (>90%). Similar trend was seen for in IgG3. The degree of anti-D fucosylation correlated significantly with CD16 (FccRIIIa)-med-

iated ADCC, in agreement with the increased affinity of defucosylated IgG to human FccRIIIa. Additionally, low anti-D fucosylation correlated significantly with low fetal-neonatal hemoglobin levels, thus with increased hemolysis. Conclusions: This skewing of IgG-responses against D-positive RBCs in pregnancy indicates that the level of fucosylation may be antigen dependent. In addition, the lowered Fc core-fucose in anti-D IgG1 was associated with increased FccRIIIa-mediated ADCC and decreased hemoglobin levels at birth, suggesting this type of glycosylation to be an important biomarker and therapeutic target in HDFN.

Organising a National Blood Service with Limited Resources 5A-S40-01

BLOOD SERVICE IN NEPAL WITH LIMITATIONS AND POSSIBILITIES Rajkarnikar M Nepal Red Cross Society, Kathmandu, Nepal Background: After establishment of blood transfusion service in the initial years the service was made possible through collection of blood from professional donors but since 1982 blood collection was emphasized from voluntary non-remunerated donors only. Motivated individuals are significant blood donors and still 10% is replacement blood donation. Nepal Red Cross Society with the sole authority given by Nepal Government conducting blood program. Therefore, a great responsibility has fallen on the Society and to prove its capability, it is systematically strengthening itself with resources available nationally and exploring resource possibilities internationally. As it stands today, there are 1 central, 4 regional, 21 district level blood banks, emergency units in 31 and 29 hospital units of the services throughout the country. Aims: With the view of providing safe blood and blood products to the needy patients all collected blood is in voluntary based. All donated blood is routinely tested for transfusion transmission infections. Blood component facility is limited and available only in few centers. In the year 2013 nationwide collection 1,89,123 units of blood and supplied 2,74,627 units of blood to the needy patients. Almost all blood transfusion service runs in the cost recovery basis in which Patients are paying for material charges for the unit of blood. Results: The blood program runs with the blood policy and guidelines for blood transfusion services given by the Nepal Government. Nepal Red Cross Society is managing blood program throughout the country in cost recovery which is difficult to cover all expenses of blood program. Every year blood collection is increasing about 6.7%. The ratios of male and female donors are of 85% and 15% Nationwide. Conclusions: National Blood program is run with the cost recovery and there is an inadequate fund for blood program. Voluntary concept for blood donation is well developed throughout the country. Entire blood centers are managing service with own resources but some of the limitations in blood program are covered with on and off support from stakeholders and different agencies which fulfill the gaps. It also support for the expansion as well as up gradation of the blood program to some extent. It is felt that there should be regular funding for blood program for its sustainability.

5A-S40-02

ORGANISING A NATIONAL BLOOD SERVICE WITH LIMITED RESOURCES – DEVELOPMENTS IN THE SOUTH AFRICAN NATIONAL BLOOD SERVICE Mpuntsha L and Reddy RV South African National Blood Service, Johannesburg, South Africa Background: South Africa has a population of about 52 million and is classified as a Middle HDI Country with historical disparities within the various population groups. The Health System is two tier with Government funding health care in the public sector and medical insurance schemes funding healthcare in the private sector for those who can afford it. Fiscal allocation for the public sector has been averag-


