Development And Clinical Application Of A Novel Flow Cytometric Immunobead Array Assay For Quantitative Dectection Of Platelet-specific Autoantibodies

Objective:To establish a standard curve and develop a novel flow cytometric immunobead assay for quantitative detection of platelet membrane glycoprotein-specific autoantibodies,and to evaluate its application in the diagnosis and treatment of primary immune thrombocytopenia(ITP).Methods:Immnuo-microbeads with 6 kind fluorescent intensities were firstly coated with a polyclonal antibody and then used for human IgG capture at 6 different concentrations.After that,the microbeads were collected,pooled and analyzed by flow cytometry using an FITC-labeled antibody.A standard curve was obtained by calculating the ratio of mean fluorescence intensity(MFI)and the corresponding human IgG level.Immnuo-microbeads with 5 kind fluorescent intensities were coated with monoclonal antibodies against human platelet GPIX(SZ1),GPIb(SZ2),GPIIIa(SZ21),GPIIb(SZ22)and P-selectin(SZ51)respectively.Plasma samples were incubated with normal platelets,then the opsonized platelets were lysed and incubated with coated microbeads,finally FITC-labeled goat anti-human IgG polyclonal antibodies were added and analyzed by flow cytometry.We subsequently collected and analyzed plasma samples from 201 ITP patients,126 non-ITP patients and 207 healthy controls by using the newly established quantitative flow cytometric immunobead assay.Receiver operating curve(ROC)was constructed to assess the specificity and sensitivity of this method,which was further compared with that from qualitative indirect FCIA assay.Moreover,The efficacy of immunomodulatory therapy in 8 patients with newly diagnosed ITP was evaluated by the quantitative levels of platelet-specific autoantibodies.Results:Platelet autoantibodies against GPIX,GPIb,GPIIIa,GPIIb and P-selectin were quantitatively detected.Coefficients of variations(CVs)of these antibodies in intra-assay were 3.54,3.65,4.66,6.43 and 6.97%,respectively,and CVs in inter-assay were 10.89,7.57,10.34,6.95 and 10.72%,respectively.Significantly elevated platelet-bound GPIX,GPIb,GPIIIa,GPIIb and P-selectin autoantibodies were found in ITP patients compared to non-ITP patients or healthy controls(anti-GPIX: 92.67 ± 56.41 vs.59.98 ± 23.46 or 52.89 ± 21.27 ng ?ml,P < 0.01;anti-GPIb: 130.68 ± 73.42 vs.95.32 ± 27.44 or 86.28 ± 26.91 ng ?ml,P < 0.01;anti-GPIIIa: 103.05 ± 47.34 vs.72.41 ± 20.93 or 70.26 ± 23.29 ng ?ml,P < 0.01;anti-GPIIb:114.81 ± 82.76 vs.74.36 ± 21.15 or 69.83 ± 23.23 ng ?ml,P < 0.01;antiP-selectin: 132.95 ± 69.82 vs.73.29 ± 24.18 or 72.29 ± 25.31 ng ?ml,P < 0.01).According to the results of ROC curve,the cut-off values of platelet autoantibodies GPIX,GPIb,GPIIIa,GPIIb,and P-selectin were 95.21,139.40,110.09,111.05 and 122.18 ng ?ml,respectively,and the corresponding areas under curve(AUC)were 0.80,0.70,0.76,0.77 and 0.79,respectively.The sensitivity,specificity and accuracy of our quantitative when using all five antibodies combined was 73.13%,81.98% and 78.65%,respectively,which was similar to that of the qualitative FCIA assay(P > 0.05).A total of 8 patients with newly-diagnosed ITP were followed up,significantly decreased levels of autoantibodies were found in four ITP patients who had response to treatment.While the change of platelet specific autoantibodies varied in the other 4 patients who did not respond to treatment.Conclusions:A novel quantitative flow cytometric immunobead assay with a good specificity and sensitivity was successfully established for detection of autoantibodies from ITP patients,which may provide a powerful tool for dynamic monitoring the diagnostic and therapeutic process in ITP patient.