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Individuals with primary or secondary immune-deficiency diseases may be at risk for vaccinia infection if widespread smallpox-immunization programmes are implemented in the United States of America (USA) for bioterrorism preparedness. The objective of this study was to determine whether commercial immune globulin (intravenous, human) products contain biologically active antibodies to vaccinia that have the potential to protect people, with immune deficiencies, from complications of vaccinia. Eight currently United States (US)-licensed and two European intravenous immunoglobulin (IVIG) products were tested in a vaccinia plaque-reduction neutralization assay. The in vivo activity of five of these lots was assessed in severely immune-deficient mice. All tested products contained neutralizing anti-vaccinia activity, in vitro and in vivo. The use of IVIG by individuals with inherited or acquired humoral immune deficiencies may provide some protection if they are inadvertently exposed to vaccinia. [ABSTRACT FROM AUTHOR]

In cold agglutinin disease, monoclonal red blood cell autoantibodies, termed cold agglutinins, induce haemolysis in patients exposed to the cold. Commonly, these autoantibodies are directed against the developmentally regulated I/i blood groups. A second blood group system, the Pr system (located on glycophorins), is involved less frequently. Anti-Pr cold agglutinins recognize either alpha 2,3- or alpha 2,6-linked N-acetylneuraminic acid as the immunodominant group. Cold agglutinins of anti-I/i specificity show a remarkable restriction in their genomic repertoire of the immunoglobulin heavy and light-chain immunoglobulin-variable domain (i.e. exclusive use of VH4-34 in heavy chains). For anti-Pr cold agglutinins, preliminary data on the repertoire of the light-chain variable domain indicate a preference for the subgroup Vkappa IV. To elucidate restrictions in the light-chain variable-domain subgroup repertoire of anti-Pr cold agglutinins systematically, and to discuss these results in the context of their anti-Pr1–3 subclassification and immunodominant sialic acid, light chains in 13 anti-Pr cold agglutinins were investigated. The anti-Pr light chains were isolated using temperature-dependent absorption/elution techniques. Subsequently, they were subjected to N-terminal Edman degradation, and the light chain Vkappa subgroup was affiliated using the Kabat database. Five of 13 (38%) light chains belonged to Vkappa IV, five of 13 (38%) to Vkappa I and three of 13 (23%) to Vkappa III. Anti-Pr with Vkappa IV subgroup light chains exclusively recognized alpha 2,3-linked N-acetylneuraminic acid. Including data from the literature, the repertoire of the light-chain variable domain in pathological anti-Pr cold agglutinins exhibits a clear bias towards the use of the single germline gene-derived subgroup, Vkappa IV (eight of 17 or 47%). The association of Vkappa IV subgroup light chain-containing anti-Pr cold agglutinins with binding to alpha 2,3-, but not alpha 2,6-linked N-acetyneuraminic acid raises speculations about a possible role of subgroup-derived determinants in anti-Pr binding. [ABSTRACT FROM AUTHOR]

This study was conducted to analyse the usefulness of hepatitis C virus (HCV) core antigen tests for the confirmation of HCV infection in a donor presenting as nucleic acid amplification technology (NAT) positive but negative for antibodies to HCV (anti-HCV). Blood donations were screened, in parallel, for anti-HCV using the Abbott PRISM HCV Chemiluminescent immunoassay (ChLIA) and an ‘in-house’ HCV NAT (pools of up to 95 donations). An HCV NAT-positive antibody-negative donor was identified. Twelve follow-up samples were obtained and tested with various HCV antigen (including the recently marketed Trak-C second-generation assay) and HCV antibody assays. The single HCV NAT-positive, antibody-negative donation was identified from 1 117 681 donations screened in the 4-year period, July 1999 to June 2003. The index donation was positive by Ortho HCV core antigen enzyme immunoassay (EIA) and Ortho Trak-C (second-generation HCV core antigen EIA). An archive sample, taken 127 days prior to the index donation, was negative for all HCV markers. Subsequent samples demonstrated a loss of reactivity in the Ortho HCV core antigen EIA and reduced activity in the Ortho Trak-C until day 69. Immunoblot (Ortho RIBA-3) and HCV PRISM became positive on day 62, whilst Ortho HCV ELISA was not positive until day 132 or Biorad HCV ELISA until day 160. An alternative immunoblot (Innogenetics Innolia III) was positive from day 55. RNA levels fluctuated considerably during the follow-up period, being completely undetectable by routine screening methods at the time-point around seroconversion; subsequently, antibody was detected using all assays investigated. This HCV-converting blood donor provided a unique panel of samples for using to assess current (and future) HCV assay systems. The overall test results led to the conclusion that individual HCV antigen testing should not be considered as equivalent to HCV NAT minipool screening. Trak-C antigen testing may be considered as a suitable confirmatory assay for isolated HCV NAT reactivity. [ABSTRACT FROM AUTHOR]

Administration of diclofenac may lead to immune haemolytic anaemia (IHA) owing to the presence of drug-dependent antibodies and/or autoantibodies. A relationship with oral or intramuscular drug administration is unknown. Here, we describe a patient who apparently tolerated oral diclofenac but developed severe IHA following intramuscular injection of the drug. A 66-year-old-female was admitted to hospital because of jaundice and nausea, which were initially presumed to be manifestations of a postcholecystectomy syndrome. The patient soon developed haemolysis and renal failure. Although the symptoms and signs were suggestive of autoimmune haemolytic anaemia (AIHA), the patient had diclofenac-induced IHA. Serological testing, including detection of drug-dependent antibodies, was performed using standard techniques. The patient's serum was found to contain a highly reactive diclofenac-dependent red cell antibody of the immune complex type (titre 256 000). She recovered after 7 weeks of treatment with prednisolone, blood transfusions, haemodialysis and plasma exchange. Diclofenac-induced IHA should always be considered when a patient on diclofenac develops haemolysis. [ABSTRACT FROM AUTHOR]

Background and Objectives: The RHAG blood group system contains five antigens: Duclos (RHAG001), Ola (RHAG002), DSLK (RHAG003), Kg (RHAG005) and SHER (RHAG006). Individuals who are DSLK‐negative and Kg‐positive have the same allele RHAG*01.–3, with a single‐nucleotide variation (rs144305805), c.490A>C (p.Lys164Gln), in exon 3 of the RHAG gene. We aimed to confirm whether DSLK and Kg are antithetical antigens. Materials and Methods: Blood samples of the original DSLK‐negative proband with anti‐DSLK, her son and another DSLK‐negative individual were examined. The RHAG gene was analysed by polymerase chain reaction and Sanger sequencing. Immunocomplex capture fluorescence assays (ICFAs) and monocyte phagocytosis assays were performed to characterize the anti‐DSLK antibody. Cross‐testing of alloanti‐DSLK and monoclonal anti‐Kg (OSK46) was performed using transduced HEK293 cells by inducing the construct of expression vectors encoding wild‐type RHAG*01 or the variant RHAG*01.–3. Results: ICFA using monoclonal anti‐RHAG (LA18.18) revealed that the anti‐DSLK and anti‐Kg antibodies reacted with the wild‐type and variant RhAG (Rh‐associated glycoprotein), respectively. The proband and a DSLK‐negative individual appeared to be homozygous for variant RHAG*01.–3, and the proband's son was typed as RHAG*01/RHAG*01.–3 heterozygote. HEK293 cells with wild‐type RhAG reacted with the anti‐DSLK but not anti‐Kg antibody, whereas HEK293 cells expressing the variant RhAG reacted with the anti‐Kg but not anti‐DSLK antibody. Monocyte phagocytosis assays indicated that 64% of red cells sensitized with anti‐DSLK were phagocytosed by monocytes. Conclusion: Our results demonstrate that DSLK and Kg are antithetical antigens in the RHAG blood group system. Anti‐DSLK may be a clinically significant antibody. [ABSTRACT FROM AUTHOR]

