anti-β2GPI IgM Research Articles

Establishment of ELISA-comparable moderate and high thresholds for anticardiolipin and anti-β2 glycoprotein I chemiluminescent immunoassays according to the 2023 ACR/EULAR APS classification criteria and evaluation of their diagnostic performance.

Recently published 2023 ACR/EULAR APS classification criteria emphasize the importance of quantifying single-, double-, and triple-antiphospholipid antibody positivity, distinguishing between IgG and IgM isotypes, and delineating moderate/high levels of anticardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies. We aimed to establish clinically important moderate/high thresholds for aCL and anti-β2GPI IgG/IgM chemiluminescent immunoassays (CLIA), in particular QUANTA Flash, comparable toour in-house ELISAs used for over two decades, and to evaluate their diagnostic performance. QUANTA Flash CLIA and in-house ELISAs were used to measure aCL and anti-β2GPI IgG/IgM. Moderate thresholds for QUANTA Flash CLIA were determined using a non-parametric approach, calculating a 99th percentile on serum samples from 139 blood donors, and by mirroring the diagnostic performance of in-house ELISA on 159 patient samples. Thresholds for QUANTA Flash CLIA achieving diagnostic performance equivalent to in-house ELISAs were 40 CU for moderate and 80 CU for high levels for aCL and anti-β2GPI IgG and IgM. The assays showed good qualitative agreement, ranging from 76.10 to 91.19 %. When considering in-house ELISA results, 14 out of 80 (17.5 %) patients did not fulfill the new ACR/EULAR laboratory classification criteria, while 27 out of 80 (33.8 %) did not when considering QUANTA Flash CLIA results. We determined moderate and high thresholds for aCL and anti-β2GPI IgG and IgM detected with QUANTA Flash CLIA, aligning with long-established in-house ELISA thresholds. These thresholds are crucial for seamlessly integrating of the new 2023 ACR/EULAR classification criteria into future observational clinical studies and trials.

Reporting and Establishment of Reference Intervals for Antiphospholipid Antibody Immunoassays: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

Misdiagnosis of antiphospholipid syndrome can occur owing to the wide diversity of antiphospholipid (aPL) assays and a lack of international calibrators and harmonized reference intervals. To assess laboratory practices regarding reporting and establishing reference intervals for immunoglobulin (Ig) G/IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (anti-β2GPI) assays. Supplemental questions related to reporting and establishing reference ranges for aPL assays were sent as part of the Antiphospholipid Antibody (ACL)-B 2019 College of American Pathologists (CAP) proficiency testing survey. The response rate and methods assessment details were determined, as well as qualitative and quantitative results for 3 test samples. The number of participants reporting results for IgG aCL (n = 489), IgM aCL (n = 476), IgG anti-β2GPI (n = 354), and IgM anti-β2GPI (n = 331) varied by antibody type. The enzyme-linked immunosorbent assay (ELISA) (up to 58.6%, 260 of 444) was the most used method; others included multiplex (from 18.9% to 23.9%), fluorescence enzyme immunoassay (14.4%-17.6%), and chemiluminescence immunoassay (6.5%-9.0%). More respondents reported quantitative than qualitative results, and manufacturer cutoff ranges were used by 92.9% and 94.2% of respondents for aCL and anti-β2GPI, respectively. Despite variation in the use of semiquantitative ranges, qualitative negative/positive reporting of the test samples achieved almost 100% consensus. Qualitative consensus was met in contrast to the wide range of quantitative results obtained for each analyte across different kits. ELISA remains the most used method for detecting aPL antibodies, with most laboratories reporting quantitative results based on manufacturers' suggested reference ranges. The categorization of quantitative results as equivocal, weak positive, or positive for responders using kits from the same manufacturer was variable.

Arterial stiffness tested by pulse wave velocity and augmentation index for cardiovascular risk stratification in antiphospholipid syndrome.

