title: Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies.
abstract: BACKGROUND
  Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described.
  
  
  METHODS
  Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis.
  
  
  RESULTS
  Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination.
  
  
  CONCLUSIONS
  In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.
authors.affiliation: Department of Laboratory, Japanese Red Cross Tokyo Blood Center, 2-1-67 Tatsumi, Koto-ku, Tokyo, Japan. ko-fujiwara@tokyo.bc.jrc.or.jp
authors.forename: K
authors.initials: K
authors.lastname: Fujiwara
authors.forename: K
authors.initials: K
authors.lastname: Shimano
authors.forename: H
authors.initials: H
authors.lastname: Tanaka
authors.forename: M
authors.initials: M
authors.lastname: Sekine
authors.forename: K
authors.initials: K
authors.lastname: Kashiwase
authors.forename: M
authors.initials: M
authors.lastname: Uchikawa
authors.forename: M
authors.initials: M
authors.lastname: Satake
authors.forename: K
authors.initials: K
authors.lastname: Nakajima
mesh_terms: D016824:Antigens, Human Platelet / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D006680:HLA Antigens / Q000032:analysis*; D006801:Humans; D008863:Microspheres; D012680:Sensitivity and Specificity
chemical_list: D016824:Antigens, Human Platelet; D006680:HLA Antigens