Diagnostic Performance of ANCA IIF in Relation to PR3 and MPO Antibodies: Impact of Formalin Reactivity in a Large Real-World Cohort

Background: The diagnostic interpretation of antineutrophil cytoplasmic antibody (ANCA) testing remains challenging due to variable concordance between indirect immunofluorescence (IIF) patterns and antigen-specific assays targeting proteinase 3 (PR3) and myeloperoxidase (MPO). The additional value of formalin fixation in distinguishing true perinuclear ANCA (P-ANCA) patterns from redistribution artifacts in routine laboratory practice remains incompletely defined. Methods: We conducted a retrospective real-world analysis of 7276 patients who underwent concurrent ANCA IIF (ethanol- and formalin-fixed substrates), anti-PR3, and anti-MPO testing between 2022 and 2024. Concordance between IIF and antigen-specific assays was assessed using cross-tabulation and Cohen’s kappa statistics. Associations were evaluated using chi-square tests and odds ratios (ORs) with 95% confidence intervals (CIs). The impact of formalin reactivity within P-ANCA patterns and the relationship between ANCA titer strength and antigen positivity were analyzed using the chi-square test for trend. Results: Concordance between ANCA IIF and PR3 antibodies was poor (κ = 0.109; p < 0.001), with 59% of PR3-positive patients being IIF-negative. Agreement between ANCA IIF and MPO antibodies was stronger but remained modest (κ = 0.221; p < 0.001); 77% of MPO-positive patients were IIF-positive. Formalin-resistant P-ANCA patterns showed a strong association with MPO positivity (OR = 67.3; 95% CI 33.3–136.6; p < 0.001), whereas MPO positivity was uncommon in formalin-sensitive patterns. Increasing ANCA titers were associated with progressively higher MPO positivity (chi-square test for trend, p < 0.001), rising from 0.4% in ANCA-negative cases to 33.3% in the highest observed titer category. Dual PR3/MPO positivity was rare (0.16%). Conclusions: In this large cohort, ANCA IIF demonstrated differential diagnostic performance depending on antibody specificity. IIF showed limited sensitivity for PR3-associated autoimmunity, whereas MPO positivity correlated more strongly with IIF patterns and formalin resistance. Integration of antigen-specific assays with formalin-based pattern interpretation may improve the interpretative accuracy of ANCA testing and should be considered in routine laboratory algorithms.