Viewpoint: Lupus anticoagulant detection and interpretation in antiphospholipid syndrome.

Lupus anticoagulant (LA) is a well-established risk factor for the clinical manifestations of antiphospholipid syndrome (APS). Accurate LA detection is an essential prerequisite for optimal diagnosis and management of patients with APS or aPL carriers. Variability remains a challenge in LA testing, with reliable detection influenced by multiple factors, including pre-analytical conditions, anticoagulation treatment, choice of tests and procedures performed, as well as interpretation of results, that can lead to false-positives or negatives. A standardised approach to LA testing, following current guidance, based on published data and international consensus, and with attention to detail, is required to underpin accurate detection of LA. Future work should focus on better characterisation of the nature of LA, which may ultimately lead to improved diagnosis and management of patients with APS and aPL carriers. This article reviews current practice and challenges, providing an overview on detection of LA.

Real-world evidence of lupus anticoagulant testing: simultaneous positivity of diluted Russell's viper venom time and silica clotting time increases thrombotic risk prediction.

Lupus anticoagulant (LA) is composed of heterogeneous autoantibodies, which have a close association with thrombotic events. Due to its heterogeneity, two methods for increasing sensitivity are recommended for LA. An investigation of the thrombotic risk and anticardiolipin (aCL) and anti-ß2-glycoprotein I (aB2GPI) antibody profiles was conducted based on the results of using two parallel methods (dilute Russell viper venom time (dRVVT), silica clotting time (SCT)) in a real world clinical laboratory. Of 5120 patients, 684 patients (13%) were LA positive, and 422 patients (8%) experienced thrombotic events including pregnancy complication. Development of thrombotic events was more likely to occur in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. In addition, significantly higher positive rates of aCL and aB2GPI and the persistently positive rate of LA at intervals of 12 weeks or longer were observed in patients who were positive for both dRVVT and SCT compared with those who were positive for dRVVT or SCT only. Considering three laboratory tests (LA, aCL, and aB2GPI), high thrombotic risk was observed for patients with both dRVVT and SCT positive LA results. A report on LA results that divides LA positive into two types (LA-single positive and LA-both positive) may be beneficial to clinicians in detection of high-risk thrombotic patients.

Mixing studies for lupus anticoagulant: mostly no, sometimes yes.

Mixing tests have long been a mainstay in the lupus anticoagulant (LA) testing armoury of screen, mix and confirm assays. If a sample with an elevated screening test does not evidence inhibition in the mixing test, the search for an LA is halted and a different diagnostic pathway embarked upon. Recent years have seen studies evidencing sometimes high frequencies of false-negative mixing tests with perhaps sinister implications for missed diagnoses and skewed patient management. Issues such as the dilution effect, between-reagent sensitivity and specificity differences, variability of normal pooled plasma (NPP) quality and suitability and interpretive inconsistencies all contribute to questioning the reliability of mixing tests and their pivotal place in the LA assay hierarchy. The advent of integrated testing, where phospholipid-dependence is demonstrated or excluded prior to any attempt to evidence inhibitory properties with a fallible analytical principle, provides an alternative path to LA detection. In the absence of other causes of elevated clotting times, LA assay screen and confirm discordance is sufficient to secure a laboratory diagnosis of the presence of an LA, leaving the mixing test in a supplementary yet valuable role when further diagnostic discrimination is required.

Evaluation of global laboratory methods and establishing on-therapy ranges for monitoring apixaban and rivaroxaban: Experience at a single institution.

