Donors 1

ISBT Academy Day Transfusion Transmitted Infectious Diseases

the gold standard. The absence of a true confirmatory test for anti-HBc complicates confirmation of chronic HBV (HBsAg non-reactive/anti-HBc reactive). Since NAT for HIV, HCV and HBV have demonstrated very high specificity they now form an independent method of confirming infection. In some countries, samples concordantly reactive on serology and NAT screening tests are considered ‘confirmed’ without the need for immunoblotting. Summary/conclusions: Even in resource constrained settings optimal screening and confirmatory strategies can significantly reduce the risk of transfusion-transmissible viral infection.

1A-H1-01

INFECTION TRANSMISSION RISK AND DIFFERENT TEST STRATEGIES

1A-H1-03

Dodd RY

Tadokoro K and Satake M

American Red Cross, Rockville, MD, United States of America

Japanese Red Cross Society, Tokyo, Japan

Testing donated blood for markers of infectious disease plays a major role in establishing and maintaining a safe blood supply. Minimal expectations worldwide are that blood should be tested for syphilis, HIV, HCV and HBV. The minimal norm for the viral testing is the use of serologic procedures but increasingly, nucleic acid testing is being considered or implemented where resources allow. The appropriate mix of tests and testing algorithms will also depend upon the local epidemiology of the infections and may also be affected by societal issues. In some parts of the world, additional testing may be indicated in order to manage localized threats, such as Chagas disease in South and Central America. In developing test strategies, it is important to understand the actual risk of transfusion-transmission and the impact of the test system on that risk. Approaches for the selection of test strategies and for estimation of risk will be discussed, with particular reference to the developing world.

It is estimated that there are 2 billion HBV-infected people including 350 million HBV-carriers in the world. It is highly endemic in South Africa, Amazon, and Southeast-Central Asia. The genotype of HBV is geographically characteristic, e.g. genotype B and C are the major in East Asia. HBV transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. The residual risk is mainly related to donations either in the pre-sero (or pre-DNA)-conversion window period or occult HBV infection (OBI) where blood test is HBV-DNA-positive and HBs-Ag-negative. Infectivity of HBV depends on the transfused blood (viral load, phase of infection, genotype, and anti-HBs in the concurrent blood) and immune status of the recipients (anti-HBs, immunocompetence). It was shown that infectivity is dependent on viral load. Allain et al reported that FFPs is more infectious than PCs or RBCs. The minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately 10 times higher than that of pre-acute phase. Satake et al reported that transmission rate of OBI-derived components with low titer anti-HBc was 1/33(3%), whereas that of anti-HBc-negative components was 11/22(50%), which was verified in the lookback programme conducted in Japan. Allain et al showed in the study conducted in Europe that adjusted transmission rate of OBI blood was 28%, and the rate was higher without anti-HBs(63.8%) and lower with anti-HBs(15.4%). Discrepancy of transmission rate of OBI-derived blood between above two reports might be related to the different cutoff levels of anti-HBc and presence or absence of anti-HBs. DNA-positivity rate among OBI-derived components is higher in those with the higher levels of anti-HBc and lower in those with the presence of anti-HBs. There has been no report of transmission by OBI-derived blood with anti-HBs of 200mIU/ml or more. Screening test for HBV is different between countries. Low endemic countries screen blood for HBs-Ag, anti-HBc and mini-pooled NAT, while highly endemic countries test for HBs-Ag without anti-HBc, because high prevalence of anti-HBc-positive donation hamper securing necessary blood. Japan as a moderately endemic country had tested for HBs-Ag, mini-pool NAT and anti-HBc/anti-HBs where anti-HBs of more than 200 mIU/ml irrespective of anti-HBc and low anti-HBc with agglutination-inhibition titer of no more than 25 is qualified. Transfusion-transmitted HBV cases related to window period donations declined by increasing the sensitivity of mini pool NAT, whereas those related to blood with low titer anti-HBc remained stable with around 10 cases annually. In order to decrease such transmission Japanese Red Cross implemented a novel strategy to eliminate all anti-HBc-positive donations with anti-HBs 10 others. Specificities for Vel negativity and Scianna have been included into HFA typing, recently. Conclusion: Analysis for Kell, Kidd and Duffy showed that genotyping worked qualitatively better and to costs comparable to serology. Consequently, genotyping Kell, Kidd and Duffy instead of routinely performing a second round of serotyping as mandatory for donors in Switzerland, is recommended. Ahead of comparable suggestions with regard to Rh and MNSs, a more detailed statistical analysis of existing raw data is needed. However, genetically identified donors with rare blood phenotypes, e.g. such as Yt(a b+), are already selected for respective transfusions and are a strong indicator for the value of the presented project.

2A-S02-02

GP.KIPP AND GP.YAK BAB HYBRID GLYCOPHORINS: NO DIFFERENCE IN SEQUENCE OR SEROLOGY Flower RLP1, Wei L2, Ji YL2, Luo GP2, Lopez G1 and Hyland CA1 1

Australian Red Cross Blood Service, Brisbane, Australia 2Guangzhou Blood Centre, Guangzhou, China Background: The MNS blood group system is complex, with at least 46 antigens, some of these arise from genetic exchange between Glycophorin A and Glycophorin B producing both A-B-A and B-A-B hybrids. These hybrid glycophorins display characteristic profiles of neoantigens that are the basis of identification of the hybrid present. Aim: To compare the serological profile and sequence for Gp.Kipp with Gp.Yak to define any differences. Methods: Reported antigen profiles, including description of the typing system used, were obtained from reference databases and publications. The exon three region of interest was amplified and analysed via standard Sanger sequencing carried out at the Australian Genome Research facility. Results: The profile of neoantigens for Gp.Kipp and Gp.Yak appeared to be identical with Mut, Mur and Hil positive, Hop negative and positive with ‘Anek-like’ antisera with Hop+Nob activity. For some typing sera a result was only reported for one or other of the hybrids. Molecular studies revealed a GPB-A-B hybrid, GPB(1-26)GPwB(27-54)-GPA(55-57)-GPB(58-103) for the Australian donor. Conclusion: GP.Kipp, has been reported for German and Australian blood donors GP.Yak amongst Japanese donors. Uchikawa et al reported Gp.Yak as a GPB*B-A-B hybrid, GPB(1-26)-GPwB(27-54)-GPA(55-57)-Bs(58-103). The serological data suggest that retention of the 208-210 TCC (51S in glycophorin Bs) codon from the GYPB pseudoexon, prior to the gene conversion insertion of GYPA sequence and coding for serine in the hybrid glycophorin, is the basis of ‘Anek-like’ activity for both hybrid glycophorins. It should be considered that these antisera react with a new Kipp-related MNS system antigen. Genetic studies revealed the same crossover for GP.Kipp and GP.Yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin.

2A-S02-03

MOLECULAR GENOTYPING OR SEROLOGICAL PHENOTYPING, WHICH IS MORE COST-EFFECTIVE Mohamed Sidik SB Health Sciences Authority, Singapore, Singapore Background: With the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. Molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one’s phenotype. There are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. Molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. However, there are several misgivings about the cost of molecular methods used to genotype antigens. Aim: To evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. Methods: A batch of 30 donor samples was typed using both serological and molecular methods. The test kit used for molecular methods, from Gen-Probe, included the typing for the following antigens – Kell (K, k, Kpa, Kpb, Kpc, Jsa and Jsb), Kidd – (Jka, Jkb, Jk), Duffy (Fya, Fyb, Fyx, FyGATAsil), MNS (M, N, S, s, S-s-

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

Uvar), Rh (C, c, E, e) and Dombrock (Doa, Dob). Negatives that are obtained using this method were confirmed using serology. Serological typing was performed with available antisera according to the various manufacturers’ instructions. This includes Kell (K, k), Kidd (Jka, Jkb), Duffy (Fya, Fyb), MNS (M, N, S, s) and Rh (C, c, E, e). The cost for the different tests were tabulated and compared. The cost includes labour, consumables and equipments. Results: The results show that molecular method of typing red blood cells is more expensive than the traditional serological method. The cost of consumables is comparable for both methods. The consumables make up about 80% of the total cost for serological methods, and about 82% for molecular methods. The cost of labour is about 9% for serological methods and C of the RHCE allele while probes targeted to 676C>G was utilised for Ee detection. For the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an East-Asian population. Results were analysed and integrated into the blood donor software system. Results: The 48C>G probe designed for identification of C was unsuccessful with poor discrimination between genotype calls. The remaining probes showed satisfactory discrimination between genotypes, with 462 of the 505 (91%) samples analysed, fully genotyped for the five alleles studied. Relatively rare genotypes were successfully identified using this strategy: ccEE (15/505, 3.0%), FYA-/FYB+ (19/505, 3.8%), SS (5/505, 1.0%). The CO2 allele responsible for Cob was identified in the heterozygous state in four of 494 evaluable samples giving an allele frequency of 0.4% in our population. The estimated reagent and consumables cost for sample DNA preparation and SNP testing was USD12.00 compared to USD27.15 for phenotyping using commercial anti-sera. We did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. Conclusions: Results of this study indicate that SNP genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell

T TID – Blood Safety and Pathogen Inactivation 11

inventory and genotyped whole blood donor pool. The cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. In addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera.

