Autoimmune Thrombocytopenia: Flow Cytometric Determination of Platelet-associated Autoantibodies against Platelet Specific Receptors

Abstract number: P0313

Tomer¹ A, Koziol² J, McMillan² R

¹¹Blood Bank and Transfusion Medicine, Soroka University Medical Center, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel ²²Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by antibody-induced platelet destruction. Despite its clinical importance, the diagnosis of ITP is one of exclusion, thus, inevitably associated with potential difficulties. We here describe a feasible diagnostic method using the commonly available technique of flow cytometry. An antigen-specific assay for platelet-associated antibody was developed and tested in 62 adult patients with chronic ITP, 14 patients with thrombocytopenia of decreased production and 60 healthy controls. The method is based on flow cytometric (FCM) detection of autoantibodies reacting with specific platelet receptors immobilized on microbeads. The binding of the patient's autoantibodies is detected by fluorescein-labeled secondary antibody. The average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8 to 31.0), in the non-ITP thrombocytopenic patients 0.9 (range 0.7, 1.2), and in the healthy controls 1.0 (range 0.7, 1.3). The average assay coefficient of variation was 0.218 (95% confidence interval 0.213 to 0.221). The difference between the ITP patients and both the non-ITP thrombocytopenic patients and the normal controls was highly significant (P < 0.001), using a stringent non-parametric analysis. A comparison of the FCM assay with the radioactive immunobead assay previously reported on the same cohort of patients showed significant correlation (R-square 0.71, 95% confidence interval 0.39 to 0.53). The overall performance of the FCM assay in discriminating between ITP patients and normals was estimated by the receiver operating characteristic (ROC) plot, showing an area under the curve of 0.96 (maximal value 1.0), with standard error of 0.033. We conclude that the present FCM assay is clinically useful for routine diagnosis and follow up of ITP.