BACKGROUND: Apixaban and rivaroxaban are approved for the prevention and treatment of deep vein thrombosis (DVT), pulmonary embolism (PE), and embolic stroke in atrial fibrillation (AF) patients. The aim of this study was to find appropriate methods of monitoring the anticoagulant effects of are direct oral anticoagulants (DOACs) and establish on-therapy ranges using conventional tests. METHODS: A total of 184 samples were collected from 91 patients receiving DOACs. Concentrations of apixaban and rivaroxaban in plasma were accessed by an anti-factor Xa chromogenic assay. PT, APTT, antithrombin, D-dimer, dRVVT screen/confirm, FDP, and fibrinogen levels were measured. On-therapy ranges were calculated by substituting previously reported trough plasma concentrations of DOACs. RESULTS: Anti-factor Xa chromogenic assay-based DOACs levels were 26.0-279.5 (115.9 ± 56.5) ng/mL for apixaban at 2.5 mg BID, 19.9-565.1 (205.3 ± 162.4) ng/mL for apixaban at 5 mg BID, 2.3-395.3 (205.3 ± 162.4) ng/mL for rivaroxaban at 15 mg OD, 3.6-494.8 (119.6 ± 95.1) ng/mL for rivaroxaban at 20 mg OD, and 9.6-431.4 (140.8 ± 113.6) ng/mL for rivaroxaban at 15 mg BID. PT (%), antithrombin, and dRVVT confirm tests showed good correlation with plasma apixaban levels. Plasma rivaroxaban concentrations were correlated well with PT (sec), PT (%),and dRVVT confirm results. On-therapy ranges established for dRVVT confirm test by linear regression were as follows: 1.32-1.52 for apixaban 2.5 mg BID, 1.12-1.75 for apixaban 5 mg BID, 1.11-1.78 for rivaroxaban 15 mg OD, 1.09-1.64 for rivaroxaban 20 mg OD, and 1.22-1.81 for rivaroxaban 20 mg BID. CONCLUSIONS: Apixaban concentrations were well correlated with PT (%), antithrombin, and dRVVT confirm test. Rivaroxaban concentrations showed good correlation with PT (sec), PT (%), and dRVVT confirm test.

Newly developed dilute Russell's viper venom reagents for lupus anticoagulant detection with improved specificity.

Background Dilute Russell's viper venom time (dRVVT) is indispensible in lupus anticoagulant (LA) detection yet commercial reagents from different suppliers perform variably, no gold standard assays exist and therapeutic anticoagulation interference is problematic. Objective The objective of this study was to compare a new formulation dRVVT with two currently available dRVVTs. Materials and methods Life Diagnostics (LD) dRVVT and Stago PTT-LA were routinely used for lupus anticoagulant detection, plus Taipan snake venom time/ecarin time (TSVT/ET) for patients on warfarin or rivaroxaban. Siemens dRVVT and the new HYPHEN BioMed (HBM) dRVVT were tested with 193 patient samples. Group 1, 59 non-anticoagulated patients (NAPs) LA-positive in LD dRVVT; Group 2, 15 PTT-LA-positive/dRVVT-negative NAPs; Group 3, 24 LA-positive warfarinized patients; Group 4, 13 patients on rivaroxaban; Group 5, 62 LA-negative thrombotic NAPs; Group 6, 20 warfarinized, non-antiphospholipid syndrome patients. Results Accepting that the Life Diagnostics reagents were acting as a pseudo-gold standard, Siemens dRVVT detected 56/59, (95%) Group 1 LA and HBM dRVVT 46/59, (76%), one each from Group 2, and Siemens dRVVT detected one in Group 5. The lower HBM dRVVT detection rate mainly concerned weaker LA, where between-reagent concordance is problematic. All Group 3 patients appeared LA-positive in undiluted plasma with Siemens dRVVT, as did 16/24 (67%) with HBM dRVVT but the fewer LA-positives in mixing tests better mapped to clear LA-positives with LD dRVVT. LD and Siemens dRVVTs exhibited 87% and 95% false-positivity for Group 6 whilst HBM dRVVT had none. Increasing the cut-off improved accuracy. Applying higher cut-offs improved accuracy in Group 4 patients. Conclusion HBM dRVVT exhibited improved specificity, mainly due to less interference by anticoagulation, but reduced sensitivity, compared to the other dRVVTs employed.

Is lupus anticoagulant testing with dilute Russell's viper venom clotting times reliable in the presence of inflammation?

