title: A novel method for simultaneous analysis of specific platelet antibodies: SASPA.
abstract: Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.
authors.affiliation: Institute of Transfusion Medicine and Immunology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Red Cross Blood Service of Baden-Württemberg-Hessen, 68167 Mannheim, Germany. x.nguyen@blutspende.de
authors.forename: Xuan Duc
authors.initials: XD
authors.lastname: Nguyen
authors.forename: Alex
authors.initials: A
authors.lastname: Dugrillon
authors.forename: Christian
authors.initials: C
authors.lastname: Beck
authors.forename: Mohammad
authors.initials: M
authors.lastname: Kerowgan
authors.forename: Harald
authors.initials: H
authors.lastname: Klüter
mesh_terms: D000906:Antibodies / Q000032:analysis*; D016824:Antigens, Human Platelet / Q000276:immunology*; D001792:Blood Platelets / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D013921:Thrombocytopenia / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000906:Antibodies; D016824:Antigens, Human Platelet; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D010980:Platelet Membrane Glycoproteins