Laboratory Diagnosis Of Lupus Anticoagulant Research Articles

Effects of direct oral anticoagulants on lupus anticoagulant assays in a real-life setting.

Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russell's viper venom time - dRVVT or activated partial thromboplastin time - aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs - dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2-1.4 LA could not be ruled out. Plasma concentrations varied between 8-172 µg/l for DAB, 8-437 µg/l for RIV and 36-178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.

The paradox of the lupus anticoagulant: history and perspectives.

A unique coagulation inhibitor prolonging whole-blood clotting time was described more than 50 years ago in two patients with systemic lupus erythematosus (SLE). The immunoglobulin nature of the inhibitor and its interaction with antiphospholipid antibodies was later demonstrated and the term "lupus anticoagulant (LA)" was coined to describe this laboratory finding. It soon became apparent that LA was a misnomer as it is often found in plasma from patients with clinical conditions other than SLE and is associated with thromboembolic events that may occur in otherwise healthy individuals. Individuals with LA have circulating autoantibodies that inhibits blood coagulation. These are mostly of IgG or IgM class and mainly directed against a phospholipid (PL)-binding plasma protein, β2-glycoprotein I (β2GPI). The presence of β2GPI-dependent LA represents a well-recognized risk factor for venous and arterial thromboembolism, as well as pregnancy loss and morbidity. β2GPI-dependent LA in the presence of documented previous thromboembolism, or history of pregnancy loss/morbidity, identifies definite anti-PL syndrome. Laboratory diagnosis of LA is thus of particular importance, as it may assign patients with a common event (thrombosis) to a group with a high risk for recurrence, which is a prerequisite for long-term oral antithrombotic treatment.

Evaluation of commercial normal pooled plasma used in the laboratory diagnosis of lupus anticoagulants

Article history: Received 18 September 2009 Received in revised form 28 December 2009 Accepted 8 January 2010 Available online 27 January 2010 screening registered at our department were included in Plasmas were qualified as LAC positive, by considering all cri ISTH using a three-step procedure [2]. In our laboratory w activated partial thromboplastin time (aPTT) (PTT-LA, Diagno Asnieres, France), and a diluted Russell's viper venom time (d Screen, Gradipore Ltd., Australia) based test system [1,3]. R expressed as normalized ratios dividing the clotting time sample by the clotting time of NPP analysed in the same r

Laboratory Testing for Lupus Anticoagulants: Preexamination Variables, Mixing Studies, and Diagnostic Criteria

The latest international guidelines for the laboratory diagnosis of lupus anticoagulants (LA) date back to 1995 and are in need of revision. Various patterns of practice and interpretation by clinical laboratories have altered the intent or usage of the criteria and recommendations made at that time. Moreover, various topics in the preexamination phase of LA testing (such as the role of the clinician in test ordering, the impact of direct thrombin inhibitors or oral anticoagulant therapy, and patient biologic variation) need to be considered in future guidelines. In like fashion, examination issues relating to mixing studies will need critical review. Mixing studies have been applied to a variety of low-phospholipid LA screening assays, and no uniformity or standardization exists as to how these test results should be interpreted or if in fact they are suitable for identifying weak lupus anticoagulants.

Laboratory diagnosis of lupus anticoagulant associated by coagulation factors(F VIII,F IX and F XI) deficiency:report of 13 cases

