Establishment Of The Method For The Determination Of Autoantibodies Against Platelet Glucoprotein Ⅱb/Ⅲa By Cytometric Bead Array And Its Clinical Application

Background Idiopathic 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 GPⅡ/Ⅲa or GPⅠb/Ⅸ.Measurement of platelet glocoprotein-specific autoantibodies have different approach,including radioactive immunobead and monoclonal antibody immobilization of platelet antigen technique.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.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 substance are transformed into detecting signals shown by flow cytometry.CBA is more and more widely used in detecting contents of soluble protein or cytokines of serum,plasma,culture fluid supematant,tears or aqueous humor.Objective To 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 Methods 1,56 patients with idiopathic thrombocytopenic purpura and 35 patients with non-immune thrombocytopenia were studied.The platelet counts of all patients were lower than 100×10~9/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),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 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 were 3.36(range 0.84-22.94)in the ITP group,1.16(range 0.67-5.59)in the non-immune thrombocytopenic patient and 1.08(range 0.72-1.76)in the health controls.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,ratios of larger than 1.76 were considered positive,cytometric bead array had a sensitivity of 71.43%,a specificity was 94.28%and a positive predictive value of 95.24%for the diagnosis of ITP.The sensitivity of cytometric bead array was higher than modified indirect MAIPA's(51.79%)(λ~2=4.57,P＜0.05).The ROC curve showed the discriminative validity of cytometric bead array was 0.916.Conclusion The 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.