Antineutrophil cytoplasmic antibody (ANCA) testing is central to diagnosing ANCA-associated vasculitides (AAV), which include microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis. International testing guidance evolved from a 1999 consensus that prioritized indirect immunofluorescence (IIF) screening with antigen-specific follow-up, to a 2017 consensus that recommended antigen-specific immunoassays (for PR3 and MPO) as the preferred first-line approach.
The 2017 recommendations were based largely on data from tertiary referral centres with relatively high AAV prevalence in tested populations. Because prevalence affects positive predictive value (PPV) and pre-test probability, the authors evaluated how the consensus approach performs in a large teaching hospital — a secondary care setting with lower AAV prevalence — and tailored testing strategy recommendations accordingly.
The study retrospectively included all patients screened for ANCA between 2012 and 2023 at the authors’ centre. Clinical data were collected from records. IIF was performed using the EUROPLUSTM Granulocyte Mosaic 25 IIF platform. MPO and PR3 antibodies were measured using the ImmunoCAP®250 antigen-specific immunoassay. Statistical analyses were conducted in R. Three temporal periods were distinguished for practice patterns: the era following the 1999 consensus, a period validating automated read-out, and the period following the 2017 consensus.
A total of 5,518 patients were tested for ANCA over the study period. Of these, 286 patients (5.2%) had a positive ANCA result. Among ANCA-positive patients, 63% did not have a diagnosis of AAV, indicating that in this secondary care population the majority of ANCA positivity did not reflect AAV.
Non-AAV causes among ANCA-positive patients included other autoimmune diseases, malignancies, infections and drug-induced ANCA. The authors emphasize that vasculitis can also occur in many of these conditions, complicating differentiation from primary AAV and contributing to the non-specific nature of ANCA positivity.
When used as a first-line test in this setting, both IIF and antigen-specific ELISA assays provided good negative predictive value (NPV). However, antigen-specific ELISA showed higher positive predictive value and higher specificity compared with IIF when used first. The authors report that performing a second, confirmatory test increases specificity and thereby PPV. These findings echo the rationale behind the 2017 consensus while adding context for a lower-prevalence secondary care population.
Measured MPO and PR3 concentrations were, on average, higher in patients with AAV than in those without AAV; however, the concentration ranges overlapped substantially. The authors therefore caution against relying solely on antibody level cutoffs for definitive diagnosis, and they reiterate the consensus recommendation to consider a second test when antibody concentrations are low or when clinical suspicion persists despite initial negative results.
A figure in the study illustrates how disease prevalence in the tested population strongly influences PPV while NPV changes little. Using sensitivity and specificity estimates derived from the EUVAS multicentre data for the ELISA (sensitivity ≈86.5%, specificity ≈96.9%), the authors show that PPV rises markedly as prevalence increases (for example, from roughly 35% at 2% prevalence to about 88% at 20% prevalence). This underlines why test strategy validated at tertiary centres may perform differently in secondary care.
Based on their findings, the authors propose the following adaptations for secondary care settings with lower AAV prevalence:
Use an antigen-specific ELISA as the preferred first-line ANCA test to both rule out and to rule in AAV, given its higher specificity and PPV in this setting.
Consider a second test when clinical suspicion remains high despite a negative first result, when antibody concentrations are low, or when there is clinical doubt despite a positive first test. A second test increases specificity and reduces false positives — an important consideration where pre-test probability is lower.
These recommendations align with the 2017 consensus but emphasize selective second testing to improve PPV in secondary care.
The authors report all relevant data in the paper and supporting files. They note that ANCA are not specific for AAV and can arise in multiple other conditions, which accounted for the majority of positive tests in their cohort. In this large teaching hospital with lower AAV prevalence, antigen-specific ELISA performed best as first-line testing. Selective confirmation with a second assay is advised in settings of diagnostic uncertainty or low antibody concentrations to increase specificity and reduce false-positive diagnoses.
Overall, the study supports adapting the 2017 consensus principles to secondary care by prioritizing ELISA-first testing and using confirmatory testing selectively to address the lower PPV inherent to lower-prevalence populations.