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Clinical significance of anti-dense fine speckled 70 antibodies in dense fine speckled immunofluorescence positive patients, systemic autoimmune rheumatic diseases, and controls
For correspondence: Dr Liza Rajasekhar, Department of Clinical Immunology and Rheumatology, Nizams Institute of Medical Sciences, Hyderabad 500 082, Telangana, India e-mail: lizarajasekhar@gmail.com
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Received: ,
Accepted: ,
How to cite this article: Mylavarapu M, Gavali M, Puranam K, Parejiya BK, Rajasekhar L. Clinical significance of anti-dense fine speckled 70 antibodies in dense fine speckled immunofluorescence positive patients, systemic autoimmune rheumatic diseases, and controls. Indian J Med Res. 2026;164:120-8. doi: 10.25259/IJMR_2840_2025
Abstract
Background and objectives
The dense fine speckled (DFS) anti-nuclear antibody (ANA) pattern, caused by anti-DFS70 antibodies, is often misunderstood due to its low association with systemic autoimmune rheumatic diseases (SARD) and presence in healthy individuals. Few studies suggest monospecific anti-DFS70 antibodies may exclude SARD. Given limited data from India, we aimed to assess the prevalence and clinical associations of anti-DFS70 antibodies in undefined disease, SARD, and controls.
Methods
Sera from patients with undefined disease and non-rheumatic disease controls were screened for DFS pattern during the evaluation for ANA by indirect immunofluorescence (IIF); positive samples were confirmed using anti-DFS70 ELISA and line immunoassay (LIA). SARD sera were tested by ANA IIF, ELISA, and LIA.
Results
DFS IIF pattern was observed in 0.25%, 0.57% and 0.4% of undefined disease referrals, SARD patients, and controls respectively. ELISA anti-DFS70 and LIA were positive in 90% of undefined disease sera, 15.4% vs. 5.7% of SARD cases, and 100% of controls, respectively. ELISA and LIA showed almost perfect agreement in undefined disease (κ= 0.861). In SARD group, anti-DFS70 ELISA positivity was predominantly observed in SLE (P=0.003); higher ELISA optical density, higher systemic lupus erythematosus disease activity index (SLEDAI) scores (P<0.001), anti-dsDNA (P=0.0001), and renal involvement (P=0.014) being significant observations. Monospecific anti-DFS70 antibodies by LIA were significantly common in undefined disease (P<0.001).
Interpretation and conclusions
DFS pattern with monospecific anti-DFS70 antibodies suggests lower likelihood of SARD. Anti-DFS70 antibodies are uncommon in SARD and coexist with other disease-specific autoantibodies. These findings highlight the complexity of interpreting anti-DFS70 antibodies and underscore the need for comprehensive serological testing and clinical correlation.
Keywords
Antinuclear
Autoimmune diseases
Immunoassay
India
Lupus erythematosus
Anti-nuclear antibodies (ANAs) are the most used test in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The standard method for its detection is the human epithelial HEp-2-cell-based indirect immunofluorescence (IIF) assay.1 ANA IIF may be positive in as many as 20% of individuals who appear healthy. Often, additional testing is requested to determine the specific antigenic targets of ANA to enhance the diagnostic value of a positive ANA-IIF test result.2
The International Consensus on Antinuclear Antibody Patterns (ICAP) has categorised ANA IIF patterns into 31 anti-cell categories, facilitating standardised interpretation across laboratories.1,3,4 The anti-cell (AC)-2 pattern, also known as the DFS IIF pattern, is defined by the presence of nuclear dense fine speckles (DFS) and pronounced metaphase chromatin staining. The antigenic target is a 70 kD protein referred to as DFS70,5-7 also known as LEDGF/p75, TCP75, and PSIP1.8,9 It is a transcription-associated protein activated by cellular stress and has implications in autoimmunity, cancer, AIDS, and ocular diseases.8-13 The prevalence of AC-2 pattern has been reported to be 0.8% to 12.3% in standard ANA populations.14-19
Data on the prevalence and clinical relevance of AC-2 pattern and anti-DFS70 antibodies in the Indian population is limited.20,21 Pattern interpretation in the ANA IIF assay may influence clinical decision-making. Assessing the prevalence of DFS70 antibodies in sera tested for ANA is crucial for informing rheumatology referrals, mitigating overdiagnosis, and enhancing test interpretation. Using ANA IIF, enzyme-linked immunosorbent assay (ELISA) and immunoblot/line immuno assay (LIA), we aimed to study the prevalence of anti-DFS antibodies in different disease settings.