Background and Objectives: Australian Red Cross Lifeblood (Lifeblood) performs red blood cell (RBC) antibody screening on every whole blood donation. An alternate strategy has been proposed whereby an antibody screen is performed on the first donation and only repeated following pregnancy, transfusion or a significant break between donations (>2 years). We assess the blood safety risks associated with removing antibody screening for every whole blood donation. Materials and Methods: A retrospective desktop analysis included all whole blood donations collected by Lifeblood between 01 May 2018 and 30 April 2019 to quantify the antibodies that would have been undetected with the alternate strategy. The strategy was further assessed using the Alliance of Blood Operators Risk‐Based Decision‐Making framework. Results: One hundred and seventy‐one routine donors had antibodies for the first time, but reported no sensitizing event since their last donation. Forty‐seven of these had antibodies of a clinically significant specificity and titre that have the potential to cause a haemolytic transfusion reaction (HTR). The calculated risk of undetected antibodies being transfused to an incompatible recipient is 1 in 82,200. Conclusion: The estimated risk of HTRs with the alternate strategy results in an increased risk. While the alternate strategy is identified as the most cost‐effective option within the Australian setting, this additional residual risk was not deemed to be acceptable. Blood services would need to determine whether the increase in residual risk stemming from implementation of such a strategy is tolerable. [ABSTRACT FROM AUTHOR]

Background and Objectives: The use of immunoglobulin (IG) solutions as an immunomodulatory therapy in certain neurological conditions has become an established modality and represents a significant proportion of total IG use. The estimation of the evidence‐based potential demand designated as the latent therapeutic demand (LTD) for IG in these diseases is required for adequate planning of the plasma supply required to manufacture the product. Materials and Methods: The diseases studied included chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain–Barré syndrome (GBS) and multifocal motor neuropathy (MMN). The LTD for IG was assessed using a decision analysis model, using Microsoft Excel. The model analysed the epidemiological and clinical factors contributing to IG usage. One‐way sensitivity analysis and probabilistic sensitivity analysis derived the LTD in grams per 1000 inhabitants. The key variables included the treatment schedule and the prevalence of the disease. Results: The model estimates that an average annual IG demand and standard deviation for CIDP, GBS and MMN in the United States is 83.05 ± 24.5, 6.1 ± 3.2 and 36.1 ± 25.5 g/1000 inhabitants, respectively. Conclusion: Together with previous work on the LTD for IG in immunodeficiencies, these results indicate that current IG usage reflects the estimated LTD for the main indications for IG in the United States The wide range of LTD found in all these studies emphasizes the need for more precise assessment of the underlying variables, particularly disease prevalence and dosage. Further studies on other indications such as secondary immunodeficiencies will augment these results and will assist in guiding demand planning for IG use and plasma collection in the United States and inform blood policy in other countries. [ABSTRACT FROM AUTHOR]

Background and Objectives: The significance of antibodies to red blood cells (RBCs) is variable and cannot be predicted solely by serological testing. A flow cytometry‐based erythrophagocytosis assay was established using phorbol 12‐myristate 13‐acetate (PMA)‐treated THP‐1 cells and RBCs labelled with PKH26 to assess allo‐ and autoantibodies to RBCs. Materials and Methods: THP‐1 cells were differentiated into macrophage‐like cells by treatment with PMA. RBC samples coated with alloantibodies or autoantibodies were obtained from 16 patients with autoimmune haemolytic anaemia of warm type (wAIHA) as well as from five pregnant women with warm autoantibodies. RBCs from healthy blood donors were used as controls. RBCs were labelled with the red lipophilic fluorescent dye PKH26 and incubated with PMA‐treated THP‐1 cells. After removal of nonadherent RBCs by washing and haemolysis of adherent RBCs, erythrophagocytosis was quantified by flow cytometry. Results: We observed significant phagocytosis of RBCs coated with clinically relevant alloantibodies (i.e. anti‐D and anti‐K) or autoantibodies from patients with active wAIHA, but not of those coated with alloantibodies (anti‐Ch) or autoantibodies from patients and pregnant women without haemolysis. Conclusion: The flow cytometry‐based erythrophagocytosis test described here is quantitative, highly reliable, and may be helpful for the assessment of the clinical significance of antibodies to RBCs. [ABSTRACT FROM AUTHOR]

Background and objectives: In 2010, an intravenous immunoglobulin (IVIG) product was removed from the market due to an association with serious thromboembolic events. Investigations revealed that factor XIa (FXIa) was present as a process‐related impurity. This study investigated the ability of two commercial FXIa assays to measure FXIa in immunoglobulin preparations and conducted a survey of FXIa activity in marketed immunoglobulin products. Materials and methods: Factor XIa assays were modified to include spiking of samples with FXIa before testing. An immunoglobulin product and its excipient were used to assess the ability of the assays to recover the spiked FXIa levels. Results: The Biophen FXIa assay required a high pre‐dilution of the sample to obtain statistically valid results and complete FXIa recovery. The ROX FXIa assay was more sensitive, giving statistically valid results at a lower sample pre‐dilution and FXIa spike level. This modified ROX FXIa assay was used to assay 17 lots of immunoglobulin products for FXIa. Two product lots had measurable FXIa levels without the need for spiking. A further 3 lots produced detectable but not statistically valid FXIa results when left unspiked. Spiking produced statistically valid assays and recoveries above 100%, demonstrating inherent FXIa. Conclusion: This study shows marketed immunoglobulin products can contain detectable levels of FXIa. Spiking brings the FXIa levels into the quantifiable range of the assay, allowing measurement of inherent FXIa. Accurate measurement is important to inform on 'safe' levels of FXIa in these products and allow future safety guidelines to be set. [ABSTRACT FROM AUTHOR]

Comments on the paper 'Rapid Detection of Antibodies to Immunoglobulin A Molecules by Using the Particle Gel Immunoassay,' published in a 2002 issue of 'Vox Sanguinis.' Limitations of the method proposed in the paper; Effective methods for the detection of antibodies against immunoglobulin.