Cardiovascular disease is a major cause of morbidity and mortality in Antiphospholipid syndrome (APS). Arterial stiffness (ArS) has emerged as a predictor of future cardiovascular events in the general population. We aimed to assess ArS in patients with thrombotic APS versus diabetes mellitus (DM) and healthy controls (HC) and identify predictors of increased ArS in APS. ArS was evaluated by carotid-femoral pulse wave velocity (cfPWV) and augmentation index normalized to 75 beats/min (AIx@75) using the SphygmoCor device. Participants also underwent carotid/femoral ultrasound for atherosclerotic plaque detection. We used linear regression to compare ArS measures among groups and assess ArS determinants in the APS group. We included 110 patients with APS (70.9% female, mean age 45.4 years), 110 DM patients and 110 HC, all age/sex matched. After adjustment for age, sex, cardiovascular risk factors and plaque presence, APS patients exhibited similar cfPWV [β = -0.142 (95% CI -0.514, 0.230), p = 0.454] but increased AIx@75 [β = 4.525 (95% CI 1.372, 7.677), p = 0.005] compared with HC and lower cfPWV (p < 0.001) but similar AIx@75 (p = 0.193) versus DM patients. In the APS group, cfPWV was independently associated with age [β = 0.056 (95% CI 0.034, 0.078), p < 0.001], mean arterial pressure (MAP) [β = 0.070 (95% CI 0.043, 0.097), p < 0.001], atherosclerotic femoral plaques [β = 0.732 (95% CI 0.053, 1.411), p = 0.035] and anti-β2-glycoprotein I IgM positivity [β = 0.696 (95% CI 0.201, 1.191), p = 0.006]. AIx@75 was associated with age [β = 0.334 (95% CI 0.117, 0.551), p = 0.003], female sex [β = 7.447 (95% CI 2.312, 12.581), p = 0.005] and MAP [β = 0.425 (95% CI 0.187, 0.663), p = 0.001]. APS patients exhibit elevated AIx@75 vs HC and similar to DM patients, indicating enhanced arterial stiffening in APS. Given its prognostic value, ArS evaluation may help to improve cardiovascular risk stratification in APS.

Determinación del percentil 99 de los anticuerpos anticardiolipina y anti-β2 glicoproteína I en una institución en la ciudad de Medellín, Colombia

IntroductionAntiphospholipid syndrome diagnosis requires abnormal results of lupus anticoagulant and high titles of anticardiolipin (aCL) and β2glycoprotein I (anti-β2GPI) antibodies. The latter immunological tests lack a standard threshold in clinical practice. ObjectiveTo determine the 99th percentile of aCL and anti-β2GPI in healthy volunteers. Materials and methodsThis cross-sectional study reviewed antibody titles of anticardiolipin and β2glycoprotein I (IgG and IgM by enzyme-linked immunosorbent assay) in forty-nine healthy blood donors in Medellin, Colombia. Sociodemographic and immunological variables are also assessed. Antibody titles are described in median and interquartile range and the 99th percentile was estimated. ResultsWe analysed samples from 16 men and 33 women. We found that the upper limits of the reference range (99th percentile) of aCL and anti-β2GPI were: aCL IgM: 18.0, aCL IgG: 16.1, anti-β2GPI IgM: 16.4, and anti- β2GPI IgG: 6.9. ConclusionsThe upper limits obtained differ greatly from the arbitrary classification values suggested in the international guidelines, taking into account that values greater than 40 international units are usually required, and the values identified in this study are between 6.9 and 18 international units. We suggest conducting additional studies that validate cut-off points according to the percentiles explored in this work.

Assessment of antiphospholipid antibodies profiles based on severity of COVID-19 pneumonia.

Introductionthrombotic events are the most severe complications of the coronavirus disease 2019 (COVID-19). It is known that anti-phospholipid antibodies (APL) could be involved in thrombosis mechanism. Thus, APL profiles were studied particularly in patients with severe and critical COVID-19, and their clinical impact.Methodsa retrospective study of 54 COVID-19 hospitalized patients (34 in intensive care unit (ICU) and 20 in non-ICU) was conducted. These COVID-19 patients were tested for the presence of LAC (lupus anticoagulant) using the ACLTOP750®, anti-cardiolipine (ACL) and anti-β2glycoprotéine I (anti-β2GPI) IgG/IgM/IgA by enzyme-linked immunosorbent assay (ELISA). IgA isotype was tested in only 25 patients.Resultsanti-phospholipid antibodies were present in 74.1% of tested patients. LAC positivity was the highest (60.8%) among all patients, followed by IgM aCL (18.5%) and IgM anti-β2GPI (14.8%). Besides, LAC and anti-β2GPI IgA were the most predominant APL regarding the 25 patients tested for IgA isotype (52% and 24% respectively). Nine patients had thrombotic events, among them 6 were positive in APL and 5 were positive in LAC. However, there was any significant association between APL positivity or titers and thrombosis. There was also no significant difference between the two COVID-19 groups regarding APL profiles.Conclusiongiven the relatively high frequency of APL and especially LAC, and given the multitude of thrombotic risk factors in these severely and critically ill COVID-19 patients, a prophylactic anticoagulation remains essential.