52 Orals

ing at about 8.5% of the country’s Gross Domestic Product (GDP) and is utilised by 84% of the population. There is inadequate value contribution due to high disease burden, increasing demand for services from previously disadvantaged groups and inefficiencies in service delivery. The Blood Services operate on a fee-for-service basis supplying about 60% of blood to the state hospital and about 40% to private health facilities. The South African National Blood Service (SANBS) supplies 85% of the population in 8 of the 9 provinces and the Western Province Blood Transfusion Service (SABTS) almost 15%, both on a ‘vein-to-vein’ basis. SANBS has organised a national blood service over a period of 12 years since the amalgamation of six (6) regional Blood Service organisations. The mission of SANBS is ‘to provide all patients with sufficient, safe, quality blood products and medical services related to blood service in a sustainable manner’. Blood safety and sufficiency is the sole mandate carried both by blood establishments (BEs), sourcing, testing, processing, storing, delivering and supplying blood components (BCs) to all hospitals. Aim: To present historical developments and achievements of SANBS in pursuit of its mandate over the last 12 years. The objective is to share outcomes of various strategies that enabled and strengthened delivery in the South African resource-constrained environment and challenging socio-economic context. Methods: A review of the Strategic plans, Governance framework, Operational service delivery and efficiency, Human Resource Management, Financial Efficiency and Stakeholder Management was performed to highlight the strategy, challenges and successes of organising a National Blood Service. Results: SANBS has over the past 12 years developed into a world class organisation despite challenging social, political and economic environments. Key successes include: Governance framework that is comparable to that of any commercial organisation. Sustainable business model with good stakeholder relationships with customers. Providing adequate blood to patients at 17 units of red cells per 1 000 population. High levels of blood safety due to 100% voluntary donors and testing strategies. Talented pool of retained human resources due to developmental programmes and opportunities for career development. Strong research, educational and haemovigilnce programmes. Conclusions: SANBS managed to organise an efficient and sustainable blood service over time, with limited resources, in very difficult circumstances by following business management principles, stakeholder management and good governance. To follow similar approaches will help developing countries in similar situations to organise their blood service on for sustainability.

5A-S40-03 Abstract not available.

5A-S40-04

IMPLEMENTATION OF BLOOD SAFETY SYSTEMS REFORMS IN PAKISTAN Waheed U and Zaheer HA Safe Blood Transfusion Programme, Government of Pakistan, Islamabad, Pakistan Background: Pakistan is the sixth most populous country in the world with a population of 180 million. The Blood Transfusion System in Pakistan is fragmented and has evolved over the last 66 years essentially as a result of local initiatives. The net outcome is a proliferation of various types and size of blood establishments, relying predominantly on family replacement donations, and functioning according to various ethical principles, from non-profit to a service charge dependant on the standard of facilities available. The back-bone of the system remains the ‘multifunctional’ hospital blood banks, complemented by an ever increasing number of private blood bank laboratories. Aims: The establishment of core elements of an independent rational structure of a national blood transfusion service that will ensure adequate, efficient and safe blood supply, in a cost effective manner through a network of new Regional Blood Centres linked to existing Hospital Blood Banks. Methods: The Government of Pakistan has embarked on an ambitious reform process through the Safe Blood Transfusion Programme. The reform agenda, defined in the National Blood Policy and Strategic Framework, is supported by the German Government. Results: Since its establishment in 2010, the SBTP has been able to deliver a series of outputs which have significantly contributed to improvement in blood safety


standards and establish strong bonds among the national stakeholders. The close collaboration with the assorted stakeholders has deepened the Programme’s understanding of the existing ‘diversity’ in terms of structures, technology, human resource capacity and overall organization. Working groups and task forces, comprising of eminent local experts, have been formed to develop key policy and operational documents including Model SOPs, National Standards and Guidelines, National CUB Guidelines, Business Plan, Functional Brief for MIS, Functional Brief for BTA, Functional Brief for RBC and HBB, Inventory of BDO and BT Laws, etc. The inclusive approach has enhanced local ownership and integration of experience and evidence, thus increasing local users’ expertise and capacity. The SBTP is also implementing a capacity building plan aimed at creating a suitably qualified workforce, well equipped to adequately operate both in the new system and in the current reality. Conclusion: Despite constitutional and operational challenges, the reforms process is proceeding in the envisaged direction. With the successful completion of the first phase of the project in the current year, the new infrastructure will be completed and functional by the end of the year. The preparatory planning will be put to test in the new system and reviewed and revised in the light of experience gained. Simultaneously, the planning for the second phase of the project envisaging expansion in the scope of coverage and enveloping the experience of the current phase is well underway in close collaboration with the German partners.

