aβ2GPI IgG Research Articles

A multicenter study to assess the reproducibility of antiphospholipid antibody results produced by an automated system

Background Inter-assay variability is a well-known problem in antiphospholipid antibody testing, because of the lack of standardization. Inter-laboratory reproducibility for the same assay is similarly important. Objectives Testing repeatability and reproducibility of HemosIL® AcuStar for anticardiolipin (aCL) and antiβ2-glycoprotein I antibodies (aβ2GPI) IgG and IgM. Patients/Methods In this observational study, out of 420 samples from the thrombophilia centers of Ghent and Geneva, 100 samples were randomly selected and successively analyzed in three centers: Ghent (C1, in duplicate for repeatability evaluation), Geneva (C2) and Frankfurt (C3). Results Results from 99 samples were available, including 25 from patients with antiphospholipid syndrome (APS) and 74 from non-APS patients. The intra-center repeatability expressed as intra-class correlation coefficient (ICC) was higher than 0.99 for each parameter. Differences between two measurements rarely exceeded 1 U mL-1 for values below 100 U mL-1 , except for aβ2GPI IgG, where differences varied from -4 to 4 U mL-1 . The inter-center ICCs were higher than 0.99, except for aCL IgM (ICC = 0.961). These ICCs remained high even when considering values below 100 U mL-1 (0.943, 0.964 and 0.977 for aCL IgG, aCL gM and aβ2GPI IgM, respectively), except for aβ2GPI IgG (ICC = 0.652). Qualitative comparison showed less than 5% discordant classification between centers, with somewhat more discordant results for aβ2GPI IgG. Conclusions In terms of discriminating properties, the HemosIL® AcuStar has excellent intra-center repeatability and a good inter-center reproducibility for aCL IgG, aCL IgM and aβ2GPI IgM. Some concern may arise for aβ2GPI IgG.

Role of anti‐domain 1‐β2glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome

Essentials Antibodies to domain 1 of β2 glycoprotein I (aD1) are a subset of antiphospholipid antibodies. We evaluated the added diagnostic value of an automated aD1 assay in antiphospholipid syndrome. AD1 IgG correctly classifies patients at risk for thrombosis. Agreement between aD1 and aβ2GPI IgG is high, limiting the added value of aD1 in our setting. Click to hear Professor de Groot's perspective on new mechanistic understanding in antiphospholipid syndrome Background Laboratory diagnosis of antiphospholipid syndrome (APS) includes lupus anticoagulant (LAC), anticardiolipin (aCL) or anti-β2 glycoprotein I (aβ2 GPI) antibodies. Antibodies targeting domain 1 of β2 GPI (aD1) constitute a pathogenic subset of autoantibodies. Objectives In this cohort study, we determined the clinical performance characteristics, additional diagnostic value and the contribution to APS risk stratification of an automated aD1 assay. Patients/Methods LAC, aCL, aβ2 GPI and aD1 IgG were measured in 101 APS patients, 123 patients with autoimmune disorders, 82 diseased controls and 120 healthy controls. aD1 antibodies were detected by QUANTA Flash(®) Beta2GPI-Domain 1 chemiluminescence immunoassay. Results With a cut-off value of 20.0 CU, the aD1 IgG assay identifies APS patients in a clinically affected patient cohort with a sensitivity of 53.5% and specificity of 98.8%. It implied a high odds ratio (OR) for clinical events (OR, 17.0; 95% confidence interval [CI], 7.1-40.5). aD1 IgG did not add diagnostic value to the formal aPL panel because aβ2 GPI IgG was nearly as specific but more sensitive for APS (sensitivity 56.4%) with a higher OR for clinical events (36.2; 95% CI, 11.1-117.9). High aD1 titers identify triple-positive patients and patients with thrombosis in a β2 GPI-dependent LAC-positive population. Agreement between aD1 IgG and aβ2 GPI IgG was high (positive and negative agreement 91.7% and 98.4%, respectively). Conclusion Detection of aD1 IgG correctly classifies patients at risk of thrombosis. However, the contribution of aD1 IgG to APS diagnosis and risk stratification depends upon the solid phase assays used for aCL and aβ2 GPI detection.

