Antiphospholipid antibody syndrome: rapid, sensitive, and specific flow cytometric assay for determination of anti-platelet phospholipid autoantibodies.
A rapid, sensitive and specific flow cytometric assay has been developed for the determination of autoantibodies directed against platelet anionic phospholipids in antiphospholipid antibody syndrome (APLAS). 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 and factors Va and Xa). The method is practical and rapid, uses readily available reagents, and involves standard equipment. The assay is inexpensive and cost-effective for both single and multiple samples. Results are provided within 2 hours from obtaining blood samples, thereby supporting clinical decision-making and management. Ten serum samples from patients with the clinical diagnosis of APLAS (48 tests), 10 from normal individuals (35 tests), and 10 from patients with immune thrombocytopenia (33 tests) were tested. Platelet preparations preincubated with normal sera showed high binding of fluorescein-labeled annexin V with an average fluorescence of 202.9 +/- 22.0 (arbitrary units). Patients with immune thrombocytopenia exhibited similar results, with an average fluorescence of 192.5 +/- 32.1 (P >.05). In contrast, incubation with sera from patients with APLAS resulted in a marked decline in the binding of annexin V to an average fluorescence of only 14.6 +/- 7.4 (P <.001). Preincubation with annexin V followed by the addition of patients' sera showed displacement of annexin V to a similar degree. Because annexin V attenuates procoagulant activity by competing with factors Va and Xa on the platelet anionic phospholipids, its displacement by patients' antibodies may result in the acceleration of procoagulant activity, thereby promoting thrombogenesis in APLAS.
Tomer
Aaron
A
Blood Bank and Transfusion Medicine, Soroka University Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, P.O. Box 151, 84101 Israel. atomer@bgumail.bgu.ac.il
D000838:Anions; D017304:Annexin A5 / Q000378:metabolism; D016736:Antiphospholipid Syndrome / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000276:immunology*; D005165:Factor V / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D010743:Phospholipids / Q000097:blood / Q000276:immunology*; D011516:Prothrombin / Q000378:metabolism; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000097:blood / Q000276:immunology; D012680:Sensitivity and Specificity
D000838:Anions; D017304:Annexin A5; D001323:Autoantibodies; D005456:Fluorescent Dyes; D010743:Phospholipids; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D015951:Factor Xa; D019793:Fluorescein