Detection of platelet antigen for antiplatelet antibodies in idiopathic thrombocytopenic purpura by flow cytometry, antigen‐capture ELISA, and immunoblotting: a comparative study

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Abstract
We compared three methods of detecting platelet antigens for antiplatelet antibodies in patients with idiopathic thrombocytopenic purpura (ITP), i.e., a microtiter well antigen‐capture enzyme‐linked immunosorbent assay (AC‐ELISA), a platelet suspension immunofluorescence test using flow cytometry (PSIFT‐FCM), and Western blotting. Using PSIFT‐FCM, the reactivity of NNKY1–32, an anti‐glycoprotein (GP) IIb/IIIa antibody, and of NNKY5‐5 (anti‐GPIb) to platelets from 60 ITP patients were examined. By PSIFT‐FCM, both the peak channel and the relative fluorescence value were below the mean‐2SD for healthy control platelets in 15 patients when NNKY1–32 was used and in 2 patients when NNKY5‐5 was used. Western blotting gave an apparent molecular weight for GPIb of 160000, while GPIIb was 135000 and GPIIIa was 88000. By the AC‐ELISA, 12 patients were positive for NNKY1–32 and 4 for NNKY5‐5. Although NNKY1‐32 binding was detected by PSIFT‐FCM in 15 of the ITP patients using platelets, only 3 were positive using plasma. By AC‐ELISA and Western blotting of plasma, 12 and 10 of the patients were positive for NNKY1–32 and NNKY5‐5, respectively. Our results suggest that none of the three methods is good enough to stand alone and that they should be used together in the analysis of platelet antigens for antiplatelet antibodies in ITP.