Objective To explore laboratory diagnosis tests for lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency.Methods We investigated the coagulation factors'activity,coagulation factors'antibodies,anticardiolipin antibodies,lupus anticoagulant and thrombin generation in thirteen patients of lupus anticoagulant associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency,ten hemophilia A patients, forty-eight healthy people. In these thirteen patients,there were five patients with schizophrenia and had been taking chlorderazin for a long time,two patients with antiphospholipid syndrome, one patient with pemphigus, five patients in which causes of disease were unknown.Among these patients,two patients has haemorrhage,eleven patients don't have reduced.The antibodies of these coagulation factors and LA were positive.Six patients in this group were ACA poslhve,and seven patients were ACA negative.The lag time of eleven non-haemorrage patients with LA positive associated by F Ⅷ,F Ⅸ and F Ⅺ deficiency was(5.60±1.18)min,higher than that of control group which was(1.88±0.25)min.This differenee had statistically significant(t=10.05,P＜0.01).The time to peak was(8.11±1.91)min,significantly higher than that of control group which was (4.10±0.36)min(t=8.83,P＜0.01).The endogenous thrombin potential(ETP)was(1 640.87±higher than that of hemophilia A patients which was(38.50±6.20)%(t=9.77,P＜0.01).The time to peak of hemophilia A patients was(8.01±1.81)min,significantly higher than that of control group which Was(4.10±0.36)min(t=8.20,P＜0.01).The lag time of control group and hemophilia A patients was (1.88±0.25)min and(2.01±0.84)min,respectively.There was no significant difierence of the lag time between these two groups(t=1.26,P＞0.05).Conclusions Diseases like lupus anticoagulant associated bv coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency and inherited hemophilia should use APTT,PT,should be used as confirmation tests. LA and ACA may serve as indication test for identifying the causes of these diseases. ETP and ETP percentage are quite good parametem for predicting the bleeding risk of patients with LA positive associated by coagulation factors(F Ⅷ,F Ⅸ and F Ⅺ)deficiency. Key words: Lupus coagulation inhibitor; Hemophilia A; Hemophilia B; Factor Ⅺ deficiency; Thrombin

Evaluation of a new silica clotting time in the diagnosis of lupus anticoagulants

Introduction A new commercial silica clotting time (SCT), the HemosIL™ SCT assay (Instrumentation Laboratory, Milan, Italy) was evaluated in the laboratory diagnosis of lupus anticoagulants (LAC). This integrated test system for screening and confirmation was compared with the frequently used aPTT-based PTT-LA and Staclot-LA (Diagnostica Stago, Asnières, France) in a patient population investigated for LAC and in a subpopulation who met the clinical criteria for antiphospholipid syndrome (APS). Materials and methods 201 samples were analysed with the HemosIL™ SCT assay. Own reference values were calculated. Results are expressed as measured clotting times in seconds or as normalised ratios. Results SCT screen and PTT-LA had a sensitivity of, 61.1% and 63.8%, respectively. Normalising the results gained sensitivity up to 72.2% and 90%, respectively. The confirmation SCT and the Staclot-LA had a sensitivity of 30.6% and 63.9% with a specificity of 86.7% and 100%, respectively. Sensitivity of SCT for detecting LAC in clinical criteria positive patients was lower compared to aPTT and dRVVT (45.8% versus 66.7% and 65%). Combination of SCT/dRVVT and aPTT/dRVVT gave a sensitivity of 51.2% and 63.6%, with a specificity of 50.0% and 52.3%, respectively. Conclusions In comparison with PTT-LA as screening test, the SCT screen shows an acceptable sensitivity. However, the HemosIL™ SCT assay including the confirmation step, has a much lower sensitivity in the diagnosis of LAC in comparison with the Staclot-LA test. Combining the HemosIL™ SCT assay with dRVVT results in a better sensitivity, although lower than the combined aPTT/dRVVT based method as usually performed in our lab.

Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants

Introduction Mixing studies are part of the laboratory diagnosis of lupus anticoagulants (LA). They are used to determine the evidence of an inhibitor by demonstrating persistence of an abnormal clotting time by mixing patient plasma with normal plasma. Since there is no standardised interpretation of results of mixing studies, percent correction formulas are proposed. The sensitivity of mixing studies strongly depends on the interpretation of the results. Materials and methods A retrospective analysis was performed on 361 samples, 75 LA-positive and 286 LA-negative samples. For all the LA-positive samples and for 181/286 LA-negative samples mixing tests on one or more screening tests were performed. A percent correction formula and the Rosner index were calculated for all mixing tests on aPTT and dPT. Results The < 70% correction formula for the mixing tests on aPTT showed the highest sensitivity (95%). The Rosner index had a sensitivity (93%) comparable with the < 70% correction formula. dPT is shown to be less sensitive in the detection for LA and, even when the screening test is prolonged, interpretation of the mixing test by the percent correction formula misclassifies many samples. Rosner index in the interpretation of mixing tests for dPT is more sensitive than the percent correction formula, 49% and 57%, respectively. Conclusions This study demonstrates that the Rosner index and the percent correction formulas for interpretation of mixing studies are complementary and can help to reduce misclassification of LA-positive or LA-negative samples.

