Anti-HPA-1a-mediated platelet phagocytosis by monocytes in vitro and its inhibition by Fc gamma receptor (FcgammaR) reactive reagents

The study was undertaken to delineate mechanisms of platelet destruction by phagocytosis during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) because of maternal antibodies against human platelet antigen 1a (HPA-1a). By employing a platelet phagocytosis assay based on the ORPEGEN flow cytometric bacterial phagocytosis test, we measured monocyte ingestion of platelets mediated by anti-HPA-1a antibodies. Moreover, we tested, as potential therapeutic agents, FcgammaR reactive reagents, for their inhibition of this process. Four of six anti-HPA-1a sera tested mediated phagocytosis of HPA-1a-positive platelets in a concentration-dependent manner. Monocyte ingestion of platelets was almost completely inhibited by cytochalasin D. No anti-HPA-1a-mediated phagocytosis was observed with anti-HPA-1a-negative platelets. The humanised anti-FcgammaRI monoclonal antibody H22 at concentrations 1-100 microg/ml, completely inhibited anti-HPA-1a-mediated phagocytosis as did similar concentrations of ivIg. By contrast, a mouse monoclonal anti-FcgammaRII (IV.3, Fab) at 10 microg/ml caused little or no suppression of platelet phagocytosis mediated by two anti-HPA-1 sera. Furthermore, the addition of anti-FcgammaRII (10 microg/ml) to sub-optimal concentrations of H22 did not significantly increase the inhibitory effect of the latter compound. Monomeric IgG (0.1-10 microg/ml) failed to suppress anti-HPA-1 mediated platelet ingestion by the phagocytes, as did anti-FcgammaRIII. To our knowledge this is a rare example of an assay that measures platelet phagocytosis in vitro. The results suggest that FcgammaRI plays a major role in anti-HPA-1a-mediated platelet phagocytosis by monocytes while FcgammaRIIa, is of little or minor importance only. Moreover, the findings indicate the use of H22 as an alternative to interavenous Ig (ivIg) in the management of FAIT/NAIT.     

Severe fetomaternal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a in a mother with a rare and silenced ITGB3*0101 (GPIIIa) allele

BACKGROUND AND OBJECTIVES: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a human platelet antigen (HPA) present on fetal, but absent from maternal platelets. We identified and characterized a case of FMAIT due to anti-HPA-1a in a mother with an HPA-1a1b genotype. MATERIALS AND METHODS: The first child of a 29-year-old mother presented with a petechial rash and a platelet count of 8 x 10(9) per l. Upon routine serological investigation, a discrepancy between the HPA-1a genotype and phenotype prompted the sequencing of the 15 exons of the ITGB3 (integrin beta3, GPIIIa and CD61) gene in the mother. RESULTS: The mother was genotypically HPA-1a1b heterozygous but phenotyped as HPA-1a negative. Sequencing of the ITGB3 exons confirmed HPA-1a1b heterozygosity, but also identified a novel single nucleotide insertion in exon 10 leading to a frameshift and premature termination at amino acid 471 of ITGB3. Maternal anti-HPA-1a was detected but with a pattern typical for a low-affinity antibody. Three transfusions of HPA-1a and -5b negative neonatal platelet concentrates were required to return to a safe platelet count. CONCLUSION: A rare ITGB3 allele was uncovered by the investigation of a severe case of alloimmune thrombocytopenia in a mother with HPA-1a antibodies who genotyped as HPA-1a1b.     