Methods
This was an observational study conducted in the departments of Microbiology, Clinical Immunology, and Rheumatology, Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India between December 2021 to November 2024. This study was performed in line with the principles of the Declaration of Helsinki. The study was approved by Institutional Ethical Committee. Written informed consent was obtained from all the patients involved in the study.
Study groups and inclusion criteria
SARD group comprised 350 patients presenting to the Rheumatology department (inpatient or outpatient) with confirmed systemic autoimmune rheumatic diseases (SARD). All SARD cases were clinically diagnosed by rheumatologists and satisfied the established classification criteria.22-27
Patients with clinical suspicion of autoimmune disease/manifestation referred for ANA IIF test and having DFS pattern on the ANA IIF were called undefined group. These ANA IIF were sent from various departments of the hospital for exclusion of autoimmune aetiology. The 82 patients having DFS IIF pattern (undefined disease group) were further evaluated by the rheumatologist and or treating clinician for ascertaining the final diagnosis.
The control group included walk-in patients or patients referred from other departments where the consulting rheumatologist requested for an ANA, but the clinical diagnosis was not autoimmune rheumatic disease. Hence, they were called ‘non rheumatic disease controls’.
Exclusion criteria
Seropositivity for hepatitis B, hepatitis C, or HIV; and pregnancy.
Sample size
The sample size estimation for both the SARD and non-rheumatic disease control groups was based on reported anti-DFS70 antibody prevalence data, using the standard formula for single-proportion sample size calculation at a 95% confidence level. For the SARD group, using a reported 24% prevalence28 and a precision of 5%, the calculated sample size was 281. A practical sample size of 350 was chosen to ensure robust power. For non-rheumatic disease controls, a study29 reporting 16.4% prevalence of anti-DFS70 antibodies from 250 healthy individuals was used which yielded a sample size of 211, which was rounded up to 250 patients to ensure adequate power.
Testing strategy
Sera from all SARD patients were analysed by both ELISA and LIA. Sera from patients in the undefined disease group and from non-rheumatic disease controls were first screened by ANA IIF; those displaying an AC-2 pattern were further tested by ELISA and LIA. This approach enabled comparative evaluation of IIF, anti-DFS70 ELISA, and LIA across the three study cohorts within available resources.
ANA testing by indirect immunofluorescence (ANA IIF)
ANA IIF was performed at 1:100 serum dilution using Euroimmun Mosaic HEp-20-10 and primate liver cell substrate (Euroimmun AG, Lübeck, Germany), according to the manufacturer’s instructions. IIF patterns were interpreted independently by two microbiologists blinded to clinical diagnosis, using a fluorescence microscope at ×400 magnification and classified according to ICAP guidelines. Fluorescence intensity was graded from 1+ to 4+ relative to the manufacturer’s positive (4+) and negative controls.
Anti-DFS70 ELISA
Anti-DFS70 ELISA was performed using anti-DFS70 IgG ELISA kit (Euroimmun, Luebeck, Germany). Serum samples were diluted 1:201 with a sample buffer. 100 μL of diluted samples, calibrators, positive and negative controls were incubated in microplate wells coated with DFS 70 antigen for 30 min at room temperature. Following incubation, the wells were emptied, washed with a wash buffer and incubated with the enzyme conjugate for 30 min at room temperature. Further the wells were emptied, washed with wash buffer and incubated with the substrate for 15 min at room temperature in the dark. The reaction was stopped with the addition of a stop solution and optical density of each well was measured at 450 nm using a spectrophotometer. Samples were considered positive if the sample optical density (OD) was higher than that of the calibrator and titres were expressed as ratio of sample OD to calibrator OD.