2A-S02-05

EXTENDED ERYTHROCYTE TYPING: MOLECULAR TECHNIQUE VS SEROLOGICAL TECHNIQUE

specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained.

TTID – Blood Safety and Pathogen Inactivation

Munno E1, Dell’Aversana MR1, De Caprio G1, D’ambrosio R1, Annarumma F2, D’onofrio M2 and Misso S1

2A-S03-01

1

Servizio Trasfusionale ASL Caserta, Aversa, Italy 2Servizio di Immunoematologia e Medicina Trasfusionale, Salerno, Italy

Benjamin RJ

Background: Over the past 20 years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. Since October 2012, in our Transfusion Center (ASL Caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and Rh phenotype. Aims: The aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. We also compared each method in regards to reliability, ease of use and reproducibility of results. Methods: We selected 250 donors aged 106 log reduction of various bacterial species. Most systems are less effective at inactivating bacterial spores, a particular problem as Bacillus spp. is common platelet contaminant. Testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. Conclusions: Clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. Blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection.

Table 1:

PATHOGEN INACTIVATION – ARE WE THERE YET? American Red Cross, Rockville, United States of America

2A-S03-02

EFFICIENT INHIBITION OF MITOCHONDRIAL DNA AMPLIFICATION USING A REAL-TIME PCR ASSAY AFTER TREATMENT WITH THE INTERCEPT SYSTEM FOR PATHOGEN INACTIVATION Bakkour S1, Dupuis K2, Chafets D1, Busch MP1, Stassinopoulos A2 and Lee TH1 1

Blood Systems Research Institute, California, United States of America 2Cerus Corporation, Concord, California, United States of America

Summary/Conclusions: The results show that the frequencies of more immunogenic antigens (K, FyA, JkA) reflect the specific and ethnic frequencies of the donors. We emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of 1:1000 according to American Rare Donor Program (ARDP), Council of Europe, International Donor Panel (IDP), International Society of Blood Transfusion (ISBT), Council of Europe, Japanese Red Cross). We only found one case of a donor expressing weak Fyb which is due to mutation in Fy 265C> T. The results were confirmed in 99.6% of cases by serological technique. However for the phenotype FyB Weak, the result was discordant in serology, where the result was fyb-. In conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. The molecular technique is able to identify mutations in particular genes, especially in

Background: The INTERCEPT Blood SystemTM for pathogen inactivation (PI) using amotosalen and UVA light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing DNA replication and RNA translation. PI technology has been adopted into routine use in some European countries and is under FDA review for licensure in the US. Current documentation of PI efficacy relies on illuminator sensors that measure the UVA light dose delivered. An indirect methodology is utilized for the validation and QC of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the Compound Removal Device. The% amotosalen remaining is a direct measurement of the UVA light delivered and photochemical conversion of amotosalen. However, a functional QC method that measures a direct target within the treated blood product has not been introduced into clinical use. Residual leukocytes, platelets and potentially plasma contain mitochondrial DNA (mtDNA) which is a collateral target of the PI process.

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

12 ISBT Academy Day

Aims: The goal of this study was to quantify the impact of INTERCEPT treatment on platelet-derived mtDNA by real-time PCR. Methods: To evaluate the feasibility of detecting PI-induced mtDNA modifications by real-time PCR, we spiked purified human leukocyte DNA into human plasma, 35% plasma/65% InterSol, or PBS. Each sample (3.6 ml) was treated with 150 lM amotosalen and 3 J/cm2 UVA (N = 2). Control samples were either untreated or treated in the absence of amotosalen or UVA. DNA was extracted from each sample (0.2 ml) in duplicate and assessed by measuring the inhibition of real-time PCR amplification in duplicate wells using mtDNA-specific primers and SYBR Greenbased detection. Amplification of sequences ranging in size from 73 to 1065 bp was evaluated over 45 cycles of amplification. Subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in 35% plasma/65% InterSol (30 ml). Six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtDNA sequences. Results: Treatment of purified DNA with amotosalen plus UVA resulted in 1.3 log to >6.0 log inhibition of PCR amplification (results shown in Table). The extent of PCR reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: PBS > 35% plasma > 100% plasma. Exposure of platelets to amotosalen and UVA showed an average of 2.5 log to 3.6 log reduction in PCR signal, with increasing inhibition observed for larger amplicons. In all cases, no PCR inhibition was observed in the absence of amotosalen and/or UVA. Conclusions: A quantitative real-time PCR assay specific for mtDNA is capable of documenting PI-induced collateral nucleic acid modification in platelets. Based on this work, this assay can be developed further for use as a quality control method for PI efficacy.

2A-S03-03

PATHOGEN REDUCTION TREATMENT AND SUBSEQUENT STORAGE OF BUFFY-COAT DERIVED PLATELETS ALTERS PRODUCTION OF MONOCYTE INFLAMMATORY MEDIATORS IN A WHOLE BLOOD TRANSFUSION MODEL Loh YS, Dean MM, Johnson L and Marks DC The Australian Red Cross Blood Service, Sydney, Australia Background and aims: Pathogen reduction technology (PRT) provides a proactive approach to improving transfusion safety for platelet concentrates (PCs). However, PRT treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. Little is known regarding how PRT-treated platelets may affect the cells of the recipient’s immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient’s inflammatory cells after exposure to PRT-treated platelets using an in vitro whole blood model of transfusion. Methods: Two ABO/RhD matched buffy coat derived PCs were pooled and split to form matched pairs on day-1 post-collection (n = 11). One unit was treated with the Mirasol PRTTM system (Terumo BCT), while the other unit remained as an untreated control. All units were stored at 22°C with agitation and samples were taken on day 2 and 7 post-collection for ‘in vitro transfusion’ experiments. To represent a transfusion in vitro, 10% v/v platelets were incubated with ABO/RhD-matched fresh whole blood, with or without lipopolysaccharide (LPS; 1 mg/ml) for 6 h at 37°C/5% CO2. Protein secretion was inhibited using brefeldin (2 lg/ml) to allow detection of intra-

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

cellular cytokine production in monocytes (CD45+/CD14+) and neutrophils (CD45+/ CD16+), using multi-colour flow cytometry. The fold change of cytokine production was calculated by comparison to a ‘no-transfusion control’ (whole blood without addition of platelets). Data was analysed using a one way ANOVA with post-hoc tests for pair-wise comparisons. Results: In the absence of LPS, both PRT-treated and untreated platelets stored for 7 days significantly increased monocyte MIP-1b expression (1.5-fold; P = 0.047), whereas exposure to day 2 platelets did not result in any significant differences in MIP-1b expression. As expected, LPS stimulation significantly increased monocyte production of both IL-12 (5.0- to 7.4-fold) and MIP-1b (1.8- to 2.4-fold). However, LPS-induced monocyte IL-12 production was significantly reduced by exposure to PRT-treated or untreated platelets stored for 2 days (PRT-treated: 3.0-fold, untreated: 2.5-fold; P < 0.0001) and 7 days (PRT-treated: 4-fold, untreated: 2.1-fold; P < 0.0001). Furthermore, IL-12 production was significantly lower following exposure to day 7 PRT-treated platelets compared to untreated platelets (P = 0.006); however there was no significant difference following exposure to day 2 platelets. LPS-induced MIP-1b production was not significantly differentfollowing exposure to either day 2 or day 7PRT-treated or untreated platelets. Exposure to platelets (untreated or PRT-treated) did not significantly modulate monocyte production of IP-10, MCP-1, MIP-1a, IL-1a, IL-1b, IL-6, IL-8, IL-10, or TNF-a. Co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. Conclusion: Using an in vitro whole blood transfusion model, we have demonstrated that exposure to PRT-treated platelets stored for 7 days results in significant changes in the IL-12 production by monocytes. These changes may reflect the way PRT-treated platelets interact with immune cells upon transfusion. Therefore, the effect of stored PRT-treated platelets, especially in recipients with underlying inflammation, should be further examined.