ABO-Incompatible Transplantation 5A-S41-01

DETECTION, SPECIFICITY AND IMPORTANCE OF ABO ANTIBODIES IN ORGAN TRANSPLANTATION Holgersson J The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Living donor (LD) kidney transplantation (KTx) is associated with better patient and graft survival than transplantation of kidneys from deceased donors. The ABO blood group system used to be a historic barrier to LD KTx in cases where otherwise suitable LDs were declined because of ABO incompatibility. The introduction in several transplantation centers of ABO incompatible (ABOi) organ, especially kidney, transplantation has increased the number of potential living donors for each patient. Removal of anti-A and/or anti-B is a prerequisite for successful ABOi KTx in adult patients. This is accomplished by plasma exchange/plasmapheresis, double filtration plasmapheresis, protein A or anti-immunoglobulin immunoadsorption, or antigen-specific immunoadsorption on matrices carrying blood group A or B antigens. Anti-A and anti-B antibody titration by hemagglutination using panel or donor RBCs is used to monitor antibody removal, determine acceptable antibody titers for transplantation to proceed and as an important diagnostic test in case of rejection. Even though ABOi KTx for the most part is successful, there is room for improvements of diagnostic as well as therapeutic tools used. For example, despite the use of the same gel and tube hemagglutination techniques, there are significant center-to-center variations with regard to determined anti-A and anti-B antibody titers in distributed test serum samples. Likewise, removal of anti-A and anti-B on columns with A or B trisaccharides sometimes fails, which may be explained by the fact that some anti-A and anti-B require the fourth sugar in the carbohydrate chain for binding. Using human serum albumin neoglycoproteins or sepharose beads carryingA or B tetrasaccharides based on the type 1 (Galb3GlcNAc), type 2 (Galb4GlcNAc), type 3 (Galb3GalNAca) or type 4 (Galb3GalNAcb) core saccharide chains, we have shown that also healthy blood donors have chain-type specific anti-A and/or anti-B. Such antibodies require the fourth sugar in the carbohydrate chain for binding and bind poorly to the terminal A or B trisaccharides. This population of anti-A and anti-B may explain that adsorption on the A and B trisaccharides, respectively, sometimes fails. However, the clinical significance of such core chain type-specific anti-A and anti-B is unknown. Our work on establishing an ELISA for reproducible semi-quantification and chain type-specificity determination of anti-A and anti-B will be discussed.

Novel Blood Products 53

5A-S41-02

5A-S41-03

THE IMPACT OF ABO INCOMPATIBILITY ON SURVIVAL AND RED CELL TRANSFUSION SUPPORT IN ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTS

ABO-INCOMPATIBLE LIVER TRANSPLANTATION USING THERAPEUTIC PLASMA EXCHANGE

Chew E, Gilbertson M, Mason K, Lim A, Ritchie D, Szer J, Juneja S, Hogan C, Haeusler M and He S

Catholic University of Daegu School of Medicine, Daegu, South-Korea

The Royal Melbourne Hospital, Parkville, Vic., Australia Background: The HLA and ABO genes are independently inherited. In allogeneic haematopoietic stem cell transplants (HSCTs), the best available HLA-compatible donor may not be ABO-identical with the patients. In the current era of high resolution HLA typing, less intensive conditioning regimens and improved supportive patient care, the effects of ABO-incompatible allogeneic HSCT on clinical and transfusion outcomes have not been fully elucidated. Aims: To determine the effect of ABO incompatibility on survival outcomes of patients undergoing allogeneic HSCTs from January 2007 to July 2013 at the Royal Melbourne Hospital, Australia. To determine the effect of ABO incompatibility on red cell concentrate (RCC) transfusion requirements of this patient cohort. To report the characteristics of ABO group change in patients undergoing ABO incompatible allogeneic HSCTs. Methods: A retrospective audit was performed using clinical and laboratory information from the Royal Melbourne Hospital HSCT Database, medical records and pathology information system. Results: From January 2007 to July 2013, 379 allogeneic HSCT’s were performed at our institution (comprising 196 ABO compatible HSCTs, 64 major ABO incompatible HSCTs, 61 minor ABO incompatible HSCTs and 16 bidirectional ABO incompatible HSCTs). Kaplan-Meier analysis showed ABO compatible allogeneic HSCTs had a better overall survival compared to major ABO incompatible allogeneic HSCTs (P = 0.016). There was no difference in overall survival due to types of conditioning regimens (myeloablative compared to non-myeloablative) or stem cell donors (sibling compared to matched unrelated donor). Major ABO incompatible allogeneic HSCTs required more RCC transfusions by day 100 post-transplant compared to ABO compatible allogeneic HSCTs (mean of 11 units of RCC compared to 7 units of RCC, P < 0.05). In the ABO incompatible allogeneic HSCT cohort, 72 patients changed their ABO blood group to that of the donor’s (31 major ABO incompatible, 37 minor incompatible and 4 bidirectional ABO mismatch transplant patients). In 15 of the 72 patients (21%), the blood group change occurred within 100 days of the transplant (4 major ABO incompatible and 11 minor ABO incompatible transplant patients). Conclusions: Major ABO incompatible allogeneic HSCTs have a worse overall survival and required more red cell transfusion support compared to ABO compatible allogeneic HSCTs. The majority of ABO mismatched allogeneic HSCT patients have not changed their ABO blood group by 100 days post-transplant. These results have implications for selection of HSCT donors as well as clinical and laboratory transfusion practice. Further research on the effects of ABO incompatibility on other clinical outcomes like graft-versus-host disease is in progress.

