Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

Human platelet antigens (HPA) is located on the platelet surface and they are inherited both from the mother and the father. If a mother who is homozygous for HPA-1b carries a child who has inherited HPA-1a from the father, the mother is in danger to form antibodies against HPA-1a on the fetal platelet. This may cause the child to suffer from neonatal alloimmune thrombocytopenia (NAIT) that could lead to death. This can be prevented by platelet transfusion. EVA Biosensor Technology is a new method for detection of HPA-1 that is currently only approved for scientific research. The aim of this study was to evaluate EVAreader R6 and find HPA-1a negative platelet donors that can donate platelets to children born with NAIT. The test material consisted of blood samples from 513 male blood donors with blood group 0. The blood was lysed and tested in EVA-reader R6 from Davos Diagnostics. The result was shown on the screen after 10 min. The results that came out negative or intermediate was analyzed a second time. In total, nine HPA-1a negative donors and 503 HPA-1a positive donors were found. Approximately 2 % of the population is HPA-1a negative, which was reflected in the result. To make sure that the results are correct, a validation with an already existing method has to be made. The conclusion is that the EVA Biosensor Technology could be used for typing of HPA in the future, as long as the results from the validation is correct.