4-Panel Assay May Reverse LAC of Antiphospholipid Antibody Detection

The pathophysiology of recurrent pregnancy loss is incompletely understood, but testing for lupus anticoagulant (LAC)/phospholipid-dependent antibodies (aPL) is commonly performed to determine if an underlying prothrombotic disorder might contribute to disease pathogenesis in an individual case and, if detected, suggest a therapeutic approach (i.e., anticoagulation) to reduce the risk of spontaneous abortions. aPLs are acquired autoantibodies against phospholipids such as cardiolipin or against phospholipid-binding proteins such as β2-glycoproteins I, prothrombin, and annexin A5. In the clinical setting, they are detected by ELISAs for IgG and IgM anticardiolipin antibodies and for IgG and IgM anti-β2 glycoprotein I antibodies. Functional coagulation assays are used to identify the LAC. These assays are specially designed to take advantage of the requirement for phospholipids in ex vivo coagulation assays. For example, anti-phospholipid antibodies prolong the clotting endpoint in the dilute Russell viper venom time (dRVVT) assay, in a modified, sensitive version of the activated partial thromboplastin time assay (PTT-LA), in the kaolin clotting time (KCT) assay, and in the dilute prothrombin time (dPT) assay. The International Society of Thrombosis and Haemostasis (ISTH) recommends the dRVVT and the PTT-LA for routine clinic use.¹  Although there is support for diagnostic guidelines such as the Sapporo criteria, definitive establishment of the presence or absence of anti-phospholipid antibodies remains problematic because interpretation of testing is compromised by both false-positive and false-negative results, by temporal inconsistencies in test results, and by assay variability.

Some of the issues that bedevil the field are underscored in the report of Clark and colleagues from the University of Toronto. The investigators participate in a tertiary clinic that annually enrolls ~250 new patients with recurrent pregnancy loss, in addition to patients with systemic lupus erythematosus (SLC) and/ or antiphospholipid syndrome (APS). In a retrospective analysis that covered a six-year period, 2,257 women with recurrent pregnancy loss, SLE, and/or APS who had been screened for the presence of the LAC were characterized. Each patient in the study had samples tested using each of four different LAC assays (dRVVT, PTT-LA, KCT, and dPT) that had been operative in the clinic for 20 years. The LAC was considered present when the clotting times were prolonged in any of the four screening assays. Positive results of the screening assays were validated using both mixing studies and an assay that confirmed phospholipid dependence. To be counted as positive in the study, LAC had to be present on more than two occasions, not less than 12 weeks apart, and within a period of six years. LAC-negative patients were defined as those testing negative for LAC on all four tests. All patients also underwent immunologic testing for both IgG and IgM anti-cardiolipin antibodies.

Of the 2,257 females studied, 136 patients (6.0%) were initially LAC-positive, with 62 (2.7%) testing positive for LAC on at least two occasions (43/136 patients who were initially LAC-positive were lost to follow-up and were not tested on more than one occasion). Compared with LAC-negative subjects, LAC-positive patients were significantly more likely to have a history of thromboembolism (TE) (idiopathic or with a risk factor), stillbirth, intrauterine growth retardation, and HELLP syndrome; but they were significantly less likely to have a history of early recurrent pregnancy loss or a history of two or more early spontaneous abortions. Notably, only 5 patients (0.02%) with a history of recurrent miscarriages were LAC-positive. No significant difference was observed between the LAC-positive and LAC-negative group for a history of preeclampsia, a history of at least one live birth, and a history of no adverse obstetric events. Clark and colleagues observed a correlation between the number of positive LAC assays and the percentage of patients with a history of TE, TE recurrence, stillbirths, and fewer live births. As anticipated, more LAC-positive patients were anti-cardiolipin antibody-positive compared with the LAC-negative group (45% IgG-positive and 27% IgM-positive vs. 0.9% IgG-positive and 1.5% IgM-positive, respectively). If only the PTT-LA assay had been used to detect LAC, 40.3 percent of the 62 LAC-positive patients would have been missed; had only the dRVVT assay been used, 32.3 percent would have been missed; and if both the dRVVT and PTTLA assays had been used, 22.6 percent would have been missed. None of the four assays used in the current study were predominantly associated with any specific clinical event.