Abstract
Antinuclear autoantibodies (ANA) displaying a dense fine speckled pattern (DFS, ICAP AC-2) on HEp-2 cells are frequently observed in clinical laboratory referrals, often associated with anti-DFS70 specificity. Anti-DFS70 positive patients rarely develop systemic autoimmune rheumatic disease (SARD), especially in the absence of clinical evidence or additional anti-extractable nuclear antigen (ENA) antibodies, prompting suggestions that an isolated DFS70-specific ENA may be an exclusionary finding for SARD. In this study, the frequency and diagnostic significance of anti-DFS70 autoantibodies was investigated in a community hospital cohort of patients undergoing routine ANA testing. ANA screening was performed by HEp-20-10-based indirect immunofluorescence, followed by ENA profiling using a multiparametric line immunoassay (LIA). Of 6,511 patient samples tested for ANA in 2016, the DFS pattern was identified in 1,758 (27.0%), 720 (41.0%) of which were anti-DFS70 positive by LIA. Of these, 526 (73.1%) revealed isolated anti-DFS70 reactivity, while 194 (26.9%) showed additional ENA specificities. Among 1,038 anti-DFS70 negative or borderline samples, 778 (75.0%) were ENA profile negative, while the remaining 260 (25.0%) showed a varied presence of other ENA specificities. Chart reviews of patients with an isolated anti-DFS70 ANA affirmed that ANA-related SARD is rare in the absence of clinical evidence or other ENA specificities, there being no case thus far identified. Rheumatoid arthritis patients occasionally had an isolated anti-DFS70 ANA and were positive for rheumatoid factor and anti-cyclic citrullinated peptide antibodies. In conclusion, the recognition of a DFS ANA pattern using a mitotic-rich HEp-2 substrate, followed by confirmation of anti-DFS70 specificity should be a routine ANA testing service. Use of an expanded ENA profile and clinical correlation is necessary to affirm the “isolation” of anti-DFS70 as the cause of an ANA. Recognition of isolated anti-DFS70 ANA enables reassurance of patients that SARD is unlikely, thus avoiding referral for more extensive testing. The presence of significant elevations of other ENAs may reflect SARD and warrants close clinical correlation and follow-up.
Highlights
The presence of antinuclear autoantibodies (ANA) is one of the key diagnostic criteria of systemic autoimmune rheumatic diseases (SARD), such as systemic lupus erythematosus (SLE), Sjögren’s syndrome, systemic sclerosis, dermatomyositis/polymyositis (DM/PM), mixed connective tissue diseases, etc. [1, 2]
526 (73.1%) had isolated anti-DFS protein of 70 kDa (DFS70) reactivity, whereas the remaining 194 (26.9%) showed additional extractable nuclear antigen (ENA) specificities. 1,038 (59.0%) of the samples with a dense fine speckled (DFS) Indirect immunofluorescence (IIF) pattern were found anti-DFS70 negative by line immunoassay (LIA), including 260 (25.0%) samples with a varied presence of other ENA specificities and 778 (75.0%) ENA profile negative samples (Figure 1)
When a mitotic-rich HEp-2 cell substrate (HEp-20-10) became available, we realized that the majority of these finely speckled ANAs showed a positive mitotic fluorescence, i.e., a mixed speckled/homogeneous pattern, later termed as “dense fine speckled,” which was often associated with anti-DFS70 specificity
Summary
The presence of antinuclear autoantibodies (ANA) is one of the key diagnostic criteria of systemic autoimmune rheumatic diseases (SARD), such as systemic lupus erythematosus (SLE), Sjögren’s syndrome, systemic sclerosis, dermatomyositis/polymyositis (DM/PM), mixed connective tissue diseases, etc. [1, 2]. The International Consensus on ANA Patterns (ICAP) committee has recently classified the DFS pattern as “AC-2” competency level recognition pattern, defined by a dense and heterogeneous speckled staining in the nucleoplasm of interphase cells (sparing the nucleoli) and the metaphase chromosomal plate [14, 15]. Recognition of this pattern on HEp-2 substrates is challenging as it can be confused with other nuclear patterns or may occur in the context of another clinically relevant ANA, and because IIF interpretation is dependent on technician expertise [16,17,18,19]. A positive DFS IIF result has to be followed by a monospecific immunoassay (e.g., ELISA, CLIA, immunoblot, immunoadsorption) [20] to accurately confirm the presence of anti-DFS70 autoantibodies, as recommended in diagnostic algorithms [19, 21,22,23,24,25,26]
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