Line immunoassay (LIA)
Anti-ENA (extractable nuclear antigen) antibodies were tested using the EUROLINE ANA profile 3 plus DFS70 (IgG) kit according to the manufacturer’s instructions (Euroimmun, Luebeck, Germany). Nylon strips coated with 11 native purified proteins (nRNP/Sm, Sm, SS-A, SS-B, Scl-70, Jo-1, dsDNA, nucleosomes, histones, ribosomal P-protein, and AMA M2) and 5 recombinant antigens (Ro-52, PM-Scl, CENP B, PCNA, and DFS70) were incubated with serum samples diluted to 1:101. Bound antibodies were detected using an enzyme-labelled anti-human IgG conjugate, producing colour bands corresponding to the antigen–antibody complexes. Band intensities were evaluated by using the EUROLineScan software (Euroimmun, Luebeck, Germany) and reported as negative, borderline [(+)], positive [+, ++] and strong positive [+++].
‘Monospecific’ anti DFS 70 antibodies were defined as presence of only anti DFS antibodies on LIA without any other anti ENA or anti-dsDNA antibodies.
Statistical analysis
Statistical analysis was performed using SPSS 21.0 software (IBM-SPSS, Inc., Armonk, NY, USA). Categorical variables were expressed as percentages, while continuous variables were summarised as mean±standard deviation for normally distributed data and as median (interquartile range) for non-normally distributed data. Inter group differences were assessed using the chi-square test or Fisher’s exact test, as appropriate. Agreement between anti-DFS70 detection by ELISA and LIA was evaluated using Cohen’s kappa coefficient.
Results
Over the study period, a total of 32,285 samples were examined for antinuclear antibodies (ANA) using IIF, of which 25,515 (79%) tested positive. An overview of the results with the testing algorithm is depicted in the Figure 1.
In patients tested for ANA IIF presenting with undefined disease, 82 sera (undefined disease group) exhibited AC-2 pattern (0.25%). Of these 74 (90.2%) were positive for anti-DFS70 antibodies by ELISA and 73/74 were positive in LIA as well; one DFS70 ELISA negative patient was positive in LIA. Overall, 73 patients were positive by both the methods. In 55 of them, anti-DFS70 was the only antibody in LIA (mono-specific anti-DFS70). Department wise breakup of anti-DFS70 positive results in IIF, ELISA and LIA are provided in Supplementary Table. There was almost perfect agreement (kappa value=0.861) between ELISA and LIA in the undefined disease group and a lower agreement in the SARD group (kappa value=0.692).
The mean (SD) age of the patients in the SARD group (n=350) and non-rheumatic disease control group (n=250) was 29.7±11.8 years (89.4% females) and 38.5±16.2 years (71.6% females) respectively. SARD group (350) comprised 247 (70.5%) patients with systemic lupus erythematosus (SLE), 46 (13.1%) rheumatoid arthritis (RA) patients, 35 (10%) patients with undifferentiated connective tissue disease (UCTD), 15 (4.3%) patients with systemic sclerosis (SSc), 3 (0.9%) patients with Sjogren syndrome and one patient each with antiphospholipid syndrome (APS), anti-synthetase syndrome (ASS), mixed CTD (MCTD), and myositis. The non-rheumatic disease control group included 89 patients with osteoarthritis and 161 patients with fibromyalgia.
In the SARD group with anti-DFS70 ELISA positive patients (n=54), AC-2 pattern was seen in 2 sera (3.7%), homogeneous pattern in 39 (72.2%) (P<0.001) and speckled pattern in 13 (24.1%) (P=0.004). The prevalence of ELISA anti-DFS70 in this group was 15.4% (54/350). The two sera with AC-2 pattern were positive for anti-DFS70 antibodies by ELISA and LIA, and both were DFS70 mono-specific.
