Report on the 15th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. MATERIALS AND METHODS: Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. RESULTS: Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. CONCLUSIONS: Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.

[Differential diagnosis and treatment of thrombocytopenia]. / Differenzialdiagnose und Differenzialtherapie der Thrombozytopenie.

Thrombocytopenia is usually acquired. The laboratory artefact of pseudothrombocytopenia should always be excluded. Bone marrow insufficiency with impaired platelet production results from infiltrating tumor cells or from a myelodsplastic syndrome. In patients with splenomegaly, platelets are trapped by the spleen. An increased platelet turnover is caused by activation of the clotting cascade, e.g. due to sepsis or malignancy. Platelet binding antibodies cause thrombocytopenia by increased platelet clearance. Important differential diagnoses in patients with severe thrombocytopenia are: acute leukemia, thrombotic thrombocytopenic purpura, autoimmune thrombocytopenia and drug-dependent thrombocytopenia. Multifactorial causes are thrombocytopenia associated with pregnancy, chronic alcohol abuse, and liver cirrhosis. Treatment should focus on the underlying disease. In regard to low platelet counts only clinical bleeding and not platelet count numbers should be treated.

Autologous blood donor screening indicated a lower prevalence of viral hepatitis in East vs West Germany: epidemiological benefit from established health resources.

Prevalence data concerning viral hepatitis and human immunodeficiency virus (HIV) in the general population are usually scarce. We aimed for a large cohort representative of the general population that required little funding. Autologous blood donors are relatively representative of the general population, and are tested for viral hepatitis and HIV in many countries. However, frequently these data are not captured for epidemiologic purposes. We analysed data from well over 35,000 autologous blood donors as recorded in 21 different transfusion centres for anti-hepatitis C virus (HCV), HBsAg and anti-HIV, as well as TPHA if available. We found a lower prevalence of hepatitis B virus and HCV in East vs West Germany, 0.2%vs 0.32% and 0.16%vs 0.32% respectively, which confirms earlier data in smaller cohorts, thus supporting the value of our approach. HIV was too rare to disclose significant differences, 0.01%vs 0.02%. TPHA was higher in East (0.34%) vs West Germany (0.29%) without significant differences. HCV was more frequent in women vs men. Transfusion institutes managing autologous blood donations should be used as a resource for epidemiological data relating to viral hepatitis and HIV, if such testing is performed routinely. This approach generates data relating to the general population with special emphasis on undiagnosed cases.

An in vitro system for the determination of individualized immunosuppression.

The risk of complications of immunosuppressive treatment in organ transplantation increases with the aggregate amount of immunosuppressive medication given to the patient. As the doses of immunosuppressive agents required to achieve comparable effects show considerable variability, methods to assess individual sensitivity toward immunosuppressive regimens are urgently needed. The aim of this study was to develop such an in vitro test system. As immunological model for allogeneic transplantation, individual pairs of recipient-derived lymphocytes and of donor-derived B lymphocytes mimicking HLA expression of cells in the transplanted organ were isolated and assessed in mixed-lymphocyte cultures (MLC). Alloreactivity was readily observed and MLC consisted of CD8(+) and CD4(+) T cells as well as CD56(+) natural killer cells. A proliferation assay to measure the response of individual MLC on the immunosuppression by cyclosporine (CsA) was developed. The concentrations of CsA leading to growth reductions by 50% (inhibitory concentration 50, IC(50)) were found between 110 and 220 ng/mL, which was near the trough whole blood levels for CsA. Accordingly, the IC(90) values (660 to 1760 ng/mL) were near the target values for peak whole blood levels. We believe that these data present a simple and potentially useful in vitro technology that allows for the prediction of individual responses to immunosuppressive therapeutic regimens.

The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2).

