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.

Interference of DOACs in different DRVVT assays for diagnosis of lupus anticoagulants.

OBJECTIVE: Determination of lupus anticoagulants (LA) is an important, but still challenging test in the diagnosis of antiphospholipid syndrome (APS). This is especially the case in patients using one of the direct oral anticoagulants (DOACs). The aim of our study was to examine the influence of these drugs on DRVVT assays from two companies (in each case: screening test, confirming test and calculated ratio) and on aPTT and lupus-sensitive aPTT. METHODS: We used plasma samples from healthy volunteers spiked with the DOACs dabigatran, rivaroxaban and apixaban (0, 10, 30, 50, 100 ng/mL) for testing. Furthermore, samples from patients receiving a DOAC were investigated. The plasma concentrations of the DOACs were determined using ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (UPLC-MS/MS). RESULTS: Depending on type and concentration, all the DOACs resulted in pathological values in the DRVVT screening assays. In samples spiked with apixaban, no influence on the DRVVT normalized ratio of the two assays was observed, but 7 to 15% of samples from patients receiving apixaban displayed pathological values. In contrast, up to 71% of dabigatran-spiked samples showed normalized ratio values above the cut-off, whereas there was no influence in the patients' samples. In both spiked and patient samples containing rivaroxaban, the DRVVT assays were influenced. CONCLUSION: LA diagnostics should, under DOAC therapy, be limited to situations in which time-critical evaluation is warranted. It is crucial to take into account the finding that even samples containing DOAC concentrations below the limit of detection of the drugs may lead to false-positive DRVVT measurements.

Lupus anticoagulant acquired hypoprothrombinemia syndrome in childhood: two distinct patterns and review of the literature.

INTRODUCTION: Lupus anticoagulant associated with acquired prothrombin deficiency also known as 'lupus anticoagulant hypoprothrombinemia syndrome' (LAHS) is an entity that is well described in adults and is usually associated with autoimmune conditions (LAHS-AI). However, in children, LAHS has unique features that are distinct from the adult type. AIMS: We report two paediatric cases of LAHS, describe their distinct patterns and review the paediatric literature on LAHS. METHODS: Case studies on two patients with LAHS were reviewed, details on their presentation, work up and management were extracted. A Medline search was conducted on LAHS in children between 1960 and 2014; Articles in languages other than English were excluded. RESULTS: The case studies highlight the differences in the two patterns of childhood LAHS. Additionally the review of the literature reveals that there are 15 case reports and 5 case series that report 25 children with LAHS-AI, 9 case reports and 6 case series report 26 children of LAHS associated with viral infections (LAHS-VI). At presentation, all patients with LAHS-AI had positive laboratory tests for autoimmune diseases, most commonly for systemic lupus erythematosus while these tests were negative in LAHS-VI. All patients with LAHS-AI had a protracted course and needed prolonged treatment with immune-suppressive therapy while patients with LAHS-VI resolved spontaneously or needed short-term immune-modulating therapy. CONCLUSION: In childhood, two distinct patterns of LAHS are observed, either associated with infection or autoimmune disease. Initial diagnostic investigations are critical to differentiating these two patterns as the prognosis and outcome for each is distinct.

Comparison of monitoring methods for lepirudin: impact of warfarin and lupus anticoagulant.

