An LR-based risk stratification framework for assessing interlaboratory variability in HEp-2 IFA pattern interpretation

Abstract Objectives Interlaboratory variability in HEp-2 indirect immunofluorescence assay (IFA) interpretation remains a major challenge. Conventional agreement metrics quantify disagreement but do not distinguish discrepancies by diagnostic relevance. We aimed to develop a likelihood ratio (LR)-based framework for clinically informed assessment of interpretation variability. Methods Pattern-specific positive LRs were derived from 33,690 routine HEp-2 IFA records and used to classify patterns into four risk tiers. A pattern discrepancy score (PDS) was developed to quantify discrepancies according to risk-tier displacement. Interlaboratory variability was assessed using a standardized 200-specimen panel tested by 18 laboratories, and diagnostic performance was explored in six laboratories. Results Pattern disagreement rates ranged from 17.5 to 50.0 %. PDS strongly correlated with disagreement rate (Spearman’s ρ=0.936, p<0.001), but laboratories with similar disagreement rates differed in cross-tier discrepancy distribution. Most discrepancies remained within the same risk tier; among discordant interpretations involving high-risk reference patterns, 20.4 % crossed into another tier. Pattern concordance varied markedly, while titer agreement was moderate to almost perfect (weighted κ, 0.569–0.821). Across six laboratories, diagnostic performance varied (Youden Index, 0.391–0.617), but was not significantly associated with interpretation consistency. Conclusions HEp-2 IFA interpretation variability differs in both frequency and potential diagnostic relevance. The LR-based framework and PDS complement conventional agreement measures by identifying discrepancies that alter diagnostic risk classification, potentially supporting risk-oriented proficiency assessment and targeted quality improvement.