Imbalance of circulating CTLA4+ follicular helper and follicular regulatory T cells in obstetric antiphospholipid syndrome.

Obstetric antiphospholipid syndrome (OAPS) is a systemic autoimmune disease that is characterized clinically by a variety of obstetric manifestations (fetal death and recurrent abortions) and serologically by the presence of antiphospholipid antibodies (aPLs). Whether dysregulation of Follicular helper T (Tfh) and Follicular regulatory T (Tfr) cells contribute to the immunopathogenesis in OAPS is still unknown. We analyzed phenotypic characterizations of circulating Tfh cells and Tfr cells in OAPS patients and healthy individuals. CTLA4(Cytotoxic T lymphocyte antigen 4)+ Tfh cells and CTLA4+ Tfr cells were declined and CTLA4+ Tfr/Tfh ratio and IL-21 were increased in OAPS patients compared with healthy controls. Percentages of CTLA4+ Tfh cells and CTLA4+ Tfr cells were the lowest in OAPS patients whose antiphospholipid antibodies (aPL) were triple positive. Increased CTLA4+ Tfr/Tfh ratio was positively correlated with anti-β2 glycoprotein I (anti-β2GPI) IgM, Complement 4(C4) or IL-21 in OAPS. Increased Th17 subtype and decreased Th1, Th2 subtypes in Tfh cells and Tfr cells, increased effector memory subtype and decreased central memory subtype of Tfh cells and Tfr cells were also observed in OAPS compared with healthy individuals. Our data demonstrated that an imbalance of circulating CTLA4+ Tfh cells, and Tfr cells correlates with the immunopathogenesis of OAPS.

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

Antiphospholipid antibodies in COVID-19-associated pneumonia patients in intensive care unit.

ObjectivesAntiphospholipid antibodies (APAs) increase the risk of excessive blood clotting, but their role in COVID‐19 remains unclear. We aimed to investigate the presence of conventional APAs used in the classification of antiphospholipid antibody syndrome in patients with severe lung infection with SARS-CoV-2 and to compare these results with non-COVID-19 critically ill patientsMethodsThirty-one COVID-19 patients (COVID group) and 28 non-COVID-19 critically ill patients (non-COVID group), were included in the study. Anti‐cardiolipin (ACA) (IgG, IgM), anti‐β2‐glycoprotein 1 (Anti-β2GPI) (IgG, IgM, and IgA), and if the patient had not received any anti thrombotic agent before blood collection, lupus anticoagulant (LAC) tests were studied from the plasma of the patients. For testing ACA and Anti-β2GPI, ELISA method was used, while fully automated coagulometer device was used for LAC test.ResultsAPAs were positive in 25.81% in the COVID group (8/31) and 25% in the non-COVID group (7/28). LAC was the most common APA present in 23.08% of the COVID-19 group, who underwent measurement (6/26), while 3.57% of the non-COVID group was LAC positive (1/28) (p = .047). In the COVID group, ACA IgM, and IgG were positive in 6.45% and 0%, respectively (2/31 vs 0/31). In the non-COVID group, ACA IgM was not positive in any patient, while ACA IgG was positive in 7.14% (2/28). Anti-β2GPI IgG and IgM tests were not positive in any patient in either the COVID or the non-COVID group. Anti-β2GPI IgA were positive in 6.45% and 14.29%, respectively (2/31 vs 4/28).ConclusionIn this study, APAs were equally positive in critically ill patients among COVID-19 or non-COVID-19 patients. Only LAC was more observed in COVID-19 patients.Key messagesIn this study, APAs were equally positive in critically ill patients among COVID-19 or non-COVID-19 related conditions. Only LAC was more observed in COVID-19 patients. ‘COVID‐19‐induced‐APS‐like‐syndrome’ is a new term and it is important in long term follow-up of COVID-19 patients with positive APAs.

Longitudinal Analysis of Anti-cardiolipin and Anti-β2-glycoprotein-I Antibodies in Recent-Onset Systemic Lupus Erythematosus: A Prospective Study in Swedish Patients.