INTRODUCTION: Appropriate monitoring methods are needed for lepirudin, a direct thrombin inhibitor, as activated partial thromboplastin time (APTT) may under- or overestimate lepirudin. We compared APTT with thrombin-specific methods, also in the presence of warfarin and lupus anticoagulant (LA). MATERIALS AND METHODS: Lepirudin i.v. was assessed in five patients (35 samples) and in vitro spiked plasma pools: normal control and plasma containing warfarin and LA. Wide dose-responses to lepirudin (0-4.0microg/ml) were studied with APTT (Actin FSL), Ecarin Chromogenic Assay (ECA), chromogenic Anti-Factor IIa (Anti-FIIa, Hirudin Activity Assay), Prothrombinase-induced Clotting Time (PiCT), and plasma diluted Thrombin Time (dTT). RESULTS: APTT both under- and overestimated in vivo lepirudin doses according to ECA) and Anti-FIIa, which matched completely in various plasma pools at all lepirudin doses (r=0.99). APTT and PiCT) underestimated high lepirudin concentrations in normal plasma, and in LA-positive plasma they were invalid. In all plasma pools, dTT (1:16) indicated lepirudin well up to 1.0microg/ml. CONCLUSIONS: ECA or Anti-FIIa are preferable for lepirudin monitoring, because neither warfarin nor LA, interfered with them, and they were the most precise methods even for supratherapeutic doses. PiCT reflected co-inhibition of FIIa and FXa, but was disturbed, like APTT, by LA and high lepirudin. Further experience of laboratory monitoring is valuable in this era of new anticoagulants.

Prolonged aPTT after kidney transplantation due to transient lupus anticoagulants.

BACKGROUND: After kidney transplantation, a renal biopsy may be needed to elucidate the reasons for lack of graft function. If the activated partial thromboplastin time (aPTT) is prolonged, the biopsy will often be postponed, as increased risk of bleeding must be expected. However, aPTT prolongation is not always due to lack of coagulation factors, but can be due to the presence of lupus anticoagulants (LAs). Clinical observations in our department indicated that a large proportion of recently kidney-transplanted patients developed prolonged aPTT values without clinical complications. METHODS: A prospective study of patients receiving a kidney transplant in 2004 was conducted to investigate the frequency and cause of prolongation of the aPTT. RESULTS: Twenty-seven patients were included in the study; none had prolonged aPTT or LAs before the transplantation. In the post-transplantation period, 19 patients (70.4%) had a significantly prolonged aPTT. Further investigation showed that for all 19 patients, prolongation was due to acquired antibodies: 13 had developed LAs and six had developed unspecific antibodies. The acquired antibodies were transient and did not affect clinical outcome. CONCLUSIONS: This is the first study investigating prolonged aPTT in the post-transplantation period. All patients with prolonged aPTT had acquired transient antibodies, i.e. LA or 'LA-like'. If a renal biopsy was requested, 70.4% of the transplanted patients would presumably have their biopsy postponed due to prolonged aPTT, but as LAs do not increase the risk of bleeding, such a delay would be unnecessary. Immediate LA investigation is therefore recommended if a recently transplanted patient requiring surgical procedures has a prolonged aPTT.

Clinicohematologic spectrum in patients with lupus anticoagulant.

A retrospective analysis of clinicohematologic parameters of 25 patients with lupus anticoagulant was carried out. The hematologic tests included dilute Russel viper venom test (dRVVT), kaolin clotting time (KCT), activated partial thromboplastin time, and prothrombin time. The diagnosis of lupus anticoagulants was based on the presence of prolonged KCT/dRVVT, its absence of correction with normal plasma and correction by phospholipids. Specific factor assays and platelet aggregation studies were performed wherever required. Ten patients (40%) had thrombosis, which was venous in 5 (50%) and arterial in 4 (40%). One patient (10%) had both arterial and venous thrombosis and presented with catastrophic antiphospholipid syndrome. Eighteen female patients conceived. Four (22%) of these had recurrent first trimester abortion. Five (20%) patients had bleeding manifestations. One (4%) of these had hypoprothrombinemia and was diagnosed to have hypoprothrombinemia lupus anticoagulant syndrome. However in two of these patients, no cause of bleeding could be identified other than the presence of lupus anticoagulants. It is concluded that patients with lupus anticoagulant have a varied spectrum of hemostatic disorders. Bleeding may sometimes occur in these patients due to associated thrombocytopenia or associated factor inhibitors. Rarely, it may occur due to presence of lupus anticoagulants alone.