Significance of fully automated tests for the diagnosis of antiphospholipid syndrome

Antiphospholipid antibodies (aPLs) can vary both immunologically and functionally, thus it is important to effectively and correctly identify their presence when diagnosing antiphospholipid syndrome. Furthermore, since many immunological/functional tests are necessary to measure aPLs, complete examinations are often not performed in many cases due to significant burden on the testing departments. To address this issue, we measured aPLs defined according to the classification criteria (anticardiolipin antibody: aCL) IgG/IgM and anti-β2 glycoprotein I antibody (aβ2GPI) (IgG/IgM) as well as non-criteria antibodies (aCL IgA, aβ2GPI IgA and aβ2GPI domain I), in a cohort of 211 patients (61 APS, 140 disease controls and 10 healthy individuals). APLs were measured using a fully automated chemiluminescent immunoassay instrument (BIO-FLASH®/ACL AcuStar®) and with conventional ELISA tests. We demonstrated that both sensitivity and accuracy of diagnosis of aCL IgG and aβ2GPI IgG were high, in agreement with the past reports. When multiple aPLs were examined, the accuracy of diagnosis increased. The proportion of APS patients that were positive for 2 or more types of aPLs (47/61, 77%) was higher than that of patients with systemic lupus erythematosus (SLE)(3/37, 9%), those with non-SLE connective tissues diseases (1/53,2%), those with other diseases or healthy volunteers. Based on these findings, it was concluded that the fully automated chemiluminescent immunoassay instrument, which allows the simultaneous evaluation of many types of aPLs, offers clear advantages for a more complete, more rapid and less labor-intensive alternative to running multiple ELISA and could help in better diagnosis for suspected APS patients.

Analytical and clinical comparison of different immunoassay systems for the detection of antiphospholipid antibodies.

We evaluated analytical and clinical performances of IgG and IgM anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (a-β2GpI) antibodies and upper limit reference ranges (99th percentiles) in comparison with manufacturer's cutoff values with different commercial methods. We assayed aCL and a-β2GpI in serum samples from 30 healthy individuals, 77 patients with antiphospholipid syndrome (APS) diagnosed according to the Sydney criteria, 51 patients with autoimmune diseases, eight patients with previous thrombotic events, six patients with other diseases, and 18 patients with infectious diseases, using ELISA Inova Diagnostics; EliA Phadia Laboratory Systems; CliA Zenit-RA; and CliA Bio-Flash. Anticardiolipin and a-β2GpI IgG and IgM immunoassays showed good analytic performances with both 99th percentile and manufacturer's cutoff reference values. Our results showed fair to moderate agreement among assays. In-house cutoff values gave significantly better performances only for a-β2GpI IgG with all the immunoassays analyzed with the exception of Inova CliA Bio-Flash where we obtained the same performances with in-house and manufacturer's cutoffs. By guidelines, all laboratories are strongly advised to validate/verify the manufacturer's cutoff values. We recommend establishing low-positive, medium-/high-positive, and high-positive CliA IgG aCL and a-β2GpI ranges in order to help clinicians in the diagnosis and treatment of APS.

Lupus anticoagulant is the main predictor of adverse pregnancy outcomes in aPL-positive patients: validation of PROMISSE study results

ObjectiveWe previously reported that lupus anticoagulant (LAC) is the main predictor of poor pregnancy outcome in antiphospholipid antibody (aPL)-positive patients. We sought to confirm this finding in an independent group...

Extended Antiphospholipid Antibodies Screening in Systemic Lupus Erythematosus Patients

