Antiphospholipid antibody syndrome: Rapid, sensitive, and specific flow cytometric assay for determination of anti-platelet phospholipid autoantibodies

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.

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Antiphospholipid Antibody Syndrome: The Flow Cytometric Annexin A5 Competition Assay as a Diagnostic Tool.

Blood ◽

10.1182/blood.v110.11.3981.3981 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3981-3981

Author(s):

Aaron Tomer ◽

Shira Bar-Lev ◽

Stela Fleisher ◽

Boris Shenkman ◽

Michael Friger ◽

...

Keyword(s):

Lupus Erythematosus ◽

Vascular Complications ◽

Relative Sensitivity ◽

Annexin A5 ◽

Antiphospholipid Antibody ◽

Antiphospholipid Antibody Syndrome ◽

Flow Cytometric Assay ◽

Anionic Phospholipid ◽

Anionic Phospholipids ◽

Flow Cytometric

Abstract The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient’s aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73∴3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72∴2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97∴0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

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