The comparison of demographic and serological characteristics in SARD patients with and without anti-DFS70 ELISA positivity is shown in the Table I In the SARD subgroup, anti-DFS70 ELISA positivity was observed most frequently in patients with SLE (87%), than in RA (1.9%). Anti-DFS70 positivity was significantly associated with SLE (P=0.003), while in RA there was a significant negative association (P=0.004). Anti-DFS70 ELISA positive sera showed a higher frequency of anti-dsDNA, anti-nucleosome, and anti-histone antibodies and a lower frequency of anti-SSA antibodies compared with anti-DFS70 negative sera.
| Characteristics (n=350) | Anti-DFS70 ELISA positive [n=54; n (%)] | Anti-DFS70 ELISA negative [n=296; n (%)] | P value |
|---|---|---|---|
| Age* (yr) | 26.8±10.4 | 30.3±12 | 0.145 |
| Sex (Women) | 50 (92.6) | 263 (88.9) | 0.629 |
| SLE (n=247) | 47 (87) | 200 (67.6) | 0.003 |
| RA (n=46) | 1 (1.9) | 45 (15.2) | 0.004 |
| UCTD (n=35) | 4 (7.4) | 31 (10.5) | 0.625 |
| SSc (n=15) | 1 (1.9) | 14 (4.7) | 0.483 |
| Sjogren’s syndrome (n=3) | 0 (0) | 3 (1) | 1.000 |
| Others† (n=4) | 1 (1.9) | 3 (1) | 0.490 |
| Anti-Sm/RNP | 23 (42.6) | 144 (48.6) | 0.460 |
| Anti-Sm | 17 (31.5) | 89 (30.1) | 0.872 |
| Anti-SSA | 9 (16.7) | 107 (36.1) | 0.004 |
| Anti-Ro-52 | 12 (22.2) | 99 (33.4) | 0.113 |
| Anti -SSB | 3 (5.6) | 25 (8.4) | 0.594 |
| Anti-PCNA | 2 (3.7) | 4 (1.4) | 0.232 |
| Anti-Rib-P | 16 (29.6) | 78 (26.4) | 0.619 |
| Anti-ds DNA | 30 (55.6) | 40 (13.5) | 0.0001 |
| Anti-nucleosomes | 33 (61.1) | 49 (16.6) | 0.0001 |
| Anti-histones | 34 (63) | 51 (17.2) | 0.0001 |
| Amti-AMA-2 | 9 (16.7) | 25 (8.4) | 0.077 |
| Anti-Jo-1 | 0 (0) | 3 (1) | 1.000 |
| Anti-centromere | 0 (0) | 13 (4.4) | 0.232 |
*Mean±standard deviation; †APS, ASS, MCTD, Myositis (n=1)
Homogeneous ANA patterns predominated in DFS70-positive patients. The distribution of frequency of DFS antibodies as assayed by the two methods in disease subgroups is depicted in the Table II. DFS70 antibodies were most common in SLE, but monospecific DFS70 antibodies were observed only in UCTD. A complete correlation was observed between anti-DFS70 ELISA and LIA positivity in all patients diagnosed with UCTD, RA, and systemic sclerosis (SSc). However, DFS antibodies were more frequently picked up by ELISA in SLE compared to use of LIA.
| Diagnosis | Anti-DFS70 ELISA positive (n) | Anti-DFS70 LIA positive (n) | Mono-specific anti-DFS 70 antibodies in LIA; n (%) |
|---|---|---|---|
| SLE | 47 | 14 | 0 |
| RA | 1 | 1 | 0 |
| UCTD | 4 | 4 | 3 (75) |
| SSc | 1 | 1 | 0 |
| APS | 1 | 0 | 0 |
SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; UCTD, undifferentiated connective tissue disease; SSc, systemic sclerosis; APS, antiphospholipid syndrome
Comparison of characteristics of SLE patients, with and without DFS70 antibodies are shown in ( Table III) DFS70 positivity correlated with anti-dsDNA positivity and greater disease activity with significantly higher median systemic lupus erythematosus disease activity index (SLEDAI) scores compared to DFS70-negative patients. Renal involvement was significantly higher in anti-DFS70 positive SLE patients (P=0.014).
