Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.
Juping Zhai, Mengyuan Ding, Tianjie Yang, Bin Zuo, Zhen Weng, Yunxiao Zhao, Jun He, Qingyu Wu, Changgeng Ruan, Yang He
Author Information
Juping Zhai: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Mengyuan Ding: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Tianjie Yang: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Bin Zuo: Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China.
Zhen Weng: Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China.
Yunxiao Zhao: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Jun He: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Qingyu Wu: Cyrus Tang Hematology Center and Ministry of Education Engineering Center of Hematological Disease, and the Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China.
Changgeng Ruan: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China.
Yang He: Ministry of Health Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, 188 Shizi St, Suzhou, 215006, Jiangsu, China. heyang1963@163.com.
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.
