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Background Pemphigus and bullous pemphigoid are two autoimmune diseases that have similar pathogenesis. Both have a genetic predisposition, which promotes the production of auto antibodies targeted against different components of the epidermal desmosome and hemidesmosome. Antiphospholipid antibodies (aPL) are heterogeneous group of antibodies found in patients with autoimmune diseases and inflammatory conditions and are associated with thrombotic events. Objective We sought to determine the expression profile of eight non classical aPLs in ABD patients. Methods A cohort of 266 serum samples of patients with pemphigus, bullous pemphigoid and controls was screened for the presence of eight aPL antibodies, using the Bio-Rad BioPlex™ 2200 system. Results Phosphatidylserine-beta-2-glycoprotein-I (aPS-β2GPI) and anti prothrombin complex (aPT-PT) serum profiles were significantly more prevalent among ABD patients; 20.7% patients with ABD compared to 5.9% of control patients were positive for aPS-β2GPI IgM. In addition, aPT-PT IgM was more prevalent among ABD patients (31% vs. 14.8%). Conclusion aPL auto antibodies are more prevalent in ABD. Any clinical association should be further determined.

One of the most intriguing characteristics of the antiphospholipid syndrome (APS) is that diagnosis requires the combined presence of clinical abnormalities (thrombosis and/or miscarriages) and at least one of the following antiphospholipid antibodies: lupus anticoagulant, anticardiolipin, or anti–β2-glycoprotein I. Clinicians occasionally have difficulty making this diagnosis in patients with a clinical picture of APS but without any of the previously mentioned antiphospholipid antibodies. Such a status has been defined as "seronegative APS." Under these conditions, antiphosphatidylethanolamine antibodies deserve particular attention, as they have been described as being associated with the main clinical events of APS. Thus, this review focuses on issues related to the characteristics of antiphosphatidylethanolamine antibodies, including the nature of antigen targets and their role in homeostasis, the methodologic problems encountered in their detection, and their clinical associations.

The aim of this study was to evaluate the influence on the results of the main variables of ELISA used for the detection of antiphosphatidylethanolamine antibodies (aPE). Forty sera from patients with either autoimmune disorders including antiphospholipid syndrome (APS) or the clinical features of APS only were assayed by ELISA performed under different conditions. Variables were sources of PE (egg yolk, soybean, bovine brain or Escherichiacoli), microtiter plates (plain or gamma irradiated) and buffer components-fetal calf serum (FCS), adult bovine plasma (ABP), adult bovine serum (ABS) or bovine serum albumin (BSA). aPE binding was decreased with PE from E. coli while the other tested PE gave comparable results. The influence of the type of plates was restricted to IgM isotype with slightly, but significantly higher optical densities with plain than with irradiated plates. Most importantly, the component buffer had the highest impact on the results as shown by a strong decrease of the signal by ABP or ABS. This inhibitory effect was confirmed by using mixtures of FCS or BSA with increasing concentrations of ABS. Partial delipidation of ABS resulted in a recovery of OD levels close to those obtained with FCS. This study is the first to demonstrate that aPE reactivity is dependent on the lipid concentration of the buffer component. These results highlight the need for standardization of aPE-ELISA for a better understanding of their clinical significance.

Background Anti-RNA polymerase III antibodies (anti-RNAP III) have been reported as potential immune markers of Systemic Sclerosis (SSc). Until now, their clinical use was disregarded because of technical difficulties to perform immunoprecipitation. Recently, ELISA kits became commercially available allowing an easy detection of anti-RNAP III. We intended to clarify the relevance of these antibodies in the diagnosis of SSc by ELISA detection. Methods The prevalence of anti-RNAP III was analyzed using two ELISA kits in 50 consecutive SSc patients from Marseilles in South of France. Controls included 66 patients with other systemic autoimmune diseases, 34 viral diseases and 50 healthy subjects. Positive results with at least one ELISA kit were controlled by immunoprecipitation which is the reference assay. Results In this study, positivity for anti-centromere and/or anti-topoisomerase I antibodies was observed in 84% of SSc patients. The prevalence of anti-RNAP III in SSc patients was 0% to 6% (3/50) depending on the ELISA kit and only 2% by immunoprecipitation. Concerning controls, two rheumatoid arthritis patients were positive using ELISA (6%), including one with immunoprecipitation confirmation. No anti-RNAP III was detected in systemic lupus erythematosus patients. Three blood donors and one viral disease control were positive using ELISA, but all were negative by immunoprecipitation. Conclusions Anti-RNAP III was rarely detected in a French population of SSc patients. Their prevalence was even lower than the one observed in rheumatoid arthritis controls. Therefore local immunologic profiles should be established before deciding a change in clinical practice for SSc immune screening.