Antiphospholipid antibodies impact the protein C (PC) pathway behavior.

Antiphospholipid antibodies may interfere with the PC pathway, displaying a resistance to the activated PC (resistant phenotype). This effect was evaluated by the APCR and the ProCG systems in 36 lupus anticoagulant samples, yielding abnormal results in 47% of APCR(original), 17% of APCR(modified), and 22% of ProCG test. ProCG values correlated with APCR(original) but not with APCR(modified). Most of lupus anticoagulants affecting the PC pathway showed abnormal APCR(original) results but not abnormal ProCG values. The different behavior between both systems may be due to the heterogeneity of the antibodies or could be attributed to the fact that, in the ProCG, a PC activator is added, while the APCR employs already activated exogenous PC.

A chromogenic substrate method for detecting and titrating anti-factor VIII antibodies in the presence of lupus anticoagulant.

BACKGROUND AND OBJECTIVES: The development of neutralizing anti-factor VIII antibodies (a-fVIII) is a major clinical complication. Lupus anticoagulant (LA) might affect detection of a-fVIII, since both inhibitors may act on the same coagulation pathway. Our aim was to accomplish unequivocal detection and titration of a-fVIII even in the presence of LA. DESIGN AND METHODS: We evaluated a-fVIII activity by a chromogenic substrate (CS) method in samples with a-fVIII (n=6), LA (n=12) and presumably both LA+a-fVIII (n=5). The inhibition index before (Ii) and after incubation at 37 C (Ii(37)) was estimated. We also performed factor VIII assays (one-stage and CS) and titration methods (Bethesda and CS) in parallel. RESULTS: Inhibition in the a-fVIII group (Ii=5-3200) was potentiated by incubation (Ii(37)=27-5200) as it was in LA+a-fVIII (Ii=9-21; Ii(37)=50-903). LA samples showed no or meaningless inhibitory effect (Ii=0-7; Ii(37)=0-4) or a-fVIII activity (0.00-0.06 CSU/ml) by the CS method; on the contrary, very low to moderate (0.52-7.00 BU/ml) a-fVIII activity was recorded by the Bethesda method. The two titration methods did not correlate (p>0.100) in the presence of LA, or LA+a-fVIII. Differences between factor VIII:C and factor VIIIcs were significant only in LA samples (p=0.005); however, patients with residual factor VIII activity from the LA+a-fVIII group also showed higher factor VIIIcs values than factor VIII:C ones. INTERPRETATION AND CONCLUSIONS: Results indicate the possibility of detecting and titrating a-fVIII without interference of LA by the CS method. This marks a difference with respect to the Bethesda method, in which a measurable effect can be expected in the presence of a strong LA.

True anti-anionic phospholipid immunoglobulin M antibodies can exert lupus anticoagulant activity.

True (cofactor-independent) anticardiolipin antibodies (aCL) are thought to lack lupus anticoagulant (LA) activity and pathogenic potential. A serum monoclonal immunoglobulin Mlambda (mIgMlambda) with aCL and LA activities found in a man with a splenicIgMlambda+ B-cell lymphoplasmacytic lymphoma (LPL) without thrombotic events has been characterized. LPL-derived hybridoma clones (designated HY-FRO) producing the serum mIgMlambda were obtained. mIgMlambda secreted by HY-FRO grown in protein-free culture medium, like that purified from serum, (i) showed binding, in a cofactor-free system, to solid-phase CL and phosphatidylserine (PS) and to the membrane of PS-expressing cells (apoptotic cells and activated platelets); (ii) failed to bind neutral phospholipids (PL), beta2Glycoprotein, histone, ssDNA, dsDNA, human IgG and umbilical vein endothelial cells. Absorption with apoptotic cells abolished its binding to anionic plate-bound CL and PS. IgMlambda-FRO used poorly mutated VH and Vlambda region genes, with a pattern that was inconsistent with an antigen-driven selection. Basic amino acids were present in the IgH complementarity determining region 3 (CDR3), which can be important for binding to anionic PL. These findings demonstrate unequivocally that true anti-anionic PL IgM antibodies can exert LA and indicate this anti-PL type does not involve thrombophilia.

