A simple and practical assay for the antigen-specific detection of platelet antibodies

BACKGROUND: The antigen-specific assays currently used for characterization of platelet (PLT)-reactive auto- and alloantibodies are too technically complex and impracticable for most routine laboratories. Here, a novel antigen-specific particle assay (ASPA) for PLTs similar to that of red blood cells is described. STUDY DESIGN AND METHODS: PLTs were solubilized and then incubated with red-dyed polystyrene particles coated with monoclonal antibodies (MoAbs) to various PLT glycoprotein complexes. These particles were directly tested for coating with autoantibodies (n = 8) or indirectly tested for serum autoantibodies (n = 33) or alloantibodies against HPA-1a (n = 4) or HPA-5b (n = 5). Serum samples from healthy blood donors (n = 100) served as negative controls. RESULTS: Negative reactions were clearly distinguishable from positive reactions, and the results of the particle assay were in concordance with those obtained by the standard MoAb-specific immobilization of PLT antigen assay (MAIPA) in all cases with alloanti-bodies. In three patients, only the ASPA was able to detect autoantibodies that were completely undetectable by the MAIPA. In contrast, in only one patient, the MAIPA detected autoantibodies that the ASPA failed to detect. CONCLUSION: In our opinion, the new ASPA is reliable, yet less complex and time-consuming than the currently available assays, and it can be implemented in any routine laboratory.     

Heterogeneity of HPA-3 alloantibodies: consequences for the diagnosis of alloimmune thrombocytopenic syndromes

BACKGROUND: Immunization against the human platelet alloantigen (HPA)-3a residing on alphaIIbbeta3 integrin accounts for approximately 2 percent of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Anti-HPA-3a alloantibodies are sometimes difficult to detect and can be overlooked by standard antigen capture assays. STUDY DESIGN AND METHODS: The reactivity of 12 anti-HPA-3a and 2 anti-HPA-3b alloantibodies from patients with FNAIT and posttransfusion purpura was analyzed by serologic (monoclonal antibody-specific immobilization of platelet antigens [MAIPA] assay, flow cytometry) and immunochemical (immunoprecipitation, immunoblotting) techniques. The influence of platelet (PLT) age, storage conditions, recombinant antigens from Chinese hamster ovary (CHO) cells, and sialic acids (treatment with neuraminidase) were analyzed. RESULTS: The most sensitive anti-HPA-3 alloantibody detection in MAIPA assay could be achieved with fresh homozygous PLTs. During a PLT storage period of 14 days before use, three types of anti-HPA-3 alloantibodies were found: 1) complete loss of reactivity (n = 6), 2) considerably weakened reaction (> or =50% reduction; n = 3), and 3) minor reduction of reactivity (< or =40% decrease; n = 5). When cryopreserved PLTs were used, 10 of 12 anti-HPA-3a and all anti-HPA-3b alloantibodies reacted positive. Only 6 of 10 serum samples reacted with recombinant HPA-3a on CHO cells. Neuraminidase treatment of PLTs showed that some anti-HPA-3a alloantibodies require the presence of sialic acids. The storage lesion seems to be related to cleavage of sialic acids. Immunochemical analysis revealed evidence that most anti-HPA-3a alloantibodies require an intact three-dimensional alphaIIbbeta3 integrin structure. CONCLUSIONS: Anti-HPA-3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the alphaIIbbeta3 integrin and protection from enzymatic degradation seem to be important during PLT storage.     

Relevance of the HPA-15 (Gov) polymorphism on CD109 in alloimmune thrombocytopenic syndromes

