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Does the Dense Fine Speckled Pattern in an ANA Test Actually Tell us Anything Important Clinically?

Mia C. Lundgren^* and John T. Crosson ^*Correspondence: Mia C. Lundgren, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis,MN, USA, Email:

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Description

In the field of Antinuclear Antibody (ANA) testing, the significance of the ANA Dense Fine Speckled (ANA-DFS) pattern, as well as the role of anti-DFS70/LEDGF antibodies, is currently one of the most discussed topics. ANA testing is an important screening test in the diagnostic workup for Systemic Autoimmune Rheumatic Diseases (SARD), such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Sjögren’s syndrome, systemic sclerosis, dermatomyositis/polymyositis, and mixed connective tissue disease. The gold standard for ANA detection is Indirect Immuno Fluorescence (IIF) microscopy, which uses Human Epithelial type- 2 (HEp-2) cells as a substrate to highlight specific antibody-antigen interactions in different parts of the cell. These interactions, or patterns, were further defined and categorized at the International Consensus on Antinuclear Antibody Pattern (ICAP) in 2014 which led to the identification of 29 distinct patterns [1]. Certain patterns, such as the homogeneous pattern, have known antigen and disease associations. To date, the only confirmed Antigen associated with the ANA-DFS pattern is DFS70/LEDGF. This protein is expressed in many cell types, including lens epithelial cells, keratinocytes, fibroblasts, and cancer cells. It may play a role in cell survival and in the protection against environmental stress through the activation of stress-related proteins. However, the role of anti-DFS70/LEDGF antibodies remains largely unknown.

There are no specific confirmed clinical associations with the ANA-DFS pattern, though it has been reported to be rare in SLE, Sjögren’s syndrome, and systemic sclerosis [1]. Since its first association with interstitial cystitis in 1994, there have been additional reports in the literature linking the ANA-DFS pattern to other non-SARD diseases, including atopic dermatitis, asthma, psoriasis, alopecia areata, Vogt-Harada syndrome, Hashimoto’s thyroiditis, ocular disease, prostate cancer, chronic fatigue syndrome, and fibromyalgia [2-7]. Recently, there has been increased interest in the potential use of anti-DFS70/LEDGF as a marker to exclude a diagnosis of SARD, an idea supported by prior studies that demonstrated the rare prevalence of anti-DFS70/LEDGF in SARD patients compared to healthy individuals [8, 9]. In our research article recently published by the Journal of Immunological Methods, we sought to determine the prevalence of SARD among a population of patients with the ANA-DFS pattern and to discern whether there were other diseases associated with this particular ANA pattern [10].

Our retrospective study included 425 patients from our university health care system with a positive ANA-DFS pattern consecutively from August 2017 to September 2018. Patient sera underwent ANA testing by IIF on HEp-2 cell substrates (Euroimmun, Germany). The study included 335 women (78.8%) and 90 men. The mean age for women was 44.0 years (range 3-92); men 43.7 (4-85). Family Practice was most frequently the ordering provider (45%), and the most common indications for ordering the ANA test were joint pain, fatigue, and a rash or skin lesion. Almost half (49%) were referred to Rheumatology. There were 102 patients with SARD (24%). The SARD patients had a higher frequency of joint pain, referrals to Rheumatology, and abnormal follow-up immunological tests. The prevalence of SARD increased with increasing titer, and the most common SARD were seronegative or seropositive RA and UCTD (Table 1). The frequency of SLE was highest in the highest titer group. The most common non-SARD diseases were atopic disorders (21.2%), some combination of fibromyalgia, chronic pain syndrome, and chronic fatigue syndrome (17.6%), and skin disorders (16.7%) (Table 2).

Table 1: Prevalence of SARD diseases in ANA-DFS positive patients by titer a,b.

Table 2: Prevalence of SARD diseases in ANA-DFS positive patients by titer a,b.

Given the high frequency of symptomatic patients and disease processes with an immunologic basis (including SARD), we argue that the ANA-DFS pattern may actually be an indicator of a proinflammatory microenvironment rather than a marker associated with healthy individuals or one that potentially excludes a SARD diagnosis. Previous studies have observed a higher prevalence of the ANA-DFS pattern in healthy individuals compared to SARD patients [8, 9]. However, on closer review of the study by Mariz et al, the frequency of ANA-positivity overall in the healthy population was only 12.9%, and the ANA-DFS pattern was observed in only 39 of 118 ANA-positive individuals (33.1%). Other studies have shown a low prevalence of anti-DFS70/LEDGF auto reactivity in patients with SARD, particularly isolated auto reactivity, compared to healthy individuals [8-11]. ICAP has acknowledged this negative association between anti-DFS70/LEDGF and SARD but states there must be isolated auto reactivity without the presence of other common extractable nuclear antigens in order to be valid [12]. More recent studies have observed higher frequencies of anti- DFS70/LEDGF auto reactivity among SARD patients, suggesting anti-DFS70/LEDGF may not be a good exclusion marker for SARD [13]. Because our study was retrospective, determination of anti-DFS70/LEDGF was not available for any of the patients.

While the significance of anti-DFS70/LEDGF antibodies is largely unknown, the ANA-DFS pattern may be indicative of an underlying antigen-antibody interaction that plays a role in or is a marker of either single or multiple inflammatory disorders. DFS70/LEDGF is the only target auto antigen identified to date. However, it is unlikely that the anti-DFS70/LEDGF antibody is the only antibody responsible for the ANA-DFS pattern. Studies have shown that the ANA-DFS pattern is not specific for anti-DFS70/LEDGF. In one study, only 14% of 94 ANA-DFS positive individuals were positive by anti-DFS70/LEDGF ELISA, and, in other studies, only 50% of ANA-DFS positive patients were positive for anti-DFS70/LEDGF by chemiluminescent or immunoblot methods.[13-15] More research is necessary to identify additional antibody specificities.

Currently, we are pursuing further studies on an expanded data base of patients with the DFS pattern by IIF ANA. We are particularly looking at the incidence of inflammatory markers in all of these patients and also looking at the frequency of the DFS pattern among all the ANAs performed during the same time period. We hope, using standard statistical methods, to be able to more precisely define the clinical significance of the ANA-DFS pattern. When final control of the SARS-CoV2 pandemic allows us to do prospective studies, we plan, in collaboration with Euroimmun, to determine the frequency of antibodies to the DFS70/LEDGF antigen in patients with the ANA-DFS pattern. We also expect to be able to begin looking for other auto antigens whose antibodies produce the DFS pattern by IIF ANA.

References

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