Background: Daratumumab (DARA), a human IgG1K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti‐granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Methods: Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow‐GIFT). Samples were tested from patients on DARA (n = 7), non‐transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti‐CD38, were evaluated. Results: Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow‐GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow‐GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow‐GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti‐CD38 neutralized the detection of human IgG observed in DARA‐treated patient serum without effecting controls. Conclusion: We established that DARA can interfere with AGA testing, leading to false positive Flow‐GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow‐GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti‐CD38 antibody was effective in neutralizing DARA interference while maintaining Flow‐GIFT sensitivity. [ABSTRACT FROM AUTHOR]

Background and Objectives: Frequent platelet transfusion may lead to the formation of alloantibodies and immune‐mediated platelet destruction. Currently, identifying economic and effective screening methods is necessary for the management of platelet transfusion while different tests were recommended. The present study aims to challenge the performance of slot blotting (SB) and flow cytometry (FC) assays in detecting immune platelet refractoriness. Materials and Methods: Sera from 118 patients who received blood components and were clinically suspected of platelet refractoriness were enrolled. Platelet‐reactive antibodies were explored in parallel by SB, FC and monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) techniques. In a further study, chloroquine‐treated platelets were incubated with MAIPA‐positive serum, and then, the results of the SB and FC techniques were compared. Results: Using MAIPA as a reference, antibodies were detected in 51 sera, with specificity for human leucocyte antigens (HLA), human platelet antigens (HPA) or both HLA/HPA, in 27, 18 and 6 patients, respectively. The sensitivity and specificity of SB and FC were 86·3%, 88·1%, 82·4% and 95·5%, respectively. The Spearman correlation revealed significant (P < 0·001) correlations between FC (r = 0·763) and SB (r = 0·738) with MAIPA. In respect to HPA antibody detection, SB had 83·3% sensitivity and 92·6% specificity compared to 91·7% and 96·3% for FC while both approaches are acceptable (P < 0·001, r = 0·69; P < 0·001, r = 0·773) and can be recommended. Conclusions: The present study acknowledges that among the used methods, the flow cytometry's performance is the most appropriate, but slot blotting, with acceptable sensitivity, can be used as an acceptable and convenient procedure for platelet antibody screening. [ABSTRACT FROM AUTHOR]

Background: Prekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed. Materials and methods: A simplified one‐step method has been developed to replace the existing Ph Eur two‐step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one‐stage method is simplified by the use of a dedicated online app. Results: The one‐stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one‐stage method. Improved methodology using true initial rate determinations and use of acid‐treated prekallikrein substrate (PKS) helped understand and reduce artefactual results. Conclusions: The one‐stage method and associated app streamline real‐time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified. [ABSTRACT FROM AUTHOR]

Background and objectives: It is sometimes difficult to obtain antigen‐negative red blood cells (RBCs) for patients with antibodies against RBCs. However, the frequency and severity of the adverse reactions have not been well elucidated. Here, we conducted a multi‐institutional collaborative study to clarify the background, frequency and clinical significance of antigen‐positive RBC transfusions to patients with the respective antibodies. Materials and methods: The survey included the background of patients, antigens on RBCs transfused, total amount of antigen‐positive RBCs transfused, results from antibody screen and direct antiglobulin tests, specificity of antibodies, adverse reactions and efficacies. All antibodies were surveyed regardless of their clinical significance. Results: In all, 826 cases containing 878 antibodies were registered from 45 institutions. The main reasons for antigen‐positive RBC transfusions included 'negative by indirect antiglobulin test' (39%) and 'detection of warm autoantibodies' (25%). In 23 cases (3% of total), some adverse reactions were observed after antigen‐positive RBC transfusion, and 25 antibodies (9 of 119 clinically significant and 16 of 646 insignificant antibodies) were detected. Non‐specific warm autoantibodies were detected in 9 cases, anti‐E in 5 cases, 2 cases each of anti‐Lea, anti‐Jra or cold alloantibodies, and 1 case each of anti‐Dib, anti‐Leb or anti‐P1. Other antibodies were detected in 2 further cases. Five (22%) of these 23 cases, who had anti‐E (3 cases) or anti‐Jra (2 cases), experienced clinically apparent haemolysis. Conclusions: Adverse reactions, especially haemolysis, were more frequently observed in cases with clinically significant antibodies than those with clinically insignificant antibodies (P < 0·001). [ABSTRACT FROM AUTHOR]

Background and Objectives: A lateral flow assay for simultaneous blood group typing of ABO, RhD, C, E, c, e, Cw and K with stable end‐point and without centrifugation is in routine use since several years (MDmulticard®). The typing of extended phenotype parameters belonging to the Duffy, Kidd, MNSs blood group systems and others, however, has not yet been demonstrated for this technique. Reliable detection of Fyx, a weak Fyb phenotype with a pronounced quantitative reduction of the number of Fyb antigens on the erythrocyte surface, remains a weakness of current serological blood grouping techniques. Material and Methods: The performance characteristics of the following reagents were evaluated in donor and patient samples in lateral flow technology (MDmulticard®): Anti‐Fya, ‐Fyb, ‐Jka, ‐Jkb, ‐S, ‐s̅, ‐P1 and ‐k. The sensitivity to detect Fyx was in addition evaluated with Fyx positive samples, which had been preselected by MALDI‐TOF MS‐based genotyping. Results: All results obtained with the MDmulticard® were in full accordance with those of the CE‐certified reference products for all the eight reagent formulations used: Anti‐Fya, ‐Fyb, ‐Jka, ‐Jkb, ‐S, ‐s̅, ‐P1 and –k. Also, all Fyx phenotypes of the selected population of 93 positive samples, originally identified by MALDI‐TOF MS‐based genotyping, were reliably detected by the lateral flow assay. Conclusion: Extended phenotype blood group parameters, including the serologically challenging Fyx phenotype, can be determined simultaneously, rapidly and accurately using the lateral flow (MDmulticard®) technology, even in cases when IgG class antibodies are the only source of diagnostic antibodies. [ABSTRACT FROM AUTHOR]

Background and Objectives Alloimmune antibodies against red-blood-cell ( RBC) antigens induced in susceptible individuals (responders) by transfusion, pregnancy or transplantation may have serious clinical consequences. The aim of this study was to investigate association of alloimmunization against selected RBC antigens with HLA-Class II. Materials and Methods A total of 230 responders (106 monoresponders and 124 multiresponders) were enrolled into the study. HLA- DRB1 and HLA- DQB1 variants were determined by PCR- SSO and their frequencies compared between the patients (patient subgroups) and 375 ethnically and regionally matched controls. Results Development of multiple RBC antibodies was associated with HLA- DRB1*15 and HLA- DQB1*06 allelic groups in the patients, with the relationship being particularly apparent in those with anti-C+D antibodies. Furthermore, DRB1*13 and DQB1*06 were more frequent in multiresponders with anti-E+c antibodies and DRB1*03 and DQB1*02 in those with anti-E+Cw. Conclusion For the first time, we confirmed the association of HLA- DRB1*15 with RBC antibody multiresponder status and found HLA-Class II associations for three frequent RBC antibody combinations. Our data support the concept that HLA restriction plays an important role in the response to RBC alloantigens. [ABSTRACT FROM AUTHOR]

We found an individual with weakened S antigen expression on red blood cells (RBCs) during routine blood grouping. The proband was typed S+s+ by polyclonal antibodies, but the RBCs demonstrated different reactivity with three monoclonal anti‐S. The proband did not have alloanti‐S. Cloning and Sanger sequencing revealed that the proband had a c.166A>T (p.Thr56Ser) mutation in exon 4 of GYPB*S. When antibody screening of 60 455 blood donors was performed using the proband RBCs, no antibodies were detected. GYPB*S with c.166T should encode an unusual S antigen but the creation of a novel antigen remains to be investigated. [ABSTRACT FROM AUTHOR]

