Alternative assays to dRVVT and aPTT for lupus anticoagulant detection.

Lupus anticoagulants (LA) are heterogeneous antibodies and no single assay will detect every LA. Consequently, testing is commonly undertaken with both dilute Russell's viper venom time (dRVVT) and LA-responsive activated partial thromboplastin time (aPTT) to maximize detection rates. Although a huge body of evidence attests to the diagnostic utility of these assays, they have limitations that can render them unreliable in certain circumstances. Other assays are available for detecting LA but unfamiliarity, variable availability and technical concerns expressed in guidelines contribute to less usage than dRVVT and aPTT. However, assays such as Taipan snake venom time and Textarin time are insensitive to anticoagulants that compromise dRVVT and aPTT, and assays such as dilute prothrombin time can detect LA unreactive in dRVVT and aPTT. The pros and cons of alternative assays to dRVVT and APTT for LA detection are discussed.

Altered platelet proteome in lupus anticoagulant (LA)-positive patients-protein disulfide isomerase and NETosis as new players in LA-related thrombosis.

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE-) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.

Performance evaluation of Revohem™ FVIII chromogenic and Revohem™ FIX chromogenic in the CS-5100 autoanalyser.

INTRODUCTION: Chromogenic substrate assay (CSA) reagents Revohem™ FVIII and Revohem™ FIX are now available as in vitro diagnostic reagents for autoanalysers in Japan. In this study, we evaluated the performance of these reagents in the CS-5100 automated coagulation analyser. METHODS: We assessed within-run and between-day imprecision, on-board stability and frozen-storage stability of Revohem FVIII and FIX. Sensitivity to lupus anticoagulant (LA) was examined using LA-positive patient plasma. Correlations were analysed using plasma samples from normal individuals and patients with haemophilia A (HA) or B (HB) or von Willebrand disease (VWD). RESULTS: Imprecision was <2% for Revohem FVIII and <6.5% for Revohem FIX. On-board storage of Revohem FVIII resulted in a <10% decrease in FVIII levels from baseline at 24 hours, whereas Revohem FIX showed a >10% decrease at 8 hours. Revohem FVIII showed good stability while frozen for 22 days. Although Revohem FIX showed degradation due to freeze-thawing, a new calibration improved stability up to 22 days. Interference from LA was not observed with Revohem FVIII or FIX. The FVIII CSA-CSA correlation was excellent in normal (r = 0.9924), HA (r = 0.9945) and VWD (r = 0.9914). The FVIII CSA-OSA correlation was good in normal (r = 0.8468) and excellent in HA (r = 0.975) and VWD (r = 0.9936). The FIX CSA-OSA correlation was fair in normal (r = 0.4791) and excellent in HB (r = 0.9501). CONCLUSION: Revohem FVIII and FIX both showed excellent performance in the CS-5100 analyser. These reagents could be useful in routine laboratory testing for diagnosing and treating haemophilia.

Renal protective effect of antiplatelet therapy in antiphospholipid antibody-positive lupus nephritis patients without antiphospholipid syndrome.

OBJECTIVE: We sought to evaluate the effect of antiplatelet therapy in addition to conventional immunosuppressive therapy for lupus nephritis (LN) patients positive for antiphospholipid antibodies (aPL) without definite antiphospholipid syndrome (APS). METHODS: Patients with biopsy-proven LN class III or IV were retrospectively evaluated. We selected patients positive for anticardiolipin antibody (aCL) or lupus anticoagulant (LA) who did not meet the criteria for a diagnosis of APS. The patients were divided into two subgroups according to whether antiplatelet therapy was received. The cumulative complete renal response (CR) rate, relapse-free rate, and change in estimated glomerular filtration rate (eGFR) over 3 years after induction therapy were calculated. RESULTS: We identified 17 patients who received antiplatelet therapy and 21 who did not. Baseline clinicopathological characteristics and immunosuppressive therapy did not show a significant difference between the two groups except for a higher incidence of LN class IV in the treatment group (p = 0.03). There was no difference in cumulative CR rate, relapse-free rate, or eGFR change between these subgroups. However, when data on LA-positive patients were assessed, an improvement in eGFR was found (p = 0.04) in patients receiving antiplatelet treatment. CONCLUSION: Addition of anti-platelet therapy was associated with an improvement of eGFR in LA-positive patients with LN class III or IV.