The antiphospholipid syndrome (APS) is one of the most encountered autoimmunity in systemic lupus erythematosus (SLE) patients and pathogenesis of these two seems to be intricate. To investigate the association of antiphospholipid antibodies (APLAs) titer with the presence of secondary APS diagnosis in SLE patients. 65 patients fulfilling the 2012 Systemic Lupus Collaborating International Clinics (SLICC) SLE's criteria were included. The APS diagnosis was sustained according to the 2006 Sydney APS's criteria. Three groups of patients were defined: SLE patients with secondary APS, SLE with history of positive "criteria" APLAs but without APS clinical features, respectively SLE patients without positive APLAs or clinical APS criteria. An extended APLAs panel was searched in all cases: both IgM and IgG of anticardiolipin antibodies (aCL), anti-P2 glycoprotein I antibodies (aβ2GPI), antiphosphatidylethanolamine antibodies (aPE), antiphosphatidylserine antibodies (aPS), respectively antiprothrombin antibodies (aPT). Results. Only the aβ2GPI, both IgM and IgG serotypes, had significantly higher titers in patients with SLE and secondary APS compared to no APS (with/ without positive APLAs): median (min; max) 7.0 (0.0-300.0) vs. 1.0 (0.0-28.0) vs. 1.0 (0.0-12.0), respectively 3.0 (0.0-79.0) vs. 1.0 (0.0-3.0) vs. 1.0 (0.0-12.0) (p<0.001, Kruskal-Wallis test)]. Also, in regression logistic models, only the aβ2 GPI (IgG and IgM ) were identified as risk factors for secondary APS diagnosis in the SLE patients: OR(95%CI) 5.9 (2.2-15.7), respectively 1.3 (1.1-1.5). In regard with the SLE markers, the IgG serotypes of the "non-criteria" APLAs analyzed (aPS, aPT, aPE) were correlated with the antiDNA titers while the IgM serotypes inversely associated with the complement C3 levels. IgG aβ2 GPI are accompanied by almost 6-fold increase risk of secondary APS when screening SLE patients. On the contrary, the "non-criteria" APLAs do not seem associated with the APS diagnosis in SLE patients. Some correlates of the "non-criteria" APLAs with the antiDNA and complement C3 levels were also observed.

Clinical features and pregnancy outcome in antiphospholipid syndrome patients with history of severe pregnancy complications

Objective. To clarify the clinical significance of antiphospholipid antibody (aPL) profile in patients with obstetric antiphospholipid syndrome (APS).Methods. Clinical records of 13 pregnant patients (15 pregnancies) with obstetrical APS were reviewed over 10 years. Patients who met the Sapporo Criteria fully were studied, whereas those with only early pregnancy loss were excluded. In addition to classical aPL: lupus anticoagulant (LA), anticardiolipin antibody (aCL), and anti-β2-glycoprotein I (aβ2GPI); phosphatidylserine-dependent anti-prothrombin antibody (aPS/PT) and kininogen-dependent anti-phosphatidylethanolamine antibody (aPE) were also examined in each case.Results. Cases were divided into two groups according to patient response to standard treatment: good and poor outcome groups. All cases with poor outcome presented LA, with IgG aβ2GPI and IgG aPS/PT were also frequently observed. IgG aPE did not correlate with pregnancy outcome.Conclusion. aPL profile may predict pregnancy outcome in patients with this subset of obstetric APS.

THU0504 Regulatory T Cells in Peripheral Blood of Patients with Antiphospholipid Syndrome

BackgroundAntiphospholipid syndrome (APS) is characterized by thrombosis, fetal loss and presence of antiphospholipid (APL) antibodies, the most studied of which are anticardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) IgG and IgM...

High Frequency of Antiphospholipid Antibodies in Primary Biliary Cirrhosis

To evaluate, retrospectively, the frequency of autoantibodies of antiphospholipid syndrome (APLS) in Tunisian patients with primary biliary cirrhosis (PBC). We analyzed 80 PBC sera and 80 sera from blood donors. ELISA was used to determine the frequency of antibodies against cardiolipin (aCL IgG, IgA, and IgM) and beta 2 glycoprotein I (aβ2GPI IgG, IgA, and IgM). The frequency of antiphospholipid antibodies (aCL and/or aβ2GPI) was significantly higher in PBC patients than in controls (70 vs. 5%, P < 10(-6)). The frequency of aCL antibodies (IgG, IgA or IgM) was significantly higher in PBC patients than in the control group (23.7 vs. 3.7%, P = 0.0005). The frequencies of aCL IgA and aCL IgM in PBC patients' sera were significantly higher than those in the control group (10 vs. 0%, P = 0.003 and 20 vs. 2.5%, P = 0.001, respectively). Two patients of eighty (2.5%) had aCL IgG, aCL IgA and aCL IgM. The frequency of aβ2GPI antibodies (IgG, IgA, or IgM) was significantly higher in PBC patients than in the control group (70 vs. 1.2%, P < 10(-6)). The frequencies of aβ2GPI IgG, aβ2GPI IgA, and aβ2GPI IgM in PBC patients' sera were significantly higher in patients than in the control group (12.5 vs. 0%, P = 0.003; 62.5 vs. 1.2%, P < 10(-6); and 21.2 vs. 0%, P < 10(-4), respectively). Autoantibodies related to APLS (aCL and aβ2GPI) were present in the majority of patients with PBC, reflecting the ability of these antibodies to engage mediators of damage.

