Introduction. Indirect immunofluorescence (IIF) is the gold standard method for the detection of antinuclear antibodies (ANA) which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) based on the calculation of a fluorescence index (FI). Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92), even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's , ). Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. Conclusion. Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization. 1. Introduction Antinuclear antibodies (ANA) are essential biological markers for the diagnosis [1], classification, and disease activity monitoring [2] of systemic autoimmune rheumatic diseases. Given this central role, ANA screening should be accurate and reproducible. For several decades, indirect immunofluorescence (IIF) on HEp-2 cells has been the reference technique for ANA testing. Although new available techniques [3, 4] such as ELISA or multiplexing solid phase technologies have been proposed to replace IIF, the American College of Rheumatology (ACR) still recommends IIF as the gold standard method for ANA detection [5]. The main drawback of this technique is IIF reading subjectivity, intra- and interlaboratory variabilities complicating the standardization expected in modern laboratories. Recently, commercial automated systems for ANA IIF reading and interpretation have become available and were described in the literature [6–11]. Most of them are based on data mining and supervised machine learning methods [12]. In addition to their complexity, they share a common weakness in the detection of weak positivity. In this work, we describe an original algorithm named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) for automation of IIF ANA evaluation offering excellent analytical performance and an attractive quantitative
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