Lupus anticoagulant screen on VKA or direct FXa inhibitor (dAPTT, dRVVT & TSVT)

Lupus anticoagulants (LA) are classified as antiphospholipid antibodies (APA), although they are in fact directed against phospholipid-binding proteins, in particular, β2 glycoprotein I and prothrombin. The presence of persistent LA has a greater association with thrombosis, pregnancy morbidity and recurrence than the criteria antibodies detected in solid-phase assays (aCL & aβ2GPI). LA are a heterogeneous group of autoantibodies that are detected by inference based on their behaviour in phospholipid-dependent coagulation assays after other possible causes of elevated clotting times have been excluded. LA detection involves use of screening, confirmatory and mixing tests.

Screening tests commonly employ dilute phospholipid to accentuate the in vitro anticoagulant effect of LA, which if present, will prolong the clotting time. Screening tests can be prolonged for reasons other than LA, (i.e. factor deficiencies, anticoagulant therapy), so all elevated screening tests receive follow-up analyses to help define the nature of any abnormality. The confirm test generally involves performing the screening test in an identical fashion except that the phospholipid concentration is markedly increased. This has the effect of partially or completely overwhelming the LA and thus leads to a shorter clotting time than the screening test, thereby evidencing phospholipid dependence. Clotting times are converted to ratios to mitigate for issues of analytical variability. Correction of the screen ratio by the confirm ratio by ≥10% is considered consistent with the presence of a LA, providing that other causes of elevated clotting times are excluded. Diagnostic specificity is improved by performing the screen and confirmatory tests on 1:1 mixtures of test and normal plasma to evidence inhibition and reduce interferences, although the inevitable dilution effect can compromise this aspect of analysis.

Antibody heterogeneity and reagent variability necessitate use of at least two assays, of different analytical principle, to achieve acceptable detection rates. First-line assays are dilute Russell's viper venom time (dRVVT) and LA-responsive APTT, a pairing that will detect most clinically significant antibodies. Our laboratories employ a dilute APTT (dAPTT) in this respect, which employs a silica activator and a low concentration of phospholipid comprised of a composition of phospholipid types that is LA-responsive. The confirm test involves addition of concentrated platelet-derived phospholipid. dRVVT analysis employs diluted FX activator from the venom of Russell's viper (Daboia russellii), a low concentration of phospholipid comprised of a composition of phospholipid types that is LA-responsive and calcium ions. The confirm test involves an identical reagent except that the same phospholipid preparation is employed at a higher concentration. All elevated dAPTT & dRVVT screen ratios are reflexed to receive the confirm test, and the screen and confirm mixing tests, and are reported with interpretive comment. Patients with LA may be postitive in one or both of the dAPTT & dRVVT test medleys.

A limitation common to both dRVVT & dAPTT analysis is that they are compromised by the VKA anticoagulant effect and results are not always reliable. LA can be detected in the mixing studies providing that the normal plasma fully compensates for the multiple acquired factor deficiency of VKA therapy and the LA itself is sufficiently potent to overcome the dilution effect. Thus, a positive result is diagnostic but apparently negative mixing tests cannot exclude the presence of a weaker antibody. LA detection is enhanced with additional tests employing other snake venoms that are insensitive to VKA and direct-FXa inhibitor therapy. The prothrombin activator present in the venom of the Coastal Taipan (Oxyuranus scutellatus) can activate the des-carboxyprothrombin generated on VKA anticoagulation to the intermediate, meizothrombin, and facilitate in vitro clot formation. The prothrombin activator requires phospholipid and calcium ions as co-factors, so dilution of a suitable phospholipid preparation renders the Taipan snake venom time (TSVT) assay LA-responsive, yet it gives normal clotting times in VKA anticoagulated patients without LA. The ecarin time (ET) is used in place of a high-phospholipid confirmatory test. The ecarin fraction is obtained from the venom of the saw-scaled viper (Echis carinatus) and is a direct prothombin activator with no co-factor requirements, so an assay without phospholipid is totally insensitive to LA. Ecarin is also insensitive to the VKA anticoagulant effect. As both venoms are prothrombin activators, they are completely unaffected by inhibitors of FXa.