Contribution of anti-β2glycoprotein I IgA antibodies to the diagnosis of anti-phospholipid syndrome: potential interest of target domains to discriminate thrombotic and non-thrombotic patients

Although the last international guidelines for aPL recommended determination of IgA aCL and anti-β2glycoprotein I (aβ2GPI) antibodies for the evaluation of APS in the absence of conventional IgG or IgM aCL and aβ2GPI antibodies, the clinical value of these antibodies remains controversial. We evaluated the clinical utility of IgA aPL and of the determination of target domains of aβ2GPI IgA antibodies. A retrospective analysis was performed on sera from 439 patients referred for routine detection of aPL IgA by in-house ELISA. Sera positive for aβ2GPI IgA were subsequently tested for aβ2GPI domain 1 (D1) and domain 4/5 (D4/5) antibodies using ELISAs. The prevalence of aβ2GPI IgA antibodies was 16% in patients, significantly different from controls (1%, P < 0.0001). These antibodies were associated with clinical contexts related to APS as thrombosis (28.6% vs. 15%, P = 0.009) and SLE (42% vs. 15%, P < 0.0001). Interestingly, determination of their target domains revealed a significant association between aβ2GPI IgA directed against D4/5 and SLE without thrombosis (66.7 vs. 16.7%, P = 0.002). In contrast, aCL IgA were not more prevalent in patients than in controls. Our study confirmed the interest of aβ2GP1 IgA in the exploration of APS and suggests that identification of target domains of aβ2GP1 IgA may be useful in the evaluation of thrombotic risk in SLE patients.

Clinical performance of anticardiolipin and antiβ2 glycoprotein I antibodies using a new automated chemiluminescent assay

The anticardiolipin (aCL) and antibeta2-glycoprotein I (aβ2GPI) antibodies are used to diagnosis antiphospholipid syndrome. It would be possible to use two or more methods to improve the diagnostic sensitivity of aCL and aβ2GPI. In this study, we investigated the laboratory performance of a new chemiluminescent assay and explored whether the combined results of aCL and aβ2GPI measured by two different methods predict thrombotic risk better. The cut-off values were assessed with 99 healthy controls. The coefficient of variations was determined using control materials. Using the plasma of 109 patients, AcuStar chemiluminescent assay and QUANTA Lite ELISA were carried out according to the manufacturers' instructions. The AcuStar cut-off points were 13.5 U/ml for aCL IgG, 19.1 U/ml for aCL IgM, 16.3 U/ml for aβ2GPI IgG and 12.6 U/ml for aβ2GPI IgM. Total coefficient of variations ranged from 4.6 to 8.8%. For both aCL and aβ2GPI, the AcuStar odds ratio for thrombosis prediction was higher than that of QUANTA Lite. When the results for both AcuStar and QUANTA Lite are positive, the odds ratios of aCL and aβ2GPI were higher than that interpreted by a single positive result each. The new automated chemiluminescent assay exhibited good precision, concordance rate, and clinical performance. The combined results of aCL and aβ2GPI as measured by two different methods are expected to increase the prediction power for thrombosis.

AB0196 Antibodies versus domain I of BETA2-glycoprotein I: A marker of systemic autoimmunity?

BackgroundAnti-β2glycoprotein I antibodies (a-β2GPI) are involved in the pathogenesis of the Antiphospholipid Syndrome (APS) and are a formal laboratory criterion for the diagnosis of APS. These antibodies however can be...

Anti-phosphatidylserine/prothrombin antibodies: an additional diagnostic marker for APS?