Testing lupus anticoagulants in a real-life scenario - a retrospective cohort study.

INTRODUCTION: Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients. MATERIALS AND METHODS: In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test. RESULTS: The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 - 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests. CONCLUSIONS: Among the parameters evaluated, aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics.

Lupus Anticoagulant Testing: Activated Partial Thromboplastin Time (APTT) and Silica Clotting Time (SCT).

Actvated partial thromboplastin time and its congener test Silica clotting time are used for the laboratory detection of lupus anticoagulants. Their performance consists of mixing test plasma with activators of the contact coagulation factors, phospholipids and calcium chloride, and recording the clotting time. Here we describe the principle, performance and extensive practical details of the two methods, the problems that may be encountered and how they can be identified and overcome.

Plasma Antiphospholipid Antibodies Effects on Activated Partial Thromboplastin Time Assays.

BACKGROUND: Activated partial thromboplastin time (aPTT) assays can be affected by plasma antiphospholipid antibodies (aPLs), but the degree of the interference is not easy to predict. This study aimed to investigate the effects on aPTT assay results of different types and combinations of aPLs, including anti-ß2-glycoprotein I antibodies, anticardiolipin antibodies and lupus anticoagulant. MATERIALS AND METHODS: We retrospectively collected clinical information and laboratory tests from aPL-positive patients. The potential influence of aPLs on aPTT assays was assessed. RESULTS: The survey included 589 aPL-positive patients. No significant differences existed in basic characteristics such as sex, age, prothrombin time, fibrinogen and alanine aminotransferase among different cases with 1, 2 or 3 types of positive-aPL markers (P > 0.05). In 113 patients with abnormal aPTT values, multivariable linear regression analysis showed a significant correlation between an abnormal degree of aPTT values and dilute Russell viper venom time (dRVVT) or silica clotting time (SCT) with a correlation coefficient of 0.437 or 0.497 (P < 0.01), whereas age, anticardiolipin antibodies-immunoglobulin G, anticardiolipin antibodies-immunoglobulin M and anti-ß2-glycoprotein I antibodies were of no significance (P > 0.05). Among blood samples with 3 types of aPLs positivity, the rate of abnormal aPTT detection values was 55.3%, which was significantly higher than that observed in patients with negative, single-positive or double-positive aPL markers (P < 0.05). Patients with a moderate to strong dRVVT or SCT had a higher proportion of abnormal aPTT assays than did patients with a low dRVVT or SCT (P < 0.05). CONCLUSIONS: When abnormal aPTT values are obtained, the influence of aPLs should be considered, especially in the presence of a moderate to strong dRVVT or SCT.

Fatal hemorrhage due to a spontaneous factor V inhibitor with lupus anticoagulant properties.

: Factor V inhibitors are rare and have varied clinical presentations. We report on a 76-year-old female admitted to the hospital for pneumonia and treated with multiple antibiotics. Her baseline prothrombin time was 15.6 s and the activated partial thromboplastin time was 35 s. On admission day 10, she developed arm weakness and brain imaging showed a subdural hematoma. The prothrombin time was now 59.1 s with an activated partial thromboplastin time of more than 160 s and a normal thrombin time. A mixing study did not correct the clotting times and coagulation factor assays showed a nonspecific inhibition pattern. Only factor V activity remained low with serial dilutions, however, and a 70 Bethesda Unit inhibitor was identified. Aggressive supportive care was initiated but the patient succumbed to the effects of the intracranial hemorrhage. Factor V inhibitors may display lupus anticoagulant properties and may cause catastrophic bleeding. Our case illustrates that these inhibitors can arise quickly and supports an association with antibiotics.

Myocardial Ischaemia, Coronary Atherosclerosis and Pulmonary Pressure Elevation in Antiphospholipid Syndrome Patients.