[Study on the mechanism of thrombosis by lupus anticoagulant inhibited protein C pathway].

OBJECTIVE: To study the mechanism of thrombosis in systemic lupus erythematosus (SLE) patients with protein C (PC) pathway inhibition by lupus anticoagulant(LA). METHODS: Phosphoethylamine(PE) dependent activated protein C (APC) inhibiting assay (modifed DRVVT) was used to observe the inhibition of APC activity after incubation of IgG from both normal and SLE patients with PE. RESULTS: The activity of anticoagulation of APC was augmented by PE. Proportionally to its concentration, LA could inhibit the PE dependent anticoagulation of APC. The inhibition of LA-IgG on APC in the LA positive SLE patients with thrombosis was stronger than that in LA negative non-thrombosis patients; but IgG has no effect on the APC activity in normal person. CONCLUSION: LA inhibiting the PC pathway by interfering with PE may be the important reason of thrombosis in SLE patients.

Interference of lupus anticoagulants in prothrombin time assays: implications for selection of adequate methods to optimize the management of thrombosis in the antiphospholipid-antibody syndrome.

BACKGROUND AND OBJECTIVE: Prolonged anticoagulation aiming at International Normalized Ratio (INR) values > 3.0 has been recommended for patients with thrombosis and the antiphospholipid-antibody syndrome. We evaluated the influence of anticoagulant antibodies in two different prothrombin time (PT) assays carried out on plasma from lupus anticoagulant patients on oral anticoagulation. DESIGN AND METHODS: INR values obtained with a combined (final test plasma dilution 1:20) and a recombinant (final test plasma dilution 1:3) thromboplastin were compared in 17 patients with persistent lupus anticoagulants (LA) receiving oral anticoagulant treatment and monitored for 69.8 patient-years. Doses of anticoagulant drugs were always assigned based on the results obtained with the combined thromboplastin, aiming at a target INR of 2.5 or 3.0 for patients with venous or arterial thromboembolic disease. Paired determinations with both reagents were also obtained throughout the study period in 150 patients on stable oral anticoagulation but free of antiphospholipid antibodies. Total IgG fractions were purified from selected patients to evaluate effect in the two PT assay systems. RESULTS: No patient experienced recurrence of thrombosis or major bleeding complications (95% confidence interval: 0.1-6.5 per 100 patient-years). INR values with the recombinant reagent were significantly higher than with the combined reagent in 8 LA patients (mean DINR ranging from 0.17 to 0.54) of the degree of anticoagulation was overestimated in all but one LA patients with the recombinant reagent when compared to the DINR observed in non-LA patients (-0.64 +/- 0.42). The anti-cardiolipin IgG titer (r(2) = 0.43, p = 0.004) and the anti-b(2)GPI IgG titer (r(2) = 0.30, p = 0.023) were positively associated with the mean deltaINR observed in LA patients. When added to plasmas with different levels of vitamin K-dependent factors, total IgG fractions from 6 LA patients with significant overestimation of the INR with the recombinant reagent (mean DINR ranging from 0.17 to 0.54, group 1) and from 7 LA patients with mean deltaINR < or = 0.0 (ranging from -0.25 to 0.04, group 2) reproduced the effects observed ex vivo in the two assay systems. However, when total IgG fractions were tested at the same final concentration in the two PT assay systems, there was no difference in the clotting times determined with total IgG fractions from group 1 and group 2 LA patients. Addition of negatively charged liposomes (0.4 and 0.8 mg/mL final concentrations) to platelet free plasma from LA-free patients on stable oral anticoagulation caused a 20% to 48% prolongation of the prothrombin time determined with the recombinant reagent. In contrast, no significant prolongation of the prothrombin time determined with the recombinant reagent was observed upon addition of negatively charged liposomes to plasma from group 1 LA patients. INTERPRETATION AND CONCLUSIONS: These results confirm previous suggestions of assay-dependency of INR values in LA patients on oral anticoagulation. For these patients, accurate INR values may be obtained using combined thromboplastin reagents that permit testing at high plasma dilution.

