A New Method For Detecting Platelet Glycoprotein-specific Autoantibodies By Flowcytometric Bead Assay And Its Clinical Application

Background Idiopathic thrombocytopenic purpura(ITP) is a common hemorrhagic disorder characterized by a low platelet count which is caused by the binding of anti-platelet autoantibodies to patient's platelets. Current diagnoses of ITP are generally based on clinical impression, primarily that of exclusion. It is very important for a patient with ITP to detect platelet-specific alloantibody. Measurement of platelet glycoprotein (GP)-specific autoantibodies has different approach, including radioactive immunobead and monoclonal antibody immobilization of platelet antigen technique(MAIPA). The high specificity (78-93%) of these assays is sufficient to confirm the diagnosis of ITP. However,these assays can not be widely used as their low sensitivity(49-66%) and methodical limitations. Therefore, it is necessary to found a set of more intelligent, rapid, accurate, and feasible technique.Objective To establish a method for the detection of platelet-associate autoanti -bodies against platelate-specific receptors by cytometric bead assay.Methods Using detecting beads coated by monoclonal antibody of SZ2, SZ22, SZ21 and 7E3 against platelet glycoproteinâ… b ,â…¡b ,â…¢a andâ…¡b/â…¢a to capture platelet glycoprotein combined platelet-associate autoantibodies, then ading fluorescein isothiocyanate labeled goat antihuman immunoglobulin antibody, finally using flow cytometer to detect bead-platelet-associate auto antibodie s-fluorescein isothiocyanate labeled polyclonal goat antihuman immuno globulin-antibody complex. Platelet-associate autoantibodies in sample plasma were detected by modified indirect MAIPA, then compare the results with that of cytometric bead assay. Reaction condition were optimized by experiments, then took methodological evaluation and statistical analysis.Results Each of the groups'mean fluorescene intensity(MFI) level was calculated as a ratio to the three controls (basic control). Using microbeads coated by monoclonal antibody of SZ2, SZ22, SZ21 and 7E3 to detect the MFI value of anti-GPâ… b,â…¡b,â…¢a andâ…¡b/â…¢a autoantibodies of ITP group, then compare with the basic contron, The median ratios were 1.49(range 0.88-16.24),1.55(range 0.84-11.04),1.50(range 0.87-11.30),1.51 (range 0.84-9.82); 1.12(range 1.00-1.33),1.13(range 0.97-1.32),1.13(range 1.01-1.27), 1.05 (range 0.86-1.12) in the non-immune thrombocytopenic group and 1.01(range 0.94 -1.37),0.99(range 0.85-1.24),1.01(range 0.85-1.48).1.01(range 0.74-1.19)in the health control group. The difference between the ITP patients and both groups was highly significant difference (P< 0.01), using stringent non-parametric analysis. If ratios of ITP group larger than 1.37, 1.24, 1.48, 1.19 of upper limit of health control group were considered positive, flow cytometric bead assay had a sensitivity of 74.12%,a specificity of 96.47%,The overall sensitivity was significantly higher than that of modified indirect MAIPA'S(P<0.05),and greater than single antibody.Conclusions Platelet-associate autoantibodies can be detected rapidly and exactly by flow cytometric bead assay, and the detection sensitivity is higher than MAIPA's. A variety of combined detection of autoantibodies can improve the positive rate, and its important for platelet-associate autoantibodies detection to be a assistant for diagnosis of idiopathic thrombocytopenic purpura.