Spontaneous Rise of Fetal Platelet Counts Despite Increasing anti-HPA-5b Antibodies

Background and objectives: Maternal anti-HPA alloantibodies are a rare cause of severe fetal thrombocytopenia. So far there have been no reports on the dynamics of maternal anti-HPA-5 during gestation and its effect on the fetus. Materials and methods: We monitored maternal anti-HPA-5b antibody titers and fetal platelet counts during gestation in a woman with known anti-HPA-5b alloimmunization. The patient was a 32-year-old woman in her third pregnancy. The 1st pregnancy and delivery of a healthy child had been uneventful. At delivery, anti-HPA-5b was detectable in the maternal serum. A 2nd pregnancy ended in early miscarriage. Results: A steady rise in the alloantibody titer was recorded throughout the 3rd pregnancy. Therefore, cordocentesis was performed at 28 weeks of gestation (platelet count 76×10⁹/l). Serial platelet transfusions were administered to the fetus at 28, 32, and 37 weeks of gestation. The platelet counts rose spontaneously thereafter and were 220×10⁹/l at delivery, despite an increase in the anti-HPA-5b antibody titer. The child developed normally during the first year of life. Conclusions: This case illustrates the spontaneous recovery of fetal platelet counts in late pregnancy despite a rise in maternal alloantibody titer.     

Maternal alloimmunization against fetal platelet antigens: a prospective study

Summary. Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies to fetal platelet antigens. This prospective study was carried out to evaluate the incidence of anti-platelet antibodies in 933 mother-child pairs where the mother and child were typed for the human platelet antigens (HPA)-l, -2,-3,-5. Sera from mismatched mother-child pairs were screened for anti-platelet antibodies, anti-HLA class I and blood group ABO IgG antibodies. Platelet-specific antibodies were anti-HPA-3a in one and anti-HP A-5b in 17 neonates, respectively. All these neonates had normal platelet counts. One woman had autoreactive antibodies. Anti-HLA class I and anti-blood group A IgG antibodies were detected in five and four neonates, respectively, born with a platelet count <150×10⁹/l. None of the 11 homozygous HP A-lb mothers became immunized against their heterozygous offspring. The maternal HLA-allotypes HLA-DR52 and -DR6, typically found in individuals immunized against HPA-la and -5b, respectively, were found in three of 11 HPA-b/b non-responders and eight of the anti-HPA-5b responders. The results indicate that a risk for NAIT due to HPA-2 and -3 alloimmunization is low. The HLA allotypes do not predict the risk for NAIT due to HPA-1 or -5 alloimmunization. Maternal anti-HPA-5b antibodies do not correlate with the platelet count in the neonate.     

A case of neonatal alloimmune thrombocytopenia in the presence of both anti-HPA-4b and anti-HPA-5b antibody: clinical and serological analysis of the subsequent pregnancy

Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies raised against fetal platelet antigens inherited from the paternal parent. In contrast to Caucasians, in Asians, predominantly in Japanese, most frequently detected antibodies in NAIT are anti-HPA-4b and anti-HPA-5b. In some NAIT cases multiple alloantibodies are detected. In such cases it is very difficult to determine which antibody is the dominant antibody in NAIT. In this case report, we describe a NAIT case (first sibling) with severe thrombocytopenia and cephalhematoma in the presence of both anti-HPA-4b and anti-HPA-5b antibodies in the maternal serum. We carefully examined titers of anti-HPA antibodies during the subsequent pregnancy with HPA-4b-positive and HPA-5b-negative fetus determined by amniocentesis at gestational week 16. We administered IVIG (1 g/kg/w) to the mother from gestational week 32 to 35. The mother subsequently delivered a second sibling with normal platelet count by cesarean section. Although we could not completely rule out the involvement of anti-HPA-4b, our findings suggested that anti-HPA-5b was implicated in the NAIT in the first sibling.     

Genomic RFLP typing of human platelet alloantigens Zw(PlA), Ko,Bak and Br (HPA-1, 2, 3, 5)

Summary. The diallelic human platelet alloantigen systems 1–5 have been found to result from single base pair substitutions in the encoding genes of platelet membrane glycoproteins IIIa, Ib, IIb and Ia. This is the basis of DNA methods for determination of platelet alloantigens. In this study, 98 blood donors were typed in the HPA-1, 2, 3 systems and, for the first time, in the HPA-5 system. Serologically obtained data (MAIPA and platelet agglutination) were compared with results from analysis of restriction fragment length polymorphisms (RFLP). Discordances were found in the HPA-2 and 3 systems and can be ascribed to false typing results in both the serological and genomic methods. In the HPA-1, 2 and 5 systems, all samples were typed correctly with RFLP analysis. Serologically, two donors were falsely typed positive with anti-HPA-2b in platelet agglutination and one donor with anti-HPA-3a in MAIPA assay. In the HPA-3 system, another four donors were misinterpreted to be HPA-3 a negative in RFLP analysis. Possible technical problems in PCR-RFLP-typing are discussed and another strategy of HPA-1 typing using the restriction enzyme Scr FI is evaluated.     