The Factor V (Leiden) test: evaluation of an assay based on dilute Russell Viper Venom Time for the detection of the Factor V Leiden mutation.

In the present study a new clotting assay for the detection of an increased resistance of coagulation factor V against degradation by activated protein C (Factor V Leiden mutation, FVLM) was evaluated. The Factor V (Leiden) Test (Gradipore, North Ryde NSW, Australia) is based on the dilute Russell Viper Venom Time (DRVVT), which is prolonged when the plasma sample is preincubated with dilute whole Agkistrodon contortrix contortrix venom for activation of protein C (PC). In contrast to the DRVVT based global assay, Protein C Pathway Test (Gradipore, North Ryde NSW, Australia) this new assay is expected to be more specific for FVLM because of optimized amounts of the venom. The test result is expressed as the ratio between the DRVVT with and without addition of the venom. The following precision values were found: intraassay coefficient of variation (CV): 5.53% (n=20) in the normal range, 4.30% (n=20) in the pathological range; interassay CV: 6.90% (n=10) and 7.64% (n=10), respectively. A normal range (5th to 95th percentile) of 2.12 to 3.08 was calculated from 50 healthy controls. A ratio below 2.12 was found in all samples from patients with FVLM (n=21), in 9 of 12 patients with PC, in 0 of 6 with protein S (PS), and in 0 of 4 with antithrombin (AT) deficiency. There was, however, a good discrimination between carriers of the FVLM (highest ratio 1.44) and patients deficient in PC (lowest ratio 1.59), in particular when samples were prediluted with factor V deficient plasma FVDP (1.16 vs. 1.96, respectively). Predilution of samples with FVDP caused a clear discrimination between controls and patients deficient in PC, PS, AT, and FVLM-positive individuals and also in patients on oral anticoagulant treatment. Our data show that the Factor V (Leiden) Test discriminates well between carriers of the FVLM and healthy controls or patients deficient in PC, PS, and AT. Individuals presenting values between the lower cutoff of controls and the range in which FVLM-positive individuals are found are highly suspicious for protein C deficiency.

Detecting APC-resistant factor V: a functional method without plasma dilution.

A method for detecting activated protein C (APC)-resistant factor V, especially factor V Leiden, is described, which uses reagents containing two unfractionated snake venoms. The procedure can be used for testing plasma samples from patients receiving oral anticoagulant therapy, heparin therapy and patients with lupus anticoagulant, and does not require the use of factor-V-deficient plasma. The sample plasma is first incubated with dilute venom from Agkistrodon contortrix contortrix (Southern Copperhead) which activates the endogenous protein C and then a dilute Russell's viper venom time test is performed. In individuals with APC-resistant factor V, especially factor V Leiden, a marginal prolongation of dilute Russell's viper venom time was noted [1.14 +/- 0.14 s (n = 16)]. Non-carriers were easily discriminated in each patient group, with a prolongation of 2.69 +/- 0.30 s for normal blood donors (n = 127), 2.61 +/- 0.38 s for patients taking oral anticoagulants (n = 102), 2.41 +/- 0.45 s for patients taking heparin (n = 96), and 2.38 +/- 0.41 s for patients with lupus anticoagulant (n = 22). Patients taking oral anticoagulants with moderate prolongation (between 1.5- and 2.0-fold) may have low levels of functional protein C and this might additionally indicate a subgroup of such patients at higher than normal thrombotic risk.

Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo.

BACKGROUND: Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. METHODS AND RESULTS: All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 microm2 for control). CONCLUSIONS: Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.