Background: Testing for lupus anticoagulant (LA) is not recommended in case of inflammation as C-reactive protein (CRP) can interfere in vitro with the phospholipids present in the activated partial thromboplastin time test used to detect an LA. However, the potential interference of an acute phase protein (ie, CRP) in LA testing using the dilute Russell's viper venom (DRVV) test is poorly studied. Objectives: To study the effect of inflammation, as evidenced by increased CRP levels, on DRVV tests. Methods: First, a retrospective analysis (2013-2023) was performed: data on all LA workups were retrieved, and the association between CRP levels and DRVV screen, mix, and confirm clotting times was studied. Second, data on DRVV panels and CRP levels were extracted from 2 prospective studies involving intensive care unit patients to study the association between both variables. Third, CRP was added to normal pooled plasma at 6 relevant concentrations (up to 416 mg/L) to study the association between CRP itself and DRVV coagulation times. Results: In the retrospective analysis, DRVV screen and confirm clotting times significantly increased as CRP increased (increase of 0.11 seconds and 0.03 seconds per 1 mg/L increase of CRP level, respectively). In the prospective analysis, only DRVV screen was prolonged with high CRP levels (increase of 0.06 seconds for a 1 mg/L increase in CRP level); DRVV screen/confirm ratio was also increased with high CRP levels. In vitro, the addition of CRP did not significantly increase any DRVV clotting times. Conclusion: LA testing should be performed with much caution in the presence of inflammation as it may be associated with prolongation of both activated partial thromboplastin time and DRVV clotting times.

Effect of residual platelets in frozen-thawed plasma on results of dilute Russell's viper venom time assay for lupus anticoagulant testing.

OBJECTIVES: To determine the impact of residual platelets on dilute Russell's viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. METHODS: We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. RESULTS: Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. CONCLUSIONS: Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

Triple-positive antiphospholipid syndrome does not guarantee positivity in each lupus anticoagulant assay.

BACKGROUND: Triple positivity for all 3 criteria antiphospholipid antibodies confers high risk of symptom development in carriers, and recurrence in antiphospholipid syndrome (APS). Most triple-positivity studies report lupus anticoagulant (LA) testing as positive without distinguishing between positivity with dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) and single-assay positivity or only perform dRVVT. Single LA assay repertoires remain in use in some centers, which risks missing some triple positives. Positivity with both assays may identify higher risk. OBJECTIVES: The aim of this study is to investigate the frequency of single LA assay positivity in triple-positive patients. METHODS: Three hundred forty-two triple-positive profiles from nonanticoagulated patients (237 APS, 45 systemic lupus erythematosus without APS symptoms, and 60 nonclinical criteria) were identified from laboratory databases and assessed for LA positivity by dRVVT and/or APTT. RESULTS: Seventy-three of 237 (30.8%) APS samples were LA-positive with 1 assay, 40/237 (16.9%) by dRVVT only, and 33/237 (13.9%) with APTT only. Nineteen of 45 (42.2%) were LA-positive with 1 assay in the systemic lupus erythematosus cohort; 12/45 (26.7%) with dRVVT only and 7/45 (15.5%) with APTT only. Thirty-three of 60 (55.0%) were LA-positive with 1 assay in the nonclinical criteria cohort; 24/60 (40.0%) with dRVVT only and 9/60 (15.0%) with APTT only. The most common solid-phase assay profile was elevated immunoglobulin G aCL and aß2GPI. CONCLUSION: Up to 55.0% of triple-positive samples were positive in 1 LA assay, representing significant potential for misdiagnosis and inappropriate management via single LA assay repertoires.

Diagnostic Challenges on the Laboratory Detection of Lupus Anticoagulant.

Lupus anticoagulant (LA) is one of the three laboratory parameters (the others being antibodies to either cardiolipin or ß2-glycoprotein I) which defines the rare but potentially devastating condition known as antiphospholipid syndrome (APS). Testing for LA is a challenging task for the clinical laboratory because specific tests for its detection are not available. However, proper LA detection is paramount for patients' management, as its persistent positivity in the presence of (previous or current) thrombotic events, candidate for long term anticoagulation. Guidelines for LA detection have been established and updated over the last two decades. Implementation of these guidelines across laboratories and participation to external quality assessment schemes are required to help standardize the diagnostic procedures and help clinicians for appropriate management of APS. This article aims to review the current state of the art and the challenges that clinical laboratories incur in the detection of LA.

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.

International multicenter, multiplatform study to validate Taipan snake venom time as a lupus anticoagulant screening test with ecarin time as the confirmatory test: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies.

