1. Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients
- Author
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Yang He, Changgeng Ruan, Bin Zuo, Zhen Weng, Jun He, Tianjie Yang, Qingyu Wu, Yunxiao Zhao, Juping Zhai, and Mengyuan Ding
- Subjects
Adult ,Blood Platelets ,Male ,medicine.drug_class ,Immunobead assay ,lcsh:Medicine ,Platelet membrane glycoprotein ,Monoclonal antibody ,Sensitivity and Specificity ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Quantitative flow cytometry ,0302 clinical medicine ,Treatment monitoring ,hemic and lymphatic diseases ,Medicine ,Humans ,Platelet ,Fluorescein isothiocyanate ,Autoantibodies ,Immunoassay ,Purpura, Thrombocytopenic, Idiopathic ,biology ,medicine.diagnostic_test ,business.industry ,Research ,lcsh:R ,Autoantibody ,Reproducibility of Results ,General Medicine ,Middle Aged ,Flow Cytometry ,Molecular biology ,Primary and secondary antibodies ,Platelet glycoprotein ,Immune thrombocytopenia ,chemistry ,ROC Curve ,030220 oncology & carcinogenesis ,Case-Control Studies ,Immunology ,biology.protein ,Female ,Antibody ,business ,030215 immunology - Abstract
Background Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Methods Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. Results The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. Conclusions A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1317-2) contains supplementary material, which is available to authorized users.
- Published
- 2017
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