2A-S03-04

THE COMPARISON OF HCV SUBTYPES BETWEEN CLINICAL PATIENTS AND BLOOD DONORS IN GUANGDONG CHINA Rong X Guangzhou Blood Center, Guangzhou, China Backgrounds: Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitic disease. HCV has six genotypes and more than 80 subtypes. The epidemiology of HCV subtypes vary with different geographic distribution. Understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of HCV and thus help to make effective precautionary measures. HCV subtypes are also closely related with clinical therapeutic effect. It is important for guiding clinical therapy and prognosis and predicting the possible burden of HCV infection and treatment in the future. Aims: To investigate and compare the prevalence of HCV subtypes in clinical patients and blood donors in Guangdong China. Methods: Of 191 samples ofclinical patients and 222 samples of blood donors whose HCV RNA were positive were collected from Guangdong province. HCV NS5B gene was amplified by RT-nested PCR and then sequenced. HCV subtypes were assigned by constructing phylogenetic trees with MEGA5 software. Moreover, SPSS16.0 software was applied to compare the difference between these two groups. Results: Of 191 clinical patients, HCV genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 2 (1.05%), 127 (66.49%), 17 (8.90%), 5 (2.62%), 5 (2.62%), 34 (17.80%) and 1 (0.52%), respectively. Of 222 blood donors, HCV genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 1 (0.45%), 92 (41.44%), 1 (6.76%), 19 (8.56%), 11 (4.95%), 83 (37.39%) and 1 (0.45%), respectively. The proportion of HCV 1b was higher in clinical patients than in blood donors(v2 = 25.866, P = 3.66E-07), while the proportion of 3a and 6a subtypes were higher in blood donors than in clinical patients(v2 = 6.602, P = 0.010; v2 = 19.398, P = 1.06E-05). One possible cause was the transmission modes varied with different HCV subtypes. HCV 1b is more related with blood transfusion while 3a and 6a are more relevant to intravenous drug abuse and sexual behavior. With the anti-HCV screen implemented from 1993, the risk of HCV infection by transfusion is diminishing. The other reason was the average time from HCV infection to serious pathological lesions is about 20 years and HCV 6a was transmitted into Guangdong later than 1b. Conclusions: In Guangdong province, HCV 1b and 6a were the predominant subtypes in clinical patients and blood donors. The proportion of HCV 1b, 3a and 6a subtypes were significantly different between clinical patients and blood donors. The reason may relate with the time of HCV transmission into Guangdong area and the HCV propagation modes. Blood safety and increased public health initiatives to reduce HCV infection from those in these high risk groups to the general population remain a priority.

Plenary Session: It’s All About Red Cells 13

Materials and methods: Data concerning to transfused patients with positive HBVmarker were collected using results of the nationwide questionnaire survey in 2007– 2011. In this questionnaire, if there is a patient showing a positive result of HBsAg and/or HBVDNA evaluated by post-transfusion-test, it is requested that detailed patient’s characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of HBV-marker-test prior to the first transfusion, should report to the JSTMCT office. A number of HBsAg and/or HBVDNA positive patients were 234 cases in 2007–2011. Among them, 19 patients were not eligible because of the absence of results of HBV-related markers. Finally, a total number of 215 patients (36, 37, 41, 43, 45 patients in 2007, 2008, 2009, 2010, 2011, respectively) were enrolled for the present study. Results: All the eligible patients showed positive results of HBsAg and/or HBVDNA in samples obtained from three months after the last transfusion. We classified the cause of these results into five categories according to results of HBV-markers tests prior to the first transfusion. (i) If a result of HBsAg and/or HBVDNA performed prior to transfusion is positive, a patient is categorized as the HBV carrier group. (ii) A patient showing both a negative result of HBsAg and positive results of antiHBs and/or anti-HBc are categorized as the HBV past-infection group. (iii) If a patient shows no HBV-related marker prior to the first transfusion and patient’s HBVDNA is identical to donor’s HBVDNA, we categorized as the transfusion transmitted infections (TTI) group. (iv) If a patient does not show any HBV-related marker prior to the first transfusion and HBVDNA is not detected in donor’s blood by single NAT, we categorized as the unknown cause group. Results were summarized in Table 1.

Caption 1: The phylogenetic trees of HCV NS5B of clinical patients and blood donors

Caption 1: Cause of HBV-positive in transfused patients

Caption 2: The comparison of HCV subtypes in clinical patients and blood donors from Guangdong

2A-S03-05

DETECTION OF HEPATITIS B VIRUS FROM SERA OF TRANSFUSED PATIENTS IS NOT ALWAYS DUE TO BLOOD TRANSFUSION

Conclusion: Although the positive result of HBsAg and/or HBVDNA of transfused patients has been considered the sequel of blood transfusion, we showed that HBV reactivation was also an important cause of it. To distinguish HBV reactivation from transfusion transmitted infection, we have to perform HBV-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient’s serum.

Plenary Session: It’s All About Red Cells

Kino S

2B-PL1

Asahikawa Medical University Hospital, Asahikawa, Japan

THE MYTHS OF BLOOD GROUPS

Background: In Japan, we routinely evaluate the presence of transmission of HBV, HCV and HIV in all transfused patients at three months after the last transfusion. Although the sensitivity of detection of hepatitis B virus (HBV) in blood donation improved during recent years, transfusion transmitted HBV infection is left as a serious problem. The Japanese Society of Transfusion Medicine and Cellular Therapy (JSTMCT) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by Ministry of Health, Labour, and Welfare, every year. In this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion HBV-marker-test. Aims: The aim of this study is to elucidate the cause of HBV positive in transfused patients.

Daniels G NHSBT, Bristol, United Kingdom The English dictionary provides several definitions for the word ‘myth’. One definition is, ‘A widely held but false belief’. This seems suitable for the purposes of this presentation. But who decides what is false? As we shall see in the course of this presentation, some ‘myths’ of blood groups may not be myths at all, and some established facts may indeed be myths. Perhaps a more scientific definition would be, ‘A widely held but unproven belief’. ABO, the original and most important blood group in transfusion and transplantation medicine, has engendered many ‘myths’. These have mostly arisen through associations between ABO groups and other characteristics such as personality, dis-

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

14 ISBT Academy Day

ease, psychological traits, and ideal diet. Although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. For example, the statistical associations between ABO type and thrombosis, where a biochemical basis involving clearance of von Willebrand factor from the blood by enzymatic cleavage appears to be affected by ABH glycosylation. In Rh, the first myth was that the human antibody, now called anti-D, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. Hence the name Rhesus, now Rh, for the blood group system. In the 1940s, early serological work with Rh antibodies led to two genetic theories, involving either one or three Rh genes. Both theories have now been rejected because molecular genetics revealed two Rh genes. But was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? Since the 1960s it has been commonly understood that there are two types of variant D antigens: weak D and partial D. Policies for transfusing patients with these variants have often been based on this dichotomy. But is this a myth? The difficulty we have in defining the terms weak D and partial D suggests that it might be. It is always tempting to dismiss anything that we don’t agree with as a myth. AS Wiener, one of the discoverers of the ‘rhesus’ antigen, wrote several papers on ‘Blood group mythology’, doing just that. Scientists should beware of falling into this trap. Perhaps ‘myth’ is a term best avoided in science. 2B-PL2

FIVE NEW BLOOD GROUPS – WHAT NEXT? Storry JR University & Regional Laboratories, Clinical Immunology and Transfusion Medicine, Lund, Sweden The past 2 years has seen the discovery of five new blood groups. At the ISBT meeting in 2012, FORS, JR and LAN were ratified as blood group systems and since then, the molecular basis of the Vel blood group antigen has been elucidated, and the complement regulator protein, CD59 has been shown itself to be a blood group antigen. These last two discoveries will no doubt lead to their elevation to blood group systems. How has this happened? It turns out to be a mixture of old and new techniques. Rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. The development of comprehensive SNP arrays, exome sequencing and rapid sequencing techniques, e.g. NextGeneration sequencing, has provide us with tools for rapid discovery. Combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens Jra and Vel. However classic biochemistry and subsequent peptide and DNAsequencing still play an important role and lie behind the (simultaneous) discoveries of Jra and Vel, but also of Lan and FORS. A rare CD59-deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the CD59 protein, which was confirmed by routine DNA-sequencing. The Jra and Lan antigens were assigned to already well-investigated ABC-transporter proteins (ABCG2 and ABCB6 respectively) whose presence on the red blood cell (RBC) had not been established previously and for which the function on RBCs is still not known. FORS antigen was shown to result from the reactivation of the human pseudogene GBGT1. This enzyme builds the carbohydrate Forssman antigen on sheep and dog RBCs but is normally inactive in humans. A mutation that reactivated the enzyme explained the unusual Apae phenotype in two families. Vel was shown to be carried on a hitherto unknown protein on the RBC, SMIM1. The function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. Absence of CD59 whose function in complement regulation is well-understood, resulted in production of an alloanti-CD59, demonstrating immunogenicity of this protein for the first time. These simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group ‘home’ continues at a rate unparalleled since the 1990s. As the ‘-omics’ fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home.