Background: Anti-phospholipid syndrome (APS) and systemic lupus erythematous (SLE) are autoimmune disorders that often co-occur. Anti-phospholipid antibodies (aPL) are typical of both conditions and may be associated with vascular events and pregnancy-related morbidities. Whereas, aPL-screening is mandatory for individuals with suspected SLE, the clinical value of longitudinal aPL analyses in established SLE is unclear.Methods: We investigated the occurrence and variation of IgG/IgA/IgM anti-cardiolipin (aCL) and anti-β2-glycoprotein-I (anti-β2GPI) antibodies, using both the manufacturer's cut-off and a cut-off based on the 99th percentile of 400 apparently healthy donors, in recent-onset SLE. Furthermore, we evaluated the relationships between aPL levels and SLE/APS manifestations, as well as the pharmacotherapy. Patients with SLE who met validated classification criteria were included in this prospective study (N = 54). Samples were obtained at 0, 6, 12, 24, 36, 48, 60, 72, 84, and 96 months after SLE diagnosis.Results: Depending on the cut-off applied, 61.1 or 44.4% showed a positive result for at least one aPL isotype or the lupus anticoagulant test over time. Median values for all six aPL isotypes numerically decreased from inclusion to last follow-up, but none of the isotypes met statistical significance. Seroconversion (from positive to negative, or the opposite direction) was occasionally seen for both aCL and anti-β2GPI. IgA and IgM anti-β2GPI were the most common isotypes, followed by IgM aCL. Presence of IgG aCL associated significantly with myocardial infarction and miscarriage, and IgG/IgA anti-β2GPI with miscarriage.Conclusion: aPL were common during the first years of SLE. Even though the levels fluctuated over time, the patients tended to remain aPL positive or negative. Repeated aPL testing in the absence of new symptoms seems to be of uncertain value in patients with recent-onset SLE.

Síndrome antifosfolípido (SAF): diferencias clínicas e inmunoserológicas entre SAF primario y secundario en una cohorte colombiana

IntroductionAntiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by recurrent thrombosis that can affect the arterial and venous circulation. ObjectiveTo analyze the immunological and pharmacological differences, as well as the clinical outcomes of a cohort of patients with primary APS and secondary APS. Materials and methodsA retrospective cohort study was conducted that included 352 records of patients diagnosed with APS and treated between 2014 and 2018. A description is presented of the sociodemographic, clinical, and immunological profile of the population. A bivariate analysis performed using the chi-squared test to determine differences between groups with primary APS and secondary APS, and finally a multivariate analysis to search for associations with thrombotic clinical outcomes in patients with APS. ResultsThe mean age was 42.4±14 years, and 84.6% were females. Two-thirds (67.6%) of the patients had a diagnosis of primary APS, and 32.4% of secondary APS, of which 84% were associated with systemic lupus erythematosus (SLE). Among the thrombotic events, the most frequent were deep vein thrombosis (17.3%) and stroke (9.9%). Obstetric events were frequent, with a prevalence of 39.4% for miscarriages. No differences were found in the sociodemographic or immunoserological profile when comparing the group of primary vs. secondary APS. Thrombotic events were more frequent in the primary APS group, although only pulmonary embolism reached statistical significance. There were no differences between the two groups as regards obstetric events, such as miscarriages. Women were found to be 5 times more likely to have a stroke and 3 times more to have deep vein thrombosis. The anti-β2GPI type IgM increased the probability of presenting miscarriages about 3 times in women with APS. ConclusionThe study contains one of the largest Colombian cohorts with APS reported so far, and although it is both clinically and sociodemographically similar to other cohorts, there is a higher prevalence of primary APS. There was a lower frequency of thrombotic complications compared to other cohorts. Patients with primary APS had a tendency to develop thrombosis, as has already been reported in the literature.