Objective: To evaluate the prevalence of serum antibodies against hepatitis C virus and other infectious agents in a large cohort of well-characterized patients with autoimmune diseases (AID). Methods: We utilized 1322 sera from patients with 18 different AID and 236 sera from healthy controls from the same countries and with similar age and sex distribution. All sera were tested for the presence of serum anti-hepatitis C virus (HCV) antibodies as well as antibodies directed at other infectious agents and autoantibodies. Results: Anti-HCV antibody was detected in 115/1322 (8.7%) of patients with AID and 0.4% of matched healthy controls (P less than 0.0001). The prevalence of anti-HCV antibody was significantly higher in 7/18 different AID (i.e. cryoglobulinemia, mixed cryoglobulinemia pemphigus vulgaris, vasculitis, secondary anti-phospholipid syndrome, Hashimoto's thyroiditis, and inflammatory bowel disease) compared to controls. Patients with AID and serum anti-HCV positivity had an increased prevalence of antibodies against hepatitis B virus, Toxoplasma gondii and Cytomegalovirus as opposed to a lower frequency of serum autoantibodies. Conclusions: The enhanced prevalence of anti-HCV serum antibodies in AID may suggest a role for HCV in tolerance to breakdown, similarly to its established role in mixed cryoglobulinemia. This immune mediated effect does not rule out the role of other infectious agents. © 2009.

Antibodies for double-stranded DNA (anti-dsDNA) and chromatin represent specific markers of systemic lupus erythematosus (SLE).(1) To evaluate the analytical performance of a multiplexed bead assay (BioPlex 2200) for the simultaneous detection of anti-dsDNA and anti-chromatin antibodies, (2) to compare the results for anti-dsDNA with those obtained using Farr assay, and (3) to analyze the clinical relevance of these antibodies when applied to the follow-up of SLE patients with active nephritis.Hundred and five clinically characterized SLE patients and 96 healthy blood donors sera were analyzed by BioPlex 2200.Prevalence of these antibodies was significantly higher (p0.0001) in SLE patients than in controls (68 and 70% for anti-dsDNA and anti-chromatin, vs. 1% for both anti-dsDNA and anti-chromatin, respectively). If you consider a sample positive if either anti-dsDNA and/or anti-chromatin is positive, then the prevalence of these antibodies reached 78% (82/105) in SLE patients. For anti-dsDNA measurements, the kappa coefficient was 0.59 between BioPlex 2200 and Farr assay. Comparison between SLE patients with and without nephritis in a follow-up study showed that patients with active nephritis were associated with an increase of anti-dsDNA and anti-chromatin levels and a reduction of CH50, whereas no variation of antibody levels was observed in SLE patients without nephritis.Our results demonstrated a benefit of simultaneously measuring anti-dsDNA and anti-chromatin in SLE patients. The BioPlex 2200 achieved good analytical performances and proved to be a useful method for monitoring and diagnosing SLE.