BACKGROUND: Alloantibodies against the human platelet (PLT) alloantigen (HPA)-15 system residing on CD109 can cause fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura, and PLT transfusion refractoriness. The detection of antibodies against HPA-15, however, is hampered by the variable low expression and instability of the CD109 molecule during preparation and storage. STUDY DESIGN AND METHODS: This study analyzed the occurrence of HPA-15 alloantibodies in 1403 patients: 930 FNAIT and 473 polytransfused (PT) patients by modified monoclonal antibody specific immobilization of PLT antigens (MAIPA) assay with well-defined phenotyped PLTs. A DNA typing technique was developed to confirm the phenotypes of PLT donors. B-cell lines were established as sources of reference DNA. RESULTS: Genotyping of 407 unrelated blood donors revealed the gene frequencies 0.512 and 0.488 for HPA-15a and -15b, respectively. Based on the selection of PLTs expressing high amounts of CD109 on the surface (mean fluorescence intensity ratio 4-5 on expression peak on Days 2-4 after apheresis) antibody screening by the MAIPA assay was performed. In total, 16 (1.1%) HPA-15 alloantibodies were found comprising four anti-HPA-15a and 12 anti-HPA-15b. Anti-HPA-15b without other PLT-reactive antibodies were detectable in three serum samples of PT patients. The incidence of HPA-15 alloimmunization in PT patients was significantly higher than in mothers with FNAIT (3.0% vs. 0.22%). In relation to all detected HPA-specific antibodies, HPA-15 is responsible for 6.2 percent of alloimmunizations. CONCLUSION: These observations indicate that alloimmunization against HPA-15 should be considered as a cause for immune thrombocytopenia, particularly in patients receiving multiple PLT transfusions.     

Platelet-specific alloantigens and antibodies in Tunisian women after three or more pregnancies

Pregnancy may allow alloimmunization against human platelet antigens (HPA), which can lead to neonatal alloimmune thrombocytopenia (NAIT). The specificities of alloantibodies are closely related to the distribution of the HPA systems. A total of 281 Tunisian multiparous women (mean number of pregnancies: 4.5) were phenotyped for the HPA-1, -3 and -5 systems, by monoclonal antibody immobilization of platelet antigens (MAIPA). We searched for antibodies against HPA-1a, HPA-3a, HPA-5b and HPA-5a in HPA-1b1b, HPA-3b3b, HPA-5a5a and HPA-5b5b individuals, respectively. The gene frequencies were: 0·83 for HPA-1a, 0·17 for HPA-1b, 0·78 for HPA-3a, 0·22 for HPA-3b, 0·82 for HPA-5a and 0·18 for HPA-5b. Anti-HPA-5b antibodies were present in eight sera and anti-HPA-3a antibodies were present in one serum. The anti-HPA-5b system is the most frequently involved in platelet alloimmunization in Tunisian multiparous women. However, prospective trials are required to confirm this result and to determine the exact frequencies and clinical relevance of platelet alloantibodies in pregnant Tunisian women.     

The first case of alloantibody against human platelet antigen‐15b in Japan: possible alloimmunization by a hydatidiform mole

BACKGROUND: The involvement of the human platelet antigen (HPA)‐15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA‐15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA‐15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan. STUDY DESIGN AND METHODS: We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti‐HPA‐15 alloantibodies. RESULTS: Anti‐HPA‐15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole–coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA‐15 genotype between the mother and the baby (both HPA‐15a/a), but incompatibility with the paternal one (HPA‐15a/b). The hydatid mole's tissue was genotyped as HPA‐15b positive. Besides anti‐HPA‐15b, maternal sera contain strong HLA Class I antibody CONCLUSIONS: Here we reported the first case of anti‐HPA‐15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA‐15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti‐HLA Class I being the possible cause.     

Low-avidity HPA-1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody–based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies.
STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time.
RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (∼490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA.
CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.     

Detection of human platelet antigen-1a alloantibodies in cases of fetomaternal alloimmune thrombocytopenia using recombinant β3 integrin fragments coupled to fluorescently labeled beads

BACKGROUND: Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA‐typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant β3 integrins displaying the HPA‐1a (rHPA‐1a) or HPA‐1b (rHPA‐1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA‐1a alloantibody screening assay, using Luminex xMAP technology. STUDY DESIGN AND METHODS: A 3‐plex bead assay was developed by coupling biotinylated rHPA‐1a, rHPA‐1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb‐specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay. RESULTS: The rHPA‐1a‐ and rHPA‐1b‐coupled beads were able to detect HPA‐1a and HPA‐1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA‐1‐specific antibodies. Furthermore, HLA Class I antibodies did not cross‐react with the coupled beads. CONCLUSION: The 3‐plex bead assay can be used to detect HPA‐1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders.     