Elevated IgA antiphospholipid antibodies in healthy pregnant women in Sudan but not Sweden, without corresponding increase in IgA anti-β2 glycoprotein I domain 1 antibodies

ObjectiveThe role of antiphospholipid antibodies (aPL) during apparently normal pregnancy is still unclear. IgA aPL are prevalent in populations of African origin. Our aim was to measure all isotypes of anticardiolipin (anti-CL) and anti–β2 glycoprotein I (anti-β2GPI) in healthy pregnant and non-pregnant women of different ethnicities.MethodsHealthy Sudanese pregnant women (n = 165; 53 sampled shortly after delivery), 96 age-matched Sudanese female controls and 42 healthy pregnant and 249 non-pregnant Swedish women were included. IgA/G/M anti-CL and anti-β2GPI were tested at one time point only with two independent assays in Sudanese and serially in pregnant Swedes. IgA anti-β2GPI domain 1 and as controls IgA/G/M rheumatoid factor (RF), IgG anti–cyclic citrullinated peptide 2 (anti-CCP2) and anti–thyroid peroxidase (anti-TPO) were investigated in Sudanese females.ResultsPregnant Sudanese women had significantly higher median levels of IgA anti-CL, IgA anti-β2GPI (p < 0.0001 for both antibodies using two assays) and IgM anti-β2GPI (both assays; p < 0.0001 and 0.008) compared with non-pregnant Sudanese. IgA anti-CL and anti-β2GPI occurrence was increased among Sudanese pregnant women compared with national controls. No corresponding increase during pregnancy was found for IgA anti-β2GPI domain 1 antibodies. Both IgG anti-CL and IgG control autoantibodies decreased during and directly after pregnancy among Sudanese. Serially followed Swedish women showed no changes in IgA aPL, whereas IgG/M anti-CL decreased.ConclusionsIgA aPL are increased in Sudanese but not in Swedish women, without corresponding increase in IgA domain 1. Whether due to ethnicity and/or environmental influences the occurrence of IgA aPL during Sudanese pregnancies, and its clinical significance, is yet to be determined.

Autoantibodies' titre modulation by anti-BlyS treatment in systemic lupus erythematosus.

The objective of this study was to analyse autoantibodies' titres modulation during belimumab treatment in 50 patients with systemic lupus erythematosus (SLE). Sera were collected at belimumab start (T0) and every six months until the 24th month. Disease activity index (SLEDAI-2K) was analysed at every timepoint. High avidity anti-dsDNA was detected by radioimmunological method, anti-ENA, anti-cardiolipin antibodies (aCL), anti-β2 glycoprotein I (anti-β2GPI) were analysed by ELISA. Fifty patients with SLE (mean SLEDAI-2K: 7.18 ± :3), mean age of 39 ± 11 years and mean follow-up of 13 ± 7.8 years were enrolled. A significant decrease of anti-dsDNA and anti-β2GPI IgM titres was observed at all timepoints. IgG aCL titre showed significant decrease only at T18. Anti-dsDNA negativization was detected in 21%, anti-β2GPI IgG in 33% and aCL IgG in 30% of sera, mostly at T6. Anti-ribosomal showed a significant titre decrease at T6 and T12, with negative seroconversion at T18. Anti-Sm titre significantly dropped down at T6, then remained stable during the time. Significant correlations were found between anti-dsDNA and anti-ribosomal titre and between SLEDAI ratio (SLEDAI value/SLEDAI T0) and anti-ribosomal titre ratio (value/value T0). Belimumab treatment induced a significant reduction of SLE-specific autoantibodies titre and IgM anti-β2GPI. Anti-ribosomal titre decrease correlates with anti-dsDNA titre and disease activity improvement.

Patients With Systemic Lupus Erythematosus Show an Increased Arterial Stiffness That is Predicted by IgM Anti-β2 -Glycoprotein I and Small Dense High-Density Lipoprotein Particles.