The human GPIa/IIa complex, also known as integrin alpha 2 beta 1, serves as a major receptor for collagen in platelets and other cell types. In addition to its role in platelet adhesion to extracellular matrix, GPIa/IIa is also known to bear the clinically important Br(a) and Brb alloantigenic determinants, which can result in antibody-mediated platelet destruction. Immunochemical studies showed that the Br antigenic epitopes reside solely on the GP Ia subunit and do not depend on sialic acid residues. To define the polymorphism responsible for the Br alloantigen system platelet RNA PCR technique, was used to amplify GPIa mRNA transcripts. Nucleotide sequence analysis of the amplified platelet GPIa cDNA from Br(a/a) and Brb/b individuals revealed a single A<-->G polymorphism at base 1648. MnlI RFLP analysis of cDNA from serologically determined individuals confirmed that this polymorphism segregates with Br phenotype. This single base change results in a substitution of Lys (AAG) in Br(a) to Glu (GAG) in Brb at amino acid residue 505 In spite of the reversal in charge at this position, however, we found no difference in the ability of Bra and Brb homozygous platelets to adhere to collagens types I, III, or V, nor did anti-Bra or anti-Brb alloantibodies interfere with platelet adhesion to any of these fibrillar collagens. The identification of the nucleotide substitution that defines the Bra/Brb alloantigen system will now permit both pre- and postnatal diagnosis for Br phenotype.

Fluorescence resonance energy transfer as a new method for the epitope-specific characterization of anti-platelet antibodies.

The detection and characterization of anti-platelet antibodies which are directed to HLA class I molecules or platelet-specific glycoproteins is of great importance in the diagnosis and treatment of thrombocytopenia. In this paper a simple and rapid flow cytometric assay for the epitope-specific characterization of anti-platelet antibodies is described using fluorescence resonance energy transfer (FRET). Patient platelets or test platelets preincubated with patient serum were analyzed for surface-bound immunoglobulins using R-phycoerythrin-conjugated polyclonal anti-human IgG antibodies (excitation 488 nm, emission 585 nm). In a second step, HLA class I structures, platelet-specific glycoproteins (gpIIb/IIIa, gpIb), and the Fc gamma receptor II were stained with murine monoclonal antibodies and Cyan 5-labelled polyclonal anti-mouse IgG antibodies (excitation 585 nm, emission 670 nm). Upon monochromatic fluorescence excitation with a 488 nm argon laser the efficiency of light transfer from R-phycoerythrin to Cyan 5 is a direct measure of the distance between the human platelet-bound antibody and the epitope detected by the murine monoclonal antibody (mab). The assay permits discrimination between human antibodies directed to different platelet-specific glycoproteins or HLA class I structures without interference from non-specific Fc gamma receptor-bound immune complexes and also between antibodies directed to different epitopes on glycoprotein heterodimers (e.g., gpIIb/IIIa).

Redistribution of platelet glycoproteins induced by allo- and autoantibodies.

The redistribution of platelet glycoproteins (GP) Ib, IIb and IIIa in response to stimulation with human platelet specific alloantibodies, autoantibodies and a quinidine-dependent antibody was investigated using immunofluorescence and a quantitative radioimmunoassay. The platelet GPs carrying the corresponding epitopes were determined using immunoblot technique or radioimmunoprecipitation. In accordance with previous results obtained with murine monoclonal platelet specific antibodies, redistribution upon stimulation with human platelet reactive antibodies was observed only due to reactions with epitopes on GP IIb, IIIa, respectively, but not on GP Ib. We therefore believe that membrane redistribution of human platelets induced by various antibodies is a function of the GP recognized by those antibodies rather than the source of the platelet reactive antibody.

Blood group A and B determinants are expressed on platelet glycoproteins IIa, IIIa, and Ib.

We report the localization of A, B blood group determinants on intrinsic glycoproteins using anti-A, B IgG antibodies. By radioimmunoprecipitation, anti-A and anti-B precipitated three bands with apparent molecular masses in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of 159, approximately 120 and 85 kDa under non-reduced conditions and 145, approximately 126 and 97 kDa under reduced conditions. In two-dimensional gel electrophoresis (isoelectric focussing/SDS-PAGE) these three bands could be resolved into six spots and fulfilled previously defined criteria for platelet membrane glycoprotein complexes I a/IIa, Ic/IIa, I b/IX and II b/IIIa. The results were supported by data obtained by an assay employing antibody-specific immobilization of platelet antigens (MAIPA). By this technique, blood group A, B determinants were shown to be immobilized by monoclonal antibodies specific for GPIa, Ic', IX, IIb/IIIa and strongly by mab specific GPIIa, but not by mab specific for HLA class I molecules. The more precise localization on platelet glycoproteins was achieved by immunoblotting technique by which blood group A determinants could be assigned to GPs IIa, IIIa and Ib.

