Assays For Antiphospholipid Antibodies Research Articles

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-β2-glycoprotein I antibody titres for all three LA assays. TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Discouraging Non-ELISA antiphospholipid antibody assays in antiphospholipid syndrome classification may hinder clinical research

Discouraging Non-ELISA antiphospholipid antibody assays in antiphospholipid syndrome classification may hinder clinical research

271 Diagnosis and Detection of Thrombosis in PCOS

OBJECTIVES/GOALS: Identify and stratify clinical presentations of thrombotic pathology in PCOS patients. This will be accomplished by 1) evaluating clinical assays for the detection of hypofibrinolysis, and 2) analyzing clinical symptomology of thrombosis in PCOS via Symptom-Disease Pair Analysis of Diagnostic Error (SPADE). METHODS/STUDY POPULATION: Preliminary study populations include n=3 for each of the following groups PCOS with thrombotic complications, PCOS without thrombotic complications, healthy controls, and healthy control samples treated ex vivo with PAI-1. Coagulation assays include ACL coagulation panel, antiphospholipid antibody assays, viscoelastic testing with TEG and Quantra devices, and global fibrinolytic capacity. Coagulation assays will be performed on three samples taken at 4-week intervals. The SPADE techniques will be used to evaluate symptomology of thrombosis in the study period and patient electronic medical history. Molecular testing will be performed for pro-thrombotic polymorphisms (PAI-1 and Apo E) and mutations (Factor V Leiden and Prothrombin). RESULTS/ANTICIPATED RESULTS: We anticipate PCOS and ex vivo PAI-1 samples to show signs of hypofibrinolysis on TEG and Quantra devices outside of reference ranges and with statistical significance. We also anticipate seeing a statistical significance with ACL coagulation panels, however, these results are expected to still be within the reference ranges, as seen in previous studies. We believe the SPADE method will identify clinical presentations of hypofibrinolysis in PCOS patients coinciding with patterns in laboratory tests that were misdiagnosed due to being within reference ranges. We hope to stratify clinical presentations predictive of thrombosis in PCOS patients with standard clinical assays and with increased precision using viscoelastic assays. DISCUSSION/SIGNIFICANCE: The incidence rate of thrombosis is 40x higher in PCOS compared to healthy populations. However, the mechanisms of thrombosis in PCOS are unknown and are undetectable with current clinical assays. We hypothesize that chronic cellular stress in PCOS disrupts the regulation of interconnected immune pathways, causing hypofibrinolysis.

Determination of diagnostic threshold in harmonization and comparison of clinical utility for five major antiphospholipid antibody assays used in Japan.

Anticardiolipin antibodies (aCL) and anti-β2 -glycoprotein I antibodies (aβ2 GPI) are essential in diagnosing antiphospholipid syndrome (APS) according to the international APS guideline. Five commercial assays for aCL and aβ2 GPI are available in Japan, but their test results are quite discordant. For harmonization of diagnosing APS, upper reference limit (URL) and diagnostic accuracy of each assay were evaluated and compared by testing common sets of specimens across all assays. We evaluated two manual and three automated assays for aCL and aβ2 GPI of IgG- and IgM classes. 99%URL (the upper limit of reference interval: as per guideline) together with 97.5%URL were determined by testing sera from 198 to 400 well-defined healthy subjects. Both URLs were compared with the cutoff values, which were determined based on ROC analysis by testing 50 each of plasma specimens from patients with/without APS. Diagnostic accuracy was evaluated as area under curve (AUC) of the ROC curve. A variable degree of discrepancy between URLs and the cutoff values was observed, which was partly attributable to between-year assay variability. 97.5%URLs were set lower and closer to the cutoff values than 99%URLs. For all assays, diagnostic accuracies of both aβ2 GPI-IgG and aCL-IgG were generally high (AUC: 0.84-0.93); whereas those for IgM-class assays were low (AUC: 0.57-0.67), implicating its utility is limited to rare IgG negative APS cases. To ensure harmonized APS diagnosis, the diagnostic thresholds of the five assays were evaluated by common procedures. Contrary to the guideline, 97.5%URL is rather recommended for diagnosing APS, which showed a closer match to the cutoff value.

