Autoimmune thrombocytopenia: flow cytometric determination of platelet‐associated autoantibodies against platelet‐specific receptors

Summary.  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 average fluorescence level in the ITP group calculated as a ratio to normal was 4.07 (range 0.8–31.0), 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 (CI) 0.213, 0.221]. The difference between the ITP patients and both groups 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 (R2 = 0.71, 95% CI 0.39, 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.

[1]  F. Millard,et al.  Platelet-associated and plasma anti-glycoprotein autoantibodies in chronic ITP. , 1987, Blood.

[2]  I. Khouri,et al.  Immune Thrombocytopenic Purpura , 2018 .

[3]  M. Tavassoli,et al.  In vitro platelet phagocytosis by splenic leukocytes in idiopathic thrombocytopenic purpura. , 1974, The New England journal of medicine.

[4]  B. Chong,et al.  Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in idiopathic thrombocytopenic purpura and other immune thrombocytopenias. , 1996, Blood.

[5]  J. George,et al.  Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology. , 1996, Blood.

[6]  J. Kelton,et al.  A prospective study of protein‐specific assays used to investigate idiopathic thrombocytopenic purpura , 1999, British journal of haematology.

[7]  I. Peake International Society on Thrombosis and Haemostasis , 1974, Thrombosis and Haemostasis.

[8]  R. Mcmillan Antigen-specific assays in immune thrombocytopenia. , 1990, Transfusion medicine reviews.

[9]  V. Kiefel,et al.  Monoclonal antibody--specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. , 1987, Blood.

[10]  N. Shulman,et al.  The role of the reticuloendothelial system in the pathogenesis of idiopathic thrombocytopenic purpura. , 1965, Transactions of the Association of American Physicians.

[11]  P. Tani,et al.  Complement activation in vitro by antiplatelet antibodies in chronic immune thrombocytopenic purpura , 1986, British journal of haematology.

[12]  S. Lux,et al.  Platelet membrane glycoprotein IIIa contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. , 1984, The Journal of clinical investigation.

[13]  P. Tani,et al.  Prospective evaluation of the immunobead assay for the diagnosis of adult chronic immune thrombocytopenic purpura (ITP) , 2003, Journal of thrombosis and haemostasis : JTH.

[14]  B. Chong,et al.  Advances in the diagnosis of idiopathic thrombocytopenic purpura. , 2000, Seminars in hematology.

[15]  R. Montgomery,et al.  Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura. , 1984, Blood.

[16]  R. Mcmillan,et al.  Fixation of C3 to Platelets in Vitro by Antiplatelet Antibody from Patients with Immune Thrombocytopenic Purpura , 1981, British journal of haematology.

[17]  R. Mcmillan,et al.  Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP. , 1984, Blood.

[18]  Andrews Hl,et al.  The role of the reticuloendothelial system in the pathogenesis of idiopathic thrombocytopenic purpura. , 1965, Transactions of the Association of American Physicians.

[19]  S. Hanson,et al.  Autologous platelet kinetics in patients with severe thrombocytopenia: discrimination between disorders of production and destruction. , 1991, The Journal of laboratory and clinical medicine.