Background and Objectives To evaluate standard intravenous immunoglobulin ( IVIG) as an alternative to intravenous cytomegalovirus hyperimmune immunoglobulin ( CMVIG) for prophylaxis and therapy of cytomegalovirus ( CMV) disease, we measured the ELISA and neutralizing titres of CMV-specific antibodies in CMVIG and IVIG preparations. Materials and Methods Anti- CMV-IgG ELISA and neutralizing titres (fibroblast-based test) in CMVIG CG (Cytogam®, n = 20), CMVIG CT (Cytotect® CP, n = 3), IVIG P (Privigen®, n = 32) and IVIG K/G (Kiovig®/Gammagard®, n = 5) were compared, and IgG subclasses 1-4 were determined by nephelometry. Results Cytomegalovirus hyperimmune immunoglobulins contained more than fourfold higher CMV ELISA and CMV-neutralizing activity per gram of IgG than the standard IVIGs. Pooled data for all four products showed a significant correlation between anti- CMV-IgG ELISA and neutralizing titres ( r = 0·93, P < 0·001). There was a good correlation between the IgG3 content and CMV-neutralizing antibodies amongst lots of CMVIGs ( r = 0·91, P = 0·01), but this did not extend to the IVIGs. CMVIG CG contained the highest CMV-neutralizing activity (3497 ± 395 PEIU/g IgG) of any product tested. Conclusion The higher anti- CMV neutralization capacity of CMVIG per gram of IgG vs. standard IVIG suggests that standard IVIGs are not equivalent to or interchangeable with CMVIG. [ABSTRACT FROM AUTHOR]

The article presents abstracts on topics related to hemovigilance and transfusion safety including transfusion reactions in donor antibodies, platelet transfusion, and errors in transfusion of transplant recipients.

The article presents abstracts on topics related to management and organization including the immunohematological reference network in India, the quality improvement in Caribbean blood bank, and the communication with clinical teams for patients with irregular antibodies.

Background and Objectives Post-transfusion reactions with dyspnoea ( PTR) are major causes of morbidity and death after blood transfusion. Transfusion-related acute lung injury ( TRALI) and transfusion-associated circulatory overload ( TACO) are most dangerous, while transfusion-associated dyspnoea ( TAD) is a milder respiratory distress. We investigated blood components for immune and non-immune factors implicated in PTR. Material and Methods We analysed 464 blood components (RBCs, PLTs, L-PLTs, FFP) transfused to 271 patients with PTR. Blood components were evaluated for 1/antileucocyte antibodies, 2/cytokines: IL-1β, IL-6, IL-8, TNF-α, sCD40L, 3/lysophosphatidylcholines (LysoPCs), 4/microparticles (MPs) shed from plateletes (PMPs), erythrocytes (EMPs) and leucocytes (LMPs). Results Anti- HLA class I/ II antibodies or granulocyte-reactive anti- HLA antibodies were detected in 18·2% of blood components ( RBC and FFP) transfused to TRALI and in 0·5% of FFP transfused to TAD cases. Cytokines and Lyso PCs concentrations in blood components transfused to PTR patients did not exceed those in blood components transfused to patients with no PTR. Only EMPs percentage in RBCs transfused to patients with TRALI was significantly higher ( P < 0·05) than in RBCs transfused to patients with no PTR. Conclusion Immune character of PTR was confirmed mainly in 1/5 TRALI cases. Among non-immune factors, only MPs released from stored RBCs are suggested as potential mediators of TRALI. Our results require further observations in a more numerous and better defined group of patients. [ABSTRACT FROM AUTHOR]

Background and Objectives Red cell antibodies may complicate blood provision and liver transplantation outcome. The aim of this survey was to document red cell antibodies in adults undergoing liver transplantation and make recommendations for clinical practice. Materials and Methods We completed a 10-year retrospective review of adults undergoing liver transplant, in a single UK centre using 4- to 6-weekly red cell antibody screening. Results Seven hundred and thirty seven patients were reviewed: 58 (7·9%) had antibodies. In 50 (6·8%) patients, the antibodies were clinically significant, and the commonest were Rhesus (49·5%) and Kell (11%). 33 patients had a single antibody, and the rest had multiple antibodies (range 2-5). Two-thirds of patients (38) had antibodies at presentation; 22% of these developed additional antibodies while on the waiting list or postoperatively. Conclusion Consideration should be given to the proactive use of Rh- and K-typed blood in end-stage liver disease in order to reduce alloimmunization. In addition, regular antibody screening would enable staff to identify those with atypical antibodies and plan their transfusion support. [ABSTRACT FROM AUTHOR]

Background and Objectives A human recombinant monoclonal anti-RhD IgG may be useful to prevent RhD allo-immunization. Roledumab is such an antibody with a glycosylation pattern optimized for biological activity. The objective of the study was to assess the safety and pharmacokinetics of roledumab in healthy RhD-negative volunteers. Materials and Methods A total of 46 subjects received doses of 30-3000 μg i.v. of roledumab or placebo using a double-blind escalating single-dose design; 12 of these subjects also received 300 μg i.m. of roledumab. Subjects were followed for 6 months after administration. Serum roledumab concentrations were determined using flow cytometry. Results Fourteen treatment-emergent adverse events related to treatment were reported in nine subjects, with no apparent difference in their frequency or nature after placebo or roledumab administration. No anti-roledumab antibodies were detected. AUClast increased from 4·4 ng/ml.day at 30 μg i.v. to 2257 ng/ml.day at 3000 g i.v. The t½ ranged from 18 to 22 days, and the absolute bioavailability after i.m. administration was between 73% and 80%. Conclusion Roledumab is safe and well tolerated in healthy RhD-negative volunteers and shows a pharmacokinetic profile similar to that of polyclonal anti-RhD immunoglobulin. [ABSTRACT FROM AUTHOR]

Background and Objectives Transfusion-related acute lung injury (TRALI) is associated with the passive transfusion of leucocyte antibodies in blood products. Blood Transfusion Services have adopted a number of different strategies for reducing the incidence of TRALI, but, while these have been successful, TRALI has not been completely eliminated. Many Transfusion Services have introduced leucocyte antibody screening of donors to further reduce TRALI. This report describes the results of donor leucocyte antibody screening within NHS Blood and Transplant and the guidelines that have been developed for Transfusion Services within the United Kingdom (UK) to reduce the incidence of TRALI. Materials and Methods Blood samples from newly recruited female apheresis donors were tested for human leucocyte antigens (HLA) class I and class II antibodies and granulocyte-specific antibodies. Results A total of 1157 female donors were evaluated. Three hundred and fifteen (27·23%) donors had HLA class I or II antibodies and were returned to red cell component donation. Fifty-seven (6·77%) of the remaining 842 donors were found to have granulocyte-specific antibodies of which 11 (1·31%) had HNA-specific antibodies. A total of 818 donors (70·70%) were accepted for platelet apheresis, 336 donors (29·04%) were returned to red cell component donation, and three donors with HNA-3a antibodies (0·26%) were deferred from therapeutic donation. Conclusions Female donors with leucocyte antibodies were identified in a stratified screening programme. Donors with antibodies were either directed to red cell donation or deferred. This process, combined with other measures that have already been introduced, is anticipated to further reduce the incidence of TRALI. [ABSTRACT FROM AUTHOR]

