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ObjectivesThe Certified Reference Material (CRM) ERM®-DA477/IFCC is a new polyclonal IgG anti-beta2-glycoprotein I (anti-β2GPI) material for the harmonization of the laboratory diagnosis of antiphospholipid syndrome (APS). We evaluated CRM’s ability to represent the heterogeneity of APS patient anti-β2GPI antibodies and to calibrate IgG anti-β2GPI methods.MethodsWe characterized CRM for its reactivity against domain-1, using the QUANTA Flash® β2GPI-domain-1 assay, and against domains-4-5 of β2GPI, and single-domain-deleted β2GPI molecules using in-house ELISAs. We used QUANTA Lite® ELISA, QUANTA Flash® CLIA, and EliA™ FEIA methods to evaluate the CRM’s anti-Cardiolipin (anti-CL) activity. Four anti-β2GPI IgG methods (in-house and QUANTA Lite® ELISA, QUANTA Flash® CLIA, and EliA™ FEIA) were also used to evaluate the CRM’s calibration efficacy, alongside 133 clinical samples (CSs) and 99 controls.ResultsThe CRM showed high anti-domain-1 activity and no anti-domain-4-5 activity at the recommended assay dilution. The domain-dependent-β2GPI reactivity profiles were comparable with full-blown APS. There was acceptable dilution linearity for anti-CL assays with R2 ranging from 0.957 to 0.997. For the four anti-β2GPI IgG assays, calibration with the CRM led to a good comparability of the average result of CSs for two of the assays. New cut-offs calculated from this work improved comparability in quantitative results between three of the assays: 85 % concordance with CRM compared to 66 % concordance with assay-specific-calibration.ConclusionsThe CRM is representative of patient anti-β2GPI/CL heterogeneity and should improve anti-β2GPI IgG method harmonization. However, the level of achievable method harmonization is affected by differences in the selectivity among the assays.
17Clinical evaluation of a novel chemiluminescent immunoassay for the detection of anti-citrullinated peptide antibodies
Clinical evaluation of a novel chemiluminescent immunoassay for the detection of anti-citrullinated peptide antibodies
343Guidelines on the investigation and management of the antiphospholipid syndrome.
Guidelines on the investigation and management of the antiphospholipid syndrome.
68Laboratory Diagnosis of Antiphospholipid Syndrome: Insights and Hindrances.
Diagnosis of antiphospholipid syndrome (APS) requires the presence of a clinical criterion (thrombosis and/or pregnancy morbidity), combined with persistently circulating antiphospholipid antibodies (aPL). Currently, laboratory criteria aPL consist of lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) IgG/IgM, and anti-β2 glycoprotein I antibodies (aβ2GPI) IgG/IgM. Diagnosis and risk stratification of APS are complex and efforts to standardize and optimize laboratory tests have been ongoing since the initial description of the syndrome. LAC detection is based on functional coagulation assays, while aCL and aβ2GPI are measured with immunological solid-phase assays. LAC assays are especially prone to interference by anticoagulation therapy, but strategies to circumvent this interference are promising. Alternative techniques such as thrombin generation for LAC detection and to estimate LAC pathogenicity have been suggested, but are not applicable yet in routine setting. For aCL and aβ2GPI, a lot of different assays and detection techniques such as enzyme-linked immunosorbent and chemiluminescent assays are available. Furthermore, a lack of universal calibrators or standards results in high variability between the different solid-phase assays. Other non-criteria aPL such as anti-domain I β2 glycoprotein I and antiphosphatidylserine/prothrombin antibodies have been suggested for risk stratification purposes in APS, while their added value to diagnostic criteria seems limited. In this review, we will describe laboratory assays for diagnostic and risk evaluation in APS, integrating applicable guidelines and classification criteria. Current insights and hindrances are addressed with respect to both laboratory and clinical implications.
Read more3Neural Network Diagnoses the Antiphospholipid Syndrome without Interrupting Anticoagulant Therapy
Neural Network Diagnoses the Antiphospholipid Syndrome without Interrupting Anticoagulant Therapy
16Characteristics of purified anti-β2GPI IgG N-glycosylation associate with thrombotic, obstetric and catastrophic antiphospholipid syndrome.
Anti-β-2 glycoprotein I (anti-β2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-β2GP1 IgG with clinical features of APS. We purify anti-β2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. Both purified anti-β2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-β2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aβ2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aβ2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-β2GPI IgG subclasses, increased bisection and core fucosylation of anti-β2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). We comprehensively characterize the N-Glycans landscape of both anti-β2GP1 and total IgG in APS. Altered N-glycan profiles of anti-β2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-β2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.
Read more18Identification of patients with triple antiphospholipid antibody positivity is platform and method independent.