The laboratory diagnosis of lupus anticoagulant in patients on oral anticoagulation

Laboratory-specific cut-off lupus ratios (LR), above which a plasma is judged positive for lupus anticoagulant (LA), were established for both activated partial thromboplastin time-based and dilute Russell viper venom time-based methods. The validity of using these cut-off values to determine the presence of LA in patients on oral anticoagulation (OAC) was assessed. A cohort of 40 patients (23 male and 17 female), aged 22-84 years (mean 52 years) were tested for LA at the time of a thrombotic event. Repeated testing was performed after the same patients were treated with OAC (international normalized ratio 2.0-3.5). For 36 patients (90%), LA status was unchanged pre- and on-OAC. Thirteen of the 40 patients (32.5%) were positive for LA both pre- and on-OAC. Of the 27 patients negative for LA pre-OAC, 23 remained negative on-OAC. The four discordant results were interesting in that LA positivity was demonstrated only after the patient was stable on-OAC. In our cohort of 40 patients, there was a trend for LRs to decrease on-OAC, but this did not reach statistical significance. The subset (4) went against this trend and became positive after the thrombotic event.

Use of a new silica clotting time for diagnosing lupus anticoagulant in patients who meet the clinical criteria for antiphospholipid syndrome

The silica clotting time (SCT) is a phospholipid-dependent coagulation assay used for the laboratory diagnosis of lupus anticoagulant (LA) antibodies. The sensitivity and specificity of a new commercial SCT for identifying LA in patients who meet the clinical criteria for antiphospholipid syndrome (APS), and its association with thrombotic events were evaluated here. Forty-five patients who met the clinical criteria for APS according to the Sapporo International Consensus Statement were examined. Sixty-nine patients who did not meet the clinical criteria for APS, and 20 blood donors were used as controls. Plasma samples from the patients and controls were tested for LA using a new commercial SCT with low and high synthetic phospholipid concentrations. The results were compared with those obtained by diluted Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT). SCT's sensitivity for identifying LA in patients who met the clinical criteria of APS was higher compared to APTT and dRVVT (53.3% vs. 31.1% and 31.1%), and the specificities of these assays were 96.6%, 100%, and 98.9%, respectively. When dRVVT was combined with SCT, and dRVVT was combined with APTT their sensitivities were 57.7% and 48.8%, and their specificities were 96.6% and 98.9%, respectively. A stepwise logistic regression analysis indicated that the combination of dRVVT with SCT was associated with total thrombotic events (odds ratio (OR)=11.5, 95% confidence interval (CI)=1.25-106.3, P=0.031) as well as with venous thrombosis (OR=4.09, 95% CI=1.16-14.43, P=0.028). According to our results, SCT is the most sensitive assay for identifying LA in patients who meet the clinical criteria for APS. Moreover, the highest sensitivity was reached with a combination of SCT and dRVVT. The method's association with total thrombotic events and venous thrombosis was in fact significant.

The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific reference ranges are applied

Introduction One of the recommended criteria for the laboratory diagnosis of lupus anticoagulants (LA) is demonstration of inhibitory activity. This is confirmed by performing mixing tests with normal plasma, usually in a 1:1 ratio, and demonstrating persistence of an abnormal clotting time in the screening test with significant confirmatory test reduction. However, the mixing with normal plasma can dilute the antibodies to undetectable levels and generate apparent negative results. No guidelines or consensus exist in how to interpret mixing study results. Patients and methods The present study assessed the 1:1 mixing study results from 600 patients with a thrombotic history positive for LA demonstrated in neat plasma by individual assays, or combinations, of dilute Russell's viper venom time, dilute activated partial thromboplastin time, activated seven lupus anticoagulant assay and Taipan snake venom time, plus confirmatory tests. Mixing tests were assessed initially using locally derived neat plasma reference ranges and subsequently with mixture specific ranges. Results The mixture specific ranges had lower upper limits. Of the total LA positive results, 32.5% were positive in the mixing studies when neat plasma reference ranges were applied, and a further 11.2% demonstrated LA activity when using the mixture specific ranges. The remaining 56.3% had been diluted such that they did not elevate the screening test above the upper limit of normal or generated minimal prolongation with an insignificant difference between the screen and confirmatory test result sufficient to confirm LA activity. Conclusions The significant impact of the dilution effect in 1:1 mixing studies emphasises the limitations of mixing studies as a vehicle for confirmation of inhibition by LA antibodies.