BACKGROUND: Lupus anticoagulant (LA) assays are compromised in anticoagulated patients, and existing strategies to overcome the interferences have limitations. The prothrombin-activating Taipan snake venom time (TSVT) screening test and ecarin time (ET) confirmatory test are innately insensitive to vitamin K antagonists (VKA) and direct factor Xa inhibitors (DFXaI). OBJECTIVES: Validate standardized TSVT/ET reagents for LA detection, in a multicenter, multiplatform study. PATIENTS/METHODS: Six centers from four countries analyzed samples with TSVT/ET from 81 nonanticoagulated patients with LA, patients with established antiphospholipid syndrome (APS), and proven persistent LA who were either not anticoagulated (n = 120) or were anticoagulated with VKAs (n = 180) or DFXaIs (n = 71). Additionally, 339 nonanticoagulated LA-negative patients, and 575 anticoagulated non-APS patients (172 VKA, 403 DFXaI) were tested. Anticoagulant spiking experiments were performed and 112 samples containing potential interferences (i.e., direct thrombin inhibitors) were tested. Results were evaluated against locally derived cutoffs. Imprecision was evaluated. RESULTS: Cutoffs were remarkably similar despite use of different analyzers and donor populations. Cutoffs for TSVT ratio, ET ratio, percent correction, and normalized TSVT ratio/ET ratio ranged between 1.08 and 1.10, 1.09 and 1.12, 9.3% and 14.8%, and 1.10 and 1.15, respectively. Coefficients of variation for TSVT and ET ratios were ≤5.0%. TSVT/ET exhibited sensitivity, specificity, and negative and positive predictive values of 78.2%/95.0%/86.3%/91.5%, respectively, with established APS as the LA-positive population, and 86.9%/95.0%/76.8%/97.4%, respectively, with triple-positive APS. Interference was seen with direct thrombin inhibitors, unfractionated heparin, and low molecular weight heparins, but not VKAs or DFXaIs. CONCLUSIONS: TSVT/ET are validated for LA detection in nonanticoagulated patients and those on VKAs or DFXaIs.

Unveiling the complex effects of direct oral anticoagulants on dilute Russell's viper venom time assays.

INTRODUCTION: Dilute Russell viper venom time (dRVVT) assays can be affected by direct oral anticoagulants (DOACs), which may cause false-positive results. However, there are conflicting results indicating significant differences between different reagents and DOACs. OBJECTIVES: To evaluate the effect of DOACs on dRVVT assays. MATERIAL AND METHODS: Samples were prepared by adding DOAC (dabigatran, rivaroxaban, apixaban, or edoxaban) to pooled normal plasma in the concentration range 0 to 800 µg/L. Six integrated dRVVT reagents were used, all composed of a screen assay (low phospholipid content) and a confirm assay (high phospholipid content). The screen/confirm dRVVT results were expressed as normalized ratios. To further evaluate the observed differences between tests and DOACs, addition of synthetic phospholipids was used. RESULTS: The dRVVT ratios increased dose dependently for all DOACs, with four of the six tests and the DOAC rivaroxaban having the greatest effect. With one test, the ratios were almost unaffected with increasing DOAC concentration, whereas another test revealed a negative dose dependency for all DOACs. Variable DOAC effects can be explained by different effects on dRVVT screen and confirm clotting time. Adding synthetic phospholipids to samples containing rivaroxaban resulted in greatly reduced screen clotting times and thereby lower calculated dRVVT ratios. CONCLUSIONS: There is a great variability in the dRVVT test result with different DOACs. The dRVVT ratios are unaffected for some reagents and this can be explained by an equal dose-dependent effect on both screen and confirm assays. The phospholipid type and content of the different reagents may contribute to the observed differences.

Lupus anticoagulant assay cut-offs vary between reagents even when derived from a common set of normal donor plasmas.