Objective Based on the frequency of immune-mediated and non-immune-mediated transfusion-related acute lung injury (TRALI), the effect of risk-minimization measures was evaluated during a period of 5 years (2006-2010). Risk-minimization measures were implemented in 2008/2009, consisting of exclusion of female donors with a history of pregnancy or exclusion of female donors with human leucocyte antigen (HLA)/human neutrophil alloantigen (HNA) antibodies. Methods TRALI was confirmed according to the criteria of the International Haemovigilance Network. Based upon the results of donor testing of white-blood-cell antibodies (WBC-Ab) against HLA or HNAs, confirmed cases were classified as immune- or non-immune-mediated TRALI. Reporting rates were calculated on the basis of the annually transfused blood components, and pre- and post-implementation periods were compared. Results In total, 60 immune-mediated (75%) and 20 non-immune-mediated (25%) TRALI reactions were confirmed. A total of 68 (64 women and four men) donors were involved: seven red-blood-cell concentrates donors (13%), six platelet concentrate donors (10%), and 48 fresh frozen plasma (FFP) donors (77%). The reporting rate of immune-mediated TRALI caused by FFP decreased continuously; from 12·71 per million units in 2006/2007 to 6·81 per million units in 2008/2009 and no case in 2010. Conclusion The comparison of the pre- and the post-implementation period demonstrated a significantly reduced risk of TRALI events comparing 2006/2007 with 2010 ( P-value: < 0·01). Furthermore, no case of TRALI-induced fatality occurred after the implementation of risk-minimization measures. [ABSTRACT FROM AUTHOR]

Background and Objectives Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells. Materials and Methods We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004-2008) and period-2 (2009-2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE™ transformed red cells (kodecytes) were used. Results Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2. Conclusion Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population. [ABSTRACT FROM AUTHOR]

Background and Objectives The occurrence of thromboembolic events (TEEs) with intravenous immunoglobulin lots (IVIGs) raised the question of the causative agent for these adverse events. We investigated the predominant plasma proteases in 19 IVIG lots from five manufacturers including three lots associated with adverse events. Material and Methods The inhibitor profile of the amidolytic activity in IVIG lots was investigated with substrates S-2302 and S-2288. In immunocapture assays, prekallikrein and FXI antigen and respective active proteases were quantified. Non-activated partial thromboplastin time (NAPTT) and a modified FXIa PTT served as global and FXIa-specific clotting assays, respectively. Results Kallikrein was identified as one major contaminant activity in IVIGs. A second activity was seen in some IVIGs with substrate S-2288, but not with S-2302. Inhibition studies excluded FXIIa, thrombin or plasmin as contaminant activity. FXI antigen was seen in all 19 IVIG lots, and FXIa activity was found as second major impurity in some IVIGs, including all lots involved in TEEs. FXIa highly correlated with a short clotting time in NAPTT. Conclusions Kallikrein and FXIa are the major contaminants in IVIGs. FXIa was highly procoagulant, with highest level in TEE-associated IVIGs. Since the NAPTT unambiguously identified FXIa procoagulant activity in IVIGs, its implementation as a release test would improve the safety of IVIGs. [ABSTRACT FROM AUTHOR]

Background and Objectives In the production process of a new 5% liquid intravenous immunoglobulin (IVIG-L) product (Nanogam®), a combined pepsin /pH 4∙4 treatment /15-nm filtration (pH 4∙4 /15NF) step and a solvent-detergent (SD) treatment step were incorporated to improve the virus inactivating / reducing capacity of the manufacturing process. Two prospective uncontrolled multicentre studies were performed to evaluate the safety and efficacy of this product. Materials and Methods Efficacy, including pharmacokinetics, of IVIG-L was studied for 6 months in 18 primary immunodeficiency (PID) patients, succeeded by a long-term follow-up study (mean 2∙2 years, n = 17). Second, in 24 patients with idiopathic thrombocytopenic purpura (ITP), IVIG-L was studied for efficacy for 14 days. In both studies, adverse events and vital signs were recorded to study safety. Results In PID patients treated with IVIG-L, 0∙60 and 0∙38 severe infections per patient per year were reported during, respectively, the short-term and long-term follow-up. Pharmacokinetic studies resulted in an IgG half-life of 30∙9 ± 11∙3 days and a mean IgG trough level of 6∙8 ±1∙2 g/ l. In the ITP study, all patients showed an increase in platelet counts after infusion with IVIG-L, and 20 / 24 patients responded with a platelet count > 50 × 109 / l (83∙3%) within 1 week. IVIG-L infusions did not cause clinical relevant changes in laboratory parameters or vital signs. Conclusions In clinical studies, IVIG-L (Nanogam®) demonstrated to be efficacious, well tolerated and safe [ABSTRACT FROM AUTHOR]

John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status. Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies. Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7A and variant Sema7A proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7A protein and not the variant one. A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein. [ABSTRACT FROM AUTHOR]

Background and Objectives Foetal alloimmune thrombocytopenia (FNAIT) is often treated transplacentally with maternally administered i.v. immunoglobulins, but not all foetuses show a consistent platelet increase during such treatment. Materials and Methods We retrospectively analysed data from a cohort of ten foetuses with FNAIT treated by direct foetal immunoglobulin infusion. Foetal treatment was begun between 17 and 25 weeks and continued until 36 weeks with weekly cordocenteses and foetal immunoglobulin infusions. Results While foetal IgG levels increased steadily during weekly IgG infusions, foetal platelet counts remained unchanged. Conclusion Our retrospective study presents a unique analysis of a historical cohort, contributing to the ongoing debate about the treatment of choice for foetal alloimmune thrombocytopenia. [ABSTRACT FROM AUTHOR]

Background and Objectives As cytomegalovirus (CMV) DNA is frequently detectable in the plasma of recently infected sero-positive blood donors, information concerning primary CMV infection is important for the identification of possibly infectious donors. Materials and Methods Monitoring of 17 982 donors for CMV antibodies and DNA in plasma identified 14 subjects with ongoing primary CMV infection. Thirteen donors were interrogated for possible sources of infection and CMV-related symptoms, and monitored for CMV antigens, CMV DNA in plasma, leucocytes and urine, course of IgG and IgM antibodies as well as markers of systemic infection and parameters of organ function. Results CMV antigens and DNA were detectable in peripheral blood for up to 54 and 269 days respectively. Clearance of CMV DNA from blood correlated with clearance of IgM antibodies, development of IgG antibodies against the membrane glycoprotein gB and development of high avidity IgG antibodies. Eighty-five percent of subjects with primary CMV infection, but even 69% of matched controls reported possibly CMV-related symptoms. Sixty-two and 23%, respectively, had contact with possible sources of infection. One donor developed a febrile illness accompanied by increased levels of CMV DNA in peripheral blood 2 to 3 weeks after seroconversion. In other donors, neither markers of systemic infection nor parameters of organ function correlated with the course of CMV DNA and antigens. Conclusion Potentially infectious donors can be identified by measuring CMV DNA, IgM antibodies or avidity of IgG antibodies. Alternatively, blood products donated during the first year after seroconversion should not be used for immunocompromised patients. [ABSTRACT FROM AUTHOR]

Background and Objectives In fetal alloimmune thrombocytopenia (FAIT), transplacental maternal antibodies cause destruction of fetal platelets. FAIT is similar to fetal Rhesus haemolytic disease, but half of the affected fetuses are born to primiparous women. In 10–20% of cases, prenatal and perinatal intracranial haemorrhages are reported. Different therapeutic approaches have been described, including maternally administered high-dose intravenous immunoglobulin (high dose IVIG) without or with steroids or intrauterine transfusion (IUT) of compatible platelets. For the latter, the use of plasma-free maternal and donor platelets has been described, but a comparison of these two sources of platelets has not been reported. Materials and Methods We retrospectively analyzed the clinical courses of cases with FAIT treated with IUT of either HPA-matched donor platelets or maternal platelets, done by a single team between 1990 and 1997. In 57 pregnancies, FAIT was treated by repeated IUT with either maternal (15 fetuses) or donor platelets (42 fetuses). Results There was no procedure-related fetal or neonatal loss. Platelets from both sources reliably raised the fetal platelet counts. Donor platelet preparations contained more platelets and yielded higher fetal post-transfusion platelet counts, but maternal platelets were clinically equally effective. Conclusions Donor and maternal platelet concentrates are effective sources for the treatment of FAIT. [ABSTRACT FROM AUTHOR]

