Laboratory identification of lupus anticoagulants: results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/Antiphospholipid Antibodies of the ISTH.

Lupus anticoagulants (LAs) are antibodies that interfere with phospholipid dependent coagulation reactions in vitro. This workshop was designed to provide the participants with an experience in identification of LAs, to evaluate different criteria for mixing studies, to assess the performance of recently introduced confirmatory studies and to assess the performance of two potential surrogate LA control plasmas. The results demonstrate that there continues to be significant variation in the sensitivity and responsiveness of APTT reagents to the presence of LAs, confirming the need for more than one screening assay before the presence of a LA can be ruled out. In this workshop, the best distinction between factor deficiency and inhibitors was obtained using a 1:1 mix of normal plasma with patient plasma and the criterion defining correction as shortening of the APTT to within 5 s of the APTT of pooled normal plasma. A 4:1 mix of patient to normal plasma did not work well in distinguishing factor deficiency from inhibitors. The platelet neutralization procedure, DVV confirm and StaClot LA all gave positive results with the LA samples. False positive platelet neutralization procedures were seen with the samples from patients on oral anticoagulants and a factor V inhibitor. False positive StaClot LA results were obtained with high titer factor VIII inhibitors. Both of the potential surrogate plasmas gave variable results with multiple assays; they can not be recommended for routine use at present.

Effects of repeated freeze-thaw cycles on anticardiolipin antibody immunoreactivity.

The effect of repeated freeze-thaw cycles on anticardiolipin antibody levels was evaluated using an enzyme-linked immunosorbent assay. Normal human serum was spiked with known quantities of freeze-dried human polyclonal anticardiolipin antibody IgG and IgM (19 samples each) or IgA (11 samples). Each spiked sample was split into four identical aliquots; one aliquot was never frozen, and the remaining three were taken through successive freeze-thaw cycles. All aliquots from each sample were evaluated on the same day using the same plate and reagents. A significant decline in mean anticardiolipin IgG levels occurred between the aliquot which had never been frozen and the one which had been through three freeze-thaw cycles (Student's t-test, P = .04). Although mean IgM and IgA values declined as well, the differences were not significant. When individual samples were evaluated the decline appeared to occur most often between the second and third freeze-thaw cycle. Eight anticardiolipin IgG and three IgM-containing samples which had been positive initially became negative by the third freeze-thaw cycle. These data show that handling and storage of serum used to perform anticardiolipin antibody assays are important potential sources of assay variability.

The Textarin/Ecarin ratio: a confirmatory test for lupus anticoagulants.

Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, IgA or a mixture) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, KCT, dilute Russell Viper Venom Time). LA are heterogeneous; consequently, the laboratory diagnosis is difficult and relies on multiple tests. We have developed a sensitive and relatively specific confirmatory test system based on fractions of two snake venoms. Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern brown snake), activates prothrombin in the presence of PL, factor V and calcium ions. Ecarin, a protein fraction of Echis carinatus venom, will activate prothrombin in the absence of any cofactors. The activation of prothrombin by Textarin yields thrombin while Ecarin yields meizothrombin. In the presence of LA, the Textarin time is prolonged and the Ecarin time is unaffected. The test results are reported as a ratio of Textarin/Ecarin times (abnormal greater than 1.3). We have evaluated this test system in the following patient populations: LA positive, therapeutically heparinized, stable oral anticoagulated, liver disease, routine preoperative, anticardiolipin antibody positive LA negative, hemophilia A, various other hereditary factor deficiencies or dysfunctional proteins, and specific inhibitors of factor V and factor VIII. The LA positive patients represented a mixed population of autoimmune disease, drug-induced and post-infectious states. Our findings indicate the sensitivity of the Textarin/Ecarin system in the confirmation of LA. In order to use the test system most effectively, it is recommended to incorporate polybrene with Textarin when evaluating heparinized samples. Factor V deficiency and specific inhibitors of factor V yielded, in some instances, false positive results.

A hexagonal (II) phase phospholipid neutralization assay for lupus anticoagulant identification.

Lupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e. g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.

A report on the First International Workshop for Lupus Anticoagulant Identification.

Recognition that the lupus anticoagulant (LA) is associated with an increased risk for arterial and venous thrombosis, recurrent spontaneous abortions and fetal loss has led to increased laboratory requests for identification of LA. It is of interest not only to laboratory medicine and obstetrics but also to rheumatology, neurology and cardiology. Due to the antibody's heterogeneous expression, no single test confirms its presence. Recent criteria for LA identification include the performance of one or more screening tests to demonstrate LA interference with phospholipid-dependent tests, differential studies to determine the presence of an inhibitor, and confirmatory procedures to prove that the inhibitor is phospholipid dependent. An international workshop was conducted to study a panel of tests for LA identification and to select those most helpful for diagnosis.