Retrospective Identification and Quantitative Image Analysis of the <scp>ICAP</scp> Nuclear Pattern <scp>AC</scp> ‐30 in Antinuclear Antibody Testing

BACKGROUND: The International Consensus on ANA Patterns (ICAP) recently codified the AC-30 nuclear pattern, characterized by fine speckled nuclear texture and metaphase chromatin staining. Although formally recognized, its clinical significance remains unclear. Quantitative methods and objective outcome measures for characterizing AC-30 have not been established. METHODS: A retrospective analysis was conducted using archived HEp-2 ANA images. AC-30 was defined by unanimous scoring from three blinded experts. AC-1 and AC-2 served as comparator sets. Clinical diagnoses and serological data were retrieved from hospital records. Pixel classification was performed using ilastik, and per-nucleus intensity features were extracted with CellProfiler. Image-level separation was assessed by partitioning nuclei into high- and low-intensity groups via Isolation Forest. Statistical comparisons used Welch's t-tests and Spearman's rank correlation. RESULTS: The AC-30 group included 183 images (AC-1, n = 183; AC-2, n = 207). Within AC-30, 57.4% had non-AID or unknown diagnoses, 26.8% had other autoimmune diseases, and 15.8% had ANA-associated rheumatic disease (AARD). In ENA-negative AC-30, AARD accounted for 7.7% (6/78), compared to 18.3% (21/115) in ENA-negative AC-2. RA accounted for 16.9% (31/183) in AC-30, consistent across ENA strata. AC-30 displayed lower per-nucleus intensities than AC-2 (all p < 0.0001) and reduced per-image ΔMaxIntensity (0.177 vs. 0.252, p < 0.0001), while ΔMeanIntensity was similar (0.068 vs. 0.067, p = 0.549). CONCLUSIONS: AC-30 is quantitatively dimmer than AC-2, with reduced nuclear brightness and peak-intensity separation. In this retrospective cohort, ENA-negative AC-30 was associated with fewer AARD cases. RA accounted for ~17% of AC-30, and anti-CCP and RF remained informative markers. The ilastik-CellProfiler workflow enables auditable ANA quantification.