Blockade of maternal anti-HPA-1a–mediated platelet clearance by an HPA-1a epitope–specific F(ab')2 in an in vivo mouse model of alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by transplacental passage of maternal human platelet-specific alloantigen (HPA)-1a antibodies that bind to fetal platelets (PLTs) and mediate their clearance. SZ21, a monoclonal antibody (MoAb) directed against PLT glycoprotein IIIa, competitively inhibits the binding of anti-HPA-1a alloantibodies to PLTs in vitro. The purpose of this investigation was to determine whether SZ21 F(ab')₂ fragments might be therapeutically effective in inhibiting or displacing maternal HPA-1a antibodies from the fetal PLT surface and preventing their clearance from circulation.
STUDY DESIGN AND METHODS: Resting human PLTs from HPA-1ab heterozygous donors were injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purified F(ab')₂ fragments of SZ21 or control immunoglobulin G (IgG) were injected intraperitoneally 30 minutes before introduction of HPA-1a antibodies. Blood samples were taken periodically and analyzed by flow cytometry to determine the percentage of circulating human PLTs.
RESULTS: Anti-HPA-1a IgG from NAIT cases were able to efficiently clear HPA-1a–positive PLTs from murine circulation. Administration of SZ21 F(ab')₂ fragments not only inhibited binding of HPA-1a antibodies to circulating human PLTs, preventing their clearance, but also displaced bound HPA-1a antibodies from the PLT surface.
CONCLUSION: F(ab')₂ fragments of HPA-1a–selective MoAb SZ21 effectively inhibit anti-HPA-1a–mediated clearance of human PLT circulating in an in vivo NOD/SCID mouse model. These results suggest that agents that inhibit binding of anti-HPA-1a to PLTs may have therapeutic potential in the treatment of NAIT.     

The detection of platelet antibodies by simultaneous analysis of specific platelet antibodies and the monoclonal antibody–specific immobilization of platelet antigens: an interlaboratory comparison

BACKGROUND: Glycoprotein (GP)‐specific platelet (PLT) antibodies can cause allo‐ or autoimmune thrombocytopenia. Their detection is of high diagnostic value. The simultaneous analysis of specific PLT antibodies (SASPA) assay is based on simultaneous detection of various PLT‐specific antibodies by flow cytometry and has entered routine use in our Mannheim institution. In this study, we performed an interlaboratory comparison investigation of PLT‐specific antibodies using SASPA versus the “gold standard,” the monoclonal antibody–specific immobilization of PLT antigen (MAIPA) assay. STUDY DESIGN AND METHODS: Sera from 194 patients with suspected PLT allo‐ or autoantibodies were tested against GPIIb/IIIa, IX, Ia/IIa, IV, and HLA Class I by SASPA (in Mannheim) and MAIPA (in Vienna). All data were reported blinded to those from the respective other method. Sensitivity studies included dilution studies with known antibodies against HPA‐1a, ‐1b, ‐3b, ‐5b, and ‐15b and HLA Class I. RESULTS: Overall, results were concordant in 78.9%. The specificity and sensitivity of SASPA, based on the MAIPA results, were 97.3 and 86.3%, respectively, for the detection of alloantibodies. The respective results for the detection of autoantibodies were 95.3 and 44.9%. Serial dilution experiments with sera containing anti‐HPA1a, ‐1b, ‐3b, ‐5b, and ‐15b and anti‐HLA Class I revealed a higher sensitivity of the SASPA assay with all alloantibodies. CONCLUSION: In this first blind interlaboratory comparison, SASPA yielded similar results to those of MAIPA. The SASPA assay may be superior to the MAIPA assay for the detection of weak alloantibodies while simultaneous detection of a variety of antibody specificities or immunoglobulin classes and the need of fewer PLTs are obvious advantages.     

