Design of a modified platelet immunofluorescence test to assess platelet-reactive antibody burden and its association with platelet functional exhaustion and clinical features in chronic immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by platelet destruction and dysfunction associated with anti-platelet antibodies. This study evaluated the relationship between total anti-platelet antibody burden, measured using a modified platelet immunofluorescence test (PIFT), platelet functional responses and clinical features in chronic ITP. Thirty-one patients with chronic ITP and 20 healthy controls were included. Bleeding severity was assessed, and platelet function was analysed in peripheral blood by measuring P-selectin expression, PAC-1 binding (antibody against active conformation of GPIIb/IIIa) and reactive oxygen species (ROS) generation at baseline and following agonist stimulation. Relative platelet-reactive antibody burden was assessed using a ratio-based PIFT assay. ITP patients demonstrated significantly higher antibody burden compared with controls. Increased platelet-reactive immunoglobulin G (IgG) signals were associated with reduced platelet responsiveness to agonist stimulation. Antibody burden correlated with bleeding severity (p < 0.01) but not with platelet count. Non-responders exhibited significantly higher antibody levels than responders. receiver operator characteristic (ROC) analysis demonstrated discrimination between responder groups at a PIFT cut-off ≥3.85 (area under the curve [AUC] 0.877, sensitivity 80%; specificity 87%). Patients above this threshold showed attenuated platelet functional responses. Taken together, this study concluded that quantitative assessment of total antibody burden against platelets using modified PIFT is associated with platelet dysfunction, bleeding severity and treatment response status in chronic ITP.