Laboratory Diagnosis of Lupus Anticoagulants

Residual platelets in plasma are considered detrimental after freezing-thawing, as phospholipids released from ruptured platelets may quench lupus anticoagulants (LA). We aimed at assessing the effect of residual platelets after freezing-thawing plasmas tested with two procedures for LA. Blood from 52 patients suspected of having LA were centrifuged at 2,500 g. Plasmas were subdivided into 2 aliquots. One was filtered to remove residual platelets and both were frozen and stored at -70 degrees C. Silica clotting time (SCT) at low and high phospholipid concentrations and Staclot LA with and without Hexagonal phospholipids were performed on thawed plasmas. Plasmas were considered LA-positive when both SCT and Staclot LA performed on filtered plasmas were diagnostic for LA. Forty-two of 52 plasmas fulfilled the diagnostic criteria and were retained for subsequent analysis. SCT on non-filtered plasmas was diagnostic for LA in 42 of 42 plasmas. Though the median (range) percentage correction recorded after phospholipids addition for filtered plasmas, i.e., 67% (36%-83%) was reduced to 54% (25%-81%) for non-filtered plasmas (p <0.001), it was still above the cut-off (i.e., 20.9%). Staclot LA on non-filtered plasmas was diagnostic for LA in 42 of 42 plasmas. Though the median (range) clotting time difference recorded after phospholipid addition for filtered plasmas, i.e., 40.8 (10-103.5) s was reduced to 31.7 (2.8-88.8) s for non-filtered plasmas (p <0.001), it was still above the cut-off (i. e., 1.7 s). In conclusion, residual platelets do not affect the diagnostic efficacy of SCT and Staclot LA. However, the fact that the percentage correction for SCT and the clotting time difference for Staclot LA are reduced by residual platelets, suggests that weak LA may be lost upon freezing-thawing non-filtered plasmas.

Laboratory Diagnosis of Lupus Anticoagulants for Patients on Oral Anticoagulant Treatment

The detection of lupus anticoagulant (LA) in plasmas from patients on oral anticoagulants is problematic because of their prolonged clotting times. Mixing of patients and normal plasmas prior to testing for LA is employed to overcome this problem. We investigated the diagnostic efficacy of silica clotting time (SCT) and dilute Russell viper venom test (dRVVT) performed at low and high phospholipid concentrations, to diagnose LA in patients on oral anticoagulants, in comparison with Staclot LA (Stago) performed with and without hexagonal phospholipids and normal plasma. Case materials were 114 filtered plasmas from patients on oral anticoagulants with (n = 62) and without (n = 52) the antiphospholipid syndrome. Plasmas were considered LA-positive when Staclot LA (taken as the "gold standard") was diagnostic for LA. Forty-four plasmas were positive with Staclot LA. Forty and 39 of these were also positive with SCT and dRVVT (sensitivity relative to Staclot LA was 91% and 89%, respectively). Seventy plasmas were negative with Staclot LA. Three of these were positive with both SCT and dRVVT (specificity relative to Staclot LA was 96%). Kappa values for measure of agreement were 0.87 and 0.85 (p <0.001), respectively. In conclusion, SCT and dRVVT performed at low and high phospholipid concentrations without normal plasma can be considered as reliable as Staclot LA performed with hexagonal phospholipids and normal plasma to diagnose LA in patients on oral anticoagulants. Advantages of SCT and Drvvt over Staclot LA are easy automation, no need for normal plasma and relatively low cost.

Monoclonal Antibodies against Beta-2-Glycoprotein I: Use as Reference Material for Lupus Anticoagulant Tests

The laboratory diagnosis of lupus anticoagulants (LA) remains difficult despite internationally accepted guidelines for its detection. Several interlaboratory surveys have shown poor agreement between laboratories. Further standardization of LA testing will to a large extent depend on better insights on the mechanisms by which LA affect phospholipid-dependent coagulation assays as well as on the availability of well characterized and internationally accepted reference materials and control specimens. We recently raised a series of murine monoclonal antibodies against human beta2GPI (anti-beta2GPI moabs), a phospholipid-binding protein directly involved in the interaction between certain lupus anticoagulants and phospholipids. In this study we report on the use of LA positive anti-beta2GPI moabs as easy to handle reference and control material. The relative LA responsiveness of various phospholipid-dependent clotting assays was determined on plasmas spiked with such moabs and compared well with that determined on LA positive plasma samples. Plasmas spiked with LA positive anti-beta2GPI moabs were also used as control specimens to study the interlaboratory precision of LA testing. With these control specimens, low interassay coefficients of variation were obtained. Our results indicate that LA positive anti-beta2GPI moabs have potential for the production of control specimens that could be made available to routine hemostasis laboratories to assess intra-laboratory precision of LA testing and to manufacturers to control batch-to-batch variability of their reagents.

