The alloimmune thrombocytopenic syndromes

We have summarized five thrombocytopenic syndromes caused by platelet-reactive alloantibodies. Increased awareness of these five syndromes, together with the greater availability of highlyspecialized laboratory methods to detect and to characterize platelet-reactive alloantibodies, will lead to their more frequent diagnosis. It is important for the clinician to consider unusual alloimmune thrombocytopenic disorders in clinical settings in which alloantigens that could be limited to just one family could cause important disease (ie, NAT caused by a private alloantigen; theoretically, PAT or TAT related to directed donations of blood or bone marrow). These considerations underscore the need for serological investigations to involve the family members rather than to rely on standard platelet typing donor pools.     

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.     

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.     

Auto‐ and alloantibodies against factor XIII: laboratory diagnosis and clinical consequences

Summary

Acquired FXIII deficiencies caused by autoantibodies against FXIII subunits represent rare but very severe bleeding diatheses. Alloantibodies in FXIII‐deficient patients also cause life‐threatening bleeding complications, but they develop extremely rarely. In this review we provide an overview of the diagnosis and classification of anti‐FXIII antibodies and analyze 48 patients with autoimmune FXIII deficiency and four additional FXIII‐deficient patients who developed anti‐FXIII alloantibody. The patients were collected from peer‐reviewed publications from which relevant data could be extracted. With the exception of two cases the antibodies were directed against FXIII‐A. The difficulties in the diagnosis of FXIII deficiency in the presence of anti‐FXIII antibodies are discussed and a scheme for the functional classification of the anti‐FXIII antibodies is recommended. The three main categories are neutralizing and non‐neutralizing antibodies and antibodies with combined effect. The methods being used for detecting and quantifying the inhibitory effect on FXIII activation and on the transglutaminase activity of activated FXIII are summarized and techniques for the classification of neutralizing anti‐FXIII antibodies are outlined. The importance of clearance studies in these cases is emphasized. Binding assays, useful for the identification of non‐neutralizing and combined type antibodies, were collected from the literature and their informative power is demonstrated by examples. The most frequently occurring bleeding symptoms in patients with anti‐FXIII antibodies were soft tissue bleeding; intracranial bleedings also occurred, but less frequently than in inherited FXIII deficiency. Treatment of such patients is extremely challenging; the main aim should be eradication of the antibody.

     

Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is one of the most common bleeding disorders in neonates. It occurs when alloantibodies from an immunized mother react with paternally inherited alloantigens, mostly human platelet antigen 1a (HPA-1a), on the fetal platelets (PLTs). Currently, monoclonal antibody-immobilized PLT antigen (MAIPA) assay represents the standard technique for the serologic diagnosis of NAIT. MAIPA is time-consuming, however, and limited by the availability of monoclonal antibodies (MoAbs). Here, a gel antigen-specific assay (GASA) was developed, which allows rapid detection of HPA-1 alloantibodies without the use of MoAbs. STUDY DESIGN AND METHODS: Glycoprotein (GP) IIb/IIIa was purified by affinity chromatography from outdated PLT concentrates derived from HPA-1aa or HPA-1bb donors. Purified GPs were biotinylated, immobilized onto streptavidin beads, and used for the analysis of HPA-1a alloantibodies by a microtyping system. HPA-1a serum samples derived from mothers with NAIT (n = 36) and from posttransfusion purpura patients (n = 2) as well as HPA-1b (n = 4), HPA-5b (n = 2), HPA-3a (n = 4), and HLA Class I (n = 2) alloantiserum samples from multitransfused patients were investigated in GASA and MAIPA assays. RESULTS: GASA was able to detect all HPA-1a and -1b alloantibodies recognized by MAIPA. Cross-reactivity with other PLT-reactive alloantibodies was not observed. Interestingly, 3 of 36 serum samples, which showed only moderate reactivity in MAIPA, reacted strongly in GASA. CONCLUSION: GASA has proved to be a rapid method for the detection of HPA-1a alloantibodies and maybe useful for PLT antibody screening, especially in initial assessment of suspected NAIT cases.     

抗HPA-3a抗体所致新生儿同种免疫性血小板减少性紫癜一例并文献复习

目的 探讨抗血小板特异性抗原(HPA)-3a抗体所致新生儿同种免疫性血小板减少性紫癜(NAITP)的诊断和治疗.方法 采用多重PCR及基因测序技术检测1例出血伴血小板减少新生儿及其父母HPA-1~21bw系统基因型,采用流式细胞术(FCM)和血小板抗原单克隆抗体特异性免疫固定检测技术(MAIPA)检测患儿及其母亲血清血小板特异性抗体并进行特异性鉴定.结果 患儿出生后2 h出现全身多发皮下出血点、血尿及咖啡色呕吐物.基因分型显示患儿为HPA-3ab、母亲为HPA-3bb、父亲为HPA-3aa;患儿及母亲血清中均含与患儿父亲血小板反应的特异性抗体,经MAIPA技术鉴定为抗HPA-3a抗体.结论 发现1例抗HPA-3a抗体所致NAITP患者,通过临床特征分析为该病的诊断和治疗提供借鉴与参考.     

Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NATP) caused by fetomaternal mismatch for human platelet (PLT) alloantigens (HPAs) complicates approximately 1 in 1000 to 1 in 2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage, and sometimes death of the fetus or neonate. As a national reference center for NATP investigations, our experience with this entity over a 12-year period was reviewed. STUDY DESIGN AND METHODS: The laboratory records of all cases of suspected NATP referred for evaluation from January 1, 1990, to December 31, 2002, were analyzed. The spectrum of PLT alloantibody specificities identified was compared with an earlier reported series of serologically verified NATP cases. RESULTS: HPA-specific alloantibodies were identified in 1162 (31%) of 3743 sera of mothers of infants with clinically suspected NATP. Maternal HPA-1a (PlA1) alloimmunization accounted for the majority (79%) of confirmed NATP cases, with HPA-5b (Bra), HPA-3a (Baka), and HPA-1b (PLA2) alloantibodies accounting for 9, 2, and 4 percent of cases, respectively. In addition, an increase in the number of cases in which multiple HPA-specific alloantibodies were present in maternal sera was observed during the study period. CONCLUSION: Although, as with the earlier series, maternal HPA-1a alloimmunization was the dominant cause of NATP, the identification of an increasing number of cases due to alternative HPA polymorphisms suggests that investigation for HPA-1 incompatibility alone is no longer sufficient to fully evaluate clinically suspect NATP cases.     

Developing recombinant HPA-1a-specific antibodies with abrogated Fcgamma receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin beta3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcgamma receptor-dependent platelet destruction (G1Deltanab). B2G1Deltanab saturated HPA-1a+ platelets and substantially inhibited binding of clinical HPA-1a-specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Deltanab-sensitized platelets was substantially less than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Deltanab inhibited chemiluminescence induced by B2G1 and HPA-1a-specific sera. In a chimeric mouse model, B2G1 and polyclonal Ig preparations from clinical HPA-1a-specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia. As the Deltanab constant region is uninformative in mice, F(ab')2 B2G1 was used as a proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a-specific antibodies. These results provide rationale for human clinical studies.     

HPA-13bw neonatal alloimmune thrombocytopenia and low frequency alloantigens: case report and review of the literature

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) linked to rare or private antigens is not a rare event. STUDY DESIGN AND METHODS: Such a case discovered during the follow-up of a second child with jaundice with mild thrombocytopenia is reported here. Platelet (PLT) genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. Glycoprotein Ia-specific amplification and sequencing were performed for the polymorphism 807 (exon 7). RESULTS: The mother was found to be HPA-13aaw, and the father HPA-13abw. A maternal alloantibody directed against HPA-13bw has been characterized, leading to the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: This report provides further evidence that NAIT associated with low-frequency antigens is not restricted to single families. Therefore, laboratory investigation of a suspected case should be carried out in a specialist laboratory well experienced in optimal testing to propose appropriate management for the index case and subsequent pregnancies.     

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies.Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.     

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.     

Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.     

HPA-5b (Bra) neonatal alloimmune thrombocytopenia in Quebec: incidence and clinical outcome in 31 cases

BACKGROUND: Clinical detection of neonatal allo-immune thrombocytopenia (NAITP) is often less than the incidence predicted in prospective studies. The aims of this study were to calculate the observed and expected incidence of NAITP in Quebec, Canada, and to evaluate the clinical outcome of infants with anti-HPA-5b NAITP. STUDY DESIGN AND METHODS: Records from 1998 to 2002 of the only PLT serology laboratory for the province of Quebec were reviewed, as were hospital charts of all HPA-5 cases. The number of expected NAITP cases was estimated with known alloantigen gene frequencies. RESULTS: Ninety cases of NAITP were identified. The clinical detection rate was 1 in 4100, with 9 to 30 percent of expected HPA-1a and 11 to 23 percent of expected HPA-5b NAITP cases detected. Seventy-eight percent of cases of HPA-5b NAITP were asymptomatic. Sixty-three percent of all deliveries but 94 percent of HPA-5b cases occurred in hospitals performing cord blood PLT counts. CONCLUSION: The NAITP detection rate was 25 to 50 percent of the rate reported in prospective trials. The less severe clinical presentation of NAITP owing to HPA-5b was confirmed. The frequent routine determination of cord blood PLT counts may explain increased detection of asymptomatic NAITP involving anti-HPA-5b.     

A naturally occurring LeuVal mutation in beta3-integrin impairs the HPA-1a epitope: the third allele of HPA-1

BACKGROUND: Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.     