A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia.

Heparin-associated thrombocytopenia (HAT) is a severe complication of heparin therapy. Its diagnosis is difficult. Conventional assays employ platelet aggregometry (PAA) and/or 14C-serotonin release (SRA) which are either insensitive (PAA) or require radioactive tracers (SRA). We here describe a newly developed sensitive and rapid assay based on visual evaluation of heparin-induced platelet activation (HIPA) in microtiter wells. Using sera of 34 patients with clinically suspected HAT we found the HIPA assay to be as sensitive as the SRA and superior to PAA. The HIPA assay allows investigation of crossreactivity with different types of heparins, low molecular weight (LMW) heparins and heparinoids. Three patients who required further parenteral anticoagulation and in whom the HIPA assay was negative before treatment with the LMW heparinoid Org 10172, were treated with this new heparinoid without adverse reactions. We conclude that the HIPA assay may be a useful tool for differential diagnosis and therapy in patients with HAT.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the Br polymorphism on a 144 bp exon of the GPIa gene and its application in platelet DNA typing.

Alloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes. Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the intron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using MnlI endonuclease obtained from 15 donors (2 Bra/a, 2 Bra/b and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

Human platelet alloantigens Bra/Brb are expressed on the very late activation antigen 2 (VLA-2) of T lymphocytes.

We report the molecular localization of the human platelet alloantigens Bra/Brb on activated T lymphocytes. By radioimmunoprecipitation anti-Bra precipitated two proteins from T lymphocytes of Br(a+) donors after long-term activation (24 days), but not from resting or short-term-activated T lymphocytes (4 days). No bands could be precipitated with anti-Bra from T lymphocytes of Br(a-) donors, but the same two bands were seen with anti-Brb. The apparent molecular weights of unreduced molecules were 155 and 130 kDa, respectively. Monoclonal antibody Gil4 directed against an epitope on the platelet membrane glycoprotein Ia/IIa complex precipitated the same two bands with long-term-activated T lymphocytes, whereas MoAb TS2/7 directed against very late antigen (VLA)-1 molecules precipitated two bands with Mr 180 and 130 kDa (alpha 1 beta heterodimer). The physicochemical properties of these two bands precipitated by anti-Bra, anti-Brb or monoclonal antibody Gil4 with molecular weights of 155 and 130 kDa, respectively, correspond to VLA alpha 2 and beta chains and fit defined criteria for the VLA-2 antigen. These results were corroborated by an assay employing monoclonal antibody for antigen immobilization showing that Bra and Brb antigens were present only on long-term activated T lymphocytes. Our results provide evidence for the expression of the Bra/Brb platelet alloantigen system on VLA-2 antigens of activated T lymphocytes and demonstrate a genetic polymorphism of VLA-2 molecules on both cell lines.

Incidence and specificity of HLA-DP antibodies in pregnancy sera.

A total of 630 human pregnancy sera were investigated for HLA-DP antibodies (ab) by monoclonal antibody-specific immobilization of leukocyte antigens (MAILA) using monoclonal antibody (mAB) B7/21.2 and selected B-lymphoblastoid cell lines from the Tenth International Histocompatibility Workshop reference panel. DP-specific abs were detected in 86 of 330 sera (26.1%) of a retrospective series selected for positive reactions in lymphocytotoxicity screening, and in 29 of 300 unselected sera (9.7%) of a prospective series. Approximately 80% of DP-reactive sera were also positive in lymphocytotoxicity test for class I and/or class II ab. On the other hand, sera containing lymphocytotoxic ab for both class I and class II ab revealed the highest incidence of DP ab (35%). Out of 115 DP-reactive sera, 28 clearly presented one or more DP specificities. Absorption/elution studies revealed complex patterns of reactivity in some sera which were similar to those of known mABs and possibly reflect supertypic DP specificities and/or serological cross-reactions. Serological DP typing of individuals by the MAILA technique appears promising.