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

TTID – Emerging Infections 2C-S04-01

EMERGING AND IMPORTED INFECTIONS IN THE REGION – WHAT’S BOTHERING US TODAY? Teo D Blood Services Group, Health Sciences Authority, Singapore, Singapore An emerging infectious disease (EID) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. In 1992, an Institute of Medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. East and Southeast Asia, with 30% of global population, has a reputation as a hot spot for EID. Within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. The emergence of severe acute respiratory syndrome (SARS) exactly 10 years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional EID threats. The re-emergence of highly pathogenic avian influenza A(H5N1) virus in 2004, isolation of novel bat-associated reoviruses in 2006, emergence of Artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in 2009 are some examples of EID emerging within the region since SARS. At the time of writing, the situation involving human cases infected with avian influenza A(H7N9) virus is evolving, and there has been a recent report of human infection with avian influenza A(H6N1) virus as well. Additionally, there are imported EIDs such as influenza A(H1N1) virus, West Nile virus, and the present cause for concern in Middle East Respiratory Syndrome Coronavirus. Climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. 40% of the world’s population is now at risk of dengue, with the majority living in the Asia-Pacific region. The scourge of dengue is sufficiently high in the region for the Association of Southeast Asian Nations (ASEAN) in 2011 to designate 15 June as ASEAN Dengue Day. Hepatitis E is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. The impact of EIDs on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. Blood products such as hyper-immune plasma preparations may be useful treatment options in some EIDs. There is a need for constant surveillance and the capacity to identify, assess and manage EID risks to the blood supply. In recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. Nonetheless, the volatile and ever-changing nature of EIDs will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community.

2C-S04-02

VIRAL TRANSMISSION RISK ASSESSMENT ON INDIVIDUALDONATION NUCLEIC ACID TESTING IN KUALA LUMPUR, MALAYSIA Lam STP1, Bon AH1, Mat Radzi M1, Mah E1, Abdul Halim MH1, Hassan R1 and Lelie N2 National Blood Centre, Kuala Lumpur, Malaysia 2Lelie Research, Paris, France

1

Background: The individual-donation nucleic acid amplification testing (ID-NAT) was introduced firstly in November 2007 at National Blood Centre, Kuala Lumpur (NBC-KL), Malaysia using automated Tigris System (Novartis). With the ID-NAT, a zero risk transmission is yet to be achieved. Aims: To determine the prevalence and residual transmission risk of HIV-1, HCV and HBV on blood donor screened from year 2008 to 2012. Methods: All blood donations were serologically screened with PRISM Analyzer (Abbott Laboratories, USA) for anti-HIV, anti-HCV and Hepatitis B Surface Antigen (HBsAg) and ID-NAT with Procleix Ultrio Assay on Tigris System, while Rapid Plasma Reagin (RPR) for syphilis screening. Immunoblotting and HBsAg Neutralization assays were used to confirm repeatedly reactive sample for anti-HIV, anti-HCV and HBsAg respectively. Reactive Ultrio samples were then subjected to discriminatory test to determine viral specific reactivity. Potential NAT yield cases were further

T TID – Emerging Infections 15

tested by alternate NAT and other supplementary tests. The prevalence rate of HBV, HCV and HIV are calculated from the first time donors. The residual transmission risk for repeat and lapsed donations were estimated using recently refined mathematical model of Weusten et al. 2011. Results: NBC-KL had screened 1,014,821 blood donations within 5 years (321,641 first time donations and 412,426 from repeat and lapsed donations). The prevalence of HIV-1, HCV and HBV was 0.04% (1:2803), 0.11% (1:921) and 0.31% (1:322) respectively. With ID-NAT, we have identified 18 HIV WP NAT yields (1:56,379), 6 HCV WP NAT yields (1:169,137), 51 acute phase HBV WP NAT yields (1:19,898), and 110 chronic OBI NAT yields donations (1:9226). The residual transmission risk per million donation were estimated at 2.0 (1 in 503,407) for HIV-1, 0.2 (1 in 4,395,251) for HCV and 8.45 (1 in 118,338) for HBV. Summary: The residual transmission risk is relatively high in HBV followed by HCV and HIV-1. This finding is not new as Malaysia is a country of medium seroprevalence for HBV which ranges from 1.5% to 9.8% in the general population, but is relatively low in blood donor population (0.04%). The OBI NAT yield was higher than acute phase WP in HBV-NAT yield. These OBI positivity have shown inconsistent NAT results on repeat testing and also low viral loads ranging from 46 years.

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

16 ISBT Academy Day

HAV seroprevalence was highest in the Black donors (86%), lower in Coloured donors (62%) and lowest White donors (36%). All were HAV IgM negative. There was an age-related increase from 44.8% (52/116) in those 16–25 years old to 81% (47/58) in those >46 years. Although no active HEV infection was identified by PCR on pooled samples, 25% of donors were HEV IgG positive. Rates were highest in Whites at 33%, followed by Coloured at 23% and lowest in Black at 19%. Again prevalence increased with age from 12.1% in those 16–25 years to 48.3% in those >46 years. Discussion: The results show a lower HAV seroprevalence in the White population compared to the Black and Coloured population groups, likely to be due to socioeconomic living conditions Such a contrast is striking given the introduction of democracy in South Africa almost 20 years ago. The reduction in anti-HBc (as a marker of past HBV infection) with age is reassuring, suggesting that HBV vaccination is impacting HBV population prevalence. The pattern of HEV exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. Given the subclinical nature of HEV infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors.

2C-S04-05

DISTRIBUTION OF HEPATOCELLULAR CARCINOMA-RELATED (HCC) MUTATIONS AND DRUG RESISTANCE RELATED MUTATIONS OF HBV IN CHINESE BLOOD DONORS Liu Y1, Wang J1, Huang Y1, Yao F2, Wen X3, Lv Y4, Bi X5, Xu J6, Liu J7, Ness P7 and Shan H7 1 Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China 2Yunnan Kunming Blood Center, Kunming, China 3Mianyang Blood Center, Mianyang, China 4Yunlai, Luoyang Blood Center, Luoyang, China 5Xinhong, Urumqi City Blood Center, Urumqi, China 6Guangxi Blood Center, Liuzhou, China 7Johns Hopkins University, Baltimore, United States of America

Background: HBV demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. In addition, mutations in the polymerase region may lead to drug resistance and changes in the PreS region (including deletions and mutations such as T31C and T53C) and PreC/BCP region (mutations including A1762T/G1764A, G1896A, T1896A, C1766T, T1768A) are associated with higher risk of hepatocellular carcinoma (HCC). Aims: To study the HBV subgenotype distribution and analyze the changes in PreS region, PreC/BCP and the polymerase region of HBV in Chinese blood donors. Methods: Of 245 blood samples were selected randomly from HBsAg positive blood donors from five blood centers in China. The PreS plus S region or the whole genome of HBV was amplified and sequenced and HBV subgenotype was determined. The amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. The nucleotide sequences of PreS region and PreC/BCP region were aligned and the mutations related to HCC were determined. Distribution of genotype, subgenotype, and mutations by different regions were examined using Chi-square statistics. Results: Of 200 samples (81.6%)were subgenotyped successfully. The predominant subgenotypes were B2, C2, D1 and A1 which accounted for 50.5%, 19.2%, 5.3%, and 3.4% respectively. Deletions and mutations (T31C and T53C) in PreS region were found in 28 (28/200, 14.0%) samples. Five of these 28 samples (2.5% of all samples) have deletions and no deletions specific to genotype D was detected. The prevalence of mutations in PreS region was significantly higher in genotype C than in genotype B (P < 0.001). Mutations in polymerase region were found in 14 samples (7%, 14/ 200), most of which were related to resistance to Adefovir and Lamivudine. Mutations in PreC/BCP region were found in 68 samples (29.8%, 68/228). The prevalence of HBeAg was significantly lower in samples with mutations in PreC/BCP region than that in the samples with no mutation (P = 0.02). More A1762T/G1764A mutations were found in C than B genotype while the opposite was observed for G1896A mutation (P’s < 0.01). Conclusions: Subgenotype B2 was the most frequent strain circulating in HBV infected Chinese blood donors, followed by C2. HCC related mutations were found less in PreS region but more in PreC/BCP region in blood donors. The prevalence of mutations in PreS region and A1762T/G1764A mutations was higher in genotype C than in genotype B while the opposite is the case for G1896A mutations. This is consistent with the distribution of HCC related mutations in general HBV carriers in China. Since all donors in this study reported not having received HBV treatment, it is not clear whether drug resistance mutations occurred spontaneously in the HBV-

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

infected blood donors or were acquired by the donors from HBV infected patients who underwent antiviral therapy.