The risk of clinical complications in antiphospholipid syndrome (APS) increases when a patient is positive for all 3 types of antiphospholipid (aPL) antibodies. However, there is a considerable disagreement between various platforms for aCL and anti-β2-glycoprotein I (anti-β2GPI) measurement, which leads to discrepancies between these platforms in assessing aPL antibody positivity. The aim of this retrospective cross-sectional study was to assess whether 2 different platforms, the QUANTA Lite enzyme-linked immunosorbent assay and the QUANTA Flash chemiluminescent immunoassay, identify the same subjects as triple positive in a group of patients with APS and comorbid autoimmune diseases. The study included 220 patients with systemic autoimmune diseases (74 with primary APS; 47 with secondary APS; and 99 with systemic lupus erythematosus without APS). All patients were tested for IgG and IgM aCL and anti-β2GPI antibodies using both platforms. The agreement between the positive results for individual antibodies obtained using both platforms was not full, ranging from 81.8% to 90.9% in a pair-wise comparison. However, the number of patients with triple aPL antibody positivity was similar (80 by QUANTA Lite and 86 by QUANTA Flash); the agreement between the 2 platforms for the identification of patients with triple antibody positivity was 95.5% (Cohen's kappa coefficient = 0.90). This resulted in a similar risk for APS-related clinical complications: an odds ratio of 24.9 for QUANTA Lite and of 24.7 for QUANTA Flash. Our results confirm a strong association between triple aPL antibody positivity and APS and indicate that the identification of patients with triple antibody positivity is platform independent. When aPL antibody profiles are assessed, the agreement between various methods is much higher than that for individual antibodies.
Read more62Anti-phospholipid antibodies following vaccination with recombinant hepatitis B vaccine
This study was undertaken to evaluate the possible role of hepatitis B recombinant vaccine inducing the synthesis of IgG and IgM anti-cardiolipin antibodies (aCL), antibodies against beta(2)GPI (anti-beta(2)GPI), lupus anti-coagulant (LA), anti-nuclear antibodies and antibodies against extractable nuclear antigens (anti-ENA). The study population consisted of 85 healthy students (63 female, 22 male; mean age 20.8 years), vaccinated with three doses of recombinant DNA hepatitis B vaccine. One month after vaccination with the first dose of hepatitis B vaccine a minority of vaccinated individuals showed changes in IgG or IgM aCL or anti-beta(2)GPI or LA activity (P < 0.001). Among subjects in whom changes of IgG anti-beta(2)GPI were observed, a significantly higher number of increased (8/85) than decreased (2/85) values were found (P < 0.01). Analyses of paired data showed that differences in aCL or anti-beta(2)GPI levels before vaccination or 1 month later did not reach statistical significance. In two people aCL transitorily reached medium positivity after the first dose of hepatitis B vaccine with a drop 5 months later. Similar evident anti-beta(2)GPI fluctuation was also observed in one person. Another participant was initially low positive for IgG anti-beta2GPI and the levels were increasing after vaccination. Two participants became positive for anti-nuclear antibodies during 6 months' follow-up. There were no sex-dependent differences in tested antibodies observed and no associations between levels of aPL and levels of anti-HBV antibodies. We conclude that HBV can induce aPL, although rarely. In genetically susceptible individuals or together with some other triggers such combination might confer the risk of developing a continuous autoimmune response in an individual.
Read more7Association Between Novel Antiphospholipid Antibodies and Adverse Pregnancy Outcomes.