| Characteristic | Anti- DFS70 ELISA positive (n=47); n (%) | Anti- DFS70 ELISA negative (n=200); n (%) | P value |
|---|---|---|---|
| Age* | 25.2±9.07 | 26.11±10.16 | 0.803 |
| Sex (women) | 43 (91.4) | 179 (89.5) | 0.794 |
| ds DNA (positive) | 40 (85.1) | 100 (50) | <0.0001† |
| Low complements | 42 (89.3) | 158 (79) | 0.146 |
| SLEDAI-2K (median-IQR) | 16 (10-23) | 10 (5-15) | 0.01† |
| Anti-Sm/RNP | 22 (46.8) | 122 (61) | 0.09 |
| Anti-Sm | 17 (36.1) | 86 (43) | 0.416 |
| Anti-SSA | 8 (17) | 91 (45.5) | 0.0003† |
| Anti-Ro-52 | 10 (21.2) | 74 (37) | 0.041† |
| Anti -SSB | 3 (6.4) | 18 (9) | 0.773 |
| Anti-PCNA | 2 (4.3) | 3 (1.5) | 0.242 |
| Anti-Rib-P | 16 (34.0) | 74 (37) | 0.739 |
| Homogeneous ANA pattern | 36 (76.5) | 98 (49) | 0.0006† |
| Speckled ANA pattern | 11 (23.4) | 93 (46.5) | 0.0049† |
*Mean±standard deviation; †statistically significant
Mean anti-DFS70 ELISA titres in SLE were higher in those with LIA-confirmed anti-DFS70 antibodies (3.38±1.61; n=14) compared to those negative in LIA (1.55±0.63, n=33).
The mean (SD) age of patients in the undefined disease group with AC-2 pattern (n=82) was 37.5±14 years (range 12 to 76 yr) with female preponderance (n=65;79.2%). Anti-DFS70 ELISA was positive in 74 patients (90.2%) with varied diagnoses; Polyarthralgia was observed in 18.2% (15/82) of them, while recent infection and chronic kidney disease were each seen in 9.7% (8/82) of patients. Mean (SD) anti-DFS70 ELISA titres in the undefined disease group were higher compared to the SARD group (2.8±1.22 vs. 2.15±1.32). Figure 2 illustrates the referral diagnoses of 74 anti-DFS70 ELISA positive patients in the undefined disease group.
Discussion
We found that the AC-2 pattern was relatively rare, observed in only 0.25% of all samples, aligning with reported rates ranging from 0.8%–12.3% in general ANA-tested populations.14-19 In undefined disease referrals, the AC-2 pattern was uncommon, but 90% of AC-2 pattern positive cases were ELISA positive, with 74.3% demonstrating monospecific anti-DFS70 antibodies on LIA. Among confirmed SARD patients, the AC-2 pattern was also extremely uncommon (0.57%), whereas anti-DFS70 antibodies were detected by ELISA in a substantially higher proportion (15.2%). The prevalence of anti-DFS70 antibodies in SARD has been reported between 2% and 19% in earlier studies,29-32 aligning with our findings. SLE patients most frequently exhibited anti-DFS70 antibodies, followed by UCTD, which is consistent with reports from other centres.14,29-32
We replicate the rarity of monospecific anti-DFS70 antibodies reported in other studies in SLE, 0.4–3.1% in prior studies.14,30,33,34 A pooled analysis of 1,396 SLE patients reported 2.7% prevalence with only 0.7% monospecificity.19 While we report correlation of anti-DFS antibodies with anti-dsDNA antibodies, higher SLEDAI scores and renal involvement, a multicentre study33 showed a negative association of anti-DFS70 antibodies with anti-dsDNA in early stage SLE (<15 months from diagnosis), suggesting that disease duration may influence antibody expression. Another Chinese study30 reported a positive association of anti-DFS70 antibodies with dsDNA even on longitudinal follow up of SLE patients. The contradiction in studies with some correlating their presence to milder disease and others reporting the contrary could be due to differences in disease duration, ethnicity of the cohort.