The aim of this study is to get new insight into the relevance of IgG anti-prothrombin antibodies in patients with thrombosis and to determine whether human prothrombin alone (aPT) or complexed to phosphatidylserine (aPS/PT) should be preferentially used for measuring these antibodies by enzyme-linked immunosorbent assay (ELISA). To this end, prevalence of anti-prothrombin antibodies, their characteristics in terms of avidity and heterogeneity, and their relationship with anti-beta2 glycoprotein I antibodies (abeta2GPI) were studied in 152 patients with thrombosis. Patients were divided into two groups according to the presence or absence of antiphospholipid antibodies (aPL), called aPL+ or aPL-, respectively. In the aPL- group (n=90), the prevalence of anti-prothrombin antibodies was substantial (10%) but not significantly different from that of control (5%). In the aPL+ group (n=62), lupus anticoagulant (LA) or anticardiolipin antibodies (aCL) positive, 61% were positive for anti-prothrombin antibodies with no statistical difference between aPT and aPS/PT prevalence (42% vs. 55%, respectively). In the whole thrombotic population, 19% were only aPT and 34% only aPS/PT suggesting the presence of different antibodies. Absorption experiments confirmed the heterogeneity of aPT and aPS/PT. No difference in their avidity was demonstrated. From the aPL+ group, 60 were LA positive. Among them, 18% were negative for abeta2GPI and anti-prothrombin antibodies showing that the detection of these antibodies could not substitute for LA determination. In conclusion, our data show that the screening of the different anti-prothrombin antibodies is not warranted in the aPL+ group since these antibodies do not provide additional information compared to aCL, LA and/or abeta2GPI measurement. Nevertheless, the substantial prevalence of anti-prothrombin antibodies in the aPL- group should be further explored in a large prospective study.

The measurement of autoantibodies specific for double-stranded DNA (anti-dsDNA) is a useful tool for the diagnosis and the prognosis of systemic lupus erythematosus (SLE). A new quantitative enzyme-linked immunosorbent assay (ELISA), ORG anti-dsDNA, is recently available for the determination of anti-dsDNA antibodies. The aim of this study was to evaluate the clinical performance of this new assay in a cohort of SLE patients. Seventy-five sera from SLE patients were tested by two methods for anti-dsDNA determination, ORG anti-dsDNA, and EliA anti-dsDNA. Normal controls were 60 sera from healthy subjects. Moreover, 37 sera from patients with non-SLE connective tissue diseases were tested in parallel. The levels of complement components (C3, C4, CH50) were measured by nephelometry. From SLE patients, 91% were positive against 9% in non-SLE patients and 2% in healthy subjects. The sensitivity, specificity, and Youden test for SLE were 90%, 98%, and 88%, respectively. The Yule test (1%) indicated a close association with the disease. The comparison with EliA anti-dsDNA showed a moderate concordance between the two tests in the group of SLE (kappa = 0.51) and a good concordance in the non-SLE group (kappa = 0.89). A significant inverse correlation was found with complement components levels, biological markers associated with disease activity. Our results show this new assay as sensitive and specific for the diagnosis of SLE. Moreover, the correlation with markers associated with disease activity makes it promising for clinical use.

SummaryThe aim of this study was to evaluate the agreement in assay results between commercial kits for the measurement of anti- β2glycoprotein I antibodies. Ten manufacturers provided one IgG and one IgM kit to three testing centres. Samples from patients with primary (n=13) or secondary (n=3) antiphospholipid syndrome (APS), from lupus patients without APS features (n=6) and from normal individuals (n=2) were tested in the three centres according to manufacturers’ instructions. Dilutions in normal serum of a pool made from positive patients’ samples (Forum Calibrators) and dilutions of humanized monoclonal antibodies (MoAbs) were used as additional calibrators. The calibration curves obtained with each calibrator differed widely between kits. The rate of positivity of patients’ samples varied from 7 to 16 for IgG and from 2 to 17 for IgM, depending on the kit. Perfect agreement occurred in 12/22 samples for IgG and 5/22 samples for IgM. Samples from normals were found negative by all kits. Between kits, cutoff values varied up to five fold when expressed in Forum Calibrators arbitrary units and up to three fold when expressed in MoAbs equivalents. Examination of discrepant samples indicated that about half of the discrepancies, scoring 8:2 and 9:1, involved the same few kits. In highly discrepant samples, some kits appeared as high responders as compared to others. In conclusion, with the exception of a few kits, agreement in assay results was acceptable. In conclusion, additional efforts are however necessary, especially concerning the way to assess the cutoff point and the adoption of a reference calibrator, in order to improve standardization of the assays.