To investigate the value of anti-β2 glycoprotein-I domain 1 (aD1) and antiphosphatidylserine-prothrombin antibodies for predicting adverse pregnancy outcomes in an at-risk population and to describe the relationship among aD1, antiphosphatidylserine-prothrombin, lupus anticoagulant, and other antiphospholipid antibodies (aPL). Data were obtained from a prospective cohort of pregnant patients with aPL, with systemic lupus erythematosus (SLE) (n=59) or without SLE (n=106), or SLE without aPL (n=100) (PROMISSE [Predictors of Pregnancy Outcome in Systemic Lupus Erythematosus and Antiphospholipid Syndrome] study; NCT00198068). Levels of aD1 and antiphosphatidylserine-prothrombin were quantified with the QUANTA Flash and QUANTA Lite systems, respectively, in sera collected at less than 18 weeks of gestation. Adverse pregnancy outcome was defined as delivery at before 34 weeks of gestation for preeclampsia or placental insufficiency or fetal death after 12 weeks of gestation. Receiver operating characteristic (ROC) analysis assessed the diagnostic properties of aD1 and antiphosphatidylserine-prothrombin for adverse pregnancy outcomes. Bivariate comparisons were made between each biomarker. Multivariable regression modeling of adverse pregnancy outcomes was performed, and backward selection determined variables for a final model for adverse pregnancy outcomes. Logistic regression of lupus anticoagulant quantified the association with aD1 and antiphosphatidylserine-prothrombin. The rate of adverse pregnancy outcomes was described by combined results of lupus anticoagulant, aD1, and antiphosphatidylserine-prothrombin immunoglobulin G (IgG). Of 265 individuals, 45 (17.0%) experienced adverse pregnancy outcomes. Area under the curve from ROC analysis for aD1 was 0.734 (95% CI, 0.664-0.805), for antiphosphatidylserine-prothrombin IgG was 0.83 (95% CI, 0.751-0.899), and for antiphosphatidylserine-prothrombin immunoglobulin M (IgM) was 0.612 (95% CI, 0.520-0.703). Markers associated with adverse pregnancy outcomes were aD1 (P<.001), anticardiolipin IgG (P<.001), β2-glycoprotein I IgG (P=.003), antiphosphatidylserine-prothrombin IgG (P<.001), antiphosphatidylserine-prothrombin IgM (P=.03), and lupus anticoagulant (P<.001). Backward selection identified lupus anticoagulant, aD1, and antiphosphatidylserine-prothrombin IgG for final adverse pregnancy outcome modeling: lupus anticoagulant odds ratio (OR) 7.0 (95% CI, 3.4-14.4), aD1 OR 12.1 (95% CI, 3.64-40.2), and antiphosphatidylserine-prothrombin IgG OR 11.4 (95% CI, 5.2-25.2). Both aD1 and antiphosphatidylserine-prothrombin IgG remained significant when lupus anticoagulant was removed from the model. Both aD1 and antiphosphatidylserine-prothrombin IgG performed the best in ruling in adverse pregnancy outcomes. With a likelihood ratio less than 0.1, aD1 or antiphosphatidylserine-prothrombin IgG performed well for ruling out adverse pregnancy outcomes. Both aD1 and antiphosphatidylserine-prothrombin IgG were associated with lupus anticoagulant positivity: OR 27.9 (95% CI, 12.1-64.0) if both were positive. Adverse pregnancy outcomes were highest in those with positive lupus anticoagulant, aD1, and antiphosphatidylserine-prothrombin IgG (47.6%). In pregnant individuals with aPL, with or without SLE, aD1 and antiphosphatidylserine-prothrombin IgG are significant independent predictors of adverse pregnancy outcomes and are strongly associated with lupus anticoagulant. Combined use may identify patients at greatest risk for severe obstetric complications.
Read more11Performance Characteristics of Commercial Immunoassays for the Detection of IgG and IgM Antibodies to β2 Glycoprotein I and an Initial Assessment of Newly Developed Reference Materials for Assay Calibration.
To investigate the performance characteristics and impact of newly developed reference calibrators on the commutability between anti-β2 glycoprotein I (anti-β2 GPI) immunoassays in antiphospholipid syndrome (APS) and/or systemic lupus erythematosus (SLE). Immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-β2 GPI immunoassays from four manufacturers were evaluated. Serum samples from 269 patients (APS only, n = 31; SLE and APS, n = 83; SLE only, n = 129; pregnancy-related clinical manifestations without APS, n = 26) and 162 women with histories of successful pregnancies were tested. Results were expressed in kit-specific arbitrary units and in the calibrator reference units (RUs) based on 99th percentile cutoff values. Diagnostic accuracies, correlation between kits, and specific clinical manifestations in APS were investigated. The sensitivities of the assays ranged from 15.8% to 27.2% (IgG) and 12.3% to 15.8% (IgM) while specificities ranged from 79.4% to 86.5% (IgG) and 80.6% to 84.5% (IgM). There was moderate to almost perfect interassay reliability (Cohen κ, 0.69-0.98), and Spearman correlation coefficients were generally improved when results of the IgG determinations were expressed in RUs. Although qualitative agreements between immunoassays for both antibody isotypes are acceptable, correlations with APS clinical manifestations were kit dependent. Only the use of IgG reference material improved quantitative correlations between assays.
Read more123IFCC Working Group Recommendations for Assessing Commutability Part 2: Using the Difference in Bias between a Reference Material and Clinical Samples.