Blood Coagulation and Haemostasis. A Practical Guide

Familial thrombophilia, J.Conard laboratory diagnosis of proteins C and S, A.Tripodi and P.M.Mannucci the serpin inhibitors of blood coagulation - antithrombin and heparin co-factor II, D.A.Lane and M.D.Boisclair the laboratory diagnosis of lupus anticoagulants, D.A.Triplett and L.Barna laboratory investigation of the fibrinolytic system, N.A.Booth the formation, measurement and clinical value of fibrinogen derivatives, W.Nieuwenhuizen laboratory aspects of thrombolytic therapy, G.D.Lowe and A.Rumley an approach to the infant with impaired haemostasis, M.Andrew and M.Johnston the implementation of International Normalized Ratios for standardization of the prothrombin time in oral anticoagulant control, J.M.Thomson.

Laboratory diagnosis of lupus anticoagulants

Recently the significance of a finding of iupus anticoagulant (LA) in a patient has changed. Previously regarded merely as a laboratory curiosity LA are now recognised as potential risk factors for thrombosis and in women appear to be responsible for recurrent fetal loss. Though it was initially suggested that LA and anticardiolipin antibodies (aCL) may be identical phosphotipid-binding immunoglobulins, more recent studies have shown the correlation between aCL and LA to be incomplete. Many patients have raised aCL without LA though the reverse situation is less common. The most sensitive screening tests for LA utilise low concentration of phospholipid for example in the kaolin clotting time, the dilute Russell’s viper venom test or the dilute tissue thromboplastin inhibition test. Prolongation of such tests by LA can usually be corrected by the addition of “excess” procoagulant phospholipid Conversely LA are not corrected by mixing with normal plasma. A LA test appropriate for a particular purpose should be selected. Thus hospital patients often present with other coagulation complications, e.g. anticoagulants, or liver disease, and the LA defect needs to be carefully distinguished from conditions likely to contribute to bleeding. Thus greater specificity is required for this group whereas sensitivity is the most important consideration for detecting LA in otherwise normal females being investigated e.g.for recurrent miscarriages. It is important to carry out LA tests on correctly processed samples particularly avoiding platelet activation which may ‘bypass” the LA defect.

Laboratory Diagnosis of Lupus Anticoagulants

Laboratories are now receiving requests to evaluate patients for the presence of LA. This review will focus on the characterization, clinical associations, and laboratory diagnosis of LA

Laboratory Diagnosis of Lupus Anticoagulant: Comparison of Three Methods of Antibody Neutralization

The lupus anticoagulant (LAC) is an antiphospholipid antibody initially identified by prolongation of in vitro phospholipid-dependent coagulation tests. Because of its association with thromboembolic disease1-3 and recurrent pregnancy loss,4,5 there is interest in developing a convenient test for confirming the presence of LAC activity. The purpose of this study was to compare three methods for detecting LAC, one a recently marketed test. Twenty plasma specimens were identified as suspicious for LAC by isolated prolongation of the activated partial thromboplastin time (aPTT) that failed to correct with a 1:1 mix with pooled plasma. First, all plasma specimens were tested for LAC by the platelet neutralization procedure (PNP) as previously described by Triplett.6 In this test, freeze-thawed platelets are used as a source of phospholipid to neutralize the antiphospholipid antibody and correct the aPTT. Second, the plasma specimens were tested utilizing Platelet Extract ReagentTM, a recently available reagent that consists of phospholipid in the form of lyophilized platelets and was designed to confirm LAC. Third, the specimens were tested with a concentrated solution of a standard aPTT reagent as a source of phospholipid to neutralize the antibody. PNP confirmed 100% of the suspected LACs, Platelet Extract Reagent detected 35%, and the concentrated aPTT reagent solution detected 30%. This study confirms the diagnostic utility of the PNP in identifying LACs. The Platelet Extract Reagent was convenient to use but was less effective in confirming LACs; it performed similarly to a concentrated solution of a standard aPTT reagent.