Two-hundred ninety five patients with the antiphospholipid syndrome (APS) were studied for the presence of antibodies against six anti-beta2GPI-related peptides Abs. The prevalence of a wide spectrum of clinical and laboratory parameters of APS was evaluated in all patients, and correlated with the presence of each anti-beta2GPI peptide antibody. The rates of the various antipeptides Abs ranged from 18.0 to 63.7%. Altogether, 87.1% of the patients had antibody reactivity against at least one of the six beta2GPI-related peptides. A high degree of simultaneous reactivity against several beta2GPI-peptides was found. Positive and negative correlations were found between several antipeptides Abs and the rates of thrombosis and fetal loss. Our results point to a heterogeneous activity of antiphospholipid Abs in APS patients, directed, often concurrently, against various epitopes of the beta2GPI molecule. Evaluation of APS patients for the presence of specific antipeptides Abs may be of a value in predicting the risk for future thrombotic and obstetrical complication, as well as for specific therapeutic purposes.

The usefulness of immumoglobulin (Ig) A antibodies to gliadin (AGA-IgA) in addition to IgA anti-endomysium and tissue transglutaminase antibodies was evaluated in 4122 children younger than 2 years with a suspicion of coeliac disease (CD). Eight percent (312/4122) displayed IgA anti-endomysium and/or IgA anti-tissue transglutaminase, whereas 2.1% (85/4122) displayed only AGA-IgA. Clinical data were obtained for 62 of 85 children with isolated AGA-IgA, and 33 children underwent a duodenal biopsy. Histologically proven CD was established for 5 patients, whereas 57 children were diagnosed to experience other diseases. The systematic detection of AGA-IgA using native gliadin conferred no additional diagnostic benefit for the diagnosis of CD in children younger than 2 years of age, except for rare cases.

To determine the prevalence of autoantibodies in patients with epilepsy and to find a possible relationship between antinuclear antibodies (ANA) and/or anticardiolipin (aCL) antibodies and epilepsy.One hundred sixty-three consecutive, unselected patients followed at the Centre Saint-Paul, a French medical center specialized in epilepsy, were included in the study. IgG and IgM class aCL antibodies were measured by an enzyme-linked immunosorbent assay (ELISA). IgG class ANA was detected by an indirect immunofluorescence technique with Hep2 cells as the substrate. Sera from 100 healthy blood donors, matched for age and sex, were used as controls.In 31 sera, IgG class a aCL antibodies were detected at a value higher than 17 GPL unit (19%, P = 0.0003); 10 of them had a value higher than 35 GPL unit. IgM class aCL antibodies were not detected at a significant value. For 6 of the 31 sera, there was a beta 2-glycoprotein I dependence. None of the patients with aCL antibodies in the serum had a past history of deep venous or arterial thrombosis. ANA were detected in the sera from 41 patients (25%, P0.005). The presence of autoantibodies in the serum was not statistically dependent on the type of epilepsy, the kind of antiepileptic drug, or the age or sex of the patients.Our study suggests that there is a relationship between epilepsy and aCL antibodies, even in the patients without systemic lupus erythematosus. Large prospective studies are needed to define the role of the aCL antibodies and ANA in pathophysiology of epilepsy.

