Reflex testing of speckled cytoplasmic patterns observed in routine ANA HEp-2 indirect immunofluorescence with a multiplex anti-synthetase dot-blot assay: a multicentric pilot study

Immunofluorescence on HEp2-cells is the standard diagnostic assay for the detection of anti-nuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding, and are associated with various antibodies, including anti-synthetase antibodies. However, classic ENA testing generally identifies only anti-Jo-1. Moreover, anti-synthetase syndrome is increasingly recognized as a pleomorphic entity, possibly presenting as isolated arthritis or interstitial lung disease. Sera referred for routine ANA testing were selected on the basis of the presence of a fine dense speckled cytoplasmic pattern (254 samples) and compared to control sera with negative cytoplasm (239 samples). All 493 samples were tested with a commercial synthetase profile dot-blot (D TEK - Alphadia-Alifax) including anti-Jo1, anti-PL7, anti-PL12, anti-EJ, anti-OJ, anti-KS, anti-ZO, anti-HA, anti-SRP, and anti-Ribosome P0. Retrospective clinical data was searched for positive patients. Dot-blot identified 18/254 (7.1%) positive sera in the samples with a cytoplasmic fluorescence pattern and 4/239 (1.7%) in the control group (χ2 = 8.4627; p = 0.003625). Blot intensity was more intense in samples with concordant cytoplasmic staining (cytoplasmic negative 27 ± 12.4; cytoplasmic positive 53.9 27 ± 27.7; p = 0.0027). In the positive samples, 8/18 had a highly compatible diagnosis (myositis, interstitial lung disease, arthritis), 7/18 an uncharacterized connective tissue disease, and 3 a diagnosis not associated with the presence of anti-synthetase antibodies. We evaluated the performance of a dot-blot assay for anti-cytoplasmic antibodies in a serologic cohort presenting a cytoplasmic speckled pattern found during routine ANA testing. This algorithm enabled the identification of a significant quota of patients with rare anti-synthetase antibodies and an incomplete or atypical clinical picture. Reflex testing strategies of speckled cytoplasmic patterns with multiplex assays containing cytoplasm-specific antigens, as opposed to standard ENA testing, may yield important data and for this reason should be implemented in routine ANA testing.