Research paperA better definition of the anti-DFS70 antibody screening by IIF methods
Introduction
The presence of autoantibodies directed against cell nuclear and cytoplasmic antigens (ANAs) (Agmon-Levin et al., 2014) is the serological hallmark of ANA-associated rheumatic diseases (AARD) (Mahler and Fritzler, 2010) and is included in the classification criteria for systemic lupus erithematosus (SLE) and systemic sclerosis (SSc) (Tan et al., 1982; Johnson et al., 2012) The indirect immunofluorescence (IIF) assay on HEp-2 cell substrates is one of the most commonly used routine tests for the ANA screening and has also been recently recommended by a task force of the American College of Rheumatology (ACR) (Meroni and Schur, 2010). Nevertheless, IIF still faces many limitations mainly due to its restricted specificity for AARDs (Fritzler and Fritzler, 2006) which leads to a more difficult interpretation of the results in some cases (Slight-Webb et al., 2016). In fact, some fluorescence patterns are specifically linked to some AARD but others have a weaker association, such as dense fine speckled (DFS) pattern (Tan et al., 1997).
The characteristic features of the DFS IIF staining pattern is a dense and heterogeneous fine speckled pattern distributed throughout interphase nucleus and on metaphase chromatin with speckles that differ characteristically in size, brightness and density in the nucleus (Ochs et al., 1994). Hence, DFS should be considered a distinct pattern and different from other similar patterns, such as the quasi-homogeneous pattern. However, depending on the titer levels and the overlapping of other ANA patterns, the interpretation can be a changelling issue (Bentow et al., 2016a).
The antigen was named DFS70 because of the 70-kDa protein found by immunoblotting and later was identified as the lens epithelium-derived growth factor (LEDGF) (Shinohara et al., 2000) or DNA binding transcription coactivator p75. This protein is highly expressed in prostate tumor tissues (Daniels et al., 2005) and has a multirole function such as interaction with viral integrase in human immunodeficiency (Maertens et al., 2003). Recently, epitope mapping analysis has shown that one of the most prevalent autoepitope was conformational in nature and located on the C-terminus of the DFS70 autoantigen (Ogawa et al., 2004). Among the 28 various patterns described by the International consensus on ANA pattern (ICAP) committee (Chan et al., 2015), the DFS pattern, called AC-02, has gained attention because of its high prevalence in the general population and its low prevalence in AARDs (Conrad et al., 2017; Watanabe et al., 2004; Mahler et al., 2012a; Miyara et al., 2013). In patients with AARD, anti-DFS70 antibodies are typically accompanied by other ANA, but in healthy individuals and in non-AARD conditions they are commonly present in isolation (i.e. without any disease specific antibodies), hence becoming an attractive clinical biomarker to rule out the presence of systemic autoimmune disease (Seelig et al., 2016; Fabris et al., 2014). In fact, the high frequency of the DFS pattern in individuals that do not have AARD and in healthy individuals makes its recognition important for appropriate clinical assessment. For this reason the ICAP committee strongly suggested reporting the DFS pattern. Recent published studies have suggested the idea that the DFS pattern by ANA IIF testing could be interpreted in this context as ANA negative result without causing inappropriate referrals to specialists and anxiety in patients (Mahler and Fritzler, 2012; Mahler et al., 2012b; Infantino et al., 2016; Mariz et al., 2011; Infantino et al., 2018). In a real life laboratory ANA positive results, regardless of the ENA results, often induce clinicians to repeat ANA testing, advise specialists visits and potentially lengthy follow-ups because they suspect AARD. Therefore, anti-DFS70 antibodies as discriminator of ANA positive results with and without AARDs may nowadays represent one of the most challenging issues. The anti-DFS70 specific commercial assays currently available are: immunoblotting (IB) (Bizzaro et al., 2016), enzyme-linked immunosorbent assay, chemiluminescent assay (CIA) (Mahler et al., 2012b) and two novel HEp-2-cell IIF assays: one allowing for the immunoadsorption of anti-DFS70 antibodies and the other based on a DFS70/LEDGF knock-out cell line (Mahler et al., 2012a; Malyavantham and Suresh, 2017). Broad ranges of correlation between IIF DFS patterns and anti-DFS70 specific assays (Mercado et al., 2017; Basu et al., 2015) and between the specific assays have been recently reported (Bizzaro et al., 2016; Bonroy et al., 2018). The variability of the specific assays is linked to the antigen selection (full length LEDGF, major antigenic region), the recombinant expression system used for antigen production (E. coli, Baculovirus system, mammalian cells), diagnostic sensitivity/specificity, and the assay cut-off. Taking all this into account, and considering the recent advances in diagnostic technologies for autoimmune diseases, it is crucial to support the traditional test algorithm for ANA testing with novel approaches such as new tests for anti-DFS70 antibodies detection (Mahler et al. 2012c; Gundín et al., 2016). This new diagnostic approach, in which the detection of antibodies to this antigen could then be used to address clinician alike that the patient does not have a systemic ARD, is interesting especially if we consider the modern laboratory scenario characterized by cost containment, growing consolidation, and increased demand for ANA testing. The aim of the study was to analyze the the inter-method agreement among three different HEp-2 cell substrates in anti-DFS70 detection. As a second aim, we evaluated the discrepancies among two novel IIF searching for isolated anti-DFS70 antibodies.