To investigate the prevalence, significance, and specificity of IgG isotype anti-β2-Glycoprotein I antibodies (a-β2-GPI) in antiphospholipid syndrome (APS), we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of IgGa-β2-GPI and tested sera from 61 patients with autoimmune disorders (AID), 39 patients with APS and 22 patients with systemic lupus erythematosus without APS, 139 patients with various infectious diseases (hepatitis C virus infection, human immunodeficiency virus infection, Q fever, Mediterranean spotted fever, syphilis) and 97 healthy control subjects. Using irradiated plates coated with human β2-GPI, we showed that in the sera of patients with AID, optical densities from the coated wells were significantly higher than those from the noncoated ones (p = 0.0001). In this assay, intra-assay and inter-assay variation coefficients ranged between 4% and 10%. Clinical evaluation showed that IgG-a-β2-GPI were found in 23 of 61 patients with AID but in only one patient with an infectious disease. The presence of the IgG-a-β2-GPI in association with APS (p = 0.005) was statistically significant with high specificity (98%) and positive predictive value (87.5%) but with low sensitivity (54%), and was significantly associated with venous thrombosis (p = 0.0025). In addition, the IgG-a-β2-GPI levels were highly correlated with those of anticardiolipin antibodies (aCL) (p < 0.001). In contrast to a-β2-GPI, aCL were found with a high prevalence (40%) in patients with infectious diseases. Because of their high specificity, anti-β2-GPI antibodies appear to be useful tools in the evaluation of the risk of APS. However, because of their low sensitivity, their detection needs to be associated with that of aCL.

A Task Force of experts in the Antiphospholipid Syndrome (APS) field met prior to the 13th International Congress on Antiphospholipid Antibodies and addressed a number of critical questions regarding the antiphospholipid antibody (aPL) tests included in current international consensus criteria for the classification of APS (anticardiolipin antibodies (aCL), anti-β2glycoprotein I antibodies (aβ2GPI), and lupus anticoagulant (LA) test, namely, the “criteria” aPL tests). The Task Force was divided into the following three subgroups: (1) aCL and aβ2GPI tests, (2) LA test, and (3) the role of aPL as thrombotic risk factors. Subgroup 1 reviewed and critiqued the current state of aCL/aβ2GPI testing and recommended developing international consensus guidelines for performance of these assays and establishing universal units of measurement for anti-β2GPI. Subgroup 2 reviewed recent guidelines for LA testing published by the International Society of Thrombosis and Haemostasis Standardization Subcommittee (ISTH-SSC) on Lupus Anticoagulants and Phospholipid-dependent Antibodies; the subgroup recommended continued close collaboration with ISTH-SSC to address remaining issues, including the role of TCT and PT in LA testing and the validity of integrated test systems that lack mixing studies. Subgroup 3 emphasized the need for more data on the risks associated with aPL and recommended collaborative studies using existing large, population-based, prospective cohorts with available data on thrombosis and/or pregnancy outcomes. Such studies should include a comprehensive panel of current and newer aPL assays, assess the effect of antibody titer, analyze the risks associated with combinations of aPL, and use state-of-the-art statistical approaches for assessment of risk factors.

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity associated with the presence of laboratory criteria such as antibodies directed towards cardiolipin or beta(2)-GPI and lupus anticoagulant. Recently, the term "seronegative APS " has been proposed to define patients with the typical clinical manifestations but with negative serologies. One explanation for such a context could be that some APS patients may only have antiphospholipid antibodies (aPL) other than the admitted laboratory criteria. This review is focused on antibodies directed against phosphatidylethanolamine (aPE) and underlines the interest of their investigation in the different clinical manifestations of APS.

The aim of this prospective study was to assess the prevalence of antiphospholipid antibodies (aPL) in women who had undergone in vitro fertilization (IVF) and the relationship between aPL and IVF outcome. A total of 101 infertile women with at least three unsuccessful IVF attempts were consecutively included in this study. Samples were collected in the follicular phase of a spontaneous ovarian cycle 2 months after the last ovulation induction treatment. Age-matched healthy fertile women (n = 160) were included as controls. All were evaluated for the presence of lupus anticoagulant (LA), antibodies (IgG, IgA, IgM) to cardiolipin (aCL), beta 2 -glycoprotein I (aβ 2 GPI), and phosphatidylethanolamine (aPE). Out of the 101 infertile women, 40 were persistently positive for aPL, showing a prevalence significantly higher than in controls (39.6% versus 5%, P < 0.0001). Among aPL, aPE were found with a significantly higher prevalence compared with LA, aCL, and aβ 2 GPI (67.5% versus 0%, 15%, and 40%, respectively). Interestingly, aPE were found in 70% of the cases in the absence of the other aPL. The predominant isotype of aPL was IgA, in particular for aβ 2 GPI. Finally, no significant association was found between the presence of aPL and IVF outcome. This prospective study shows aPE as the most prevalent aPL in infertile women and IgA as more common than IgG and IgM. However, our results do not support an association between aPL and IVF outcome.