BACKGROUND: Thrombotic events in antiphospholipid syndrome (APS) involve venous and arterial circulation with the possible involvement of coronary or pulmonary microcirculation. OBJECTIVES: To evaluate the influence of antiphospholipid antibodies (aPL) and on myocardial ischaemia assessed by single-photon emission computerized tomography (SPECT), coronary atherosclerosis assessed by multidetector computerized tomography (MDCT) and pulmonary pressure assessed by transthoracic echocardiography (TTE) in patients with primary antiphospholipid syndrome (PAPS). MATERIAL AND METHODS: TTE, SPECT (Tc 99m sestamibi) and MDCT-based coronary calcium scoring were performed in 26 consecutive PAPS patients (20 females, 6 males, aged 20-61, mean 39.7) without any signs of other autoimmunological disease and without clinical symptoms of heart disease. RESULTS: Out of 26 patients, TEE showed normal left and right ventricle function in 25 (96.2%) and elevated (≥ 30 mm Hg) right ventricle systolic pressure in 7 (26.9%) patients. SPECT revealed myocardial perfusion defects in 15 (57.7%) patients: exercise-induced in 6 (23.1%) and persistent in 11 (42.3%). MDCT revealed coronary calcifications in 4 (15.4%) patients. The number of plaques ranged from 1 to 11 (median 2), volume 3-201.7 mm³ (median 7), calcium scores 1.3-202.6 (median 5.7). In the group with perfusion defects or coronary calcifications (n = 15), all the patients showed elevated aCL IgG. CONCLUSIONS: In most of the relatively young APS patients, without any symptoms of ischemic heart disease, SPECT showed myocardial perfusion defects, and coronary calcifications in 1/6 of them. Right ventricle systolic pressure was elevated in 1/4 of APS patients. These pathologies, well known as cardiovascular risk markers, were associated with elevated levels of the IgG class of both anti-cardiolipin and antiB2 GPI antibodies. Thus, in a high percentage of APS patients, clinically silent myocardial ischaemia, pulmonary pressure elevation and coronary atherosclerosis are present and related to the presence of antiphospholipid antibodies.

Differences in lupus anticoagulant final conclusion through clotting time or Rosner index for mixing test interpretation.

BACKGROUND: Lupus anticoagulant (LAC) testing includes a screening, mixing and confirmation step. Although recently published guidelines on LAC testing are a useful step towards standardization, a lack of consensus remains whether to express mixing tests in clotting time (CT) or index of circulating anticoagulant (ICA). The influence of anticoagulant therapy, e.g. vitamin K antagonists (VKA) or direct oral anticoagulants (DOAC) on both methods of interpretation remains to be investigated. The objective of this study was to contribute to a simplification and standardization of the LAC three-step interpretation on the level of the mixing test. METHODS: Samples from 148 consecutive patients with LAC request and prolonged screening step, and 77 samples from patients non-suspicious for LAC treated with VKA (n=37) or DOAC (n=30) were retrospectively evaluated. An activated partial thromboplastin time (aPTT) and dilute Russell's viper venom time (dRVVT) were used for routine LAC testing. The supplemental anticoagulant samples were tested with dRVVT only. We focused on the interpretation differences for mixing tests expressed as CT or ICA and compared the final LAC conclusion within each distinct group of concordant and discordant mixing test results. RESULTS: Mixing test interpretation by CT resulted in 10 (dRVVT) and 16 (aPTT) more LAC positive patients compared to interpretation with ICA. Isolated prolonged dRVVT screen mix ICA results were exclusively observed in samples from VKA-treated patients without suspicion for LAC. CONCLUSIONS: We recommend using CT in respect to the 99th percentile cut-off for interpretation of mixing steps in order to reach the highest sensitivity and specificity in LAC detection.

Effects of storage and thawing conditions on coagulation testing.

INTRODUCTION: Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. METHODS: Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. RESULTS: Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. CONCLUSIONS: Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

Inhibition of Binding of ß2-Glycoprotein 1 to Phosphatidylserine by Polymyxin B, a Lupus-Like Anticoagulant.

