Megakaryocyte- and Platelet-Derived Microparticles as Novel Diagnostic and Prognostic Biomarkers for Immune Thrombocytopenia

Altered cell-derived microparticles (MPs) have been reported in multiple autoimmune diseases. However, the roles of megakaryocyte- and platelet-derived MPs (MKMPs and PMPs) in immune thrombocytopenia (ITP) have not been investigated. In this study, we examined plasma MKMP and PMP levels in patients with ITP and evaluated their potential diagnostic values. Plasma MKMP and PMP levels were analyzed by flow cytometry in a discovery set of ITP patients (n = 78), non-immune thrombocytopenia (TP) patients (n = 69), and age- and gender-matched healthy controls (n = 88). Samples from a therapy set of ITP patients (n = 21) were used to assess the response to thrombopoietin receptor agonist (TPO-RA) treatment. Spearman correlation analysis was performed between MP levels and disease parameters. Receiver operator characteristic (ROC) curves were generated to evaluate the diagnostic values of the MPs. We found that plasma MKMP and PMP levels were significantly lower in ITP patients than those in healthy controls (p values p p p values p r = 0.558, p < 0.01). Our results indicate that plasma MKMP and PMP levels are decreased in ITP patients and that plasma MKMP and PMP levels may serve as biomarkers for ITP diagnosis and prediction of TPO-RA treatment response

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

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