Antiphospholipid Antibody Assays in 2021: Looking for a Predictive Value in Addition to a Diagnostic One.

Antiphospholipid antibodies (aPL) are mandatory for the diagnosis but are also a risk factor for the antiphospholipid syndrome (APS) clinical manifestations. Lupus anticoagulant (LA), anticardiolipin (aCL), and anti-beta2 glycoprotein I (β2GPI) assays are the formal laboratory classification/diagnostic criteria. Additional nonclassification assays have been suggested; among them, antiphosphatidylserine-prothrombin (aPS/PT) and antidomain 1 β2GPI antibodies are the most promising ones although not yet formally accepted. aPL represent the example of a laboratory test that moved from dichotomous to quantitative results consistent with the idea that reporting quantitative data offers more diagnostic/prognostic information for both vascular and obstetric manifestations. Although the general rule is that the higher the aPL titer, the higher the test likelihood ratio, there is growing evidence that this is not the case for persistent low titers and obstetric events. LA displays the highest diagnostic/prognostic power, although some isolated LAs are apparently not associated with APS manifestations. Moreover, isotype characterization is also critical since IgG aPL are more diagnostic/prognostic than IgA or IgM. aPL are directed against two main autoantigens: β2GPI and PT. However, anti-β2GPI antibodies are more associated with the APS clinical spectrum. In addition, there is evidence that anti-β2GPI domain 1 antibodies display a stronger diagnostic/prognostic value. This finding supports the view that antigen and even epitope characterization represents a further step for improving the assay value. The strategy to improve aPL laboratory characterization is a lesson that can be translated to other autoantibody assays in order to improve our diagnostic and prognostic power.

Functional lupus anticoagulant testing in a large retrospective cohort of thrombosis patients with direct oral anticoagulants

Functional tests for lupus anticoagulants (LA) as part of a thrombophilia workup are commonly performed in patients under anticoagulant therapy that may interfere with assay results. There is no consensus on how these tests should be assessed in patients on direct oral anticoagulants (DOACs). In this retrospective cohort study, we analysed data from patients with a history of thrombosis in whom dilute Russell viper venom time (dRVVT), LA-sensitive aPTT, and solid phase assays for antiphospholipid antibodies (aPL) were performed (n = 3,147, thereof 588 on rivaroxaban, 144 on apixaban, 1,179 on other anticoagulant drugs). The dRVVT ratio was correlated with rivaroxaban (r = 0.30, P < 10–4) but not with apixaban plasma levels. The LA-sensitive aPTT/aPTT ratio showed no correlation with DOAC levels. Correspondingly, the rate of patients with abnormal dRVVT test was significantly higher (P < 10–4) under rivaroxaban (88%) than in thrombosis patients without anticoagulant medication (6%), independent from their aPL plasma levels. No isolated positive results of functional LA testing in patients on anticoagulants could be confirmed in repeated testing after discontinuation of the medication (n = 40). These data indicate that rivaroxaban should be discontinued before functional LA testing is performed. However, viable interpretation of these tests appears to be less affected in patients on apixaban.

Rituximab‐associated posterior reversible encephalopathy syndrome

Rituximab‐associated posterior reversible encephalopathy syndrome

Real-world practice of obstetricians in respect of assays for antiphospholipid antibodies

Real-world practice of obstetricians in respect of assays for antiphospholipid antibodies

Determination of clinically significant tests for antiphospholipid antibodies and cutoff levels for obstetric antiphospholipid syndrome.