Quantification of human platelet antigen-1a antibodies with the monoclonal antibody immobilization of platelet antigens procedure

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia results from a maternal alloimmunization against fetal platelet (PLT) antigens. In Caucasian persons, human PLT antigen-1a (HPA-1a) is the most frequently implicated antigen. Our aim was to develop a method supported by a mathematical approach to quantify HPA-1a antibodies. STUDY DESIGN AND METHODS: The monoclonal antibody immobilization of PLT antigens (MAIPA) protocol was applied on serial dilutions of a serum with high concentration of HPA-1a antibodies used as reference. Two mathematical procedures were used: the standard curve was constructed with a linear and a logistic regression. These regressions were used to calculate the amount of antibody in 14 test serum samples from mothers with detectable anti-HPA-1a. RESULTS: Similar quantifications were obtained with both mathematical procedures. In 11 serum samples, the results, expressed in arbitrary units (AU), ranged from 62 +/- 4 to 1096 +/- 19 with the linear regression and from 62 +/- 4 to 1117 +/- 38 with the logistic regression. In addition, the linear regression allowed us to measure 3 test serum samples with low antibody concentrations from 4 +/- 2 to 44 +/- 3 AU, whereas the logistic regression was not suitable for the quantification of antibodies in these serum samples. CONCLUSION: A simple quantification method was developed for anti-HPA-1a, based on the MAIPA procedure, allowing further studies concerning correlations between anti-HPA-1a quantification and clinical relevance.     

External quality assessment of platelet serology and human platelet antigen genotyping: a 10-year review

BACKGROUND: In this review, the results of an external quality assessment (EQA) over 10 years of platelet (PLT) serology and of human platelet antigen (HPA) polymorphisms genotyping are shown. The detection and identification of PLT antibodies and the distinction between PLT-specific antibodies and HLA Class I antibodies are evaluated. STUDY DESIGN AND METHODS: Each year, serum samples from five patients and four donor blood samples for DNA typing were distributed. Laboratories could participate as a screening laboratory (SL; n = 7) or as an identification laboratory (IL; n = 8). RESULTS: SLs scored 57 to 100 percent correct positive and 91 to 100 percent correct negative results in the detection of PLT-specific antibodies. SLs only using a PLT immunofluorescence test (PIFT) scored less well than those using a PLT glycoprotein-based antibody detection method. ILs scored 70 to 100 percent correct positive and 87 to 100 percent correct negative results for, respectively, the detection and identification of PLT-specific antibodies. Both the specificity and the sensitivity for the detection and identification of PLT-specific antibodies were not as good in ILs using solid-phase enzyme-linked immunosorbent assay methods as in those using the monoclonal antibody immobilization of PLT antigens (MAIPA) assay. For HPA-1, -2, -3, and -5 genotyping, incorrect results were observed only twice in 280 genotyping assays. CONCLUSION: The data underscore the necessity of using the most accurate methods with a high level of knowledge, experience, and technical training. For screening purposes, it is not sufficient to use only the PIFT, whereas for identification of PLT-specific antibodies, the MAIPA assay is the superior assay.     

Maternal human platelet antigen-1a antibody level correlates with the platelet count in the newborns: a retrospective study

BACKGROUND: Maternal plasma and/or serum levels of anti-HPA-1a at delivery were compared to neonatal platelet (PLT) counts. STUDY DESIGN AND METHODS: Samples from HPA-1bb women who gave birth to children with thrombocytopenia or had anamnestic information about a previous child with neonatal alloimmune thrombocytopenia (NAIT) were collected at delivery. A modified monoclonal antibody immobilization of PLT antigen method was used for quantification of anti-HPA-1a. RESULTS: The anti-HPA-1a level in women with severely thrombocytopenic children was higher than the corresponding level in mothers of children born with moderate thrombocytopenia or normal PLT counts. CONCLUSION: Our data show a significant correlation between maternal anti-HPA-1a level and the neonatal PLT count and indicate strongly that this may be a reliable predictive measure for NAIT. Suitable test systems for quantitative measurements of anti-HPA-1a must be developed and evaluated for this particular purpose.     