Polymyxin B is a cationic peptide that inhibits phospholipid-dependent coagulation tests including activated partial thromboplastin time and to a lesser degree prothrombin time. Thrombin clotting time is insensitive to polymyxin B. ß2-glycoprotein 1 (ß2GP1) is a cofactor of antiphospholipid antibodies. Antiphospholipid autoantibodies also poses lupus anticoagulant activity through interactions with ß2GP1. Using affinity chromatography, polymyxin B can effectively decrease the binding of ß2GP1 to immobilize phosphatidylserine. Since then, anticoagulant effect of polymyxin B is most likely due to the binding to negatively charged phospholipids, preventing formation of coagulation complexes.

Interference from lupus anticoagulant on von Willebrand factor measurement in splenic marginal zone lymphoma: a case report.

We present a case concerning a patient with splenic marginal zone lymphoma (SMZL) and isolated prolonged activated partial thromboplastin time (aPTT) caused by lupus anticoagulant. Von Willebrand factor (VWF) activity and antigen were immeasurable by latex particle immunoturbidimetric assays, and several coagulation factor levels were decreased. However, VWF activity and antigen were normal when analyzed by other methods. Also, coagulation factor levels were normal if an aPTT reagent with low lupus anticoagulant sensitivity or a chromogenic method was applied. Altogether, the initial findings were because of lupus anticoagulant interference and in fact, the patient had normal VWF activity and coagulation status. Interference of lupus anticoagulant in clot-based assays is well known but has not previously been described in VWF assays. This is furthermore the first report in which lupus anticoagulant activity in SMZL cannot be ascribed to a monoclonal immunoglobulin. In our study, aPTT normalized after treatment, suggesting resolution of lupus anticoagulant. APTT could thus be a marker of treatment response in SMZL. Whether treatment decreases the thrombosis risk due to lupus anticoagulant remains unknown.

Antiphospholipid antibody and risk of retinal vein occlusion: a systematic review and meta-analysis.

BACKGROUND: Retinal vein occlusion (RVO) is a common retinal vascular disease and it is one of the most frequently reported causes of visual damage and blindness in the elderly. The current study investigated the potential association between antiphospholipid antibodies (APLA) and RVO risk by conducting a meta-analysis of case-control studies. METHODS: A systematic literature search of Pubmed and Embase databases was conducted in August 1st, 2014. Odds ratios (ORs) were used to evaluate the associations between APLA and the incidence of RVO. A random-effects model was obtained for the quantitative synthesis. RESULTS: A total of 11 studies were included in this meta-analysis. A meta-analysis of all studies assessing the risk of RVO revealed that APLA was associated with a statistically increased risk of RVO incidence (OR = 5.18, 95% CI = [3.37, 7.95]). The association between anticardiolipin antibodies (ACA) and the risk of RVO was significant (n =8, OR = 4.59, 95% CI = [2.75, 7.66]). However, the association between lupus anticoagulants (LA) and risk of RVO was non-significant (n = 5, OR = 3.90, 95% CI = [0.99, 15.37]). No significant publication bias was found in the 11 selected studies. CONCLUSION: APLA was significantly associated with the risk of RVO. Advanced analyses showed that ACA rather than LA affected the risk of RVO. Additional well-designed and well-conducted epidemiological studies are required to further our understanding of the relationship between APLA and RVO risk.

Drug-induced lupus anticoagulants and antiphospholipid antibodies.

Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, and/or IgA) which interfere with one or more of phospholipid-dependent in vitro coagulation tests, eg, activated partial thromboplastin time (aPTT), kaolin clotting time (KCT), dilute Russell viper venom time (dRVVT), and dilute prothrombin time (dPT). LAs may be seen in a variety of clinical settings including the primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE), other autoimmune diseases, secondary to infections, malignancies, and in association with certain drugs. LAs associated with the antiphospholipid syndrome and other autoimmune disease recognize certain phospholipid-binding proteins (ß(2)-glycoprotein I [ß(2)GPI] or prothrombin). Many drugs have been implicated as possibly causing LAs, although the majority of such cases are limited to a select few. Drug-induced LAs are heterogeneous, differing in laboratory findings as well as related clinical complications. This paper reviews the English medical literature on drug-induced LA and potential mechanisms of induction.