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| Antinuclear antibodies (ANAs) are a diverse group of autoantibodies that recognize nuclear macromolecules and their complexes. ANAs represent key biomarkers in the evaluation of rheumatic diseases, most prominently systemic lupus erythematosus (SLE), and ANA testing is commonly performed in the clinical setting. In addition, ANA testing is now used to assess eligibility for participation in clinical trials of new therapeutic agents for SLE. ANAs can be assayed by various techniques, with the fluorescent ANA assay often viewed as the gold standard. Whereas a positive ANA test represents a classification criterion for SLE, up to 20-30% of the healthy population, depending on the assay used, is positive for an ANA, complicating the use of this test for diagnosis or the detection of preclinical autoimmunity. Furthermore, ANAs might be expressed in SLE less commonly than often thought. This Perspectives article discusses important questions about the use of ANA testing in both the clinical and research settings.