BACKGROUND: Multicenter studies reveal that diagnostic efficacy of lupus anticoagulant (LA) assays is enhanced if cut-offs are locally generated. However, a potential confounder is the inevitable use of separate normal donor populations. OBJECTIVES: Generate cut-offs for multiple LA reagents with the same analyzer and normal donor plasmas. METHODS: Cut-offs for screen ratio, confirm ratio, percent correction of screen ratio by confirm ratio, and normalized screen/confirm ratio (NSCR) were derived from the same 50 normal donor plasmas for screen and confirm pairs for two dilute Russell's viper venom time reagents, LA1/LA2 and HEMOCLOT™ LA-S/LA-C, and two APTTs, Actin FSL/FS and Cephen LS/Cephen. The cut-offs were challenged with plasmas from 20 triple-positive APS patients and 25 plasmas from LA-negative, thrombotic patients. RESULTS: Cut-offs for screen ratio, confirm ratio, percent correction, and NSCR, respectively, were 1.12/1.08/8.3/1.09 for LA1/LA2; 1.17/1.10/13.6/1.13 for HEMOCLOT™ LA-S/LA-C; 1.12/1.13/9.7/1.10 for Actin FSL/FS; 1.09/1.13/11.0/1.11 for Cephen LS/Cephen. LA1 and LA-S screens were elevated in 19/20 and 16/20 triple-positive plasmas, respectively, while 20/20 were detected with both via integrated interpretation ie, percent correction or NSCR irrespective of screen elevation. Actin FSL and Cephen LS screens were elevated in 17/20 and 19/20 triple-positive plasmas, respectively, while one more LA was detected with Actin FSL via integrated interpretation, but not for Cephen LS. Integrated interpretation suggested 5/25 LA-negative plasmas contained weak LA (two with Actin FSL/FS, two with LA1/LA2, one with LA-S/LA-R). CONCLUSIONS: Employing the same normal donor plasmas and analytical platform does not compensate for between-reagent differences when generating LA assay cut-offs.

Impact of different preanalytical conditions on results of lupus anticoagulant tests.

INTRODUCTION: The currently recommended preanalytical conditions for lupus anticoagulant (LA) analysis require analyzing samples in fresh or freshly frozen platelet-poor plasma. The aim of this study was to evaluate whether alternative and less cumbersome preanalytical procedures for LA testing give significantly different results compared to recommended conditions. MATERIALS AND METHODS: Citrated blood samples were drawn from 29 study participants, 15 with negative and 14 with positive LA results. The samples were processed according to the ISTH guideline for LA testing and compared to several alternative preanalytical conditions. Measurements were performed using the dilute Russell's viper venom time (DRVVT) and silica clotting time (SCT), both screen and confirm, on a STA-R Evolution analyzer. Stability criteria were based upon biological variation. RESULTS: All DRVVT tests (normalized screen, confirm, and screen/confirm ratio) met the stability criteria for all the preanalytical conditions. The SCT tests (normalized screen, confirm, and screen/confirm ratio) met the stability criteria only when treated according to the ISTH guideline, except for SCT normalized screen/confirm ratio which also met the stability criteria for double-centrifuged aliquoted plasma stored in room temperature for 24 hours and then analyzed "fresh" or after being frozen. One warfarin-treated patient was reclassified from positive to negative for DRVVT after the preanalytical modifications, while 2 of 29 participants became falsely positive for 2 of 8 conditions for SCT. CONCLUSIONS: The DRVVT assays met the criteria for stability for all preanalytical conditions tested, while the SCT assays should be interpreted with caution if the preanalytical guidelines from ISTH are not followed.

Lupus anticoagulant diagnosis in patients receiving direct oral FXa inhibitors at trough levels: A real-life study.

