|Ahead of print publication
Antibody to dense fine speckled 70 and its significance in a Sub-Himalayan population: A hospital-based study
Deepjyoti Kalita, Amit Mangla, U Sashi Rekha, Anusha Krishnaraj, Sangeeta Deka
Department of Microbiology, AIIMS, Rishikesh, Uttarakhand, India
|Date of Submission||19-Mar-2021|
|Date of Acceptance||20-Oct-2021|
|Date of Web Publication||21-May-2022|
Department of Microbiology, AIIMS, Veerbhadra Road, Rishikesh - 249 203, Uttarakhand
Source of Support: None, Conflict of Interest: None
Background: In antinuclear antibody (ANA) testing, antidense fine speckled 70 (DFS70) antibodies are commonly detected as a specific indirect immunofluorescence assay pattern (IIFA in HEp-2-based substrates) or as bands/blot lines in immunoblotting. A negative association between anti-DFS70 antibodies and the presence of autoimmune diseases is often reported. However, some studies found that this may not be true in females. We conducted this study to see the positivity rate of anti DFS70 antibodies and their association with other autoantibodies in the predominantly hilly population of our state.
Methods: Serum samples sent for ANA testing between February 2018 and December 2020 were included. ANA IIFA-positive cases underwent immunoblot profiling with 16 targets (including DFS70) blotting kit. Those with DFS70 band in the immunoblot) underwent tests for other autoantibodies such as antineutrophil cytoplasmic antibodies, and anti-dsDNA. Association analysis was performed in a software package (SPSS version 23).
Results: Out of 1770 samples, about 15.3% yielded a meaningful IIFA pattern. About 10.7% of ANA positives (1.6% of total samples) had the DFS70 band on immunoblotting. Isolated DFS70 was found in the predominantly male population (6 out of 7), while most females had this antibody in the presence of other autoantibodies (17 of 22).
Conclusion: Male population had predominantly isolated anti DFS70 antibody detection (with the likely possibility of using it as a marker for the absence of autoimmune disease), while in females, mostly, it was along with other autoantibodies.
Keywords: Antinuclear antibody, antinuclear antibody-associated autoimmune diseases, dense fine speckled 70, ENA, DFS, immunoblot
|How to cite this URL:|
Kalita D, Mangla A, Rekha U S, Krishnaraj A, Deka S. Antibody to dense fine speckled 70 and its significance in a Sub-Himalayan population: A hospital-based study. Indian J Rheumatol [Epub ahead of print] [cited 2022 Oct 1]. Available from: https://www.indianjrheumatol.com/preprintarticle.asp?id=345783
| Introduction|| |
Although the International Consensus on antinuclear antibody (ANA) Pattern (ICAP) recommends using indirect immunofluorescence assay (IIFA)-based ANA patterns for the diagnosis of autoimmune rheumatic diseases, ANAs are also detected in healthy individuals. Antidense fine speckled 70 (DFS70), also known as the lens epithelium-derived growth factor (LEDGF), is usually associated with an ANA IIFA pattern called DFS70. The ICAP code for the DFS70 IIFA pattern (i.e., AC2) indicates that both interphase and metaphase nuclei/chromatin are filled with irregular, fine granular fluorescence.,, Here, the target antigen is a transcription coactivator that can upregulate some antistress and anti-inflammatory genes (coding regions). Hence, this antigen may support cellular survival under environmental stress both in healthy individuals and patients with autoimmune rheumatic disease. Any modification in the target function or structure may elicit disease pathogenesis and autoantibody formation (IgG class). The prevalence of this autoantibody ranges from 0.8% to 16.6% in different studies with likely heterogeneous patients and employment of multiple diagnostic techniques.,,,
Isolated detection of anti-DFS 70 antibodies can be considered an indicator/marker for excluding ANA-associated rheumatic disease (AARD) in healthy individuals, as concluded by earlier studies.,,,,, Hence, anti-DFS70 testing can prevent unnecessary expenditure in the follow-up diagnostic procedures for clinically suspected cases of autoimmune rheumatic diseases.
Isolated antiDFS70 was found to be more in males, while a higher proportion of mixed autoantibody detection (including anti DFS70 antibody) in the female population complicates the picture.,,
Given the situation, we examined the prevalence of anti-DFS70 antibodies in our diagnostic laboratory setting and determined their association with other serum autoantibodies in our hilly population.
| Methods|| |
Serum samples for ANA testing, sent by various outpatient and inpatient units (General Medicine, Pulmonary Medicine, Cardiology, Dermatology, Obstetrics/Gynecology, Hematology, Neurology, Oncology, and Ophthalmology), were included in our study. The Institutional Ethics Committee approved this retrospective study protocol (vide letter no. AIIMS/IEC/20/834 dated 12/12/2020) carried out from February 2018 to December 2020.