Among the diagnostic assays for anti-phospholipid syndrome (APS), lupus anticoagulant (LA) is the strongest predictor of thrombosis; however, it presents several limitations as interference with anticoagulant therapy and poor inter-laboratory agreement. Two-thirds of LA activity is apparently due to antibodies against prothrombin (PT), usually detectable by ELISA. Binding of PT to phosphatidylserine (PS) has been shown to enhance solid-phase anti-PT assay sensitivity. To determine the prevalence of antibodies against PS/PT (aPS/PT) in APS, we tested the semiquantitative QUANTA Lite(®) aPS/PT ELISA in a cohort of 80 APS patients. The prevalence of aPS/PT was 81.3%, rising to 87.6% when considering LA-positive subjects only. We observed a strong correlation between aPS/PT and LA (p = 0.006). To note, APS patients with thrombotic manifestations displayed significantly higher IgG aPS/PT titers compared to 20 aPL asymptomatic carriers (p = 0.012). To rule out a possible cross-reactivity of anti-β2 glycoprotein I antibodies (aβ2GPI) with PS/PT complex, we tested two monoclonal aβ2GPI antibodies and an affinity-purified (AP) polyclonal aβ2GPI IgG obtained from the serum of a patient reacting against both β2GPI and PS/PT. The two monoclonal antibodies did not show any reactivity against PS/PT complex, similarly the AP IgGs did not react toward PS/PT antigen while preserved their aβ2GPI activity. Our findings suggest that aPS/PT are a definite antibody population in APS. Moreover, the good correlation between aPS/PT ELISA and LA may support its use as a surrogate test for LA, particularly useful to overcome the technical limitations of the functional assay.

A5.5 Antibodies against Domain I of β2 Glycoprotein I in Antiphospholipid Antibody Syndrome

<h3>Background and Objectives</h3> antibodies anti-phospholipids (aPL) react to proteins bound to PL, mainly β2glycoprotein I (β2GPI). Antibodies against β2GPI (aβ2GPI) exert a pathogenic role and represent a risk-factor for clinical manifestations of anti-phospholipid syndrome (APS). However, some aβ2GPI-positive subjects never develop APS-related clinical manifestations. This observation may be explained by the heterogeneity of aβ2GPI population, with autoantibody subgroups targeting different β2GPI epitopes. In particular, antibodies anti-domain I (aDI) but not domains IV and V (aDIV/V) of β2GPI have been associated with thrombotic events. Therefore, the aim of this study was to assess the prevalence of aDI and aDIV/V IgG in a cohort of aPL-positive patients. <h3>Material and Methods</h3> 58 patients with a diagnosis of primary APS (PAPS) according to the 2006 Sydney criteria have been included in this study. 38 PAPS patients (65.5%) presented with venous and/or arterious thrombothic events while 20 subjects (34.5%) had obstetric manifestations only. 15 aPL asymptomatic carriers were also recruited. All samples had been tested for LA and for aCL and aβ2GPI with home-made assays according to international guidelines. In the thrombotic PAPS group, 35/38 subjects (92.1%) were aβ2GPI IgG positive; aβ2GPI IgG positivity rate was 85% in the obstetric PAPS group (17/20 women); 80% of the asymptomatic aPL carriers displayed aβ2GPI IgG. IgG specificities against whole β2GPI, DI and DIV/V have been evaluated with a novel solid-phase chemiluminiscent assay (BioFlash and ELISA, INOVA Diagnostics). <h3>Results</h3> Out of the 73 aPL positive patients: 21% were positive for aβ2GPI, aDI and aDIV/V; 41% were positive for aβ2GPI and aDI but negative for aDIV/V; 4% were positive for aβ2GPI and aDIV/V but negative for aDI; 21% were aβ2GPI positive only; 4% were positive for aDIV/V; 9% were negative for antibodies against the whole molecule and the studied domains. The prevalence of aDI was 74% among patients with thrombotic pAPS and 60% among women with obstetric manifestations. 40% of aPL asymptomatic carriers were positive for aDI. We observed a strong correlation between aβ2GPI and aDI (p &lt;&lt; 0.01, r = 0.836) but not aDIV/V (p = 0.07, r = 0.216). <h3>Conclusions</h3> Most of the aβ2GPI positive sera displayed reactivity against DI, while aDIV/DV were detected in a low rate of patients. Our data suggest that DI is the immunodominant β2GPI epitope and that aDI are the main antibody population in APS patients. Future studies are warranted to better define the diagnostic and prognostic role of anti-DI in APS.

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.