Figure 1: Overall survival of ABO compatible compared to major ABO incompatible allogeneic HSCT.

Lee AJ, Kim SG, Jang HB and Kim JD

Background: ABO-incompatible (ABOi) adult living donor liver transplantations (ALDLTs) have been tried despite of the risk of antibody-mediated rejection because of shortage of ABO-matched donors. However, the standard protocol of TPE in conjunction with immunosuppressive agents to manage and treat patients with high titer ABO antibodies has not yet established. Aims: In the present study, we report the experiences with ABOi ALDLTs using a therapeutic plasma exchange (TPE). Methods: Twelve patients was administered the immunosuppressive agent, rituximab and underwent TPE during the pre-operative period according to the isoagglutinin titer. Results: The median age for recipients was 54 years (range, 32–64 years). The initial range of IgM and IgG titers was 1:8–1:256 and 1:16–>1:1024, respectively. The median number of TPE was 3 (range, 2–5) in the initial low titer group (

Abstracts of the 11th World Congress of the International Hepato-Pancreato-Biliary Association, 22-27 March 2014, Seoul, Korea.

Abstracts from the International Surgical Congress of the ASGBI, 30 April-2 May 2014, Harrogate, UK.

The Congress of Neurological Surgeons International Division 2014.

The 6th congress of the Korean Society of Neuro-Ophthalmology, Seoul, Korea.

Congress summary: 21st Congress of the International Society for Rotary Blood Pumps.

Reports from the International Liver Cancer Association (ILCA) congress 2014.

2014 ACAPS Congress: Abstracts.

Abstracts of the ILTS 20(th) Annual International Congress, June 3-7, 2014, London, United Kingdom.

The 8th International Congress of the Croatian Society of Nuclear Medicine: highlights from Šibenik, 9-12 May 2014.

Abstracts of the Fourth International IASSIDD Europe Regional Congress 14-17 July, 2014, Vienna, Austria.

Abstracts of the 12th International Congress on Obesity, 17-20 March 2014, Kuala Lumpur, Malaysia.

The International Medical Congress.

Abstracts of the WFH 2014 World Congress, May 11-15, 2014, Melbourne, Australia.

Abstracts of the IPA World Congress 2014, May 9-11, 2014, Athens, Greece.

Abstracts of the 24th Regional Congress of the International Society of Blood Transfusion in conjunction with the 6th Malaysian National Transfusion Medicine Conference of the Malaysian Blood Transfusion Society. Kuala Lumpur, Malaysia. December 1-4, 2013.

First international congress of the society for ethnopharmacology, India.

Abstracts of the 16th ESSKA Congress, 14-17 May 2014, Amsterdam, The Netherlands.

Abstracts of the 101st Annual Congress of the Swiss Society of Surgery, 21-23 May, 2014, Berne, Switzerland.

The International Congress of Hygiene and Demography.

The International Congress of Hygiene and Demography.

Discovering the pathogenesis of autoimmune diseases at the 9th International Congress of Autoimmunity, Nice, France, 2014.

Abstracts of the 49th Congress of the European Society for Surgical Research, May 21-24, 2014, Budapest, Hungary.

The Fourteenth International Congress of Medicine.

Abstracts of the Heart Failure Congress 2014 and the 1st World Congress on Acute Heart Failure, May 17–20, 2014, Athens, Greece.