title: Heterogeneity of HPA-3 alloantibodies: consequences for the diagnosis of alloimmune thrombocytopenic syndromes.
abstract: 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.
  
  
  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.
authors: Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Giessen, Germany.
authors: Ines
authors: I
authors: Socher
authors: Claudia
authors: C
authors: Zwingel
authors: Sentot
authors: S
authors: Santoso
authors: Hartmut
authors: H
authors: Kroll
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D016824:Antigens, Human Platelet / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007231:Infant, Newborn; D007518:Isoantibodies / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D013577:Syndrome; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000175:diagnosis* / Q000276:immunology; D014162:Transfection
chemical_list: C044561:3a alloantigen, human; C083448:3b alloantigen, human; D016824:Antigens, Human Platelet; D007518:Isoantibodies; D011994:Recombinant Proteins