Two cases of platelet transfusion refractoriness associated with anti‐CD36

BACKGROUND: Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non‐Caucasians. We report two cases of PLT transfusion refractoriness linked to anti‐CD36. STUDY DESIGN AND METHODS: A 5‐year‐old female of Lebanese descent and a 70‐year‐old male of Chinese descent both failed to respond to HLA‐matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid‐phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody–specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme‐linked immunosorbent assay (MACE). RESULTS: Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti‐CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36‐negative donors were available, antigen‐negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused. CONCLUSION: Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients.     

Frequency and specificity of platelet-specific alloantibodies in HLA-immunized haematologic–oncologic patients

The frequency and specificity of platelet-alloantibodies to human platelet antigens (HPA) -1, -3 and -5 was investigated in 59 multitransfused, HLA-immunized patients. Using the MAIPA test (monoclonal antibody specific immobilization of platelet antigens) platelet alloantibodies could be demonstrated in 10 (17%) patients.   In one patient the antibody was present prior to any transfusions and probably induced by multiple previous pregnancies. This antibody was directed to HPA-5b. The remaining nine antibodies were found in patients ( n  = 36) with HLA-antibodies reacting with over 95% of unselected lymphocytes. In these patients the target antigens were HPA-1b in six, HPA-3a in one and both antigens in two patients.   Our findings demonstrate platelet alloimmunization induced by transfusions to be restricted to patients with high HLA-immunization. 25% of these patients (9/36) show platelet-specific antibodies, primarily HPA-1b.     

Low-avidity anti-HPA-1a alloantibodies are capable of antigen-positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA‐1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti‐HPA‐1a could be overlooked by standard methods due to low avidity. Low‐avidity HPA‐1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low‐avidity anti‐HPA‐1a. STUDY DESIGN AND METHODS: A retrospective cohort consisting of 82 HPA‐1bb mothers of HPA‐1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low‐avidity antibodies in SPR using purified GPIIb/IIIa (HPA‐1a or ‐1b). The capability of HPA‐1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. RESULTS: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA‐1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA‐negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA‐1a (mean, 31 ± 5 RU), but not to HPA‐1b flow cell (mean, 5 ± 2 RU). In vivo, low‐avidity antibodies were capable of clearing HPA‐1ab PLTs but not HPA‐1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA‐positive antibodies. CONCLUSIONS: Low‐avidity HPA‐1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.     

Neonatal alloimmune thrombocytopenia caused by human leucocyte antigen-B27 antibody

Neonatal alloimmune thrombocytopenia (NAIT) occurs when maternal alloantibodies to antigens presented on foetal platelets cause their immune destruction. Whether human leucocyte antigen (HLA) antibodies can cause NAIT is controversial. Here, a patient was described who suffered from a NAIT caused by an HLA-B27 antibody. Sera from the mother and the newborn were tested for human platelet antigen antibodies and HLA antibodies by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, solid phase-linked immunosorbent assay (ELISA), lymphocytotoxicity assay (LCT) and flow cytometric analysis. No antibodies against cluster designation (CD)109 and platelet glycoproteins of the father were found in patient's and mother's serum. However, HLA ELISA was used to identify HLA antibody in both sera. The antibody was specified as HLA-B27 antibody. Typing results showed that the father descended HLA-B27 antigen on patient and his brother. The mother was HLA-B27 negative. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-B27 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented.     

Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus) for the detection of antibodies against human platelet antigens

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.     

Gene frequencies of eight human platelet-specific antigens in Koreans

Human platelet-specific antigens (HPAs) are found on platelet membrane glycoproteins and are the target of platelet alloantibodies that mediate platelet destruction in neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP) and refractoriness to platelet transfusion therapy. The biallelic polymorphism of all HPA systems is known to be due to a substitution of a single base pair. This study was performed to investigate the frequency of the HPA genes in Koreans, based on these substitutions. The genotypes of eight HPA systems were determined by polymerase chain reaction using sequence-specific primers (PCR–SSP) for HPA-1, -2, -4, -5, and -8 and restriction fragment length polymorphism (RFLP) for HPA-3, -6, and -7. The gene frequencies obtained from 200 unrelated Koreans were 0.99 and 0.01 for HPA-1a and -1b, 0.92 and 0.08 for HPA-2a and -2b, 0.55 and 0.45 for HPA-3a and -3b, 0.99 and 0.01 for HPA-4a and -4b, 0.98 and 0.02 for HPA-5a and -5b, and 0.98 and 0.02 for HPA-6a and -6b. All the individuals tested were homozygotes for HPA-7a and HPA-8a. It has been reported that the HPA-1b antigen is extremely rare (less than 0.3%) in Oriental populations, but this study suggests that the frequency of this antigen in Koreans (2.0%) is higher than in Japanese and Chinese populations.     

HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia

BACKGROUND: The antenatal management of fetomaternal alloimmune thrombocytopenia (FMAIT) due to HPA-1a antibodies remains controversial, and a test identifying pregnancies that do not require therapy would be of clinical value. STUDY DESIGN AND METHODS: The statistical correlation was analyzed between clinical outcome and 1) anti-HPA-1a potency in maternal serum samples determined by a monoclonal antibody immobilization of platelet (PLT) antigen assay with an international anti-HPA-1a potency standard and 2) anti-HPA-1a biological activity measured by a monocyte chemiluminescence (CL) assay. RESULTS: A total of 133 pregnancies with FMAIT due to anti-HPA-1a were analyzed. In 97 newly diagnosed cases, there was no difference in antibody potency or CL signal between cases with intracranial hemorrhage (ICH; n = 15), those with no ICH but a PLT count of less than 20 x 10(9) per L (n = 52), and those with a PLT count of at least 20 x 10(9) per L (n = 30). In 22 previously known pregnancies, the positive predictive value of maternal anti-HPA-1a of greater than 30 IU per mL for a PLT count of less than 20 x 10(9) per L was 90 percent, but the negative predictive value was only 66 percent. Antibody potency tended to stay stable throughout pregnancy (n = 16) and from one pregnancy to the next (n = 16). CONCLUSION: Neither severe thrombocytopenia nor ICH in HPA-1a-alloimmunized pregnancies can be predicted with sufficient sensitivity and specificity for clinical application from maternal anti-HPA-1a potency or bioactivity.     

High-throughput multiplex single-nucleotide polymorphism analysis for red cell and platelet antigen genotypes

BACKGROUND: Transfusion recipients who become alloimmunized to red cell or platelet (PLT) antigens require antigen-negative blood to limit adverse transfusion reactions. Blood collection facilities use regulated and unregulated antibodies to phenotype blood, the cost of which can be prohibitive depending on the antisera and demand. An alternative strategy is to screen blood for these antigens with genomic DNA and the associated single-nucleotide polymorphisms (SNPs). STUDY DESIGN AND METHODS: A multiplex polymerase chain reaction (PCR)-oligonucleotide extension assay was developed with genomic DNA and a SNP genotyping platform (GenomeLab SNPstream, Beckman Coulter) to identify SNPs related to D, C/c, E, S/s, K/k, Kp(a/b), Fy(a/b), FY0 (-33 promoter silencing polymorphism), Jk(a/b), Di(a/b), and human PLT antigen (HPA)-1a/1b. A total of 372 samples were analyzed for 12 SNPs. The genotypes were compared to the blood group and PLT antigen phenotypes. RESULTS: Individual sample results varied from 98 to 100 percent for 11 of 12 SNPs. D was correctly identified in 292 of 296 (98.6%) D+ donors. The RHCE exon 5 E/e SNP analysis had the lowest concordance (89.5%). Thirty-three R(1)R(1) and 1 r"r were correctly identified. PCR-restriction fragment length polymorphism (RFLP) on selected samples confirmed the presence of the FY0 silencing polymorphism in nine donors. Homozygous HPA-1b/1b was identified in four donors, which was confirmed by PCR-RFLP (n = 4) and anti-HPA-1a serology (n = 2). The two HPA-1a-negative donors were recruited into the plateletpheresis program. CONCLUSION: The platform has the capacity to genotype thousands of samples per day. The suite of SNPs provides genotype data for all blood donors within 36 hours of the start of testing.