The objective of this paper is to determine which kinds of assays for antiphospholipid antibodies (aPL) should be tested for clinical practice for patients with recurrent pregnancy loss (RPL). We studied 560 patients with a history of RPL prospectively. We determined the obstetric significance of 11 commercially available tested assays for lupus anticoagulant (LA)-aPTT StaClot, phosphatidylserine-dependent antiprothrombin (aPS/PT) IgG, IgM, classical cardiolipin (CL) IgG, IgM, CL IgG, IgM, IgA, and β2glycoprotein I (β2GPI) IgG, IgM, IgA Phadia. Obstetric significance was defined as the potential for anticoagulant therapy to improve the subsequent live birth rate, or a difference in the live birth rate between positive and negative untreated cases. The LA-aPTT StaClot assay and aPS/PT IgG assay, but not CL IgG, were found to have obstetric significance. Our conventional tests covered positive cases with the aPS/PT IgM and classical CL IgG assays. The results of the LA-aPTT StaClot, LA-aPTT and LA-RVVT assays showed different distributions, although strong or moderate correlation was observed. LA-aPTT StaClot and aPS/PT IgG might be suitable for use in routine practice for patients with RPL. Each test for aPL should be ascertained for obstetric significance, because similar assays may have different outcomes.

Real-world practice of obstetricians in respect of assays for antiphospholipid antibodies

Objective. The international classification criteria (CC) for definite antiphospholipid syndrome (APS) recommend confirmation of the sustained presence, for at least 12 weeks, of both lupus anticoagulant (LA), as determined by aPTT and RVVT, and anti β2glycoprotein I (β2GPI) or anticardiolipin (aCL) IgG and/or IgM. However, it remains unclear whether obstetricians comply with the aforementioned CC for the diagnosis of APS in daily clinical practice. We performed a nationwide survey to examine the attitudes of Japanese obstetricians toward the use of assays for antiphospholipid antibodies (aPLs).Methods. A questionnaire was sent to 2,700 obstetric facilities where maternity checkups are carried out. The types of assays conducted for aPLs, ascertainment of persistence of the antibodies for at least 12 weeks, and the cutoff points used for the assays were examined.Results. Of the facilities surveyed, 61.5% carried out the assay(s) only once. In regard to the type of assay performed, 97.1% carried out the assay for aCL IgG and/or β2GPI-dependent aCL, while 67.9% performed the LA-aPTT and/or LA-RVVT assay. Only 8.8% carried out assays for both LA. As for the cutoff points used, 98% of the facilities used lower cutoff points described in the manufacturers’ manuals rather than the cutoff values recommended in the CC.Conclusion. Thus, only a limited number of facilities adhered precisely to the CC for the diagnosis of APS. Inappropriate treatment and unnecessary expense are potentially major concerns when facilities overdiagnose APS using lower cutoff points or without ascertaining the persistence of the antibodies for at least 12 weeks. On the other hand, some patients miss the opportunity to be treated for APS because of the absence of testing for LA.

Standardization and Harmonization of Antiphospholipid Antibody Assays

Standardization and Harmonization of Antiphospholipid Antibody Assays

Clinical significance of antiphospholipid antibody measured by EliA anticardiolipin antibodies and anti-β2Glycoprotein I antibodies in antiphospholipid syndrome

Anticardiolipin antibodies (aCL-IgG/IgM) and anti-β2-glycoprotein I antibodies (aβ2GPI-IgG/IgM) are laboratory tests included in the current classification criteria for definite antiphospholipid syndrome (APS). However, not all of these assays have been commercially available in Japan. We investigated the efficacy of aCL-IgG/IgM and aβ2GPI-IgG/IgM assays using fluorescence enzyme immunoassay (Phadia:EliA(TM)) for the diagnosis of APS in Japan. This study comprised 229 sera from patients (100 with APS and 129 without APS). The diagnosis of APS was made according to Sydney revised Sapporo criteria. EliA(TM)Cardiolipin and EliA(TM)β2-Glycoprotein (Phadia AB. Uppsala Sweden) were used to detect aCL IgG/M and aβ2GPI IgG/M, respectively. Sensitivity, specificity and area under the receiver operating characteristic curve (AUC) were as follows; aCL-IgG (45%, 94%, 0.80), aCL-IgM (20%, 94%, 0.54), aβ2GPI-IgG (33%, 98%, 0.88) and aβ2GPI-IgM (16%, 99%, 0.64) respectively. aCL-IgM, aβ2GPI-IgG or aβ2GPI-IgM were detected in 10 patients (18%) with aCL-IgG negative. The use of Phadia:EliA(TM)antiphospholipid antibodies assays improve the diagnostic yield of thrombotic risk in APS patients.

