title: Detection of human platelet antigen-1a alloantibodies in cases of fetomaternal alloimmune thrombocytopenia using recombinant β3 integrin fragments coupled to fluorescently labeled beads.
abstract: BACKGROUND
  Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA-typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant β3 integrins displaying the HPA-1a (rHPA-1a) or HPA-1b (rHPA-1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA-1a alloantibody screening assay, using Luminex xMAP technology.
  
  
  METHODS
  A 3-plex bead assay was developed by coupling biotinylated rHPA-1a, rHPA-1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb-specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay.
  
  
  RESULTS
  The rHPA-1a- and rHPA-1b-coupled beads were able to detect HPA-1a and HPA-1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA-1-specific antibodies. Furthermore, HLA Class I antibodies did not cross-react with the coupled beads.
  
  
  CONCLUSIONS
  The 3-plex bead assay can be used to detect HPA-1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders.
authors.affiliation: Histocompatibility & Immunogenetics Laboratory, NHS Blood and Transplant, Colindale, London.
authors.forename: Winnie
authors.initials: W
authors.lastname: Chong
authors.forename: Paul
authors.initials: P
authors.lastname: Metcalfe
authors.forename: Rosey
authors.initials: R
authors.lastname: Mushens
authors.forename: Geoff
authors.initials: G
authors.lastname: Lucas
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: Cristina V
authors.initials: CV
authors.lastname: Navarrete
mesh_terms: D016824:Antigens, Human Platelet / Q000276:immunology*; D001681:Biological Assay / Q000379:methods*; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007518:Isoantibodies / Q000097:blood* / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000097:blood* / Q000276:immunology*
chemical_list: D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D011994:Recombinant Proteins