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

BACKGROUND: Immunization against the human platelet alloantigen (HPA)-3a residing on alpha IIb beta 3 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 (>= 50% reduction; n = 3), and 3) minor reduction of reactivity (<= 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 aIIbb3 integrin structure. CONCLUSIONS: Anti-HPA- 3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the aIIbb3 integrin and protection from enzymatic degradation seem to be important during PLT storage.