THU0167 Evaluation of two assays for antiphospholipid antibodies in 712 SLE patients; clinical associations depend on isotypes and cut-off levels

BackgroundThere are several problems with the current methods for detection of antiphospholipid antibodies (aPL) and there is an on-going discussion about the interpretation of different aPL-tests in everyday practice. (1)ObjectivesI....

Performance of two new, automated chemiluminescence assay panels for anticardiolipin and anti‐beta2‐glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome

Anticardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibodies are two of the three laboratory criteria for antiphospholipid syndrome (APS). All assays for antiphospholipid antibodies (aPL), coagulation assays as well as ELISAs, show methodological shortcomings, which makes the search for better assays everlasting. The purpose of this study was to investigate the diagnostic performance of two new, fully automated systems (Zenit RA and HemosIL Acustar) applying chemiluminescent technology for aPL detection. The study cohort consisted of a patient population presenting with thrombosis. In such patient population, the demonstration of aPL determines whether a patient has APS or not with implications for treatment. One hundred and twenty-four patients with thrombotic complications, of whom 26 were patients with definite APS, were integrated in this study. Besides, aPL titres were compared to the Sapporo standards. Results of both systems agreed well with ELISA and mutually. Analysis of the discrepant results between Zenit and Acustar finally led to one misclassification as APS. Diagnostic performances of both Zenit RA and HemosIL Acustar were comparable with odds ratios lower limits of CI of 5 for aβ2 GPI IgG for Zenit and Acustar and 6 and 5 for aCl IgG on Zenit and Acustar, respectively. However, even with these new automated systems, titres differed largely between systems, especially for aβ2 GPI IgG.

Standardization of antiphospholipid antibody assays. Where do we stand?

The laboratory criteria (lupus anticoagulants (LA), and/or anti-cardiolipin (aCL) antibodies and/or anti-β2-glycoprotein I antibodies (aβ2GPI)) that define patients with antiphospholipid syndrome (APS) were set in the Sapporo and Sydney criteria published in 1999 and 2006, respectively, and led to a substantial improvement in the recognition of APS. In addition, guidelines for LA detection were published by the Scientific Standardisation Subcommittee (SSC) of the International Society of Thrombosis and Haemostasis (ISTH) in 2009. However, a number of questions on this respect remain unresolved. Recommendations for the aCL and aβ2GPI assays intended to ameliorate the performance of these solid-phase assays. Despite efforts over the years, standardization has not been reached. This review will focus on methodological issues of the three antiphospholipid antibody (aPL) subtypes that are the subject of debate. The use of an international standard for aPL detection might help solve many of the problems caused by a lack of standardization of these assays.

Laboratory testing for the antiphospholipid syndrome: making sense of antiphospholipid antibody assays