Products and Components – Blood Components 2C-S05-01

A FRESH LOOK AT MEASURING QUALITY IN BLOOD COMPONENTS Devine D Canadian Blood Services, Vancouver, Canada Confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. The assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. However, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. This begins with the manner in which we conduct these tests. Although we describe our practice as ‘quality control’, it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. This testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. In addition, the standards used to assess blood components often have ‘wiggle room’. For example, the North American standards for the number of platelets in a whole blood-derived platelet concentrate require that at least 75% of tested products meet or exceed the required platelet count. In practice, this means that up to 25% of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. There are other examples in component quality standards of this same phenomenon. In an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. As a community, we have considerable work to do to get to this utopian ideal. First we must identify better product characteristics to use as standards for blood component production. Modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some 50 years ago, yet we have not applied these advances to quality assessment for blood products. Those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. Production methods are only one way to impact component quality; another is the actual features of the donors themselves. This biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor VIII or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. This presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality.

2C-S05-02

RESIDUAL PLATELETS POTENTIATE RELEASE OF PROCOAGULANT AND PROINFLAMMTORY RED CELL MICROPARTICLES (RMP) DURING BLOOD STORAGE Ahn YY, Gomez-Marin O, Bidot C Jr, Johansen M, Morgan J, Shariatmadar S, Horstman LL and Jy W University of Miami School of Medicine, Miami, United States of America Background: Blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. Microparticles (MP) released in packed red cells (PC) in storage have been suggested to be mediators for transfusion-related complications. However, the underlying mechanism for MP release during storage is mostly unresolved. Aims: To examine MP released in PC in storage for procoagulant and proinflammatory activity and define the role of residual platelets in PC in generation of MP during storage.

Products and Components – Blood Components 17

Methods: (i) Leukoreduced (LR) and non-LR (NLR) packed cells (PC) were stored according to blood bank standards and sampled at day 0, 10, 20, 30, and 40. Assay of MP was by flow cytometry using mAb to label CD235a, CD45, CD41, and CD62E. Thrombin generation (TG) and CD11b expression were used as measures of procoagulance and proinflammation, respectively. (ii) The impact of platelets was further evaluated by reconstituting LR PC with increasing concentrations of platelets at day 0. Results: (i) Multiple species of MP were released in a time-dependent manner. Using NLR PC, we found that, relative to day 0, red cell MP (RMP) were increased by 2.59 at day 20, and by 8.59 by day 40. Small amounts of MP from leukocytes (LMP), platelets (PMP), and endothelia (EMP) were detected, generally 30%, Eight patients (8/18, 44.4%) show 1 h PPR >60%, 10 patients (12/18, 66.7%) show 24 h PPR >20%. The mean 1, 24 h CCI and PPR values from the best donors were significantly higher than those from random donors they transfused before. Conclusion: The use of HLA-A,-B and HPA,ABO- compatible aphaeresis platelet improves posttransfusion 1, 24 h CCI values and percentage of platelet recovery in refractory patients. Transfusion with HLA-A,-B and HPA,ABO-matched platelets is mandatory to reduce the risk of bleeding in PTR patients. Refractoriness to platelet

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

30 ISBT Academy Day

transfusions developed at least in 50% of the patients we observed and to maintain a long-term platelet transfusion strategy. Establish large-sized platelet donor registry with HLA class I, HPA, ABO-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. The optimal strategy for platelet substitution in immunized patients remains a challenge.

Asian populations an international network within laboratories in South Asian region should be established in the future.

3C-S13-05

Products and Components – Blood Component Therapy

THE CLINICAL IMPACT OF ANTIBODIES AGAINST CD36 IN SOUTH CHINA

3C-S14-01

Xia W1, Xu X1, Ye X1, Fu Y1, Deng J1, Liu J1, Ding H1, Chen Y1, Shao Y1, Wang J1, LI H2 and Santoso S3

DO WE REALLY NEED FFP? THE EVOLVING ROLE OF PF24 AND PRE-THAWED PLASMA

1

Devine D

Guangzhou blood center, Guangzhou, China 2Department of Biotechnology, Guangdong Food and Drug Vocational College, Guangzhou, China 3He Institute for Clinical Immunology & Transfusion Medicine, Justus-Liebig Univ., Giessen, Germany

Background: Immunization against CD36 leads to the production of anti-Naka antibodies associated with fetal/neonatal alloimmune thrombocytopenia (FNAIT), platelet transfusion refractoriness (PTR) and post-transfusion purpura (PTP). However, no data regarding the clinical relevance of CD36 immunization is currently available for Chinese population. Study design and methods: Platelets and monocytes derived from 998 healthy blood donors were typed for CD36 deficiency using flow cytometry. In addition, four patients with suspected FNAIT (one case) and PTR (three cases) were investigated. Nucleotide sequencing was performed to identify the mutations underlying the CD36 deficiency. Transfection in mammalian cells (HEK-293T) with CD36 mutated constructs was conducted to confirm these results. Anti-Naka antibodies were screened by the use of platelet solid-phase kit (PAK-PLUS, GTI Diagnostics). Results: Of 18/998 blood donors failed to express CD36 on their platelets surface. In 5/12 individuals no CD36 expression was detected both on platelets and monocytes, suggesting that the frequencies of type I CD36 deficiency (platelets and monocytes) and type II CD36 deficiency (platelets only) were approximately 0.5% and 1.3%, respectively. Nucleotide sequencing analysis of type I CD36 deficient individuals revealed eight different mutations; four of them were not described so far. However, 1228-1239del ATTGTGCCTATT and 329-330delAC appeared to be the most common mutations related to type I CD36 deficiency in South Chinese population. Further analysis showed that the presence of anti-Naka antibodies in one healthy donor (Donor 1) as well as in three cases of PTR (Patients 2–4) and one case of FNAIT (Patient 1). These results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected CD36 cell lines. In all PTR patients, transfusion with platelets derived from CD36 negative donors resulted in good increment (24 h, PPR >20%). Table 1 shows the mutations found in these five GPIV defective individuals. Conclusions: More than 0.5% of CD36 type I deficient individuals are at risk to be immunized through blood transfusion or pregnancy in China. In this study, we could demonstrate that this immunization is of clinical relevance for the development of PTR and FNAIT. Therefore, testing of anti-Naka antibodies should be considered in suspected immune mediated thrombocytopenia. A national registry of CD36 deficient blood donors should be established to maintain bleeding disorders associated with anti-Naka antibodies. Since immunization against CD36 is conceivable for other

Table 1 Type I CD36 deficiency associated with anti-Naka isoantibodies Age/ CD36 Subjects Sex Plt/Mo

Antibody Mutations of Change in Type clinic CD36 gene amino acid

Canadian Blood Services, Vancouver, Canada Fresh frozen plasma (FFP) is defined as plasma frozen within 8 hours of collection. While this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on FFP. In many blood systems, an increasing role for plasma frozen within 24 h of collection (FP24) is seen. Such plasma shows little difference in functional protein levels when compared to FFP, with the exception of FVIII levels which a show time related decay of activity. Even factor VIII loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of FP24. In addition, the activity profiles of coagulation proteins in FP24 prepared in routine production closely resemble those of commercial pooled plasma products. Taken together, these observations have led many blood systems to move from the exclusive use of FFP to a mix of inventory of FFP and FP24, if not to the complete removal of FFP from their menu of offerings. The preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within 8 h of collection. Since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor VIII, in part owing to original role of the latter in the treatment of hemophilia A, collection of FFP has persisted as the starting material for cryoprecipitate production. In jurisdictions where hemophilia or other factor VIII deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. As data began to accumulate on FP24, similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. This led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from 24 h to up to 5 days, if stored at 4°C. Such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. From the perspective of health resources management, the use of both FP24 and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. With the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as AB plasma. This presentation will focus on a review of the relevant studies of plasma quality for FFP, FP24 and pre-thawed plasma. We will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented.