Frequencies of maternal platelet alloantibodies and autoantibodies in suspected fetal/neonatal alloimmune thrombocytopenia, with emphasis on human platelet antigen-15 alloimmunization

BACKGROUND AND OBJECTIVES: Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only approximately 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP. MATERIALS AND METHODS: In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP. RESULTS: Genotyping maternal and paternal samples (together n = 573) revealed a gene frequency of 0.496 for HPA-15a and a gene frequency of 0.504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and -15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and -15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8.4%) were more frequent than anti-HPA-15. In addition, panreactive (5.5%) or autoreactive (5.2%) anti-GPIIb/IIIa or anti-GPIb/IX were detectable in maternal samples. CONCLUSIONS: These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia.     

HPA-1a-mediated platelet interaction with monocytes in vitro: involvement of Fcgamma receptor (FcgammaR) classes and inhibition by humanised monoclonal anti-FcgammaRI H22

To gain insight into mechanisms of platelet destruction and its possible inhibition during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) the binding to monocytes (Mo) of anti-HPA-1a-sensitised platelets, the initial step in IgG-mediated destruction by effector cells, was evaluated in an in vitro assay. Neonatal Mo were compared with adult Mo as effectors in the assay. Moreover, the potential involvement of Fcgamma receptor (FcgammaR) classes during platelet destruction in the disease was tested by using FcgammaR class-specific reagents as inhibitors of the binding reaction. Neonatal Mo were 37% less active than adult Mo in their interaction with anti-HPA-1a-sensitised platelets (p < 0.05). The FcgammaRI-specific reagents human monomeric IgG and humanised anti-FcgammaRI monoclonal H22 caused virtually complete inhibition of platelet binding to Mo. When compared to an intravenous immunoglobulin preparation the inhibitory activity of H22 was 10-100 x greater than that of the latter compound. Monoclonal anti-FcgammaRII IV.3 and anti-FcgammaRIII 3G8 decreased platelet binding by 70% and 64%, respectively, but only the anti-FcgammaRII inhibition was statistically significant (p < 0.001). Finally, anti-HPA-1a-sensitised platelets bound to 131H- but not to 131R- FcgammaRIIa transfected 3T6 mouse fibroblasts (p < 0.01), in an anti-HPA-1a-concentration-dependent manner. The results suggest that FcgammaRI and FcgammaRIIa may be involved in anti-HPA-1a-mediated platelet destruction by mononuclear phagocytes during FAIT/NAIT. Moreover, the much greater potency ofmonoclonal H22 than of intravenous immunoglobulin as an inhibitor of anti-HPA-1a-mediated Mo-platelet interaction, might render it superior to the latter agent in the maternal therapy of the disorder.     