We report a lower prevalence of DFS IIF pattern in undefined disease referrals (0.25%), compared to previous reports of 0.48 - 12.3%,14-21 which included two reports from India. Differences in prevalence may reflect population, assay, or laboratory factors.9,19 The EUROIMMUN ELISA and LIA assays employ full-length (FL) DFS70 antigen (aa 1–530), 35,36 whereas the QUANTA Flash DFS70 CLIA uses a truncated antigen (TC, aa 349–435). Comparative studies36,37 have reported higher anti-DFS70 detection with FL antigen assays, likely due to missing epitopes in TC antigens, although some discordance may also result from lower assay cut-offs, as most discordant samples exhibited low positivity in FL assays. Concordance between AC-2 pattern and specific anti-DFS70 assays varies widely (14%–>90%),14,18,38,39 and in our study, 90.2% of AC-2–positive samples were confirmed by both ELISA and LIA. Additional nuclear antigens, such as MeCP2, may co-localise with DFS70 and generate DFS-like HEp-2 staining, further complicating the interpretation. 37,40
Recognition of mixed patterns comprising AC-2 and other significant nuclear patterns by IIF has low accuracy, as shown in an international internet-based survey.41 In SARD patients, coexisting disease-specific ANAs can mask anti-DFS70 detection by IIF, likely due to higher titres and greater affinity of disease-specific antibodies, which produce homogenous or speckled patterns,29 a phenomenon also observed in the present study. The almost perfect agreement between ELISA and LIA in the undefined disease group compared to SARD group could be due to comparatively higher anti-DFS70 antibody titres in the former group.
The AC-2 pattern has been updated by ICAP in February 2025 from ‘competency level’ to ‘expert level,’ reflecting the potential for misclassification as homogeneous or speckled patterns.3 Several studies have highlighted the importance of confirming AC-2 with assays such as immunoblotting or ELISA to accurately identify anti-DFS70 antibodies and coexisting SARD-specific autoantibodies. 9,35
Strengths of our study include a relatively large sample size encompassing SARD patients, ANA referrals with undefined disease, and non-rheumatic disease controls, the use of two methods to confirm the presence of anti-DFS70 antibody and correlation with clinical and laboratory characteristics in SLE patients. Choosing LIA over ELISA for anti-DFS70 confirmation is advantageous, as LIA allows simultaneous detection of antibodies against multiple antigens, whereas ELISA necessitates batch-wise testing.
This study has certain limitations. First, longitudinal follow up of patients who tested positive for anti-DFS70 antibodies was not available, which restricts the ability to assess the persistence of these antibodies over time or their potential impact on disease evolution. The relatively small RA and UCTD subgroups may not reflect the true significance in these populations. Longitudinal studies in patients with lupus may bring out true associations of this antibody with disease behaviour.
The DFS IIF pattern and anti-DFS70 antibodies show variable prevalence across patient groups with important diagnostic implications.
In SARD patients with DFS70 antibodies, they are unlikely to be monospecific on LIA. Further in undefined disease referrals, the strong concordance between DFS IIF pattern and mono-specific anti-DFS70 antibodies suggests that when AC-2 pattern is present, SARD is unlikely. Since anti-DFS70 antibodies are rare in SARD and usually coexist with other disease-specific autoantibodies, adding DFS antigen to an ANA LIA being used for studying patients with SARD offers limited utility. However, confirming isolated DFS70 positivity in ANA-positive patients with undefined disease may help reduce unnecessary referrals, repeat testing, and misdiagnosis.
Author contributions
RM: Conceptualised the study, devised the protocol, analysed and interpreted data manuscript writing; MG: Interpreted data, reviewed, manuscript writing; KP: Performed all laboratory experiments; BKP: Collected and interpreted the clinical data; LR: Conceptualised the study, devised the protocol, supervised the complete study, manuscript writing. All authors have read and approve the final printed version of the manuscript.
Financial support and sponsorship
This study was funded by a research grant provided under the Multidisciplinary Research Unit (MRU) scheme of the Department of Health Research (DHR), Ministry of Health and Family Welfare, Government of India.
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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