3C-S14-02

SOURCING CLINICAL PLASMA FROM MALE ONLY DONORS IN AUSTRALIA Chesneau S Australian Red Cross Blood Service, Melbourne, Australia

Donor 6

50/F

neg/neg I

Anti-Naka none

Patient 1

30/F

neg/neg I

Patient 2

11/M

neg/neg I

Patient 3

35/F

neg/neg I

Patient 4

22/M neg/neg I

Anti-Naka FNAIT Anti-Naka PTR Anti-Naka PTR Anti-Naka PTR

329-330delAC; Frameshift at C220Ta AA 110; Gln74Term Ser127Leu; exon C380T; 6 skipping 429+4insga Heterozygous Frameshift 329-330delAC at AA 110 Glu378Leu Homozygous T1163Aa Homozygous Frameshift 329-330delAC at AA 110

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

Background: In 2008, the Australian Red Cross Blood Service (Blood Service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of TRALI (Transfusion Related Acute Lung Injury). The challenge of sourcing all clinical plasma from male donors is exacerbated in Australia due to its adherence to the Council of Europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in Australia. Aim: The aim of the programme was to achieve a result of 100% of clinical plasma sourced from male only donors. Methods: Work began in early 2008 to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that

Products and Components – Blood Component Therapy 31

Caption 1:

could be broken down by blood group and by day of the week. This was then formulated into a suite of reports, highlighting opportunities and variance in performance. Based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: Incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. This assisted with prioritisation and workflow management. Additional deliveries of blood from collection centres to processing centres. A range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation Donor centre collection staff were trained to convert male AB donors over to plasmapheresis donation activity. Changes to Progesa to prevent manufacture of female plasma were made (after a time). Results: The first report in July 2008 showed that the Blood Service were issuing 87.71% male clinical plasma; the Group AB rate was 57.86%. The results improved dramatically by November 2010 in groups O, A and B (99.64%, 99.72% and 99.62% respectively), allowing for Progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. By March 2012, the results in group AB had improved to 97.77% (chart below) and the directive was given to manufacture male clinical plasma only as routine. January 2013 was the first month ever where 100% of all clinical plasma was sourced from male donors only. In 2012/13, 99.98% of clinical plasma was male only – the 0.02% constituted short lead time requests for IgA deficient plasma. Subsequent to a system change that allowed for a donor identification marker for IgA deficient donors, inventory levels of this sub-product have increased by 34%, negating the need to turn female production on to accommodate sporadic demand. Summary/conclusion: The multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of TRALI in Australia. This is an achievement that many thought impossible and one that many other blood services have been unable to attain.

3C-S14-03

LOWER RATE OF ADVERSE EVENTS WITH METHYLENE-BLUE TREATED PLASMA COMPARED WITH QUARANTINE STORED PLASMA Nussbaumer W, Mayersbach P and Schennach H University of Hospital Innsbruck, Innsbruck, Austria Background: Since the introduction of Methylene-Blue (MB) treated plasma (Theraflex, Macopharma, Frankfurt, BRD) in 2009 for our hospitals (University Hospitals Innsbruck, Austria) as an alternative to quarantine stored plasma(QP) number of transfused units per year has increased constantly. In 2011 French concerns about the safety of MB were published and caused a phasing out of MB treated plasma in France. Aims: Initiated by that report we reviewed all our plasma transfusions starting with 2009, analyzed adverse events (AE) separated for QP and MB and compared the frequency in total as well as split by years. Methods: We integrated a written reporting system for every released blood component in the late 1990s. Reported data are integrated in our software system and ana-

lyzed separated for MB treated plasma and quarantine plasma for the defined period. We compared number and quality of reported AE in total as well as for each year. Results: Between 2009 and June 30th 2013 44718 (17652 MB vs 27066 QP) units of plasma were requested from our clinicians. We received a written report of transfusion for 92.89% of the released units (92.65% MB vs 93.15 QP). Percentage of MB raised from 26.8% in 2009 to 64.4% in 2013. Number of reported side effects for the whole observation period was 53 for QP and 11 for MB (P < 0.01). These numbers corresponds to one reaction every 1487 units transfused for MB and one reaction every 476 units for QP or seven reactions every 10,000 units transfused for MB vs 21 reactions every 10,000 units transfused for QP. The calculated risk for an unexpected side effect using QP was three times higher (adds ratio = 3.13; 95% confidence interval: 1.63–5.99). However, splitting the data by year, MB group shows lower rate of transfusion reactions but difference reached significance only in 2009 and 2012 (P < 0.01). Nine from eleven patients showing AE for MB received between 3 and 70 more MB units after the event without any further AE, two had no need for further plasma units. One patient received on the day of his event MB and FFP simultaneously, the event was counted for the MB group. Days between event and last MB transfusion varied between 1 and 11 days. All adverse events in the MB group were qualified s allergic reactions (Pruritus 2, Urticaria 4, Flush 2, Hypotension 1, Stridor 1 and Hypertension 1), non were of serious character. Summary: Our data do not indicate a higher risk potential for MB units compared with quarantine stored units despite the increasing number of transfused MB treated units over the last 4 years. Rate of side effects is significantly lower with MB units compared with QP for the whole observation period, although significance could only be reached in 2009 and 2012 if data are split by year.

3C-S14-04

QUALITY OF PATHOGEN AND LEUKOCYTE INACTIVATED RBC COMPARED TO CONVENTIONAL AND GAMMA IRRADIATED RBC Erickson A1, Donnelly B1, Schott MA1, Palascak M2, Franco R2, Knutson F3, Lof H3 and Mufti N1 1

Cerus Corporation, Concord, United States of America 2University of Cincinnati College of Medicine, Cincinnati, United States of America 3Uppsala University, Sweden, Uppsala, Sweden

Background: The INTERCEPT Blood System for Red Blood Cells (RBCs), using the proprietary compound S-303, has been developed to inactivate pathogens and leukocytes in RBC components for transfusion. Previous studies using the first and second generation pathogen inactivaton (PI) systems have demonstrated >5 log of leukocyte inactivation, effective PI against a spectrum of pathogens found in RBC components, comparable in vitro quality to conventional RBCs and successful 24-h recovery per the FDA criteria. The INTERCEPT Blood System for RBCs is currently in advanced clinical development in the US and EU. Aims: The purpose of these studies was to demonstrate the quality of stored PI RBCs as compared to conventional and gamma irradiated RBCs. Methods: Study 1: Whole blood (450 ml) was stored overnight at 4°C, leukocytedepleted (LD) and separated into platelet poor plasma and RBCs. ABO matched pairs of AS-5 (Optisolâ) RBCs were combined and split into units of 283–306 ml (N = 6). Study 2: Whole blood (450 ml) was separated into plasma, buffy coat and RBCs after being held on cooling plates for a minimum of 2 h post-collection. ABO matched pairs of LD SAG-M RBC were combined and split into units of 284  7 ml (n = 11). Control (C) RBC units in study 1 were stored at 4°C,Test (S-303 RBC) units for both studies(T1 and T2) were treated with the second generation S-303 treatment system and stored in SAG-M. In study 2, the untreated unit of each replicate was exposed to 25 Gy of gamma irradiation (IR RBCs) on D1. Study units were sampled for in vitro analyses throughout the storage duration. Results: In Study 1, T1 units had 49  2 g of Hb; while C units had 46  1 g of Hb. In Study 2, both T2 units and IR RBCs had 45  2 g of Hb. The extracellular protein was reduced 10-fold in T1 units (74  11 mg/dl) compared to C units (775  80 mg/dl) and was reduced 7-fold in T2 units (26  7 mg/dl) compared to IR RBCs (193  33 mg/dl). For Study 1, after 35 days of storage hemolysis, ATP, K+, Na+, MCHC and MCV were not different between T1 and C units (Table 1). Rejuvenated T1 and C RBCs showed the ability to generate 2,3 DPG and ATP and they had equivalent p50 values. For study 2, on D28, the expiration for IR RBC, T2 had significantly higher ATP, Na+, and MCHC and significantly lower hemolysis, pH, plasma free Hb, K+, lactate and MCV; these significant differences were consistent over 42 days of storage (Table 2). T2 had significantly less red cell microvesicles on D28 than IR RBC (1565  1856 vs 3716  2775 vesicles/ll; P = 0.017).

