Elsevier

Journal of Immunological Methods

Volume 461, October 2018, Pages 110-116
Journal of Immunological Methods

Research paper
A better definition of the anti-DFS70 antibody screening by IIF methods

https://doi.org/10.1016/j.jim.2018.07.001Get rights and content

Abstract

Background

Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies.

Methods

Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco).

Results

The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22).

Conclusion

The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

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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    The three authors contributed equally to this work.

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