INTRODUCTION: Direct oral factor Xa inhibitors (xabans) induce false positive results for lupus anticoagulant (LA) diagnosis. Consequently, it is suggested not to perform LA testing in xabans patients although it may be useful in selected patients. In this monocentric study, we evaluated xabans impact at trough levels (ie, just before the next intake) on LA diagnosis in treated patients using dilute Russell viper venom time (dRVVT) and two LA sensitive activated partial thromboplastin time (aPTT). METHODS: Sixty patients receiving rivaroxaban (30) or apixaban (30) were included. Plasma concentrations were measured using specific anti-Xa assays. LA testing was performed using one dRVVT (LAC-Screening® /Confirm® ; Siemens) and two LA sensitive aPTT-based assays (Hemosil® Silica Clotting Time (SCT) Screen/Confirm; Werfen and Dade® Actin® Factor Sensitivity FSL/FS (Actin F); Siemens). RESULTS: Median [min-max] concentrations were 23 [<18-68] for rivaroxaban and 42 ng/mL [19-99] for apixaban. dRVVT was positive in 93% of rivaroxaban and 40% of apixaban samples. SCT was positive in 40 and 30% and Actin F in 17 and 20% of samples respectively. Xabans affected more significantly dRVVT than aPTT-based assays (P < .001) with less false positive results with apixaban than with rivaroxaban samples irrespective of the assay used. CONCLUSION: lupus anticoagulant diagnosis in rivaroxaban and apixaban samples drawn at trough levels remains questionable whenever positive results are obtained. If LA testing in apixaban samples might be useful to rule-out LA using dRVVT and/or aPTT-based assays, the wide majority of rivaroxaban samples would give false positive results.

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.

Variability of cut-off values for the detection of lupus anticoagulants: results of an international multicenter multiplatform study.

Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable. SUMMARY: Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time [APTT] and dilute Russell viper venom [dRVV]). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.

Application of different lupus anticoagulant diagnostic algorithms to the same assay data leads to interpretive discrepancies in some samples.

BACKGROUND: Gold standard lupus anticoagulant (LA) assays and reference plasmas do not exist and detection is based on inference in a medley of coagulation assays, creating potential for interpretive discrepancies when applying different algorithms. OBJECTIVES: To investigate discrepancies from applying different algorithms to a common data set. METHODS: Diagnostic data on 311 non-anticoagulated patients LA-positive by dilute Russell's viper venom time (dRVVT) and/or dilute activated partial thromboplastin time (dAPTT) assays were employed to compare algorithms. Routine testing applied interpretive criteria from guidelines endorsing classification as LA-positive despite negative mixing tests, after exclusion of other clotting abnormalities. Integrated testing without mixing tests, and the classical algorithm where negative mixing tests preclude confirm tests, were then retrospectively applied to those data. RESULTS: Initial testing showed 92/311 (29.6%) were LA-positive by dRVVT only, 156/311 (50.1%) by dAPTT only, and 63/311 (20.3%) by both assays. All dAPTT-positive plasmas remained positive with integrated testing but eight dRVVT-positives became negative. Other data suggested they were false-negatives. The classical algorithm altered 52/155 (33.5%) dRVVT and 111/219 (50.7%) dAPTT interpretations to LA-negative because of normal mixing tests, most of which were apparently weak LA in undiluted plasma. CONCLUSIONS: The classical algorithm improves diagnostic specificity and confidence but risks missing some genuine LA due to false-negative mixing tests. Integrated testing can be diagnostically accurate and logistically efficient but oversimplifies complex cases. Performing mix and confirm in response to an elevated screen with their interpretation based on clinical data, coagulation screens and the LA-assay design offers a potentially valuable option.

Current Controversies in Lupus Anticoagulant Detection.

Antiphospholipid syndrome is an autoimmune, acquired thrombophilia diagnosed when vascular thrombosis or pregnancy morbidity are accompanied by persistent antiphospholipid antibodies. Lupus anticoagulants (LA) are one of the criteria antibodies but calibration plasmas are unavailable and they are detected by inference based on antibody behaviour in a medley of coagulation-based assays. Elevated screening tests suggest the presence of a LA, which is confirmed with mixing tests to evidence inhibition and confirmatory tests to demonstrate phospholipid-dependence. At least two screening tests of different principle must be used to account for antibody heterogeneity and controversy exists on whether assays, in addition to dilute Russell's viper venom time and activated partial thromboplastin time, should be employed. A variety of approaches to raw data manipulation and interpretation attract debate, as does inclusion or exclusion of mixing studies in circumstances where the presence of a LA is already evident from other results. Therapeutic anticoagulation compromises coagulation-based assays but careful data interpretation and use of alternative reagents can detect or exclude LA in specific circumstances, and this aspect of LA detection continues to evolve. This review focuses on the main areas of debate in LA detection.