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

32 ISBT Academy Day

Conclusions: S-303 treated RBCs met current EU and AABB guidelines for LD RBCs with respect to Hct, Hb, and hemolysis. S-303 treated RBCs had improved in vitro quality compared to gamma-irradiated RBCs with respect to potassium leakage and hemolysis post-processing, throughout the storage period while containing less plasma protein. All measured in vitro parameters of S-303 treated RBCs indicate suitability for transfusion. The INTERCEPT System for RBCs is not approved for commercial use.

tocol. Five units of BCs were pooled using the ‘train’ method with addition of 280 ml of PAS. After a soft spin centrifugation in a refrigerated blood centrifuge (Beckman Model J6-MI, Rotor JS-4.2, 1300 rpm [498 g], 8 min, 22°C), PC were slowly transferred into a 450 ml platelet bag. The PC pools were treated with INTERCEPT Blood System for Platelets before the end of Day 1 according to package insert instruction. Results: Quality control (QC) parameters were measured in the prepared blood components and were listed in Table 1. By standardising BC volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in SAGM and plasma units while WBC and RBC contamination levels in PC and plasma were maintained low. All parameters were well within the blood component specifications set out in the Council of Europe Guide (16th Edition), the standard adopted by HKRCBTS. QC parameters of the pathogen reduction-treated platelet concentrates in PAS so produced were also within the HKRCBTS blood component specifications. Low contamination with red cells and white cells were demonstrated and the pH range was acceptable after 5 days of storage at 20–24°C. Conclusion: The new T&B production method for the separation of 450-ml Quadruple WB and the preparation of INTERCEPT platelet concentrates in SSP+ PAS was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates.

Donors and Donation – Donor Management

3C-S14-05

PREPARATION OF POOLED BUFFY-COAT PATHOGEN REDUCTION-TREATED PLATELET CONCENTRATES IN PLATELET ADDITIVE SOLUTION USING TOP AND BOTTOM METHOD Chan CMY, Lau TKC, Chui BTK, Lee CK, Chua EKM, Tsoi WC and Lin CK Hong Kong Red Cross Blood Transfusion Service, Hong Kong, China Background: In preparation for clinical evaluation of pathogen reduction-treated platelet concentrates (PC) in Platelet Additive Solution (PAS) by INTERCEPT (Cerus, Concord, CA), production of buffy-coat (BC) platelet concentrates in SSP+ (Macopharma, Tourcoing, France) from whole blood (WB) donations was introduced in the Hong Kong Red Cross Blood Transfusion Service (HKRCBTS). The preparation of BCremoved red cells, plasma and PC from 5 units of BC in PAS was validated using quadruple Top and Bottom (T&B) blood bag system. Aim: To validate the preparation of pooled buffy-coat pathogen reduction-treated platelet concentrates in PAS. Method: One hundred and five WB (450 ml  10%) donations were collected into T&B quadruple collection systems (JMS, Singapore). After overnight temperature-controlled storage at 22°C, WB was separated with T-ACE II+ automated blood extractors (TerumoBCT, Lakewood, CO) into BC-removed red cells, plasma and BC within 24 h of blood collection. The BCs so produced were standardised to the target parameters (volume: 50 ml; haematocrit: 36%) before addition of PAS according to the following pro-

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

3C-S15-01

CURRENT ISSUES IN DONOR HEALTH AND SAFETY Lin CK Hong Kong Red Cross Blood Transfusion Service, Kowloon, Hong Kong Blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. They should be managed in a way that ensures high standards of care. Nevertheless, there are recognized adverse reactions that can occur during blood donation. The overall incidence of complications directly related to blood donation is 1%. They are generally more common in women, in younger and in first-time donors. Although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. Adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. Vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. The majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. Other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. Staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. The incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary.

Donors and Donation – Donor Management 33

Iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. As each donation causes a loss of 213–236 mg of iron, repeat donation can lead to a continuous depletion of body iron stores. Studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. To address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. Adverse reactions such as delayed faint may occur after the donor leaves the donation venue. Donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. A system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. All adverse events and reactions in donors should be identified, documented and reported. These data should be regularly analysed for possible corrective and preventive actions. The goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. For various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. Not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly.

3C-S15-02

DONOR VIGILANCE – A GLOBAL UPDATE Wiersum-Osselton JC TRIP National hemo- and biovigilance office, The Hague, The Netherlands Background: Without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. Donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. Method: This ‘global update’ draws on work by the International Haemovigilance Network and International Society for Blood Transfusion haemovigilance working party, experience in The Netherlands, as well as a Pubmed search using terms blood donor and adverse reaction. Results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. Results and discussion: The occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. A reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. Needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. Repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. Some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. Fewer studies on acute complications in plasma and other types of apheresis have been published. Preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. International collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. Conclusion: Donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. There remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation.

3C-S15-03

DEMOGRAPHIC PROFILE OF HIV DONORS WHO DONATED DURING THE SEROLOGICAL WINDOW PERIOD Wooi-Seong K, Choo PY, Johari NL, Mah E, Hassan A, Hassan R and Ayob Y National Blood Centre, Kuala Lumpur, Malaysia Background: Blood safety is a national priority for Blood Transfusion Services in Malaysia. Since the HIV/AIDS epidemic in the 1980’s and the realization that HIV is a transfusion transmissible infection, mandatory screening of blood for HIV using enzyme immunoassay was implemented nationwide in 1986. Advances in HIV

screening have led to the discovery of Nucleic Acid Test (NAT), which has considerably shortened the HIV serological window period, thereby improving blood safety. At National Blood Centre (NBC), routine HIV screening of blood donors with NAT was implemented in 2007. Despite the recruitment of voluntary and non-remunerated donors, informing high-risk donors to self-defer and mandatory donor predonation interview, high-risk donors are still donating blood even during the HIV serological window period. Hitherto, the demographics of HIV blood donors in Malaysia who have donated during serological window period have not been studied. Assuming that these donors were newly infected, it is crucial for BTS to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. Aims: This study aimed to profile blood donors who donated during the HIV serological window period and to identify the risk factors of these blood donors. Methods: Past donor records of blood donors who had donated blood during the HIV serological window period (NAT Ultrio and discriminatory detected and negative for both anti-HIV and p24 antigen) at NBC or at blood mobiles organized by NBC from November 2007 until July 2013 were retrieved and analyzed using SPSS 15.0. Results: A total of 18 donors were NAT detected and negative for anti-HIV and p24 antigen (none in 2007, 2 in 2008, 2 in 2009, 1 in 2010, 6 in 2011, 4 in 2012 and 3 in 2013). The age of donors ranged from 18 to 50, with a mean age of 28.94 years old. Majority of them (66.6%) were younger than 30 years old, with 22.2% of the donors aged 20 and below. All except one of the blood donors were males (94.4% males vs 5.6% females). The ethnic composition was 50% Malay, 26.8% Chinese and 22.2% Indian. Fifteen donors were single (83.3%) compared to 3 (16.7%) who were married. Almost half of the donors were students (44.4%) and 27.7% were professionals. Fifteen of the donors (83.3%) were seroconvert donors, one of whom had 49 previous donations. Only 3 (16.7%) of the donors were lapsed donors. The risk factors were MSM (27.8%) followed by sexual encounter with sex workers (11.2%) and multiple sex partners (5.6%). Two donors (11.1%) denied any risk factors and eight donors (44.4%) did not return for post-donation counseling. Conclusions: Majority of the donors who had donated during the HIV serological window period were seroconvert donors, single males in the young age group and students. MSM remained the commonest risk factor. Education and awareness should be intensified and targeted towards these groups of donors. NAT should be a part of blood screening to improve blood safety. 3C-S15-04

