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We read with great interest the multicentre study of Damoiseaux et al 1–3 on the detection of antineutrophil cytoplasmic antibodies (ANCA). ANCAs are important laboratory markers to support the diagnosis of ANCA-associated small vessel vasculitis (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Traditionally, laboratories screen for ANCA by indirect immunofluorescence (IIF) and IIF positive samples are further evaluated for antibodies to proteinase 3 (PR3) or myeloperoxidase (MPO) by specific immunoassays. Such diagnostic algorithm is based on an international consensus statement on ANCA testing issued in 1999.4 Over the last two decades, the diagnostic performance of immunoassays has significantly improved. The recent multicentre study showed a …

Objective To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format. Methods Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods. Results Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P = 0.0047). Conclusion Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

Primary biliary cirrhosis (PBC) is a chronic disease characterized by portal inflammation and necrosis of small intrahepatic bile ductules (1). PBC is an irreversible condition, and destruction of the bile ductules leads to progressive cholestasis and fibrosis, and may eventually lead to the development of cirrhosis. PBC is most likely an autoimmune disorder (2), and anti-mitochondrial antibody (anti-M2) has been a diagnostically very useful marker: several studies have reported a positivity rate for anti-M2 of >95% in biopsy-confirmed PBC patients (3). The major mitochondrial antigen was identified as the 74-kDa E2 subunit of the pyruvate dehydrogenase complex (PDC), a member of the 2-oxoacid dehydrogenase complex family (4). The traditional technique for the detection of anti-M2 is immunofluorescence (5), but recently new serological assays, such as ELISA, immunoblotting, and enzyme inhibition (EI), have been developed (6)(7). A miniaturized EI assay for performance on microtiter plates has already been described (8)(9). In this study we evaluated a new commercially available and completely automated EI assay manufactured by Trace Scientific (Victoria, Australia) and compared it to the immunofluorescence and ELISA techniques performed in our laboratory. The TRACE enzymatic procedure is a unique method based on the PDC inhibitory properties of the principal anti-74-kDa antibody. PDC catalyzes the following reaction: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Pyruvate}{+}\mathrm{NAD}^{\mathrm{{+}}}{+}\mathrm{CoA}\ \begin{array}{l}\mathrm{PDC}\\{\rightarrow}\end{array}\ \mathrm{Acetyl-CoA}{+}\mathrm{NADH}{+}\mathrm{H}^{{+}}{+}\mathrm{CO}_{2}\] \end{document} In the absence of anti-M2, this reaction will proceed at the maximal rate after the addition of a serum sample. If a sample contains anti-M2, the PDC activity will be inhibited, greatly reducing the rate of reaction, typically to

Anti-Saccharomyces cerevisiae antibodies (ASCA), anti-nuclear associated anti-neutrophil antibodies (NANA) and antibodies to exocrine pancreas (PAB), are serological tools for discriminating Crohn's disease (CrD) and ulcerative colitis (UC). Like CrD, coeliac disease (CoD) is an inflammatory bowel disease (IBD) associated with (auto) antibodies. Performing a multicenter study we primarily aimed to determine the performance of ASCA, NANA and PAB tests for IBD diagnosis in children and adults, and secondarily to evaluate the prevalence of these markers in CoD.Sera of 109 patients with CrD, 78 with UC, 45 with CoD and 50 healthy blood donors were retrospectively included. ASCA, NANA and PAB were detected by indirect immunofluorescence (IIF).ASCA+/NANA- profile displayed a positive predictive value of 94.2% for CrD. Detection of ASCA was correlated with a more severe clinical profile of CrD and treatment of the disease did not influence their serum levels. ASCA positivity was found in 37.9% of active CoD. PAB were found in 36.7% CrD and 13.3% CoD patients and were not correlated with clinical features of CrD, except with an early onset of the disease. Fifteen CrD patients were ASCA negative and PAB positive.ASCA and PAB detected by IIF are specific markers for CrD although their presence does not rule out a possible active CoD. The combination of ASCA, NANA and PAB tests improves the sensitivity of immunological markers for CrD. Repeating ASCA, NANA, and PAB testing during the course of CrD has no clinical value.

This article gives a summary of the history of vasculitis through early and past descriptions. Known, reported and depicted since time immemorial those diseases are quite polymorphous, depending on type and localization of the affected vessels. Clear classification criteria were finally laid down at the end of the XXth century giving a new impulse as well to the detection and the identification of specific biological markers as to the way to treat effectively those severe diseases.