Discrimination of antibodies against antigens of different MHC loci in human sera by monoclonal antibody-specific immobilization of leukocyte antigens.

To detect human antibodies against antigens of different major histocompatibility complex loci, particularly of class II specificity, a newly developed enzyme immunoassay for platelet antibodies was adapted for the use of lymphocytes as target cells. Peripheral blood lymphocytes, phytohemagglutinin-stimulated T cells, or Epstein-Barr virus-transformed B cells were simultaneously incubated with a monomorphic class- or locus-specific monoclonal antibody and the human antibody to be investigated. After solubilization, cell lysates were transferred to an enzyme-linked immunosorbent assay tray coated with a goat anti-mouse Ig antibody. Following immobilization of the monoclonal antibody/antigen complexes, human major histocompatibility complex antibodies were detected by addition of enzyme-labeled goat anti-human Ig. By means of this technique human antibodies against different major histocompatibility complex molecules present in the same sample could be clearly distinguished. Application of the monoclonal antibody-specific immobilization of lymphocyte antigens assay is presented by several examples. Of these, identification of DP-specific antibodies as well as serological DP typing are of particular interest.

Leucine33-proline33 substitution in human platelet glycoprotein IIIa determines HLA-DRw52a (Dw24) association of the immune response against HPA-1a (Zwa/PIA1) and HPA-1b (Zwb/PIA2).

Alloantibody formation against HPA-1a (Zwa/PIA1) has, to date, only been found in HLA-DRw52(a+) (Dw24) individuals. Alloimmunization against the product of the other HPA-1 allele, HPA-1b, is rare. We have been able to evaluate ten cases of HPA-1b alloimmunization in Europe in order to study whether there is an association between HLA phenotype and anti-HPA-1b antibody formation. HLA typing of these patients was performed with particular attention to the DRw52a specificity using specific T-cell clones. No association with DRw52a or any other known HLA phenotype was found. This finding implies that the amino acid substitution leucine33-proline33 in GPIIIa, responsible for HPA-1a/b, is of primary importance for the association of anti-HPA-1a antibody formation with DRw52a. These data show that the amino acid polymorphism affects the presentation of the immunogenic oligopeptides of HPA-1a and -1b in the HLA class-II groove.

Pregnancy-maintaining antibodies: workshop report (Giessen, 1988).

To analyse the nature of antibodies which are purported to be essential for the maintenance of normal human pregnancy, six centers participated in a workshop of "blind" tests on 19 allosera. Fc-receptor dependent assays detected antibodies with specificity only for HLA. In addition to cytotoxic antibodies, the Fc-receptor dependent immune phagocytosis inhibition test revealed two non-cytotoxic alloantibodies with HLA specificity. These antibodies had high titers and may, therefore, be essentially non-cytotoxic. Murine monoclonal antibodies to HLA-A, B, C or DR (W6/32 and 2MC3) were used to evaluate the methods. These antibodies inhibited immune rosette formation as well as immune phagocytosis. Diluted to concentrations below the threshold of complement-dependent cytotoxicity, the monoclonal antibodies still inhibited the mixed lymphocyte reaction and the immune phagocytosis. A human monoclonal immunoglobulin M with specificity for monomorphic non-HLA lymphocyte antigens inhibited the mixed lymphocyte reaction. The immune rosette inhibition test exhibited several false positive reactions, e.g. three out of four with a serum that did not contain alloantibodies to blood cells. Non-cytotoxic antibodies were therefore rare in the selected sera of the workshop and they exhibited HLA specificity only. No participant was able to identify pregnancy-maintaining non-HLA-antibodies.

Drug-induced and drug-dependent immune thrombocytopenias.