Background and Objectives In 2005, the Australian Red Cross Blood Service implemented a malaria antibody testing based strategy for donors with a history of travel/residence in a malaria endemic country or a past history of malaria. This report assesses the safety and efficacy of the strategy since inception. Materials and Methods Eligible blood donors were tested using the Newmarket malarial antibody EIA at least 4 months after their last potential exposure. Where EIA non-reactive their quarantined red cells were considered for transfusion and they were re-instated for cellular component manufacture at their next donation. The efficiency and safety of this strategy were evaluated based on the additional number of components recovered for transfusion and the observed incidence of transfusion transmitted malaria (TTM) respectively. Results Of the repeat reactive donors, 2696 (> 99·99%) were PCR negative whilst one was PCR positive with very low level parasitaemia. The average number of RBCs and platelets recovered per annum was 64 967 and 7398 representing 7·9 and 5·5% respectively of their annual production. No new TTM cases were recorded and the observed TTM rate of zero was consistent with the upper 95% CI for the pretesting TTM incidence of 0·9 per million donations. Conclusion The study findings support the efficacy and safety of a targeted screening strategy combining a sensitive antibody screening test with a 4-month cellular component restriction period for donors with a declared malarial risk. The TTM risk in Australia remains low and did not measurably change after implementing the testing strategy. [ABSTRACT FROM AUTHOR]

The article presents several abstracts on medical reasearch including one on the use of imatinib for refractory chronic graft-versushost disease with fibrotic features, one on antibody identification using automated solid-phase red cell adherence assay and a tube polyethylene glycol antiglobulin method, and one on clinical outcomes of platelet transfusions in thrombotic thrombocytopenic purpura patients.

Background There are two presumed mechanisms for the pulmonary oedema in transfusion-related acute lung injury (TRALI). One is antibodies to leucocytes while the other is biologically active lipids. We evaluated the vascular injury due to the former. Methods The pulmonary vasculature was studied by light microscopy (LM) and scanning electron microscopy (SEM) in three fatal cases of TRALI and compared with that of two autopsied control patients. Lung tissue from two of the TRALI cases and both controls was studied by gas chromatography–mass spectroscopy (GC–MS) to identify crystals present in the former. Results All three TRALI cases exhibited massive pulmonary oedema by weight and light microscopy and extensive defects by SEM in the endothelium of venules of the lungs. Such endothelial defects were absent in controls. Thrombi, composed of crystals, were present in venules and small veins diffusely throughout the lungs in Case 1. Similar crystals were identified in Case 2. The crystals in the lung vessels were identified morphologically as cholesterol and were proximate to the cytoplasmic defects of the endothelial surfaces. By GC–MS, there were markedly elevated levels of cholesterol and fatty acids in the two TRALI lungs tested compared with the lungs of the two controls. Conclusions Pulmonary damage in TRALI is related to formation of cholesterol crystals that appear to pierce endothelial membranes of venules. The endothelial defects lead to plasma extravasation into the alveoli causing TRALI. [ABSTRACT FROM AUTHOR]

Background and Objectives Alloantibody formation against red blood cell (RBC) antigens is a common complication of transfusion therapy. However, the prevalence of RBC alloimmunization is hardly known in Black Africans. In Uganda, the practice is to transfuse ABO/D compatible blood without screening for immune antibodies. The aim of this study was to determine the prevalence and specificities of RBC alloantibodies in transfused Ugandans. Materials and Methods Using a cross-sectional design, transfused patients at Mulago Hospital in Kampala, Uganda were investigated. Demographic characteristics and transfusion histories were recorded. EDTA blood samples were obtained from consenting patients and RBC alloimmunization was demonstrated using immunohaematological tests. Results A total of 214 transfused patients (mean age, 30·3 years; F/M ratio, 1·0) were investigated. Thirteen patients (6·1%) possessed RBC alloantibodies whose specificities were six anti-E; three anti-S; one each of anti-D, -K and -Lea; and two samples were pan-reactive. Eleven (84·6%) of the alloimmunized patients had experienced up to 10 transfusion episodes. The number of units of blood transfused and the transfusion episodes were significantly associated with the RBC alloimmunization rate ( P = 0·01). Conclusions The prevalence of RBC alloimmunization in transfused Ugandans was 6·1% and was associated with the number of donor exposures. This immunization rate is similar to that observed in transfused Caucasians despite differences in RBC antigen distributions. Patients with malaria were less likely to develop RBC alloantibodies. Alloantibodies were mainly against E and S antigens. We recommend the introduction of pretransfusion antibody tests in Uganda depending on the recipient’s diagnosis. [ABSTRACT FROM AUTHOR]

Background and Objectives Genotyping for human platelet antigen (HPA) systems is required in the investigation of patients with suspected HPA antibodies and for the provision of compatible blood products from HPA-typed donors. Here, we set up a polymerase chain reaction sequence-based typing method for simultaneous genotyping of HPA-1 to 17w (PCR-SBT). Materials and methods The specific primers for HPA-1 to 17w were designed byprimer premier 5·0 software. The PCR amplification conditions were optimized. Amplification products were purified by enzyme digestion and sequenced by a BigDye Terminator Cycle Sequencing kit. Results We used two standard DNA samples and 16 reference samples, including six with HPA gene mutations, for the HPA-1 to 17w analysis by the PCR-SBT and obtained 100% correct genotypes in these samples. The genotype and allele frequencies of HPA-1 to 17w were analysed from 112 Han Chinese persons. Thirteen new single nucleotide polymorphisms and one microsatellite were found in the sequenced regions, in which two nucleotide polymorphisms of ITGB3 gene could cause amino acid alteration of Val419Met and Glu628Lys on the mature glycoprotein, respectively. Conclusions The PCR-SBT method is reliable and accurate for HPA genotyping, suitable for routine HPA analysis, and can screen large numbers of donors. We identified 14 new polymorphisms, two of which led to amino acid changes. [ABSTRACT FROM AUTHOR]

Background and Objective It is known that red blood cells (RBC) from healthy blood donors with a positive direct antiglobulin test (DAT) for IgG continue to circulate despite carrying elevated numbers of IgG molecules. To unravel the properties of these RBC-bound IgG, we studied them not only on whole RBC populations, but also on density-fractionated RBCs. Materials and Methods The properties of acid-eluted RBC-bound IgG and plasma IgG were studied by ELISA for binding to RBC proteins and opsonins, and by blotting. In vitro phagocytosis was studied on density-separated RBCs. Results IgG-DAT-positive blood donors carried most IgG molecules on dense RBCs and had more RBCs of high density than DAT-negative controls. Their densest RBCs were older than the oldest RBCs of DAT-negative controls, based on the band 4·1a/b ratio. In vitro phagocytosis of senescent RBCs from IgG-DAT-positive donors was 1·5 to 2 fold higher than that of senescent control cells, but the same or less in the presence of physiological IgG concentrations, implying that RBC-bound IgGs impaired complement-dependent uptake. The IgG molecules on these DAT-positive RBCs comprised anti-band 3 naturally occurring antibodies (NAbs) and were two- to fivefold enriched in anti-C3 and framework-specific anti-idiotypic NAbs as compared to controls. Correspondingly, anti-C3 and framework-specific anti-idiotypic NAbs were proportionally elevated in the plasma of two-thirds of DAT+ donors. Conclusions Extra-binding of anti-C3 together with anti-idiotypic NAbs to senescent RBC-associated C3 fragments may suppress complement-dependent RBC phagocytosis and may prolong the in vivo life span of RBCs. [ABSTRACT FROM AUTHOR]