To investigate the metabolic and immunologic factors associated with the presence of central arterial stiffness as measured by the augmentation index (AIx). We conducted a cross-sectional study of 69 female patients with systemic lupus erythematosus (SLE) compared with a control group of 34 healthy women. The anthropometrical variables, the vascular studies, and the analytic data were obtained the same day. The AIx was assessed by peripheral arterial tonometry. The analysis of lipoprotein populations was performed using nuclear magnetic resonance (NMR) spectroscopy. Arterial stiffness was increased in patients with SLE compared with control subjects (mean ± SD 20.30 ± 21.54% versus 10.84 ± 11.51%; P = 0.0021). Values for the AIx were correlated with the Framingham risk score (r = 0.481, P < 0.001), carotid intima-media thickness (r = 0.503, P < 0.001), systolic blood pressure (r = 0.270, P < 0.001), and age (r = 0.365, P < 0.001). Patients receiving antimalarial drugs had a lower AIx (mean ± SD 11.74 ± 11.28% versus 24.97 ± 20.63%; P = 0.024). The AIx was correlated with the atherogenic lipoproteins analyzed by NMR. The immunologic variables associated with the AIx were C4 (r = 0.259, P = 0.046) and IgM anti-β2 -glycoprotein I (IgM anti-β2 GPI) (r = 0.284, P = 0.284). In the multivariate analysis, age (β = 0.347, 95% confidence interval [95% CI] 0.020-0.669, P = 0.035), IgM β2 GPI (β = 0.321, 95% CI 0.024-0.618, P = 0.035) and small dense high-density lipoprotein (HDL) particles (β = 1.288, 95% CI 0.246-2.329, P = 0.017) predicted the AIx. SLE patients had increased arterial stiffness compared with healthy control subjects. Arterial stiffness was decreased in patients treated with antimalarial drugs. Age, IgM β2 GPI, and the number of small dense HDL particles predicted the AIx.

Assessment of diagnostic methods for the detection of anticardiolipin and anti-βeta2 glycoprotein I antibodies in patients under routine evaluation for antiphospholipid syndrome

BackgroundWe assessed the performance characteristics and correlations of the traditional enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CIA) for detecting IgG and IgM antibodies to cardiolipin (aCL) and beta2 glycoprotein (anti-β2GPI) antibodies in patients under routine evaluation for APS. MethodsPatients (n = 216) referred to ARUP Laboratories for lupus anticoagulant (LAC) and/or aCL or anti-β2GPI IgG/IgM antibodies evaluation were assessed by ELISA and CIA methods. Diagnostic accuracies, correlations between methods and specific clinical manifestations in APS were investigated. ResultsThe areas under the curve (%) for APS using LAC with CIA (74, 95% CI: 65–82) or ELISA (70, 95% CI: 61–79) aPLs were comparable. The overall agreements and linear regression correlations between methods for aPL antibody of the same specificity were variable: aCL IgG 87.3%; R2 = 0.7491, aCL IgM 71.6%; R2 = 0.2656, anti-β2GPI IgG 77.2%; R2 = 0.7688 and anti-β2GPI IgM 81.7%; R2 = 0.3305. ConclusionsWith inclusion of LAC, the ELISA and CIA show comparable performance for the diagnosis of APS. However, correlations of APS-specific manifestations were dependent on method of detecting the aPL antibodies suggesting platforms may not be used interchangeable.

Antı-β2 Glycoprotein I Antibodies in Children with Rheumatologic Disorders.

Anti-beta-2-glycoprotein I antibodies (anti-β2GPI) which are the main antiphospholipid antibodies that characterize the autoimmune "antiphospholipid syndrome" are pathogenic and are contributing to thrombosis. We aimed to evaluate the presence and the diagnostic importance of these antibodies in children with different rheumatologic diseases with or without thrombosis risk. A total of 100 children with different rheumatologic diseases evaluated retrospectively. The mean anti-β2GPI IgG (p=0.108), IgA (p=0.547), and IgM (p=0.807) levels showed no statistically significant difference between different diagnosis groups. But anti-β2GPI IgA and IgM levels were higher in SLE patient group. The mean anti-β2GPI IgG (p=0.375), IgA (p=0.811), and IgM (p=0.276) levels were not also showed difference between disease groups with/without predisposition to thrombosis even though concentrations were higher in thrombosis group. In children with rheumatological complaints, anti-β2GPI antibody measurements should not be the first diagnostic criteria if vasculitis is not thought as the primary defect underlying the clinical symptoms.

Differential assay reactivity of immunglobulin A anti-ß2 glycoprotein I antibodies: implications for the clinical interpretation of antiphospholipid antibody testing.

