title: Detection of anti-human platelet antibodies against integrin α2β1 using cell lines.
abstract: BACKGROUND
  Antibodies against human platelet antigens (HPA) are a cause of thrombocytopenia. Detection of rare anti-HPA antibodies using platelet preparations is difficult and would be improved by an alternative method that does not require platelets. In the present study, we describe the establishment of cell lines that stably express specific HPA associated with integrin α2β1 and the application of these cell lines for detecting anti-HPA-5a and anti-HPA-5b antibodies.
  
  
  METHODS
  Complementary DNA of the integrin α2 variants HPA-5b, -13b and -18b were individually transfected into K562 cells using retroviral vectors. Expression of integrin α2 was confirmed by flow cytometric analysis, immunoprecipitation and western blotting analysis. To verify whether the cell line panel was suitable for clinical diagnosis, we analysed its properties using monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) and well-characterised serum samples.
  
  
  RESULTS
  Exogenous integrin α2 expression was observed in the transfected cells for over 6 months. The cell line panel specifically detected previously characterised anti-HPA-5a and anti-HPA-5b antisera. No reactivity was observed with control sera, including normal sera and HLA antisera.
  
  
  CONCLUSIONS
  We successfully established a cell line panel to facilitate the sensitive and reliable detection of anti-HPA-5a and anti-HPA-5b antibodies.
authors.affiliation: Department of Preparation, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Department of Laboratory, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Amakishi
authors.affiliation: Department of Laboratory, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Nobuki
authors.initials: N
authors.lastname: Matsuyama
authors.affiliation: Department of Preparation, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Kazuta
authors.initials: K
authors.lastname: Yasui
authors.affiliation: Department of Preparation, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Rika Ann
authors.initials: RA
authors.lastname: Furuta
authors.affiliation: Japanese Red Cross Osaka Blood Centre, Osaka, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hori
authors.affiliation: Department of Preparation, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Laboratory, Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Fukumori
authors.affiliation: Japanese Red Cross, Kinki Block Blood Centre, Osaka, Japan.
authors.forename: Fumiya
authors.initials: F
authors.lastname: Hirayama
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D016824:Antigens, Human Platelet / Q000235:genetics / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D039421:Integrin alpha2 / Q000235:genetics / Q000276:immunology*; D038982:Integrin alpha2beta1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D020014:K562 Cells; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000276:immunology; D014162:Transfection
chemical_list: C055973:5b alloantigen, human; D000911:Antibodies, Monoclonal; D016824:Antigens, Human Platelet; D018076:DNA, Complementary; D000939:Epitopes; C000592916:HPA-13b antigen, human; C000592917:HPA-18b antigen, human; D039421:Integrin alpha2; D038982:Integrin alpha2beta1; D011993:Recombinant Fusion Proteins