Frozen Platelet Plates for Platelet Antibody Detection and Cross-Match

We performed 2 studies aimed at developing a frozen platelet panel suitable for platelet cross-matching. The stability of the most important platelet membrane glycoproteins and the reactivity of antigens of the human platelet antigen (HPA) and of the human leukocyte antigen (HLA) systems were evaluated with the platelet suspension immunofluorescence test (PSIFT) in a panel of platelets frozen in microplates with 6% dimethylsulfoxide. In study No. 1 we evaluated platelet reaction with a broad-spectrum weak anti-HLA and a potent anti-HPA-1a antiserum and the expression of glycoproteins Ib and IIb/IIIa complex on platelet membrane before freezing and after 0.5, 1, 2, 3, 4, 5, 6 and 12 months of storage at -80°C. In study No. 2 we examined platelet reactivity with anti-HPA-1b, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 of platelets stored frozen for 12 months in parallel with fresh platelets from the same donors. Study No. 1 showed that glycoprotein expression was stable and that the weak anti-HLA and the potent anti-HPA-1a antibodies were clearly detected during 12 months at -80°C. Of the 35 paired PSIFT performed in study No. 2 with fresh and frozen/thawed platelets incubated with anti-HPA-1b, -HPA-2a, -HPA-3a -HLA-A2, -HLA-A3 antisera and AB serum, concordant reactions were obtained in all cases with the exception of 1 case of HLA-A3-positive platelets incubated with anti-HLA-A3 antiserum, that was reactive with frozen/thawed platelets but nonreactive with fresh platelets from the same donor. The discrepant finding obtained with fresh platelets from 1 donor could be due to the well-known variable and weak association of HLA antigens to platelet membrane. We conclude that frozen platelet plates can be stored and used for at least 12 months for detecting platelet-reactive antibodies in patients' sera.     

A case of severe neonatal thrombocytopenia with schizencephaly associated with anti-HPA-1 b and anti-HPA-2a

We report a family with a neonate who was severely damaged by intracranial haemorrhages. These probably occurred before the 20th week of gestation. The neonate had a moderate thrombocytopenia. In the maternal serum anti-HPA-1b and anti-HPA-2a alloantibodies were detected. Third-generation assays were applied to identify the alloantibodies. No other cause for the bleeding was found. Probably the combination of anti-HPA-1b and anti-HPA-2a alloantibodies, directed against the platelet fibrinogen receptor and the von Willebrand receptor, respectively, induced a thrombocytopenia and a thrombocytopathy.     

Localization of the platelet-specific HPA-2 (Ko) alloantigens on the N-terminal globular fragment of platelet glycoprotein Ib alpha.

The human platelet-specific alloantigens HPA-2a and HPA-2b (= Kob and Koa) together constitute a biallelic antigen system. The HPA-2 antigens have not, to date, been located on a particular platelet membrane molecule. Here, we describe the localization of these antigens on platelet glycoprotein (GP) Ib alpha. Platelets from two patients with the Bernard-Soulier syndrome (BSS) were HPA-2(a-,b-) in the immunofluorescence test with HPA-2 alloantibodies on chloroquine-treated platelets. With monoclonal antibody (MoAb) immobilization of platelet antigen assay (MAIPA), positive reactions were obtained only when MoAbs against the platelet GPIb/IX complex were used in combination with anti-HPA-2a or -2b alloantibodies and normal donor platelets. By immunoprecipitation under nonreducing and reducing conditions a protein of 160 Kd and 145 Kd, respectively, was precipitated by the anti-HPA-2a serum. A protein migrating identically to this was precipitated by anti-GPIb MoAb. Normal donor platelets became HPA-2(a-,b-) after elastase treatment, suggesting that anti-HPA-2 antibodies bind to the N-terminal elastase-sensitive part of GPIb alpha. Anti-HPA-2a antibodies inhibited the ristocetin-induced agglutination of HPA-2a-positive platelets but not of HPA-2a-negative platelets, indicating that the epitopes recognized by these alloantibodies are localized in the proximity of the von Willebrand-factor-binding domain. Together, these data provide evidence that the HPA-2 alloantigens are located on the N-terminal globular elastase-sensitive part of GPIb alpha. Furthermore, we show that the recently described Siba antigen is probably identical to HPA-2a.     

An International Reference Reagent (minimum sensitivity) for the detection of anti-human platelet antigen 1a

Background and Objectives   The platelet-specific alloantibody anti-human platelet antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate 'in-house' controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled human plasma, coded 05/106, containing anti-HPA-1a.
Materials and Methods   Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries.
Results   As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or human leucocyte antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t -test.
Conclusions   When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.     

Thrombocytopenia caused by passive transfusion of anti-glycoprotein Ia/IIa alloantibody (anti-HPA-5b).

