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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

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Abstract

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.

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Acknowledgments

The authors thank Alphadia for kindly providing the reagents for autoantibody detection free of costs, and Dr. Chiara Derioni and Dr. Riccardo Favilli of Alifax for the technical assistance.

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Author notes

  1. Maria Infantino and Boaz Palterer contributed equally to this work.

Authors and Affiliations

  1. SOS Immunology and Allergology Laboratory Unit, Department of Laboratory Medicine, Azienda Usl Toscana Centro, S. Giovanni di Dio Hospital, Florence, Italy

    Maria Infantino, Valentina Grossi & Mariangela Manfredi

  2. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

    Boaz Palterer, Roberta Biagiotti, Fabio Almerigogna & Maria Grazia Giudizi

  3. SOS Rheumatology Unit, Department of Medicine, Azienda Usl Toscana Centro, S. Giovanni di Dio Hospital, Florence, Italy

    Maurizio Benucci & Arianna Damiani

  4. S.O.C. Clinical Chemistry and Microbiology Laboratory, Department of Laboratory Medicine, Azienda Usl Toscana Centro, S. Stefano Hospital, Prato, Italy

    Annalisa Azzurri & Patrizia Casprini

  5. Department of Biology, University of Florence, Florence, Italy

    Giovanni Bacci

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  1. Maria Infantino
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Correspondence to Maria Infantino.

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Infantino, M., Palterer, B., Biagiotti, R. et al. 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. Immunol Res 66, 74–78 (2018). https://doi.org/10.1007/s12026-017-8974-3

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