Antinuclear antibodies (ANAs) are autoantibodies that target several nuclear and cytoplasmic antigens. ANAs were first described more than a half-century ago and remain the most sensitive serologic marker for screening, diagnosing, and classifying patients with suspected systemic autoimmune rheumatic diseases (SARDs) including but not limited to systemic lupus erythematosus (SLE), Sjögren’s syndrome (SjS) and scleroderma [1]. Depending on the ANA screening results, specific autoantibodies (i.e., extractable nuclear antigen (ENA) antibody and anti-double stranded DNA (anti-dsDNA) testing may be clinically indicated. According to international recommendations, the testing for ANA is indicated only if a patient’s clinical presentation and history show signs or symptoms suggestive of SARDs. Yet, inappropriate autoimmune serology testing of ANA remains a global healthcare concern [1, 2]. The misuse and unnecessary repeats of ANA and specific autoantibodies can cause suboptimal patient care and increased costs without adding clinical value.

Determining the root cause for inappropriate test ordering and repeating is important for designing interventions. Thus, evaluating ANA, ENA and anti-dsDNA test ordering patterns and the frequency of repeats can help identify appropriate test utilization plans. Recently, a Canadian study showed that over 60% of ANA tests were ordered by family physicians with only 11% ordered by rheumatologists [2]. In this study, only 21.5% of the total ANA test orders were positive and 28% were repeat tests. Of the repeat tests, 50% were ordered within 12 months after an initial positive result, often by following up rheumatologists. These findings underscore the need for collaborative engagement of clinical laboratorians with rheumatologists to implement testing algorithms and to educate primary care practitioners on appropriate ANA and specific autoantibodies test ordering and frequency of repeats if clinically indicated.

Some clinical laboratories have proposed and implemented reflex testing algorithms to facilitate optimal ANA, ENA and anti-dsDNA test utilization [1]. Reflex testing algorithms involve the automatic addition of subsequent tests based on prespecified rules and the initial test result.

Although implementing an evidence-based reflex testing algorithm is relatively straightforward, it is challenged by test-specific considerations. For example, ANA testing can be performed by indirect immunofluorescence (IIF) or solid phase assays (SPAs). ANA-IIF is the gold standard for ANA testing due to its high sensitivity for SARDs (e.g., >95% for SLE diagnosis) as well as the ability to detect organ-specific autoimmune diseases (e.g., autoimmune liver diseases). Of note, ANA-IIF analysis can be either done manually or automated with assisted software interpretation. However, both methods are labor intensive, as the second requires a technologist to verify the accuracy and titer of the suggested pattern. Moreover, ANA-IIF interpretation is subjective, requires extensive expertise and has limited specificity. On the other hand, ANA-SPAs have higher specificity relative to ANA-IIF, objective interpretation and reduced turnaround time. However, ANA-SPAs contains a limited number of antigens which limit the assay overall sensitivity [1].

ANA reflex testing algorithms are implemented by clinical laboratories to achieve two main goals (1) to reduce the labor-intensive use of ANA-IIF and (2) to guide the appropriateness of ENA/dsDNA autoantibodies testing. Currently there are two approaches proposed for ANA and ENA/dsDNA reflex testing [1]. The first approach uses ANA-SPAs as initial screening and positive samples are reflex to ANA-IIF. Further, the positive ANA-IIF samples are reflexed to ENA/dsDNA autoantibodies testing based on the ANA-IIF titer and pattern as well as the clinical indications. When compared to an algorithm starting with an ANA-IIF, this three-step approach resulted in a reduction in the ENA/dsDNA autoantibodies reflex rate from 64% to 36%, reduction in technologist time by 33% and overall increase in the sensitivity of ENA/dsDNA autoantibodies testing (98%). The second approach uses an ANA-IIF for initial screening with positive sample reflexing to specific ENA/dsDNA autoantibodies testing based on the screening titer and patterns of ANA-IIF as well as the clinical indications. This approach led to significant reduction in inappropriate ENA/dsDNA testing and can be used to drive automated reflex testing for specific autoantibodies based on the ANA-IIF patterns identified [1].

Finally, before implementing reflex testing it is crucial that clinical laboratorians communicate and collaborate with clinical stakeholders and operations staff. This is to evaluate the impact of the proposed reflex algorithm on diagnostic accuracy, laboratory workflow, turnaround time, laboratory information system limitations, and overall cost of care. Lastly, implementing effective educational plans with clinical stakeholders prior to introducing changes is paramount to effective test utilization.


  1. Sepiashvili L, Kenyon SM. Clinical, Methodological, and Practical Considerations for Algorithmic Testing in Autoimmune Serology. The Journal of Applied Laboratory Medicine 2022;7:268-80.
  2. Lake S, Yao Z, Gakhal N, Steiman A, Hawker G, Widdifield J. Frequency of repeat antinuclear antibody testing in Ontario: a population-based descriptive study. Canadian Medical Association Open Access Journal 2020;8:E184-E90.