Section snippets
Patients and study design
Sera were collected in Immunology and Allergology Laboratory Unit, S. Giovanni di Dio Hospital, Florence (Italy) between June 2017 and September 2017. The samples were obtained from patients who were routinely referred for ANA testing. The pre-test probability for having an AARD varied between the different patients, based on the specialty of the physician ordering the test (rheumatologist, other specialists, and general practitioners). Of these, we studied the 29 patients (23 females and 6
Sensitivity of DFS pattern detection by IIF in three different HEp-2 substrates
The selected sera samples of 29 subjects positive for anti-DFS70 antibodies by CIA and IB were analyzed by IIF on the HEp-2 cells of three different substrates (Inova, Immco and Euroimmun). The Euroimmun substrate showed the highest sensitivity for detecting a DFS staining pattern (93.1%), followed by the Immco substrate (79.3%) and the Inova substrate (72.4%) (Table 2a). There were 11 samples with discrepant results on pattern recognition between different substrates (Table 2b).
Qualitative agreement between different substrates for DFS detection
The positive
Discussion
The detection of anti-DFS70 antibodies commonly follows the DFS staining pattern identified by IIF on HEp-2 cells, even if different patterns might also be linked to the presence of these antibodies (Mahler et al., 2012a; Bizzaro et al., 2015). DFS is one of the most commonly found patterns by routine diagnostic laboratories executing the ANA IIF test on HEp-2 cells (Broadfoot et al., 2016), underlining the clinical impact of a better definition of these new autoantibodies (Shovman et al., 2018
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Cited by (23)
Investigation of dense fine speckled pattern and anti-dense fine speckled 70 antibody by a single step assay
2022, Journal of Microbiological MethodsCitation Excerpt :It can provide faster access to the diagnosis compared to the traditional two-step reflex test strategy. There had been differences in correlations between IIF patterns and specific assays (Infantino et al., 2018; Vázquez-Del Mercado et al., 2017; Basu et al., 2015) and the performances of these tests showed variability (Bizzaro et al., 2016; Bonroy et al., 2018). Similar to this data, ELISA and LB tests that we used in our study gave different positivity rates which might be because of the heterogeneity of the antigen causing the DFS pattern or different sources of DFS70 antigens used in these assays.
The choice of anti-LEDGF/DFS70 assay matters: a comparative study of six assays
2022, PathologyCitation Excerpt :Indeed, we found no significant inter-reader variation when interpreting the HEp-2KO IIF. But substantial disagreement between the two ANA substrates was seen, similar in magnitude to the differences observed between other manufacturers as evaluated by Infantino et al.14 Frequent disagreement with other substrates and solid phase assays, and low positive detection rates suggest that the HEp-2KO is unsuitable as a confirmatory assay for DFS and suggest that further development is required before this method could be used as the sole detection strategy for anti-LEDGF/DFS70 autoantibodies. An optimal algorithm for LEDGF/DFS70 autoantibody detection and determination remains to be established.
Clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies testing
2020, Journal of Immunological MethodsCitation Excerpt :The reason for this is unknown; maybe it represents predisposition to autoimmunity, or the assay allows detection of many antibodies that do not reflect serious immune disturbances (Pisetsky, 2017). Although ANA production might cease in some patients with SARD, it is also possible that the finding of seronegativity results from inadequate sensitivity of the assay kits or the inherent difficulty of IIF in detecting certain antibodies (Pisetsky, 2017; Infantino et al., 2018). Due to a perceived high prevalence of “false negative or positive” results and other technologies for ANA detection continue to evolve the atypical ANA type (Mariz et al., 2011).
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The three authors contributed equally to this work.