Thrombocytopenia is a frequent comorbid condition in many in hospital patients. In some patients, drugs are the cause of low platelet counts. While cytotoxic effects of anti-tumor therapy are the most frequent cause, immune mechanisms should also be considered. This review addresses thrombocytopenias in four groups. Heparin-dependent thrombocytopenia (HIT), by far the most frequent drug-induced immune-mediated type of thrombocytopenia, has a unique pathogenesis and clinical consequences. HIT is a clinicopathological syndrome in which antibodies mostly directed against a multimolecular complex of platelet factor 4 and heparin cause paradoxical thromboembolic complications. The mechanisms through which heparin can enhance thrombin generation are discussed and treatment alternatives for affected patients are presented in detail. It is of primary importance to recognize these patients as early as possible and to substitute heparin with a compatible anticoagulatory drug, such as hirudin, danaparoid or argatroban. Patients seem to benefit from therapeutic doses of alternative treatment rather than from low-dose prophylactic doses. With the increasing use of glycoprotein (GP) IIb/IIIa inhibitors in patients with acute coronary syndromes, thrombocytopenias are increasingly recognized as an adverse effect of these drugs. Up to 4% of treated patients are affected. Most important, pseudothrombocytopenia, a laboratory artefact, is as frequent as real drug-induced thrombocytopenia and must be excluded before changes in treatment are considered. The pathogenesis of these thrombocytopenias is still debated; an immune mechanism involving preformed antibodies is likely. However, since these antibodies are also detectable in a high percentage of normal controls and of patients not developing thrombocytopenia, their impact is still unclear. Patients with real thrombocytopenia are at an increased risk of bleeding; treatment consists of cessation of the GP IIb/IIIa inhibitor and platelet transfusions in cases of severe hemorrhage. Classic immune thrombocytopenia can be induced by some drugs, e.g. gold, which trigger anti-platelet antibodies indistinguishable from platelet autoantibodies found in autoimmune thrombocytopenia. Drug-induced and drug-dependent immune thrombocytopenia is induced by antibodies recognizing an epitope on platelet GP formed after binding of a drug to a platelet glycoprotein. Still unresolved is whether antibody binding is the consequence of a conformational change of the antigen, the antibody, or both. These antibodies typically react with monomorphic epitopes on platelet GP, but only in the presence of the drug or a metabolite. Although several platelet GP have been identified as antibody target (GPIb/IX, GPV, GP IIb/IIIa), antibodies in an individual patient are highly specific for a single GP. Clinically, these patients present with very low platelet counts and acute, sometimes severe, hemorrhage. Treatment is restricted to withdrawal of the drug and symptomatic treatment of bleeding.

Impaired agglutination of IgM resulting from non-enzymatic glycation in diabetes mellitus.

Non-enzymatic glycation of circulating immunoglobulin G, A and M, measured by boronic acid affinity chromatography was found to be significantly increased in diabetes mellitus. The mean value of glycated IgM was already high in the control group: 49.58% (+/- 10.3% SD) and increased up to a mean value of 61.00% (+/- 8.5%) in the diabetic group. Similarly IgG and IgA glycation was significantly higher in the diabetic group than in controls: IgG 21.6% (+/- 3.4%) vs. 14.1% (+/- 2.9%; P less than 0.01); IgA 14.7% (+/- 4.9%) vs. 7.7% (+/- 1.3%; P less than 0.01). To investigate how glycation would alter IgM function, serum proportions from diabetic patients with blood group O were separated into glycated and non-glycated fractions by affinity chromatography and, adjusted to the same concentrations, tested against group A1 erythrocytes. Agglutination, which is mainly an IgM-mediated reaction, was significantly lower in the glycated than in the non-glycated fraction of IgM. The correlation between glycation of IgM and the reduction of agglutination titres in the glycated fraction was significant (r = 0.88, P less than 0.001). We conclude that impaired IgM function may be caused by non-enzymatic glycation in diabetes mellitus with possible consequences for host resistance in the early phase of infection.

Human platelet alloantigens.

Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.