The routine measurement of IgA anticardiolipin (aCL) and IgA anti-β2 glycoprotein I (anti-β2 GPI) antibodies remain controversial despite several studies demonstrating an association with thromboembolic disease in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). This controversy may be a contributing factor for the current under use of IgA antiphospholipid antibodies. We aimed to investigate the nature of discrepant IgA anti-β2 GPI reactivity to help define the diagnostic value of IgA antiphospholipid antibodies. Four sera selected from SLE/APS patients and positive for antiphospholipid antibodies but having discrepant IgA anti-β2 GPI reactivity on two commercial assays were studied. IgA antibodies were affinity purified to investigate anti-β2 GPI reactivity. Column wash through and eluent fractions were tested on both IgA anti-β2 GPI assays. Results were normalized to total protein. Assay conjugates and standards from the discrepant assays were interchanged. The diseased samples were strongly positive in one assay [144-388 IgA antiphospholipid (APL) units] and negative or weakly positive in another assay (9.9-53 APL units). IgA eluents from IgA anti-β2 GPI positive samples reacted 10 times stronger on the reactive assay. When normalized to protein content, the eluents showed no cross-reactivity for IgG or IgM anti-β2 GPI antibodies, confirming IgA isotype specificity. Conjugate interchange confirmed that both assays bound IgA anti-β2 GPI antibodies, but the anti-IgA conjugate from the reactive assay was 4 times stronger, suggesting that its ability to detect IgA anti-β2 GPI antibodies was partially dependent on the anti-IgA conjugate and calibration. These results confirm not only the presence of IgA anti-β2 GPI antibodies in the selected patient samples but also highlight an IgA conjugate issue for the unreactive assay, causing an underestimation of IgA anti-β2 GPI. This finding may assist in the ongoing standardization efforts of APS antibody testing. In addition, conclusions from published clinical studies may need to be revised as some assays may understate IgA significance.

International standards for IgG and IgM anti-β2glycoprotein antibody measurement.

International standards for anti-beta2 glycoprotein I (anti-β2GPI) testing are needed. We evaluated the suitability of polyclonal/monoclonal candidate reference materials (RM) for the assay. IgG/IgM anti-β2GPI were affinity-purified (AP) from high-positive antiphospholipid syndrome sera and IgG from HCAL clone supernatant. Igs were tested for purity by SDS-PAGE, pooled, concentrated, sterile-filtered and the protein concentration determined. One unit was defined as the binding activity of 1 µg/ml of AP anti-β2GPI Ig. IgG/IgM RM were each assigned a unit value using the respective AP material as a calibrator. Polyclonal/monoclonal RM and 30 samples were evaluated for linearity, unit equivalency and commutability. Polyclonal AP material was assigned a value of 100 U IgG and 15 U IgM anti-β2GPI, respectively. IgG-RM had a value of 270 IgG and the IgM-RM of 220.3 IgM anti-β2GPI U. The linearity (R (2)) of each RM curve for the various assays ranged from 0.96 to 0.99. Commutability samples fit very well within 95% prediction intervals and had excellent correlation when comparing assays. IgG and IgM polyclonal and IgG monoclonal RM displayed excellent linearity and commutability, being good candidates for better standardization of anti-β2GPI immunoassays.

Long-term persistence of anti-β2 glycoprotein I in treated leprosy patients.

β2 glycoprotein I (β2GPI) is a phospholipid binding protein that plays an important role in endothelial stability, blood coagulation, clearance of apoptotic debris and other physiologic processes. Anti-β2GPI antibodies occur in normal individuals and transiently during the course of infections, but are also associated with thrombotic events in autoimmune disease: the antiphospholipid syndrome (APS). A total of 31 out of 37 treated leprosy patients previously found to present high titers of IgM anti-β2GPI and/or anticardiolipin antibodies (aCL) remained positive for IgM antiphospholipid antibodies (aPL), and exhibited high titers of anti-β2GPI. The 37 patients were part of the 77 aPL-positive patients from a previous study that evaluated 158 leprosy patients. The median time elapsed between the first and second sample was 66 months. None of the 37 patients had any thrombotic event and 24 had a reactional state and were still requiring the use of prednisone, thalidomide or both. None of them fulfilled World Health Organization criteria for leprosy recurrence.

THU0039 Relationship between Antiphosphatidylserine/Prothrombin Antibodies and Conventional Antiphospholipid Antibodies in Primary Antiphospholipid Syndrome

BackgroundThere is a large consensus regarding the association with APS clinical features of antiprothrombin antibodies, directed to phosphatidylserine/prothrombin complex.ObjectivesAs their diagnostic value remain matter to debate we designed a study...

FRI0483 Variation of antiphospholipid antibody (APL) titers in patients with persistent antiphospholipid antibodies

BackgroundAPL have been closely associated with venous and arterial thrombosis. For the diagnosis of Antiphospholipid Syndrome (APS) clinical criteria are needed (thrombotic events and/or repeated abortions) and a persistently positive...