Applying index of circulating anticoagulant to mixing tests with lupus anticoagulant screen and confirm reagents can distinguish with high specificity between lupus anticoagulants and direct factor Xa inhibitors.

BACKGROUND: Lupus anticoagulants (LA) are detected by prolongation of clotting times for dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) screening tests. Direct oral anticoagulants (DOACs) can interfere with both screening and confirmatory tests. The present study aimed to investigate the influence of direct factor Xa inhibitors (DiXaIs) on screen, confirm and mixing tests and establish a method for differentiation from other sample types. MATERIALS AND METHODS: A total of 257 samples including nonanticoagulated LA positive, LA positive with DiXaIs, factor deficiency, FVIII inhibitors, warfarin and non-APS DiXaIs were tested. APTT reagents Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used, respectively, to screen/confirm the study group. Index of circulating anticoagulant (ICA) was calculated from clotting times based on the following formula as ICA screening and ICA confirmation. ICA= (1:1 Mix sample - Normal pooled plasma) / Screen patient x 100. An ICA matrix was established which suggested the presence of a DiXaI when both ICA screening and confirmation were above the cut-off. When only ICA screening is elevated, LA is suspected. RESULTS: Sensitivity and specificity of the ICA matrix were 52.2% and 92.8% for DiXaIs and 38.1% and 96.7% for LA in APTT, and 61.2% and 92.9% for DiXaIs and 22.2% and 88.4% for LA in dRVVT, respectively. CONCLUSION: The ICA matrix achieved high specificity with a lower apparent sensitivity for DiXaI samples comparatively to other devices but due only to less interferences: the matrix could contribute to differentiating DiXaIs from LA in samples where anticoagulation status is unknown.

Paired APTTs of low and high lupus anticoagulant sensitivity permit distinction from other abnormalities and achieve good lupus anticoagulant detection rates in conjunction with dRVVT.

INTRODUCTION: A prolonged activated partial thromboplastin time (APTT) may be indicative of a specific or multiple factor deficiency, therapeutic anticoagulation, presence of a nonspecific factor inhibitor, or lupus anticoagulant (LA). Recently, pairing of the LA-sensitive APTT and standard APTT reagents, Cephen LS and Cephen, respectively, has been shown to be effective in LA detection. The present study aimed to evaluate the usefulness of this reagent pair for discriminating between causes of APTT elevation and the detection of LA in conjunction with dilute Russell's viper venom time (dRVVT). METHODS: Plasma samples from 50 normal and 105 non-anticoagulated LA-positive patients in routine dRVVT and/or dilute APTT (dAPTT) via the percent correction formula were employed. Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used to screen/confirm, respectively. Thirty-four symptomatic LA-negative, 25 warfarinised non-antiphospholipid syndrome, 6 coagulation inhibitors, 17 samples with hereditary elevated APTT, and 24 FVIII/IX/XI/XII and 17 FII/V/X artificial single deficiency plasmas were used. RESULTS: Thirty-three samples out of 105 (31%) were LA-positive in Cephen LS/Cephen. The total percent positivity in Cephen LS/Cephen and LA1/LA2 pairs was 89.1% against samples with the routine dRVVT/dAPTT double positive. The percent corrections of Cephen LS/Cephen in the routine dAPTT/dRVVT positive group were significantly higher than those in all other groups. CONCLUSIONS: The percent correction of the APTT reagent pair showed higher values in LA-positive samples. The combination will be useful with respect to differentiating LA from other abnormal samples and is effective in LA detection when paired with dRVVT.

Anti-phospholipid syndrome: Current opinion on mechanisms involved, laboratory characterization and diagnostic aspects.

Anti-phospholipid syndrome is a complex and severe clinical situation, associated with symptoms such as recurrent thrombosis, arterial or venous, at any site, pregnancy loss, and other related syndromes. These clinical burdens, are highly variable from patient to patient, and are associated with biological abnormalities, such as the presence of the Lupus Anticoagulant or phospholipid dependent antibodies, confirmed on two occasions at least 12 weeks apart. From the diagnosis standpoint, both, functional (clotting) or immunological assays, are difficult to standardize and to optimize, due to the absence of reference material, or a characteristic clinical group, and international reference preparations. Large cohort studies are necessary for defining the usefulness of each assay, in terms of specificity, sensitivity, accuracy and for following-up the disease evolution. Clotting assays are based on Activated Partial Thromboplastin Time (APTT) and diluted Russell Viper Venom Time (dRVVT), performed at low and high phospholipid concentration, or on 1:1 mixtures of tested sample and a normal plasma pool. They allow evaluation of the paradoxal effects of LAs, which are pro-thrombotic in vivo, and anticoagulant in vivo. Use of synthetic phospholipids improves assay specificities and sensitivities, especially in patients treated with anticoagulants. Immunoassays can also be used for testing phospholipid dependent antibodies, first identified and measured as anti-cardiolipin antibodies, but now characterized as targeted to phospholipid cofactor proteins: mainly ß2GP1 (which exposes cryptic epitopes upon binding to phospholipids), and in some cases prothrombin, and more rarely Protein S, Factor XIII, Protein Z or Annexin V. Use of optimized assays designed with well-characterized anionic phospholipids, then complexed with highly purified phospholipid cofactor protein (mainly ß2GP1), offers a better link between reactivity and clinical associations, than the former assays which were empirically designed with cardiolipin. Standardization also remains complicated due to the absence of international standards and harmonized quantitation units. Validation on large cohorts of negative and positive patients remains the key approach for defining assay performance and clinical usefulness. Laboratory practice for all these methods is now greatly facilitated thanks to the use of automated instruments and dedicated software. Along with clinical criteria, laboratory assays are of great usefulness for identification and confirmation of the anti-phospholipid syndrome and they allow disease follow-up when appropriate patient management is in place.