ANA-IIFA was performed using the Euroimmun Mosaic HEp-20-10 and primate liver cell substrate (Euroimmun AG, Germany, Lübeck). After dilution (at 1:100), serum was processed following the manufacturer's instructions and examined under a fluorescence microscope at a magnification of 400×. The fluorescence intensity was semi-quantitatively scored from 1+ to 4+ relative to the positive control (4+).
As chances of false-positive ANA IIFA DFS70 cannot be ruled out, ANA-positive samples were further tested using the immunoblot assay with Euroline ANA profile with DFS 70 IgG (Euroimmun AG, Germany, Lübeck) for extractable nuclear antigen (ENA) targets. Strips were incubated with 1:100 diluted sera, and the intensity of the reactions was analyzed by using the image software EUROLine Scan (Ver. 3.4.30, Euroimmun AG, Germany, Lübeck). Here, 0 index score (i.e. signal intensity 0–5) and borderline (+) index score (i.e. signal intensity 6–10) were considered negative for a given band while score +, ++, and +++ (i.e., signal intensity 11–25, 26–50 and >50) were taken as positive (i.e., presence of the concerned antibody).
Anti-DFS700-positive samples were tested for the presence of the other autoantibodies such as anti-dsDNA (Crithidia luciliae IIFA and NcX ELISA/enzyme-linked immunosorbent assay from Euroimmun AG, Germany, Lübeck), antimitochondrial antibody (AMA), antismooth muscle antibody (ASMA), antiliver/kidney/microsome type 1 (LKM), and antisoluble liver antigen/liver pancreas antigen. Kits used for these tests were of euroimmun make (Euroimmun AG, Germany, Lübeck); other tests performed included antineutrophil cytoplasmic antibody (ANCA; determined using the IIFA technique and ELISA (proteinase-3 ELISA and myeloperoxidase ELISA), anti-Saccharomyces cerevisiae IgG/IgA (ASCA), and antipernicious anemia (PCA)/intrinsic factor (examined using the monkey stomach substrate) using IIFA-based kit (Euroimmun AG, Germany, Lübeck). A Euroimmun Analyzer-1, a walkaway 7-plate ELISA system (Euroimmun AG, Germany, Lübeck), was employed.
Statistical analysis was performed using SPSS, version 23 (IBM SPSS Statistics for Windows, version 23, IBM Corp., Armonk, N.Y., USA). Fisher's exact test and Chi-square test were employed for comparing categorical data. Nonparametric calculations were performed using the Mann–Whitney U test and rank correlation methods. P < 0.05 was taken as statistically significant.
| Results|| |
Prevalence of DFS70 antibodies
We included 1770 (502 men and 1268 women) consecutive adult patients and examined their serum samples submitted for routine ANA testing. As shown in [Figure 1] and [Supplementary Table 1], about 271 samples were ANA positive; out of this, 243 had positive immunoblot assay results, while a clear DFS70 band was observed in 29 patients. The DFS70 IIFA pattern (n = 34) and DFS70 blot (n = 29) band were conforming in 27 patients and discrepant in 9 patients [Supplementary Table 2].
Of the 29 DFS70 positive cases (immunoblot assay), two did not exhibit a DFS70 pattern in microscopy-based ANA-IIFA. Thus, from presumptive ANA-IIFA positive cases, 79.4% (27 of 34 patients) were confirmed cases, whereas 6.9% (2 of 29 patients) were missed by ANA-IIFA. Overall, DFS70 antibodies were observed in 1.6% (29/1770) of patients.
Positivity was higher in female patients (1.7%; 22 of 1268) than in male patients (1.4%; 7 of 502); but difference was not statistically significant (P = 0.6836) [Supplementary Table 3]. A marked female predominance was observed in ANA-positive samples (F/M: 210/61) and anti-DFS70-positive samples (F/M: 22/7). However, no statistically significant difference was observed between the mean age of anti-DFS70-positive patients (51.8 ± 11.3 years) and anti-DFS70-negative patients (49.9 ± 15.7 years) and between outpatients and inpatients (P = 0.836) [Supplementary Table 3].