Anti-cardiolipin and anti-beta 2-glycoprotein I antibodies in celiac disease

AimsTo determine the frequency of anti-cardiolipin (aCL) and anti-β2-glycoprotein I antibodies (aβ2GPI) in celiac disease (CD) patients. Patients and methodsSixty-three untreated CD patients and 40 healthy blood donors (HBD) were studied. IgG, IgA and IgM aCL and aβ2GPI were detected by Elisa. ResultsThe frequency of antiphospholipid antibodies (aPL) (aCL and/or aβ2GPI) was significantly higher in CD patients (12 out of 63) than in HBD (two out of 40) (19% vs 5%, P=0.04). Six CD patients out of 63 (9.5%) and one HBD out of 40 (2.5%) had aCL. Ten CD patients (15.9%) and two HBD (5%) had aβ2GPI. Only aβ2GPI-IgA was significantly more frequent in CD patients than in HBD (14.3% vs 2.5%, P=0.048). In CD patients, aβ2GPI-IgA (nine out of 63) was significantly more frequent (14.3%) than aβ2GPI-IgG (1.6%) and IgM (1.6%) (P=0.008). In CD patients, the frequency of aCL-IgA and IgM was 6.3% (four out of 63) and aCL-IgG were not detected. Simultaneous presence of positive antibodies was found in four CD patients: one patient had four aPL, one had three aPL and two had two aPL. The four patients who had aCL-IgA had also aβ2GPI-IgA and three of them had a titer higher than 50 units. Among nine patients with aβ2GPI-IgA, four had a titer higher than 100 units. The highest titers were found in adults. ConclusionsaPL and particularly aβ2GPI-IgA are frequent in CD. The significance of these antibodies has to be determined.

Subpopulations of anti-β2glycoprotein I antibodies with different pathogenic potential: fine specificity against the domains of β2glycoprotein I

Anti-β₂glycoprotein I antibodies (a-β₂GPI) are a laboratory criterion for the antiphospholipid syndrome (APS) and were demonstrated to be involved in the pathogenesis of APS. However, they can also be detected in asymptomatic subjects. It has been suggested that a-β₂GPI against Domain1 (D1) associate with thrombosis, while those recognizing Domain4/5 (D4/5) have been identified in non-thrombotic conditions. We evaluate the specificity of a-β₂GPI in different clinical situations. We studied 39 one-year-old healthy children born to mothers with systemic autoimmune diseases (SAD) (15 (38.4%) were born to mothers who were a-β₂GPI positive), 33 children with atopic dermatitis (AD) and 55 patients with APS (50 adults and 5 paediatrics). All subjects were IgG a-β₂GPI positive. IgG a-β₂GPI were performed by homemade ELISA, while IgG a-β₂GPI D1 and D4/5 were tested on research ELISAs containing recombinant β₂GPI domains antigens. One-year-old children and AD children displayed preferential reactivity for D4/5; patients with APS recognized preferentially D1. We also found a good correlation between a-β₂GPI and D4/5 in one-year-old (r=0.853) and AD children (r=0.879) and between a-β₂GPI and D1 in the APS group (r=0.575). No thrombotic events were recorded in both groups of children. A-β₂GPI found in non-thrombotic conditions (healthy children born to mothers with SAD and AD children) mostly recognize D4/5, in contrast to the prevalent specificity for D1 in the APS group. The different specificity could at least partially explain the "innocent" profile of a-β₂GPI in children.

Significant association of antiphospholipid antibodies and TNF-alpha: Marker of severe atherogenic profile of patients with type II diabetes mellitus without micro and/or macrovascular complications

ObjectiveIn vitro, stimulation of monocytes with antiphospholipid (aPL) antibodies resulted in increased secretion of TNF-alpha, but association of aPL with features of diabetes mellitus is not clarified yet. Therefore, we investigated the distribution of anticardiolipin (aCL), anti-β2gpI (aβ2gpI), anti-annexin A5 (aannxA5), and anti-oxLDL (aoxLDL) antibodies, and TNF-alpha in well-formed group of 78 patients with type II diabetes mellitus without vascular complications. MethodsInvestigated antibodies and TNF-alpha concentrations were measured by ELISA. ResultsAntiphospholipid antibodies were in positive correlation with TNF-alpha concentrations: aCL IgG (r=0.303, p=0.007), aCL IgM (r=0.386, p=0.000), aβ2gpI IgG (r=0.499, p=0.000), aβ2gpI IgM (r=0.462, p=0.000), aanxA5 IgG (r=0.479, p=0.000), aanxA5 IgM (r=0.641, p=0.000), aoxLDL (IgG+IgM, r=0.279, p=0.000). Anticardiolipin-positive and aCL-negative subgroups differed in TNF-alpha concentrations (Mann–Whitney, p=0.032). Significantly elevated LDL concentrations were noticed in aCL-positive patients with disease duration 10–15years (χ2=15.000, p=0.000) and apoB concentrations were elevated in aoxLDL-positive patients with disease duration 7–10years (χ2=3.938, p=0.047). ConclusionSignificant association of antiphospholipid antibodies and TNF-alpha might be a marker of severe atherogenic profile (suggested by increased levels of lipids in aPL-positive subgroups) and should be used for the stratification of patients with an increased risk for future deterioration of the disease.