A NATIONWIDE SURVEY ON KNOWLEDGE, ATTITUDE AND PRACTICES TOWARDS BLOOD DONATION IN PAKISTAN Zaheer HA and Waheed U Safe Blood Transfusion ProgrammeP, Islamabad, Pakistan Introduction: In Pakistan, the predominant reliance for blood supply is on the Replacement donors, as sufficient numbers of voluntary blood donors are not available. An increase in the proportion of voluntary donors following the promotion of the concept of Voluntary Non-Remunerated Blood Donation (VNRBD) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. Objective: The objective of the current study was to promote VNRBD through a Public Awareness Campaign (PAC) based on a thorough analysis of the Knowledge, Attitudes and Practices (KAP) of a key segment of the society, i.e. 18–25 years old college and university students. Material and methods: A cross-sectional, descriptive study was conducted over a period of three months (Jan–Mar 2012). Multi-stage random cluster sampling approach was followed and college and university students were targeted through 20 University based Blood Donor Organisations (BDOs) out of a total 56 BDOs identified. All the participants voluntarily participated in the study and informed consent was obtained orally. A pre-tested questionnaire comprising of 28 questions related to Knowledge, Attitudes and Practices was applied. The questionnaire was kept anonymous and each question included multiple options or statements. Statistical analysis was conducted by the assistance of Statistical Package for Social Sciences (SPSS) software version 17. Results: A majority (65%) of the students had heard about blood donation through family/friends and a minority (9%) through the internet, although 45% preferred internet as spare time activity. Majority (+85%) of the students had access to the internet and mobile phone. More than 50% of the respondents had donated blood: 22% donated for family, relatives or friends, 4% donated voluntarily as an act of altruism, and 25% donated voluntarily once and then stopped donating, but 55% of these respondents still considered themselves as volunteer blood donors. 35% indicated that important people in their environment had an influence on crucial decisions that they made. Motivation for blood donation was a desire to help other people (73%), 28% followed friends invitations, in 21% cases respondents family or friends had received blood transfusions, 16% followed the example of fellow students. Restriction for blood donation: 40% generally feared donation, 25% had a specific

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

34 ISBT Academy Day

fear of the needle, 20% had no confidence in the public (health) sector, 25% condemned blood selling practice, 14% had no confidence in the donation procedure (hygiene), 13% experienced parental discouragement. Conclusion: To overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. This strategy will help convince family replacement donors to become VNRBD and also recruit healthy individuals to become a VNRBD. The approaches and strategies for this transition can be based on the findings of the study. The reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a PAC, for which detailed study is required.

Clinical – Dilemmas in Clinical Transfusion 3D-S16-01

MANAGING THE PLATELET REFRACTORY PATIENT Murphy MF NHS Blood & Transplant, Oxford, United Kingdom Platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. There are immune and non-immune causes of platelet refractoriness. The main immune cause is HLA alloimmunisation which occurs predominantly in females with a history of pregnancy. Other immune causes include HPA alloimmunisation, ABO incompatibility, platelet autoantibodies and drug-related platelet antibodies. The incidence of alloimmune platelet refractoriness due to HLA antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. In current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, DIC and splenomegaly. If platelet refractoriness occurs, a clinical assessment should be made for clinical factors associated with non-immune platelet consumption. If there are no obvious clinical factors present, an immune mechanism should be suspected and testing for HLA antibodies conducted. If HLA antibodies are detected, HLA-matched platelet transfusions should be used. There are a number of methods including computer algorithms to determine HLA compatibility and acceptable HLA mismatches. HLAmatched platelet transfusions are not indicated when serological testing has failed to detect HLA antibodies; further consideration should be given to the presence of non-immune clinical factors, and testing for HPA antibodies. Responses to HLA-matched platelet transfusions should be carefully monitored, ideally with post-transfusion platelet counts both 1 and 20–24 h post-transfusion. If there are improved responses, HLA-matched platelet transfusions should be continued. If there are poor responses to HLA-matched platelet transfusions, the reasons should be sought including HLA incompatibility which is most likely to occur in patients with unusual HLA types with few well-matched donors, non-immune platelet consumption, and HPA and ABO incompatibility. Further serological investigations including testing for HPA antibodies may be used to differentiate between these possibilities. Depending on the results, the appropriate management could be the use of ABO-identical or HPA-matched platelet concentrates if the specificity of the HPA antibodies can be identified. Platelet crossmatching may be helpful in some patients with non-specific HPA antibodies. The management of patients with HLA and/or HPA alloimmunisation and no compatible donors may be very difficult. There is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. If bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. Other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. The management of patients with non-immune platelet consumption is similarly problematic. The usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased.

© 2013 The Author Vox Sanguinis © 2013 International Society of Blood Transfusion Vox Sanguinis (2013) 105 (Suppl. 2), 1–132

3D-S16-02

MANAGING BLEEDING IN CARDIAC SURGERY Ariff MH National Heart Institute, Kuala Lumpur, Malaysia Managing bleeding in cardiac surgery: Despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. Among all major surgical procedures, cardiac surgery with CPB still consumes a large part of the available blood supply. In England indicated that 10–15% of the blood units supplied by the National Blood Service is used in cardiac surgery units. In the USA, nearly 20% of blood transfusions are associated with cardiac surgery. During the early history of cardiac surgery, patients received large amounts of allogeneic blood. In the 50’s, most operations were performed to correct congenital heart disease. During the 60’s and 70’s, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. In the 80’s pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. With increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. Not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. Perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient’s preoperative RBC mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. The ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. Using the TRAC and TRUST scoring system predicts candidates likely for transfusion. Diminished RBC mass appears to be one of the strongest predictors of transfusion. The acceptance of a lower postoperative haematocrit (in IJN the Hct on bypass is 20–25% and post bypass is >25%) or haemoglobin concentration represent an important element in current blood conservation practice. The decision to transfuse a patient cannot be based only on haematocrit concentration. Optimizing preoperative RBC mass involves the early detection of anaemia and its correction before surgery. Preoperative autologous blood donation can be used to conserve allogeneic blood. Besides economic concerns, one essential argument against PAD is the lack of sufficient time because of the uncertainty of waiting list. Erythropoietin has also been used to augment PAD in elective cardiac surgery. Acute normovolaemic haemodilution (ANH) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. Perioperative cell salvage (CS) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. Antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotinin – now unavailable) may reduce excessive fibrinolysis and platelet dysfunction. The use of activated F VII has been reported in intractable bleeding post cardiac surgery.

3D-S16-03

UNDERLYING PRECIPITANTS DO NOT INFLUENCE CLINICAL PRESENTATION OR THERAPY FOR TTP: DATA FROM THE AUSTRALIAN REGISTRY Engelbrecht S1, Wood E2, McQuilten Z2, Best R2, Cannell P3, Hsu D4, Isbel N5, Kausman J6, Opat S7, Phillips L8, Polizzotto M2, Roxby D9, Sloane J2, Ward C10 and Cohney S11 Australian Red Cross Blood Service, Melbourne, Australia 2Monash University, Melbourne, Australia 3Royal Perth Hospital, Perth, Australia 4Liverpool Hospital, Liverpool, Australia 5Princess Alexandra Hospital, Brisbane, Australia 6The Royal Children’s Hospital, Melbourne, Australia 7Monash Health, Melbourne, Australia 8 Calembeena consulting, Melbourne, Australia 9Flinders Medical Centre, Adelaide, Australia 10Royal North Shore Hospital, Sydney, Australia 11Western Hospital, Melbourne, Australia

1

Background: Historical approaches used to distinguish thrombotic thrombocytopenic purpura (TTP), haemolytic uremic syndrome (HUS) and other thrombotic microangiopathies (TMA) included underlying conditions/precipitants, predominant clinical presentation and laboratory parameters. Tests for ADAMTS13 are now incorporated into diagnosis and classification. Plasma exchange (PEx) with fresh frozen plasma (FFP) or cryodepleted plasma (CDP) remains the standard of care for TTP.

Clinical – Dilemmas in Clinical Transfusion 35

immunosuppression was not only used in idiopathic cases, but also in secondary cases. Mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed.

3D-S16-04

INTRAOPERATIVE BLOOD REQUIREMENTS DURING LIVING DONOR LIVER TRANSPLANTATION IN PEDIATRIC RECIPIENTS Sachan D1, Shanmugam N2, Kaliamoorthy K2, Srinivasan V1, Venkataraman J2 and Rela M2 Global Health City, Chennai, India 2Institute of Hepatology, Liver Transplantation & HPB Surgery, Global Health City, Chennai, India

1

Caption 1: Plasma exchange product

Caption 2: Additional therapy

Additional immunosuppression may be used in patients with low ADAMTS13 due to autoantibodies. Aims: To investigate differences between idiopathic and secondary TTP with regard to clinical presentation, management and outcomes. Methods: The Australian TTP registry was established in 2009, and expanded in 2012 to include all TMA. Of 34 hospitals currently participate in the registry. Of 103 cases in 94 patients were registered from 18 sites; eight were excluded for incomplete data, leaving 95 episodes in 86 patients. Results: Median age was 46 years (15–83 years), 53% were female, 16% were relapses. Of 51% were idiopathic and 49% had ≥1 identified precipitant, including infection 23%, autoimmune 18%, malignancy 12%, medication 14%, pregnant/postpartum 4%, solid organ transplant 3%, and allograft 3%. ADAMTS13 levels were available in 66 cases (37 idiopathic and 29 secondary). ADAMTS13 was

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.

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. - PDF Download Free
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