Abstract
A flow cytometric assay was developed for the determination of autoantibodies directed against platelet anionic-phospholipids in antiphospholipid syndrome (APS). The method is based on demonstrable competition between the placental anticoagulant protein I, annexin V, and the patients’ autoantibodies on the platelet anionic-phospholipids (the binding site for the prothrombinase complex; prothrombin, factors Va and Xa). The method is practical and rapid, uses readily available reagents, and standard equipment. Ninety-two plasma samples, 41 from patients with clinical diagnosis of APS, 27 from patients with systemic lupus erythematosus (SLE) and 24 from healthy individuals were analyzed. Thirty-five (85%) of patients with APS (either with or without SLE), 15 (94%) of patients with APS and SLE, and 20 (80%) of APS patients without SLE were positive. Nineteen (70%) out of 27 patients with SLE alone were also positive; of whom 15 (58%) were positive for either anti-cardiolipin antibody (ACL) or lupus anti-coagulant (LAC). Comparison with ACL showed 40 (93%) out of 43 patients positive for ACL were also positive by flow cytometry (FCM). However, 13 (48%) out of 27 patients negative for ACL were found positive by FCM. Seven of these patients have primary APS and 6 have SLE; 7 of whom were positive for LAC. Three (13%) out of the 24 control samples, all negative by FCM, were positive for ACL. Comparison with LAC showed 32 (91%) out of 35 patients positive for LAC also positive by FCM. Of 18 patients negative for LAC, 7 (39%) were negative and 11 (61%) were positive by FCM. Four of the 11 patients have a diagnosis of APS and 7 of SLE; 6 of whom were positive for ACL. None of the controls, all negative by FCM, was positive for LAC. In conclusion, the annexin V competitive flow cytometric assay for the determination of anti-platelet phospholipid autoantibodies maybe useful for the laboratory diagnosis of APS.
Establishment of a flow cytometric assay for determination of human platelet glycoprotein VI based on a mouse polyclonal antibody