We describe a patient who developed transient and moderately severe thrombocytopenia (platelet count nadir 35 x 10(9)/L) after the transfusion of plasma. Using the technique of direct radioimmunoprecipitation, we showed that during the thrombocytopenia episode, the patient's platelets had IgG specifically bound to the glycoprotein (GP) Ia/IIa complex. Indirect radioimmunoprecipitation using serum from the plasma donor confirmed that anti-HPA-5b (anti-Zava) was the cause of GP Ia/IIa sensitization. The relatively mild thrombocytopenia, compared with passive alloimmune thrombocytopenia caused by anti-HPA-1a (anti-P1A1), may reflect the low copy number of HPA-5 compared with HPA-1. Direct radioimmunoprecipitation permits the detection of the GPs carrying the known platelet alloantigen systems, and this study suggests that this technique can be used to diagnose passive alloimmune thrombocytopenia.     

Human platelet activating antibodies.

Platelet antibodies identified in the plasma of three multiply transfused patients and a woman who had delivered a baby with neonatal alloimmune thrombocytopenia were investigated for their platelet activating properties. Three patients possessed multispecific HLA antibodies reactive with 90 to 100% of the cells on a lymphocytotoxic panel. These antibodies were also detected using the MAIPA assay and MAb w6/32, which recognizes an epitope common to all HLA class I molecules. In addition to HLA antibodies, three of the patients possessed platelet-specific antibodies that were identified by the MAIPA assay as anti-HPA-1a and anti-HPA-3a (one patient) and anti-HPA-1b (two patients). Each of the HLA antibodies when reacted with platelets expressing the corresponding HLA antigens, potently induced aggregation and release of ATP from dense granules. In contrast, the HPA-1b antibodies induced platelet agglutination, but failed to trigger ATP release. However, platelets coated with these latter antibodies were now refractory to subsequent stimulation by ADP. Similarly, when HLA antibodies were reacted with platelets to produce suboptimal activation, the platelets could now be stimulated only poorly or not at all by either epinephrine or thrombin. This was also true for anti-HPA-1b, which, although not inducing aggregation or ATP release by itself, was capable of almost completely blocking thrombin-induced platelet activation. The thrombin-inhibiting activity of these antibodies could partially be reversed by pretreating antibody-coated platelets with epinephrine immediately followed by stimulation with thrombin. These findings suggest that transfused platelets may either be activated or inhibited by reaction with various platelet antibodies. Therefore it is conceivable that the presence of platelet reactive antibodies in multiply transfused recipients may contribute to the increased thrombotic and hemorrhagic symptoms often observed among these patients.     

Report on the Eleventh International Society of Blood Transfusion Platelet Genotyping and Serology Workshop

BACKGROUND AND OBJECTIVES: The aims of the Eleventh International Workshop were to evaluate proficiency in platelet genotyping and antibody detection, to equip laboratories to perform Gov antigen system genotyping and antibody detection, and to evaluate the laboratory and clinical approach to cases of neonatal alloimmune thrombocytopenia (NAIT). MATERIALS AND METHODS: There were 34 participating laboratories from 22 countries on five continents. Participating laboratories were provided with 10 DNA samples, 15 unknown sera, and three monoclonal antibodies for titration, as well as primer pairs and a protocol for Gov genotyping and Gov antibody screening. They were also provided with a questionnaire on investigation and clinical management of patients with NAIT. RESULTS: Thirty-three participants reported human platelet antigen (HPA)-1, -2, -3 and -5 genotyping results, 25 reported HPA-4 typing results, 17 reported HPA-6 typing results and 24 reported Gov typing results. For HPA-1-6 genotyping, 23 laboratories were concordant with a majority vote for all allotypes tested, five laboratories reported one deviation, three laboratories reported two deviations and one laboratory reported three deviations. For Gov genotyping, six deviations occurred in three of the 24 laboratories reporting results. Antibody detection was 90% concordant for anti-HPA-1a, anti-HPA-5a and anti-HPA-5b detection. Anti-HPA-2b and anti-Gova were detected by 20 and 14 out of 33 laboratories, respectively. Approaches to the clinical management of NAIT vary widely, especially for mothers with a history of a previous infant with mild NAIT. CONCLUSIONS: The overall error rate for HPA-1-6 genotyping decreased from 2.7% in the tenth workshop to 0.8% in the eleventh workshop. The majority of laboratories were able to perform Gov genotyping, although the error rate was 7.5%. Detection of common clinically significant antibodies was good, although detection of the much rarer HPA-2b was problematic. There was considerable progress in the detection of anti-Gova. The lack of consensus over treatment of NAIT demonstrates uncertainty over optimal management of these patients.     