SummaryThe aim of this study was to evaluate the heterogeneity of IgG-anti- β2 -glycoprotein I antibodies (IgG-aβ2 GPI) as regarding their reactivity pattern against different sources of human β2 GPI,their avidity and their association with clinical events of antiphospholipid syndrome (APS).Three thousand six hundred and eightyfour consecutive patient sera were routinely tested for IgG-a β2 GPI over 1 year using an in-house ELISA with 2 different commercial preparations of human purified β2GPI. Of the 340 sera found positive, all those clinically documented were included in this study; 61 were positive with only one preparation (S1) and 59 with both (S2).The results of ELISA were confirmed by Western blot. Heterogeneity was stressed by testing sera with a human recombinant protein and 3 β2 GPI-related peptides. No contribution of glycosylation in the binding to β2GPI was found.The avidity indices for each protein were significantly higher in S1 than in S2 (p = 0.0021). S2 were more associated with antiphospholipid antibodies than S1 (75% versus 21% ; p

According to international criteria, autoimmune hepatitis (AIH) type 1 is characterized by the presence of antinuclear or anti-smooth muscle antibodies (SMA) with F-actin specificity. SMA have been found in 85% of AIH patients, but are not specific to this disease, and anti-F-actin specificity is not always verified when SMA are detected. The objective of this study was to determine the diagnostic value of anti-F-actin antibodies in a large population. A multicenter study involving 12 clinical centers was performed. Patients were selected on the basis of the presence of F-actin SMA detected by indirect immunofluorescence (IIF) on rat liver-kidney-stomach sections and was confirmed by IIF on Hep2 cells treated with colchicine, or F-actin dot-blot. The clinical status of patients was determined from their medical records. One hundred sixty-eight patients were included: 76% women, 24% men; mean age of 45 years (range, 2-88 years), with a bimodal age distribution. Sixty percent had AIH type 1, and 40% had another disease. In the group of women younger than 25 years, 90% had AIH type 1. Other pathologies associated with antiactin were other liver diseases (19%), including viral hepatitis C (7%), and non-liver diseases (21%), including connective tissue diseases (12%). Antibody titers were higher in AIH than in other diseases. Antiactin antibodies are of major diagnostic value in AIH, especially in young women; they may be found in other disease settings, but mostly at low levels.

Antiphospholipid ELISAs are part of the Antiphospholipid Antibodies Syndrome classification criteria, having the same diagnostic value as lupus anticoagulant. However, sometimes their results appear scarcely meaningful especially when wide metanalyses studies are performed, probably because of their well-known inter-laboratory variability. The application of a common protocol was shown to improve the test reproducibility, but this observation did not have any influence on the routine performances. After discussion among experts at the European level, we identified four conditions named "minimal requirements" considered useful to decrease the inter-laboratory variability: (1) to run the samples in duplicate; (2) to determine the cut off level in each laboratory analysing at least 50 samples from normal subjects, possibly age- and sex-matched with the patient population usually attending the Centre; (3) to calculate the cut-off level in percentiles; (4) to use stable external control in the tests. A collaborative study involving 36 European centres proved that the use of monoclonal anti-beta2 glycoprotein I antibodies, HCAL (IgG) and EY2C9 (IgM) as standards, can help to reduce the inter-laboratory coefficient of variation both in anticardiolipin (aCL) and anti-beta2GPI (anti-beta2 glycoprotein I) ELISA. Therefore, we propose HCAL and EY2C9 as external controls, but other monoclonal or polyclonal preparations may be considered. During an interactive workshop held last May in Italy, 16 companies producing these tests agreed to consider the introduction of the "requirements" in their products. We suggest to adopt these "requirements" particularly in clinical studies, in order to compare more easily the literature data.