The antiphospholipid syndrome (APS) is an autoimmune condition characterised by a wide range of clinical features (primarily thrombosis and/or obstetric related), associated with the presence of antiphospholipid antibodies (aPL) as detected by a diverse range of laboratory tests. APS remains a significant diagnostic challenge for clinicians across a wide range of specialities, largely due to issues related to laboratory testing as well as the expanding range of reported clinical manifestations of APS. The laboratory issues include limitations in detailed knowledge by both clinical and laboratory personnel regarding the 'complete' range of available aPL tests, as well as ongoing problems with assay reproducibility and standardisation. aPL are identified using diverse laboratory procedures based on one of two distinct test processes, namely solid phase and liquid phase assays. The former includes anticardiolipin antibodies (aCL) and anti-β(2)-glycoprotein I antibodies (aβ(2)GPI). The latter are centred on clot-based tests that are used to identify the so-called lupus anticoagulant (LA). This article will discuss: (i) issues related to laboratory testing for APS in terms of the currently available solid-phase and liquid-phase assays, and identifiable biases resulting from these tests usually being performed in different laboratories; (ii) current problems with calibration, standardisation and reproducibility of these assays; (iii) pre-analytical, analytical and post-analytical considerations and ongoing initiatives for improvement; (iv) issues related to potential combinations/panels of available aPL tests; and (v) the entities of seropositive APS, seronegative APS and non-APS aPL-positivity. In doing so, this review will hopefully help bridge the two disciplines of haematology and immunology ('representing' liquid-phase and solid-phase aPL testing, respectively), by improving the understanding of those working in each of these disciplines of the merits and limitations of the assays performed in the other discipline, and encouraging inter-discipline cooperation in the reporting of aPL test results.

Antiphospholipid syndrome

The antiphospholipid syndrome (APS) is a non-inflammatory autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL) in the plasma of patients with vascular thrombosis, recurrent complications of pregnancy, or both (1, 2). The presence of aPL in plasma of patients can be detected with either a prolongation of phospholipid dependent coagulation tests (lupus anticoagulant, LAC), or with solid phase immune assays against the protein beta2-glycoprotein I (beta2-GPI) or the phospholipid cardiolipin (anti-beta2-GPI antibody ELISA and anti-cardiolipin antibody ELISA, respectively) (3). For a long time there was a lot of confusion on who had the syndrome and who not. To solve this dispute, an international consensus meeting was organized in Sapporo in 1999 to formulate classification criteria for patients with the antiphospholipid syndrome (4). These criteria have been updated in 2004 at another international consensus meeting in Sydney (5). The classification criteria were defined for scientific purposes and were aimed to be used as inclusion criteria in patient related studies. They were specifically not defined for diagnostic purposes. However, current practice is that these criteria are used as a diagnostic tool. This is very unfortunate because the specificity of the different aPL assays to detect the clinical manifestations that characterize APS are disputable. One of the aims of defining the criteria was to initiate studies to determine the value of the different anti-phospholipid antibody assays to serve as biomarker for the risk of thrombosis and pregnancy morbidity. The recent progress made on this important topic will be discussed.

The Activated Seven Lupus Anticoagulant Assay Detects Clinically Significant Antibodies

Lupus anticoagulants are a heterogeneous group of autoantibodies detected by their effects on phospholipid-dependent coagulation assays. Persistent lupus anticoagulants are associated with thrombotic disease, but not all are clinically significant. Antibody heterogeneity and reagent and test variability dictate that at least 2 tests, of different types, should be used to screen lupus anticoagulants. The objective of this study was to investigate whether the activated seven lupus anticoagulant assay detects clinically significant antibodies. Eighty-two patients with antiphospholipid syndrome (APS) and 32 with systemic lupus erythematosus + positive for activated seven lupus anticoagulant and who were without thrombosis, who were positive by activated seven lupus anticoagulant assay, were investigated for lupus anticoagulants by dilute Russell's viper venom time, dilute activated partial thromboplastin time, and Taipan snake venom time, and for anticardiolipin antibodies. Fifty-seven of the APS patients were positive for lupus anticoagulants in multiple assays, 25 in activated seven lupus anticoagulant alone. Fourteen of the latter group were previously positive in other antiphospholipid antibodies assays, and 11 had only been positive for lupus anticoagulants by activated seven lupus anticoagulant. Twenty-eight had elevated anticardiolipin antibodies, 6 of whom were from the group that was positive in activated seven lupus anticoagulant only. Eight of the systemic lupus erythematosus + lupus anticoagulants (without thrombosis) patients were positive for lupus anticoagulant by activated seven lupus anticoagulant alone and had only been positive in activated seven lupus anticoagulant previously, and none had elevated anticardiolipin antibodies. The remaining 24 patients were lupus-anticoagulant positive in multiple assays, and 9 had elevated anticardiolipin antibodies. Dilute Russell's viper venom time and Dilute activated partial thromboplastin time are widely used to detect lupus anticoagulants and are considered to detect clinically significant antibodies. Activated seven lupus anticoagulant detected antibodies in APS patients who were positive by these assays and also lupus anticoagulants undetectable by the dilute Russell's viper venom time/dilute activated partial thromboplastin time reagents used, demonstrating its utility as a first-line or second-line assay.

