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The recent paper by Pisetsky et al,1 which already elicited some debate,2 3 reported data on the use of antinuclear antibodies (ANA) detection in patients with systemic lupus erythematosus (SLE) in the real life and raised concerns on the usefulness of the assays because of the significant percentage of samples tested negative for ANA in spite of the use of well-validated assays and the inclusion of patients with an established diagnosis. This finding may have negative implications both for the correct classification and the inclusion in clinical trials.
We report here a multicentre study carried out by an Italian interdisciplinary group (Forum Interdisciplinare per la Ricerca sulle Malattie Autoimmuni) for the validation of new automated reading systems for ANA detection by indirect immunofluorescence on HEp-2 cells (HEp-2 IFA). Ninety-one patients with well-established SLE (18/91 men, mean age 40±11 years)4 were tested for ANA, anti-extractable nuclear antigens (ENA) and anti-double stranded (ds)-DNA. Manual HEp-2 IFA (with different HEp-2 commercial preparations including HEp-2000) was performed at the recruiting centre and then sent to two core labs where three different automated ANA reading systems were used with the manufacturers’ cell substrate (1:80 screening dilution). Moreover, all the samples were tested by two commercial connective tissue disease (CTD) screening solid phase arrays (SPA). We found almost a perfect agreement between manual and automated ANA reading. As expected, SPA displayed a lower sensitivity (table 1).5 By testing all the samples with both HEp-2 IFA and SPA we reached 100% sensitivity. One sample only tested positive for DFS70 but the positivity was associated with anti-ENA antibodies, a combination that can be found in systemic autoimmune rheumatic diseases.6 The detection of antibodies against ENA and dsDNA was performed at the recruiting centres by using different commercial kits. The percentages of patients positive for anti-ENA and anti-dsDNA were similar to those of the Pisetsky’s series making the two cohorts comparable.
The results of our study show that: (1) ANA is a hallmark of established SLE; (2) ANA detection by different commercially available kits is reliable in a multicentre setting and the variability linked to the single operator does not seem to be a critical issue. The CTD screening SPAs display a lower sensitivity than HEp-2 IFA likely due to the limited number of autoantigens, however they can offer an additional diagnostic value when carried out together with IFA.5
Contributors FP contributed to data acquisition, analysis and interpretation. FP, MOB and PLM gave substantial contributions to the conception of the work and drafting of the letter, had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The FIRMA Study Group provided patients, collected data and critically reviewed the study proposal. All the contributors read and approved the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Ethics Committee of Istituto Auxologico Italiano, 22-07-2010.
Provenance and peer review Not commissioned; internally peer reviewed.
Collaborators Claudia Alpini, Alessia Alunno, Maria Romana Bacarelli, Andrea Doria, Sergio Finazzi, Franco Franceschini, Roberto Gerli, Anna Ghirardello, Luigi Giovannelli, Maria Grazia Giudizi, Milvia Lotzniker, Paola Migliorini, Gabriella Morozzi, Federico Pratesi, Antonella Radice, Valeria Riccieri, Amelia Ruffatti, Paola Sabatini, Giandomenico Sebastiani, Renato Alberto Sinico, Angela Tincani, Marta Tonello.
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