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Objectives: Detection of antinuclear antibodies (ANAs) plays an important role in the diagnosis of systemic autoimmune rheumatic disease (SARD). Our goal was to evaluate the diagnostic accuracy of three commercially available enzyme-linked immunosorbent assay (ELISA) kits and one chemiluminescent assay for ANA detection, using the clinical diagnostic as the reference standard.Methods: We evaluated serum samples from 143 patients with an established diagnosis of SARD (group 1), 166 patients with infectious diseases and other rheumatic diseases for which the ANA test is not useful in diagnosis (group 2), and 89 outpatients with suspicion of SARD (group 3).Results: The sensitivity for ANA HEp-2, calculated in group 1, was 87.4% and varied between 62.9% and 90.0% for other tests. The specificity for ANA HEp-2, calculated in group 2, was 72.3% and varied between 45.2% and 90.4% for other tests. In group 3, the negative predictive value for ANA Hep-2 was 92.5% and varied between 89.3% and 100% for other tests.Conclusions: Some ELISA kits have comparable or superior diagnostic sensitivity to ANA HEp-2 and could be used as an alternative method for ANA screening, therefore allowing the immediate report of the results with fewer false negatives than ANA HEp-2. Owing to the lower specificity, ELISA-positive samples should be submitted to ANA HEp-2 for confirmation of results.