A Consensus Approach to the Formulation of Guidelines for Laboratory Testing and Reporting of Antiphospholipid Antibody Assays

Despite numerous past and ongoing efforts, there remains significant variation in results from assays for the major antiphospholipid antibodies (aPL), namely anticardiolipin (aCL), anti-beta2 glycoprotein I (anti-beta2GPI), and lupus anticoagulant (LA). There is therefore a need to produce comprehensive guidelines on laboratory testing and reporting of aPL assays. However, because of the paucity of good-quality published evidence, there is a heavy reliance on expert opinion, and thus the existing consensus guidelines for aPL testing and reporting are largely eminence based rather than evidence based. This may potentially bias recommendations to reflect the personal preferences of those who have the greatest influence during the guideline formulation process. This article largely details the experience of the Australasian Anticardiolipin Working Party in undertaking a consensus approach to formulation of guidelines on aCL and anti-beta2GPI testing and reporting, including measures taken to minimize these issues. Despite the time-consuming nature of the process, given the paucity of good-quality published evidence, formulation of guidelines by the consensus process remains an important initiative to improve the standardization of aPL testing and reporting.

A comprehensive screening analysis of antiphospholipid antibodies in Indian women with fetal loss

Objective The present study was aimed at a comprehensive analysis of acquired thrombophilia in a large series of Indian women with fetal loss. Study design Four hundred and thirty women (median age 26 years, range 18–39 years) with unexplained fetal loss (median number of abortions 3, range 1–13) were screened for the presence of antiphospholipid antibodies (APA), i.e. lupus anticoagulant (LA), IgG/M antibodies for cardiolipin (ACA), β2 glycoprotein 1 (β2 GP1) and annexin V. We also studied 100 normal healthy women (median age 24 years, range 18–30 years) who had at least one healthy child and did not have any miscarriage or other obstetric complications. Results The prevalence of persistently positive LA was 8.1% and 1% in the patients and controls, respectively (OR 8.7; 95% CI, 1.4–51; P < 0.05). The overall prevalence of IgG and/or IgM antibodies for cardiolipin, β2 GP1 and annexin V were as follows—ACA 27.9% (OR 18.9; 95% CI, 5–70; P < 0.05), β2 GP1 12.2% (OR 6.8; 95% CI, 1.8–25; P < 0.05) and annexin V 14.6% (OR 17; 95% CI, 2.9–98; P < 0.05). The conventional LA and ACA tests were positive 23.2% of the cases as against 1% in the controls (OR 14.8; 95% CI, 3.9–55; P < 0.05). The prevalence of LA, ACA, β2 GP1 and annexin V antibodies as independent risk factors were observed in 0.5%, 16.5%, 5.4% and 7.8% in the patients as against 1% each in the controls. The overall positivity for any one of the APA studied was 42.6% (OR 10.2; 95% CI, 4.5–23; P < 0.05). Conclusion The present study thus indicates the importance of APA in women experiencing fetal loss where all the conventional causes of miscarriages have been ruled out. It also suggests that conventional APA assays (LA and ACA) are effective in the detection of a majority of APA positive cases and by the addition of other cofactor-dependent (β2 GP1 and annexin V) APA assays, there is a considerable increase in the diagnostic efficiency in the detection of APA.