Fodrin, an actin binding protein found in the cytoskeleton of most eukaryotic cells, would seem to be an important organ-specific autoantigen in Sjogren’s syndrome (SS). Fodrin is, moreover, detected in the salivary glands of patients with primary SS (pSS) but not in controls. It was thus recently claimed that antibodies against α-fodrin are a sensitive and specific serological marker for pSS.1 In this study we investigated the prevalence of autoantibodies against α-fodrin in patients with pSS, as compared with healthy subjects and patients with other autoimmune diseases. The study group included 107 patients with well defined pSS (mean age 57.5 years), 32 patients with systemic lupus erythematosus (SLE; mean age 40.1 years), 43 patients with rheumatoid arthritis (RA; mean age 59.0 years) with no signs of secondary SS, and 48 healthy blood donors (mean age 84.3 years). A diagnosis of SLE, RA, or pSS was …

The case of a patient who developed an acute dermatomyositis-like syndrome upon infection by Toxoplasma gondii is reported. Emphasis is given on the chronology of the anti-Toxoplasma IgM and IgG antibody rise, the muscle enzyme movements, the antinuclear antibody development in comparison to the clinical symptoms and their follow-up after Toxoplasma treatment. The close monitoring of the sequential clinical and biological pattern allows insights into the infectious and auto-immune diseases presented by this patient. Taking into account other reported cases and the clinical presentation of this patient some conclusions and recommendations are inferred.

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease. Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing. Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases. Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

Background The nucleosome is considered as a major antigen in SLE and nucleosome-specific antibodies (Ab) seem to be the most specific and the earliest markers of SLE. Objectives To evaluate different methods of detecting anti-nucleosome Abs and determine the diagnostic value of these Abs in recently active SLE. Methods A 2 year (1998–99) prospective multicenter study in 13 European Immunopathology Centres (Groupe d’Etude de l’Auto-Immunite). Patients: (i) 55 cases of recently active SLE (evolution IgG anti-nucleosome Abs were detected by ELISA or immunodot using different nucleosome preparations. IgG anti-ds DNA Abs were detected by ELISA (calf thymus and human recombinant plasmid DNA). Results 34/55 SLE patients had anti-nucleosome Abs reacting with 4 kits. 16/55 SLE patients had anti-nucleosome Abs reacting with 1 to 3 kits. 5/55 SLE patients were negative for anti-nucleosome Abs. 5/50 patients with other autoimmune diseases had anti-nucleosome Abs. Conclusion 50/55 (91%) of patients with recently active SLE had anti-nucleosome Abs (by at least 1 method), 46/55 (84%) also had anti-ds DNA Abs (by ELISA). The sensitivity and the specificity of the anti-nucleosome Abs for SLE depend on the method and the nucleosome preparation.

Anti-filaggrin autoantibodies (AFA) are highly specific markers for rheumatoid arthritis (RA) and can be detected by immunoblotting using human epidermal protein extracts. Furthermore, it was demonstrated that citrullination of the filaggrin epitopes is crucial for epitope recognition and that citrullinated peptides are also recognized by these specific autoantibodies. Based on these data, a line immunoassay (LIA) was developed containing as individual markers in vitro citrullinated recombinant filaggrin and two citrullinated synthetic peptides. Firstly, a comparison was made between this prototype LIA and the AFA blot using natural filaggrin. A blind serum panel consisting of 25 early RA, 25 longstanding RA, and 25 disease controls was selected. Results showed a similar performance of both tests at a specificity level of 95%, while the LIA proved significantly better (P = 0.035) than the AFA blot at 99% specificity. At the latter specificity level, 2 out of 17 RF negative samples were retrieved on LIA but not on Western blot. The LIA was further evaluated on sera obtained from 335 RA patients and 254 patients with non-RA rheumatological disorders in a retrospective study. The overall sensitivity of the LIA including three markers (LIA3) was 65.1% versus 61.8% if only the reactivity towards the citrullinated peptides was considered (LIA2). The specificity of LIA3 was 97.6% versus 98.4% for LIA2, which correlates with an estimated positive predictive value (PPV) of 87.3% for LIA3 and 90.7% for LIA2. Thirty-seven percent (30/81) of the RF-negative RA samples proved LIA2-positive, while 52 out of 55 RF positive control samples were negative for anti-filaggrin. Higher specificity and sensitivity was obtained for LIA2 in comparison with anti-RA33 immunoblot, whereas good agreement could be observed with anti-keratin antibody (AKA) testing. In conclusion, anti-filaggrin autoantibodies can be readily detected by a LIA test based on citrullinated peptides, resulting in a high specificity and hence high PPV for RA. The assay can serve as a user-friendly alternative for AKA immunofluorescent and immunoblot techniques. Together with the RF complementarity, this test provides a valuable tool in the differential diagnosis of RA.