Human platelet antigen-1a antibodies induce the release of the chemokine RANTES from human platelets

BACKGROUND AND OBJECTIVE: Binding of human platelet antigen-1a (HPA-1a)-specific antibodies to target platelets can trigger platelet activation and mediator release. Here we tested the effect of HPA-1a antibody-containing sera on platelet release of the chemokine RANTES (regulated on activation, normal, T-cell expressed, and presumably secreted) in vitro. PATIENTS AND METHODS: HPA-1a-containing sera obtained from 11 mothers delivered of an infant with neonatal alloimmune thrombocytopenia (NAIT) and from six patients with post-transfusion purpura (PTP) were incubated with HPA-1a/a target platelets. Antibody-induced release of soluble RANTES was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant release of soluble RANTES was induced by four out of the 17 sera. Two out of the four reactive sera were obtained from mothers who were delivered of a baby with NAIT and the remaining two sera were from patients with PTP. Chemokine release was specific for binding of anti-HPA-1a to the platelet membrane, as none of the reactive sera induced the release of soluble RANTES when incubated with HPA-1b/b platelets. The blockade of platelet-expressed Fc gamma receptor type II (FcgammaRII) inhibited anti-HPA-1a-mediated RANTES release when incubated with the reactive sera of patients with NAIT, but not when platelets were incubated with sera of patients with PTP. CONCLUSION: Our findings suggest that anti-HPA-1a antibody-induced release of platelet-derived RANTES can play a role in adverse reactions in alloimmunized patients.     

Collaborative study to establish the first international standard for quantitation of anti-HPA-1a

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. MATERIALS AND METHODS: Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. RESULTS: An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. CONCLUSIONS: Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).     

A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

Background

Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays.

Design and Methods

Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a.

Results

This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies.

Conclusions

This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes.     

Maternal antibodies against paternal class I human leukocyte antigens are not associated with foetal and neonatal alloimmune thrombocytopenia

The causative role of maternal, anti-human leukocyte antigen (anti-HLA) class I antibodies in foetal and neonatal alloimmune thrombocytopenia (FNAIT) remains controversial. Furthermore, in FNAIT cases caused by anti-human platelet antigen-1a (anti-HPA-1a) antibodies, the possible additive effect of maternal anti-HLA class I antibodies on outcomes is unclear. Among 817 mother–father–neonate trios of suspected FNAIT, we assessed the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, and the incidence of FNAIT caused by anti-HPA-1a antibodies. In 144 FNAIT cases caused by anti-HPA-1a antibodies, we investigated the possible association of maternal anti-HLA class I antibodies with neonatal platelet count, birth weight, and the incidence of intracranial haemorrhage (n = 16). Maternal anti-HLA class I antibodies were not associated with neonatal platelet count in suspected cases of FNAIT. There was no significant interaction between the presence of anti-HLA class I antibodies and anti-HPA-1a antibodies. In FNAIT cases caused by anti-HPA-1a antibodies, there was no association between the presence of anti-HLA class I antibodies and neonatal platelet count, birth weight, or occurrence of intracranial haemorrhage. This study’s findings do not support the concept that maternal anti-HLA class I antibodies represent a risk factor of FNAIT or disease severity.     

Collaborative studies to establish the first World Health Organization International Standard for detection of human antibody against human platelet antigen-3a

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.