Abstract: Objectives: ANA are the most extensively used test for the diagnosis of systemic autoimmune diseases. However, testing by indirect immunofluorescence assays (IIFAs) on HEp-2 cells, the gold standard test, is time-consuming and needs expertise. Thus there is a trend to replace it with other automated solid-phase assays directed against specific ANA. Nonetheless, the Hep-2 cell is an autoantigen array and ANA have been classified into 29 types, some of them with no clear association with a specificity to be detected. It is especially in these uncommon patterns where no clinical relationship is found and no antigenic specificity is detected. Here we retrospectively collected clinical data from patients with confirmed uncommon HEp-2 IIFA patterns to search for an associated clinical condition. Methods: We conducted an observational retrospective study including 608 patients with organ-specific and non-organ-specific autoimmune diseases (OSADs and NOSADs, respectively) with a confirmed rare pattern of ANA detected by IIFA on HEp-2 cells in the routine practice of the Spanish European Autoantibodies Standardization Initiative laboratories. Inclusion criteria are the existence of a minimum follow-up of 2 years and the availability of clinical data. Results: Nuclear patterns were more frequent in SLE (P = 0.001) and SS (P = 0.001), whereas the cytoplasmic ones were significantly higher in SSc (P = 0.022) and inflammatory myositis (P = 0.016). Mitotic patterns did not show any preferences for a specific disease and 62.7% of them corresponded to the nuclear mitotic apparatus pattern (AC-26). The most frequent NOSADs in patients with the AC-26 pattern were SLE (28.6%), SS (11.9%) and RA (11.9%). The cytoplasmic HEp-2 IIFA patterns were equally distributed in both groups of patients. In the OSAD patients there was no predominant pattern, except for AC-6 in primary biliary cholangitis due to Sp-100 antibodies (P < 0.001). Conclusion: Detection of infrequent ANA might be a unique finding with no disease-associated specificities and could lead to the suspicion of an autoimmune disease.

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