A single-nucleotide polymorphism in the human ITGB3 gene is associated with the platelet-specific alloantigen Va (HPA-17bw) involved in fetal maternal alloimmune thrombocytopenia

BACKGROUND: The previously reported platelet (PLT)-specific antigen, Va(a), was defined by an alloantibody detected in the serum sample of a mother who delivered an infant displaying symptoms of severe fetal maternal alloimmune thrombocytopenia (FMAIT). This PLT antigen was localized to the integrin alphaIIbbeta3 (GPIIbIIIa) but its genetic basis was not defined. STUDY DESIGN AND METHODS: Genomic ITGA2B (alphaIIb) and ITGB3 (beta3) DNA from a Va(a)-positive individual were sequenced to identify potential polymorphisms underlying the Va(a) antigen. Recombinant beta3 integrin carrying the putative mutation was then used to assess serologic reactivity with the original maternal Va(a) antiserum. RESULTS: A single-nucleotide polymorphism (SNP; C622>T) in exon 5 of ITGB3 resulting in the replacement of threonine with methionine at residue 195 of the mature beta3 was identified. Calmodulin-tagged soluble recombinant beta3 encoding Met195 was produced in S2 cells and found to react specifically with the Va(a) antiserum. CONCLUSION: With the use of a combination of DNA sequencing and recombinant antigen expression, the molecular basis of the PLT-specific Va(a) antigen (HPA-17bw), a low-frequency antigen implicated in FMAIT, has been resolved. These data further demonstrate the value of using recombinant beta3 peptides for the detection of rare but clinically relevant antibodies.     

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.     

Maternal alloimmunization against the rare platelet-specific antigen HPA-9b (Max a) is an important cause of neonatal alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NATP) is an important cause of morbidity and mortality in the newborn. Optimal management of subsequent pregnancies requires knowledge of the alloantigen that caused maternal immunization, but this is possible only in a minority of cases. This study investigated whether this can be explained in part by maternal immunization against the "rare" alloantigen HPA-9b (Max(a)), implicated previously only in a single NATP case. STUDY DESIGN AND METHODS: Archived paternal DNA from unresolved cases of NATP and normal individuals was typed for platelet (PLT)-specific antigens with real-time polymerase chain reaction and direct sequencing. PLT-specific alloantibodies were characterized by flow cytometry and solid-phase enzyme-linked immunosorbent assay. Recombinant GPIIb/IIIa was expressed in stably transfected Chinese hamster ovary cells. Clinical information was obtained directly from attending physicians. RESULTS: Six of 217 fathers were positive for the presence of HPA-9b (Max(a)), an incidence about seven times that in the general population. In each of five cases studied, maternal serum samples reacted with intact paternal PLTs and paternal GPIIb/IIIa. Only one of three serum samples tested recognized recombinant GPIIb/IIIa carrying the HPA-9b (Max(a)) mutation. These seemingly discrepant reactions may reflect different requirements for oligosaccharides linked to residues close to the mutation in GPIIb that determines HPA-9b (Max(a)). NATP in the affected children was severe and was associated with intracranial hemorrhage in three of six infants on whom information was obtained. CONCLUSIONS: Maternal immunization against HPA-9b (Max(a)) is an important cause of NATP and should be considered in cases of apparent NATP not resolved on the basis of maternal-fetal incompatibility for "common" PLT antigens.     

The first two cases of neonatal alloimmune thrombocytopenia associated with the low-frequency platelet antigen HPA-21bw (Nos) in Japan

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a disorder characterized by maternal alloimmunization against paternal fetal platelet antigens. Two healthy, unrelated Japanese women each gave birth to a child with severe NAIT. STUDY DESIGN AND METHODS: To elucidate the maternal causes of NAIT, we conducted serologic and genetic studies in these two NAIT infants. RESULTS: The serologic experiments localized the antigens to the glycoprotein (GP) IIIa subunit of the GPIIb/IIIa complex. Sequence‐based typing studies subsequently identified a G>A mutation at Nucleotide 1960 (a glutamic acid > lysine substitution at Position 628) in the 11th exon of the GPIIIa gene. This mutation was recently identified in a report as HPA‐21bw. Next, it was determined that the cause of NAIT in both cases was the HPA‐21bw antigen, as shown by the mothers' antibodies reacting with the mutated GPIIIa‐transfected cells, but not with transfectants expressing wild‐type GPIIIa. A molecular genetic screening for the HPA‐21bw allele among Japanese donors showed that its genetic frequency in the population was 0.53% (10/1888), indicating that HPA‐21bw occurs at a low but appreciable frequency in the population. Furthermore, in a retrospective study of 50 previous NAIT cases of unknown causes, we found one NAIT case associated with the HPA‐21bw antibody. The two NAIT cases in this study represent the first ones to be associated with HPA‐21bw in Japan. CONCLUSION: We identified the HPA‐21bw allele from two unrelated Japanese infants with severe NAIT. We identified 10 individuals (1.06%) positive for the HPA‐21bw allele from a genetic screening of 944 Japanese blood donors.