ANA Testing From Background Prevalence to Pretest Probability: A Recommendation for Routine Practice

Antinuclear antibodies (ANA, now correctly designated as the HEp-2 immunofluorescence test) are a commonly ordered laboratory test. In one regional laboratory that was asked to provide figures, the number of ANA tests carried out per year doubled from 2019 to 2025. This review provides a detailed explanation of the utility of ANA testing and the interpretation of positive findings.

The positive predictive value (PPV) of a positive ANA test and its clinical interpretation were studied with reference to the background prevalence of ANA and the prevalence of ANA-associated rheumatic diseases, e.g., systemic lupus erythematosus (SLE) and systemic sclerosis. This narrative review is based on publications that were retrieved by a selective search on the topics of ANA prevalence and the diagnostic value of ANA testing and its clinical indications, with special attention to population-based and clinical studies and pertinent consensus recommendations.

The estimated overall ANA positivity rate (background prevalence) ranges from 13% to 33% in population-based studies. The PPV strongly depends on the cohort studied, the clinical pretest probability of an ANA-associated disease, and the antibody concentration (titer) required for a positive test. In a rheumatological referral cohort, the PPV of the test was 2.1% for SLE and 9.1% for ANA-associated rheumatic diseases. Large cohort studies have shown that relatively few ANA-positive persons have an ANA-associated rheumatic disease; in a Korean study, for example, the figure was 4.7%. Thus, a positive ANA test alone does not establish the diagnosis. Its clinical relevance depends on the constellation of disease manifestations, antibody titer, fluorescence pattern, and (where applicable) disease-specific autoantibodies.

ANA should only be measured if there is a well-founded clinical suspicion of an ANA-associated disease, not as a general screening test for patients with nonspecific symptoms such as isolated fatigue, pain of undetermined origin, or pain in a single joint. Positive test findings require clinical contextualization first and foremost, rather than automated follow-up laboratory testing. Serial ANA tests are not suitable for determining the activity level of a rheumatic disease or for monitoring the effects of treatment. The symptom- and test-finding-based approach following the Choosing Wisely recommendations can help minimize the risk of overdiagnosis, needless consultations, and unnecessary follow-up.