The Measurement And Clinical Significance Of Platelet Membrane Glycoprotein Autoantibodies Using Flow Cytometric Bead Array On Idiopathic Thrombocytopenic Purpura

BackgroundIdiopathic thrombocytopenic purpura is a common hemorrhagic disorder characterized by a low platelet count which is caused by the binding of circulating anti-platelet antibodies to patient's platelets. Current diagnoses of ITP are generally based on clinical impression, primarily that of exclusion. Platelet-associated IgG assays were used to be considered as a potential way in the diagnoses of ITP in 1970s, but as were proved later, their specificity is too low to be a useful diagnostic tools and these assays are no longer recommended for the diagnosis of ITP. The autoantibodies of ITP patients are directed to platelet-specific receptors, primary GPⅡb/Ⅲa or GPⅠb/IX. Measurements of platelet glocoprotein-specific autoantibodies have different approach, including radioactive immunobead and monoclonal antibody immobilization of platelet antigen technique. The high sensitivity (78-93%) of these assays is sufficient to confirm the diagnosis of ITP. However, these assays can not be widely used as their low specificity (49-66%) and methodical limitations. Cytometric bead array is a new technique based on flow cytometry. Polystyrene microbeads coated with specific monoclonal antibodies can immobilize some low molecular weight substance in solution, so the contents of those substances are transformed into detecting signals shown by flow cytometry. CBA is more and more widely used in detecting contents of molecule.ObjectiveTo establish a method for detecting platelet-associate autoantibodies against platelet-specific receptors by cytometric bead array, and to compare the clinical usefulness of this method and modified indirect monoclonal antibody immobilization of platelet antigen technique(MAIPA) in the differential diagnosis of immune and non-immune thrombocytopenic purpura.Materials and Methods1,67 patients with idiopathic thrombocytopenic purpura and 35 patients with non-immune thrombocytopenia were studied. The platelet counts of all patients were lower than 100×109/L, and patients who received blood transfusion in recent three months were excluded.30 healthy volunteers were used as control, whose sexuality and age matched ITP group.2,Coating microbeads with monoclonal antibodies against glycoproteinⅡb/Ⅲa(CD41a),Ⅰb/IX. (CD42), segregating platelets from blood samples, then incubating platelet lysate with coated microbeads after solubilizing the platelets, next adding R-Phycoerythrin-conjugated goat-antihuman IgG polyclonal antibodies, finally analyzing the microbeads with flow cytometry. GPⅡb/Ⅲa.Autoantibodies and GPⅠb/IX.Autoantibodies in sample plasma were measured by modified indirect MAIPA at the same time. Then compare the results of these two methods.Results The individual fluorescene level was calculated as a ratio to the three controls. The mean ratios of GPⅡb/Ⅲa were 6.52 (range 0.22-30.20) in the ITP group, 1.20(range 0.32-3.04) in the non-immune thrombocytopenic patient and 1.19(range 0.23-3.51) in the health controls. The mean ratios of GPⅠb/IX were 4.91 (range 0.56-22.00) in the ITP group,1.24(range 0.33-3.56) in the non-immune thrombocytopenic patient and 1.31 (range 0.23-2.84) in the health controls. Nether GPⅡb/Ⅲa or GPⅠb/IX there was highly significant difference(P<0.01) between the ITP patients and both the non-immune thrombocytopenic patients and the normal negative controls. If the up limit of health controls were set as cutoff, GPⅡb/Ⅲa ratios of larger than 3.535,GPⅠb/IX ratios of larger than 2.855 were considered positive, cytometric bead array had a sensitivity of 82.09%, a specificity was 95.38%, a positive predictive value of 94.83% for the diagnosis of ITP. The sensitivity of cytometric bead array was higher than modified indirect MAIPA's (P< 0.05). The ROC curve showed the discriminative validity of cytometric bead array GPⅡb/Ⅲa was 0.856 and GPⅠb/IX 0.916.ConclusionThe way of using flow cytometric bead to detect platelet-associate autoantibodies against platelet-specific receptors can be characterized by rapidity, good reproducibility and it got a higher sensitivity than modified MAIPA did, so it has a potential value in enhancing the diagnostic level of ITP and giving a guide to treatment.