A process is described to assess the commutability of a reference material (RM) intended for use as a calibrator, trueness control, or external quality assessment sample based on the difference in bias between an RM and clinical samples (CSs) measured using 2 different measurement procedures (MPs). This difference in bias is compared with a criterion based on a medically relevant difference between an RM and CS results to make a conclusion regarding commutability. When more than 2 MPs are included, the commutability is assessed pairwise for all combinations of 2 MPs. This approach allows the same criterion to be used for all combinations of MPs included in the assessment. The assessment is based on an error model that allows estimation of various random and systematic sources of error, including those from sample-specific effects of interfering substances. An advantage of this approach is that the difference in bias between an RM and the average bias of CSs at the concentration (i.e., amount of substance present or quantity value) of the RM is determined and its uncertainty estimated. An RM is considered fit for purpose for those MPs for which commutability is demonstrated.
Read more9Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.
Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content.
Read more23Evaluation of bilirubin interference and accuracy of six creatinine assays compared with isotope dilution–liquid chromatography mass spectrometry
Evaluation of bilirubin interference and accuracy of six creatinine assays compared with isotope dilution–liquid chromatography mass spectrometry
Read more1Modulation of the activation of endothelial nitric oxide synthase and nitrosative stress biomarkers by aspirin triggered lipoxins: A possible mechanism of action of aspirin in the antiphospholipid syndrome.
Antiphospholipid syndrome (APS) is characterized by the clinical manifestation of vascular thrombosis (VT) or pregnancy morbidity (PM) and antiphospholipid antibodies (aPL) that can modify the nitric oxide production. Low-dose aspirin is used in the prevention and treatment of diverse alterations of pregnancy. One of the mechanisms of action of aspirin is to induce the production of aspirin-triggered-lipoxins (ATL). The aim of this study was to evaluate the modulatory effect of ATL over the activation of endothelial nitric oxide synthase (eNOS) and nitrosative stress biomarkers induced by aPL. We used polyclonal IgG and sera from women with aPL and PM/VT or VT only, and from women with PM only and positive for non-criteria aPL (SN-OAPS). In these sera, biomarkers of nitrosative stress (nitrites and nitrotyrosine) were measured. The protein expression of nitrotyrosine and the phosphorylation of eNOS (at Ser1177) were estimated in human umbilical vein endothelial cells (HUVECs) stimulated with polyclonal IgG with or without ATL. Women with SN-OAPS showed increased circulating levels of nitrites and nitrotyrosine. Likewise, polyclonal IgG from either SN-OAPS or VT patients stimulated nitrotyrosine expression in HUVECs. ATL decreased the nitrotyrosine expression induced by polyclonal IgG from the SN-OAPS group. ATL also recovered the reduced eNOS phosphorylation at Ser1177 in HUVECs stimulated with polyclonal IgG from women with PM/VT or SN-OAPS. Increased nitrosative stress present in serum of women with SN-OAPS is associated with IgG-mediated impaired endothelial NO synthesis in endothelial cells. ATL prevent these cellular changes.
Read more12Anti-β2GPI antibodies enhance atherosclerosis in ApoE-deficient mice
Anti-β2GPI antibodies enhance atherosclerosis in ApoE-deficient mice
2Prognostic Value of Lupus Anticoagulant and Anti-β2 Glycoprotein I Antibody in Adverse Pregnancy Outcomes.
International criteria for antiphospholipid syndrome (APS) include lupus anticoagulant (LA), anticardiolipin (aCL) IgG and IgM, and anti-β2-glycoprotein I (β2GPI) IgG and IgM. However, evidence supporting their prognostic value or treatment efficacy in improving live birth rates is limited. The Lancet series on miscarriage recommends testing only for LA and aCL, excluding β2GPI. We aimed to examine whether commercially available antiphospholipid antibody (aPL)-related tests have a prognostic value for obstetric APS. This prospective cohort study enrolled 1,237 pregnant women between July 2021 and March 2024. Women using heparin, including those with an obstetric APS diagnosis before pregnancy, were excluded. Pregnancy outcomes were followed until December 2024. The aPL-related tests comprised LA (diluted activated partial prothrombin time and diluted Russell's viper venom time [LA-RVVT]), aCL IgG, IgM, anti-β2GPI IgG, IgM, anti β2GPI domain I IgG, phosphatidylserine-dependent antiprothrombin IgG, IgM, protein S, and coagulation factor XII activity. The prevalence rates of early-onset preeclampsia, intrauterine fetal death (IUFD), and small for gestational age were 1.4% (17), 0.7% (9), and 0.9% (11), respectively. Logistic regression analysis revealed that LA-RVVT and anti-β2GPI IgG were each predictor of early-onset preeclampsia and IUFD. The area under the curve for these conditions increased to about 0.8 when combined with a history of preeclampsia or IUFD, respectively. LA-RVVT, anti-β2GPI IgG, complications including hypertension, and a history of IUFD were valuable in identifying obstetric APS. The variation in test results across facilities limits the ability to establish consistent prognostic or treatment value for each aPL.
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