Background and Objectives Complement inhibition is considered important in the mechanism of action of intravenous immunoglobulin (IVIG) in a number of inflammatory and autoimmune disorders. The capacity of different IVIG preparations to ‘scavenge’ activated C3 and thereby inhibit complement activation was assessed by a new in vitro assay. Materials and Methods Diluted human serum as a complement source, with or without addition of different concentrations of IVIG, was incubated in microtitre plates coated with heat-aggregated human IgG. Complement scavenging was measured by detecting reduced C3 binding and determining fluid phase C3b–IgG complex formation. Complement activation induced by the IVIG preparations was measured as C5a formation. Results All IVIG preparations exhibited a dose-dependent inhibition of C3b deposition, correlating strongly with binding of C3b to fluid-phase IgG, but the extent of complement scavenging varied considerably between different IVIG preparations. At an IVIG concentration of 0·9 mg/ml, the inhibition of C3b deposition ranged from 72 ± 16% to 22 ± 4·1%. The reduction of C3b deposition on the complement-activating surface was not due to IVIG-induced complement activation in the fluid phase, as shown by the low C5a formation in the presence of serum. Conclusion In vitro analysis allows comparison of the complement-inhibitory properties of IVIG preparations. The extent of complement scavenging varies between the products. [ABSTRACT FROM AUTHOR]

Background Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. Study Design and Methods Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen–antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Results Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. Conclusion In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching. [ABSTRACT FROM AUTHOR]

Background The immune processes involved in the development of alloantibodies against the human platelet antigens in alloimmune disorders remain unclear. Antibody recognition of the platelet antigens on their respective platelet glycoproteins has been shown to be dependent on glycoprotein conformation. Furthermore, the post-translational modification of glycoproteins adds complexity to the alloantigenic determinants. Methods Nine anti-HPA-3a sera along with several control sera were tested for reactivity to an 11-mer peptide straddling the HPA-3a/b polymorphism. Sera found to specifically recognize the 3a peptide were further assessed by platelet pre-exposure and immunoblotting. Results Three of the nine antisera were found to specifically recognize an 11-mer synthetic 3a peptide by ELISA. Further analysis of all anti-HPA-3a sera by Western blot showed that only those reactive to the 3a peptide were able to bind both reduced and non-reduced GPIIb. Conclusion The results presented in this study provide the first known evidence for the identification of an antibody population capable of recognizing a linear and non-glycosylated form of the HPA-3a epitope. [ABSTRACT FROM AUTHOR]

Background and Objectives The Fc function assay of immunoglobulin G is performed to ensure that the biological activity of the molecule is not compromised during purification and storage. The current British Pharmacopoeia (BP) method is cumbersome, time consuming and requires a continuous source of fresh red blood cells. We have modified the technique to improve the convenience, throughput and reproducibility of the assay by using frozen red blood cells, a microtitre plate format and electronic derivative analysis for processing results. Materials and Method Immunoglobulin G samples were assayed using either the BP Fc function procedure or a new procedure incorporating frozen cells, microtitre format and electronic derivative analysis. Results The Fc function results demonstrated that there was no significant difference between the BP and the new improved procedure. Conclusion In this study, we demonstrated that a new Fc function assay incorporating frozen red blood cells, microtitre format and derivative analysis of haemolysis curves has been shown to be comparable to the standard BP procedure. It offers a more convenient and quicker means of performing analysis of Fc function of immunoglobulins. [ABSTRACT FROM AUTHOR]

Background Antibody-mediated transfusion-related acute lung injury (TRALI) is an important cause of transfusion-associated morbidity and death. Preventive strategies are currently a matter of debate. Methods Specificities of leucocyte antibodies implicated in previous severe TRALI reactions were determined using standard techniques. Based on these results, a leucocyte antibody screening strategy for the testing of parous female donors was introduced. Results Of 36 TRALI cases, 17, 12, four and three were due to human leucocyte antigen (HLA) class II, human neutrophil alloantigen (HNA), HLA class I, and mixtures of HLA class I and II antibodies, respectively. HNA-3a antibodies accounted for 10 of 12 HNA antibody-mediated reactions and 6 of 10 fatalities including one after transfusion of red blood cells. Investigation 5332 parous female donors showed leucocyte antibodies in 473 samples, resulting in an alloimmunization rate of 8·9%. Sixty-one per cent of these donors presented HLA class I, 19% class II, 12% HLA class I and II antibodies and 5% HNA antibodies. Additional HLA class I antibodies were found in 39% of HLA class II and in 17% of HNA antibodies containing sera. Our restrictive plasma strategy did not result in a shortage of plasma or platelets. No antibody-mediated TRALI case was observed since introduction of the policy of plasma from male, nulliparous or tested multiparous donors. Conclusion Compared to HLA class I antibodies, those directed against HLA class II and HNA-3a were of greater clinical relevance. Isolated HLA class I antibody screening was found to be insufficient for leucocyte antibody screening. [ABSTRACT FROM AUTHOR]

Background and Objectives Previous studies have presented evidence that human immunoglobulin G preparations for intravenous use contain antibodies directed against the death receptor Fas (CD95). The function of these antibodies was described as either antagonistic or agonistic; therefore, inhibiting or stimulating Fas-dependent apoptosis. Based on these reports, we asked whether the proportion of antagonistic and agonistic anti-Fas activities differs between different lots of intravenous immunoglobulin (IVIG). Variations between lots would open the possibility to preselect suitable lots of IVIG for different therapeutic purposes. Materials and Methods Eleven lots of IVIG were tested for their ability to induce or inhibit Fas-dependent apoptosis. The biological significance of anti-Fas antibodies was confirmed by including anti-Fas antibodies purified from IVIG and IVIG depleted of anti-Fas antibodies in the study. Results All 11 lots inhibited FasL-induced apoptosis. In addition, five lots stimulated apoptosis in the absence of FasL. Depletion of anti-Fas antibodies from IVIG abolished the capacity of IVIG to inhibit FasL-induced apoptosis, but reduced the ability to induce apoptosis only slightly. Conclusion The inhibition of FasL-induced apoptosis by IVIG is because of the presence of antagonistic anti-Fas antibodies. The activity of these antibodies differs considerably between different lots. On the other hand, the induction of apoptosis by IVIG is probably because of the concerted action of a range of different antibodies. The variation in the proportion of stimulating and inhibiting anti-Fas activities between different lots of IVIG opens the possibility to preselect suitable lots for different therapeutic purposes. [ABSTRACT FROM AUTHOR]

