Flow cytometric immnno-bead array assay for detection of platelet-specific autoantibodies

Objective To establish a new flow cytometric immuno-bead array assay (FCIBA)to detect several platelet-specific autoantibodies simultaneously in a single serum sample.Methods A series of beads of different red fluorescent intensity were used for coating with different anti-platelet membrane protein monoclonal antibodies(anti-GP Ⅱb/a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰ b/Ⅸ and anti-HLAABC)to detect five platelet-specific autoantibodies in serum. The beads captured platelet antigenautoantiboay complex.Subsequently,different platelet-specific autoantibodies in a patient serum can be detected simultaneously by the flow cytometry.In addition,we evaluated the new FCIBA,and compared its results with modified antigen capture ELISA method(MACE).The new FCIBA was used to detect five platelet-specific autoantibodies in serums of autoimmune thrombocytopenic purpura(AITP)and nonautoimmune thromboeytopenic purpura patients.Results The new FCIBA can be used to detect five plateletspecific autoantibodies simultaneously(Anti-GP Ⅱ b/Ⅲ a,Anti-GP Ⅰ a/Ⅱ a,Anti-GP Ⅳ,Anti-GP Ⅰ b/Ⅸand Anti-HLA-ABC).The coefficient of variation(CV)of intra-repetition are 4.82%,6.09%,5.04%,5.73%and 5.30%,respectively.The dilution test results are in good logarithm linearity which are 0.997 2,0.996 6,0.998 8,0.996 5 and 0.998 2,respectively. The resuhs of the new FCIBA are highly correlated with those with MACE method,and the coefficient correlation were 0.928 9,0.922 4,0.889 4,0.910 0 and 0.913 4,respectively(P＜0.01).51.69%samples of AITP patients show positive for platelet-specific autoantibodies as detected by the new FCIBA.Among the AITP patients,the positivity of specific autoantibodies for anti-GPⅡb/ Ⅲ a,anti-GP Ⅰ a/Ⅱ a,anti-GP Ⅳ,anti-GP Ⅰb/Ⅸ and anti-HLA-ABC were 40.82%,24.45%,19.39%,32.65%and 17.35%.Among the 40 non-autoimmune thrombocytopenic purpura patients,none of platelet-specific autoantibodies in serum samples can be detected.Conclusion A new FCIBA is established successfully to detect five platelet-specific autoantibodies coefficient correlation. Key words: Purpura,thrombocytopenic;  Blood platelets; Platelet membrane glyeoproteins; Autoantibodies;  Flow cytometry; Protein array analysis