Among eight anti-DFS70-positive inpatients, 10.3% (n = 3), 6.9% (n = 2), 3.4% (n = 1), 3.4% (n = 1), and 3.4% (n = 1) were from the departments of general medicine (one was referred by a rheumatologist), pulmonary medicine, hematology, neurology, and ophthalmology, respectively [Supplementary Figure 1]. The remaining 72.4% (n = 31) patients were from outpatient departments. Among ANA IIFA-positive patients, the number of anti-DFS70-positive outpatient's cases (1.7%; 21 of 1241) were higher than that of anti-DFS70-positive inpatients (1.5%; 8 of 521) subjects.
Isolated DFS70 positivity was found in 85.7% (6 of 7) male patients. However, in 77.3% (17 of 22) female patients, DFS70 positivity was associated with other concomitant serum autoantibodies. The most common antibodies that exhibited an association with DFS70 in 21 patients were anti-ENA such as Sm, nRNP, SSA-A, SS-B, and Ro-52 (n = 11), ANCA (n = 4), anti-dsDNA (n = 3), and anti-LKM1 (n = 1), respectively. Other autoantibodies tested (anti-PCA, ASMA, AMA, and ASCA) showed negative results. One patient had positive results for both ANCA and anti-dsDNA [Supplementary Table 3].
| Discussion|| |
Previous studies have reported DFS70 autoantibodies prevalence to be around 1%–16% in ANA-IIFA results. However, the cohorts were highly heterogeneous with varied individual positivity rates, e.g., blood donors and healthy individuals had a rate of 1%–5%, while ANA-screening cases had a rate up to 16%.,,, Furthermore, studies that performed only IIFA (without conducting the immunoblot or enzyme assay for confirmation) reported higher positivity rates.
The current study analyzed 1770 consecutive serum samples received for routine ANA screening by IIFA (at a screening dilution of 1:100); immunoblotting was further performed using these samples to confirm ANA-positive patterns. Overall, 1.6% of patients were positive for DFS70 (with a 10.7% rate in ANA-IIFA–positive patients), similar to some previous studies.,, The positivity rate of 1.6% reported by Mahler et al., who analyzed the serum samples of 3263 patients, was the same as that observed in our study. We used serum samples only from inpatients and outpatients, which can be extended in the future by taking the sample from the community.
Female patients with ANA positivity and anti-DFS70 positivity were higher by 3–4 times than male patients [Supplementary Table 1], supporting the notion that increased immune reactivity in women predisposes them to develop AARD, as stated by Ngo et al. This could be multifactorial with a scope of much in-depth study for the future. The prevalence of anti-DFS70 positivity was 1.7% in outpatients and 1.5% in inpatients; however, this difference was insignificant [P = 0.8360; [Supplementary Figure 1]].
Earlier, Muro et al. found a high prevalence of disease-related autoantibodies in anti-DFS70-positive female patients. Carbone et al. observed isolated anti-DFS70 antibodies in 85.7% of male subjects in contrast to the majority (77.3%) of female subjects showing combined anti-DFS70 and disease-marker autoantibodies (similar to our findings). [Supplementary Table 3] shows the association between DFS70 and other antibodies in our study population. We found a statistically significant simultaneous presence of other autoantibodies with DFS70 in females. The association of anti-ENA (i.e., immunoblot-related targets) with DFS70 was significant [[Supplementary Table 3] for detail]. Immunoblot assay helped in ruling out false anti-DSF70 positivity in ANA-IIFA-based assay.
Compared to some previous studies, a larger sample size and inclusion of immunoblot to confirm made the current study more reliable. The present study found an anti-DFS70 ANA IIFA pattern in 34 patients, of whom 27 (79.4%) showed positive results in immunoblot (DFS70 band), while two (6.9%) blot-positive cases were ANA IIFA DFS70 negative. Lundgren et al. have suggested that nonanti-DFS70/LEDGF antibodies can also yield the DFS70 pattern in ANA IIFA. More positive cases in the male population and clinical data incorporation can make this study more robust in the future.
| Conclusion|| |
Anti DFS70 positivity rate in ANA screening cohort is around 1.6% in our population. Detection of anti-DFS70 in the male population can be a screening indicator for the absence of other circulating autoantibodies. However, in females, it may indicate further need to probe into other autoantibodies and their clinical significance. A significant association between DFS70 and ENA (immunoblot targets) is an area for further study.
We are thankful to AIIMS Rishikesh authorities for allowing us to conduct this work. We express gratefulness to all the clinicians from different departments for sending us the samples for testing. Furthermore, we convey our thankfulness to all our patients.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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