Background Human leucocyte antigen (HLA) antibodies have been implicated in transfusion-related acute lung injury, but the probability that the transfusion of a blood component containing HLA antibodies will cause a reaction is not known. This study compared the prevalence of reactions associated with the transfusion of platelet components with and without HLA antibodies. Study Design and Methods This retrospective study tested 96 consecutive apheresis platelet donors for HLA class I and II antibodies. Matched control donors without HLA antibodies were selected and records were reviewed to determine the proportion of components from each group that caused reactions. In addition, all apheresis platelet donors involved with two or more reactions were identified and tested for HLA class I antibodies. Results Five of the 96 donors had antibodies to class I or class II antigens and, of these, four had components transfused. The prevalence of reactions to components from these four donors with HLA antibodies and the 12 matched control donors without antibodies was similar (three reactions to 167 transfusions or 1·8% vs. three to 295 or 1·0%, respectively, P = 0·32). A retrospective review of the transfusion records from all platelet donors found that components from 22 caused two or more reactions and three (13·6%) had antibodies to HLA class I compared to 4·2% of the consecutively selected donors ( P = 0·12). None of the patients experienced transfusion-related acute lung injury. Conclusion Reactions associated with transfusion of apheresis platelets containing HLA antibodies are unusual. [ABSTRACT FROM AUTHOR]

Background and Objectives A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated. Materials and Methods Virus and prion removal capacities were assessed with down-scale spiking experiments, validated for equivalence to the large-scale process. Results Lipid-enveloped viruses were completely inactivated/removed by each of the three dedicated virus clearance steps, and for human immunodeficiency virus 1 (HIV-1) and pseudorabies virus (PRV), also by the upstream cold ethanol fractionation step. Relevant non-enveloped viruses [i.e. hepatitis A virus (HAV) and parvovirus B19 (B19V)] were effectively removed by nanofiltration and the cold ethanol fractionation step, and partial inactivation of non-enveloped viruses was achieved by low pH incubation. Overall log reduction factors were > 20·0 for HIV-1, > 18·1 for bovine viral diarrhoea virus, > 16·3 for West Nile virus, > 10·0 for influenza A virus subtype H5N1, > 21·8 for PRV, 12·0 for HAV, > 12·1 for encephalomyocarditis virus, 10·6 for B19V and 10·3 for mice minute virus. Prions (Western blot assay) were completely removed (≥ 3·2 mean log reduction) by a step of the cold ethanol fractionation process. Conclusions Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety. [ABSTRACT FROM AUTHOR]

Background and Objectives Kell antigens are encoded by the KEL gene on the long arm of chromosome 7. Kx antigen is encoded by the XK gene on the short arm of the X chromosome. Kell and Kx proteins in the red cell membrane are covalently linked by a disulphide bond. The McLeod phenotype is characterized by weakened expression of antigens in the Kell blood group system, absence of Km and Kx antigens, and acanthocytosis. It has an X-linked mode of inheritance with transmission through carrier females. Some males with the McLeod syndrome also have chronic granulomatous disease (CGD). It is generally believed that patients with non-CGD McLeod may develop anti-Km but not anti-Kx, but that those with CGD McLeod can develop both anti-Km and anti-Kx. Materials and Methods We present serological data, DNA genotyping and gene sequencing, monocyte monolayer assay and neutrophil oxidative burst test from a patient with the McLeod phenotype without clinical evidence of CGD. Results We report here the second example of a patient with non-CGD McLeod who developed anti-Kx in addition to anti-Km. Sequencing of our patient's XK gene confirmed the presence of a mutation resulting in a premature stop codon and lack of Kx protein in the red cell membrane, which is consistent with the diagnosis of McLeod syndrome. Neutrophil oxidative burst test was normal, indicating that our patient did not have CGD. The challenge of providing 10 compatible blood units for multiple surgeries was met. Conclusion The second case of a rare entity, a patient with non-CGD McLeod who developed anti-Kx and anti-Km, was managed successfully with a combination of autologous donations and procurement of compatible units from national and international sources. [ABSTRACT FROM AUTHOR]

Background and Objectives The platelet-specific antibody anti-human platelet antigen-3a (anti-HPA-3a) is involved in neonatal alloimmune thrombocytopenia, post-transfusion purpura, and platelet refractoriness. However, HPA-3a antibodies are often difficult to detect, probably because the antigen is labile. This report describes the production of a freeze-dried preparation of pooled human plasma, coded 03/190, containing IgG antibodies against the HPA-3a. The material is intended for use as a minimum sensitivity reagent in glycoprotein-specific assays currently used for anti-HPA-3a detection. Laboratories can use it to assess the sensitivity of their ‘in-house’ assays for anti-HPA-3a and to calibrate local controls for routine use in each batch of tests. Materials and Methods Plasma containing anti-HPA-3a was obtained from a mother of two babies both born with severe thrombocytopenia, and following dilution it was freeze dried in glass ampoules. Results Two collaborative studies demonstrated that the candidate material contained anti-HPA-3a and human leucocyte antigen (HLA) class I antibodies, but no other HPA antibodies that might confuse the detection of the anti-HPA-3a. The minimum dilution that should give a positive result was determined to be 1 : 8 by two further international collaborative studies involving a total of 49 laboratories in 23 countries. Conclusion The material also contains HLA antibodies and is suitable for use only in techniques that are glycoprotein specific (i.e. monoclonal antibody immobilization of platelet antigens and enzyme-linked immunosorbent assay) where only HPA antibodies will be detected. This standard will allow laboratories to measure their sensitivity of detection of anti-HPA-3a and will also allow those laboratories with relatively insensitive techniques to monitor their performance as they improve their methodology. [ABSTRACT FROM AUTHOR]

Background and Objectives Platelet immunology quality assurance exercises have been organized by National Institute for Biological Standards and Control since 1991 and, as of 2006, 35 laboratories participate in the serology section. Molecular human platelet antigen (HPA) typing has been included in the exercises since 1998 and, as of 2006, 29 laboratories participate in this part of the exercise. This report details the performance of laboratories in these two areas. Materials and Methods Every 6 months laboratories are sent up to four coded serum/plasma samples for testing in their in-house assays for HPA antibody detection/identification and four coded whole blood samples to be typed for HPA-1, -2, -3 -5 and (since 2003) -15 by their molecular typing assays (‘genotyping’). Results Fifty-two sera containing HPA-specific alloantibodies and 13 sera that were inert, contained only human leucocyte antigen (HLA) class I or high-titre anti-A+B antibodies were distributed; 15 sera were issued in more than one exercise. The percentage of participating laboratories that were able to detect HPA-specific alloantibodies ranged from 15·0 to 100%; the percentage that were able to correctly determine the specificities also ranged from 15·0 to 100%. Over the 20 serology exercises the percentage of laboratories classified as poor performers ranged from 3·1 to 36·7%. A total of 12 780 individual HPA genotyping results were assessed. The overall error rate was 0·7% but there was considerable variation between HPA alleles. Over 11 exercises the percentage of laboratories classified as poor performers varied from 6·3 to 26·3%. Conclusions The ability to detect and to identify platelet-specific alloantibodies varied widely between laboratories and between various examples of antibodies issued. An increase in the number of laboratories screening for HPA-15 antibodies was seen, although detection and identification of these antibodies was problematic. The majority of examples of HPA-3a antibodies and some examples of HPA-1a and -5b were also difficult to detect and identify. In addition, this scheme has shown that despite the apparent reliability of molecular typing techniques, mistakes do occur, particularly with certain systems. Approximately one in five laboratories participating in the serology exercises and one in seven participating in the genotyping exercises were classified as poor performer at one point or more during the series of exercises. [ABSTRACT FROM AUTHOR]