Antibodies to Domain I of β 2Glycoprotein I are in close relation to patients risk categories in Antiphospholipid Syndrome (APS)

Introduction Antiphospholipid Syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies in patients with thrombosis or pregnancy morbidity. Antibodies involved in these disorders are mainly those directed against β 2-Glycoprotein I (β 2GPI) with the major epitope apparently located on discontinuous antigen with several parts of Domain I (DmI) involved. The relation between anti-DmI antibodies and patients’ risk categories is unknown. Materials and Methods The synthetic full-length and correctly-folded DmI (1–64) to set up a competitive inhibition enzyme-linked immunoadsorbent assays (ELISA) was used. Plasma of 22 patients with APS and triple positivity [Lupus Anticoagulant positive (LAC+), IgG anti-cardiolipin positive (aCL+), IgG anti-β 2GPI positive (a β 2GPI +)], 15 with double positivity (IgG aCL+, IgG aβ 2GPI+), 9 with single positivity (IgG aβ 2GPI+) and 20 controls were evaluated. Results Median of percentage inhibition was 25.5% [interquartile range (IQR)17.2-33.0] in triple positive patients. Significantly lower inhibition was observed in patients with double positivity, median inhibition 5.0% (IQR 0.0-27.0) and in patients with single positivity median inhibition was 2.0% (IQR 0.5-8.0) (p < 0.0001). No inhibition was detected in control subjects or using β 2GPI peptides (40–52 and 57–70), or when antithrombin, an insignificant control protein was used. Conclusions High risk patients with APS and triple laboratory positivity as compared with double and single positivity patients have significantly higher titre of anti-DmI antibodies as evaluated by an inhibition test.

Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome

Introduction The laboratory diagnosis of antiphospholipid syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL): lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2glycoprotein I IgG or IgM antibodies (aβ2GPI), usually detected by ELISA Materials and methods We evaluated the diagnostic value of aCL and aβ2GPI measured by a new automated system using the chemiluminescence principle, the immunoanalyzer Zenit RA (Menarini). Results Results of aCL and aβ2GPI were correlated with the clinical background of the patients and with results of ELISA (n = 314). Correlated to the clinical background sensitivity/specificity ranged for aCL IgG between 7.5-45.2% / 54.2-98.8%, for aCL IgM 3.4-5.5% / 89.9-94%, for aβ2GPI IgG 5.5-25.3% / 75.6-100% and aβ2GPI IgM 3.4-4.8% / 89.9-92.3%, depending on the cut-off used. Sensitivity with manufacturer's cut-offs was comparable to ELISA, except for aβ2GPI IgG with a significantly lower sensitivity compared to ELISA (5.5% vs 11.6%). In the APS patient population (n = 30) sensitivity of aCL IgG and aβ2GPI IgG was higher measured by ELISA compared to Zenit RA (46.7% vs 30.0%, and 46.7% vs 26.7%, respectively). Agreement between Zenit RA results and ELISA results for the four parameters was moderate (Kappa-values ranging 0.509-0.565). Sensitivity was 38.5%, 53.3%, 40% and 69.2% for aCL IgG, aCL IgM, aβ2GPI IgG and aβ2GPI IgM, respectively, applying the highest cut-off value for Zenit RA, raising towards 64.3%, 100%, 57.1%, for aCL IgG, aCL IgM, aβ2GPI IgG, respectively, in a APS patient population. Conclusions The new technology of chemiluminescense for measuring aPL showed good performance characteristics. Interpretation of results with a cut-off value associated with a good discrimination for disease, resulted in a lower sensitivity for the diagnosis of APS for aβ2GPI IgG measured by Zenit RA assays compared to ELISA; sensitivity for aCL IgG was comparable to ELISA. Specificity for all parameters was high and comparable for aCL and aβ2GPI.