High serum prevalence of autoreactive IgG antibodies against peripheral nerve structures in patients with neurological post-COVID-19 vaccination syndrome.

Background: Patients suffering from neurological symptoms after COVID-19 vaccination (post-COVID-19 vaccination syndrome (PCVS)) have imposed an increasing challenge on medical practice, as diagnostic precision and therapeutic options are lacking. Underlying autoimmune dysfunctions, including autoantibodies, have been discussed in neurological disorders after SARS-CoV-2 infection and vaccination. Here, we describe the frequency and targets of autoantibodies against peripheral nervous system tissues in PCVS. Methods: Sera from 50 PCVS patients with peripheral neurological symptoms after COVID-19 vaccination and 35 vaccinated healthy controls were used in this study. IgG autoreactivity was measured via indirect immunofluorescence assays on mouse sciatic nerve teased fibers. The frequencies of autoantibodies were compared between groups using Fisher's exact test. Serum anti-ganglioside antibodies were measured in ganglioside blots. Autoantibody target identification was performed using immunoprecipitation coupled to mass spectrometry. Subsequent target confirmation was conducted via cell-based assays and ELISA. Results: Compared with controls, PCVS patients had a significantly greater frequency of autoantibodies against peripheral nervous system structures (9/50(18%) vs 1/35(3%); p=0.04). Autoantibodies bound to paranodes (n=5), axons (n=4), Schmidt-Lanterman incisures (n=2) and Schwann cell nuclei (n=1). Conversely, antibodies against gangliosides were absent in PCVS patients. Target identification and subsequent confirmation revealed various subunits of neurofilaments as well as DFS-70 as autoantibody epitopes. Conclusion: Our data suggest that autoantibodies against nervous system tissue could be relevant in PCVS patients. Autoantibodies against neurofilaments and cell nuclei with so far non-established links to this disease spectrum should be further elucidated to determine their biomarker potential.

Clinical Significance of Antibodies to DFS70 in Immunoinflammatory Rheumatic Diseases.

The relevance of the problem of immunoinflammatory rheumatic diseases (IIRD) for modern medicine is determined by their high prevalence in the population, the difficulty of early diagnosis, the rapid development of disability and poor life prognosis. Recent data on the significance of anti-DFS70 have opened up new possibilities for optimizing the step-by-step diagnosis of IIRD. The detection of these antibodies can help in the interpretation of a positive result for antinuclear antibodies (ANA) by indirect immunofluorescence assay on HEp-2 cells (IIFA-HEp-2) in the absence of autoantibodies specific for IIRD. Detection of anti-DFS70 in antinuclear factor (ANF) seropositive patients without clinical and/or serological markers characteristic of a certain disease from the IIRD group can be considered as a potential marker that excludes this group of diseases.

Neuronal autoantibodies in the cerebrospinal fluid of 148 patients with schizophrenia and 151 healthy controls.

Schizophrenia is a syndrome with multiple etiologies, one of which is the potential for an autoimmune disease of the brain such as N-methyl-d-aspartate receptor (NMDAR) encephalitis, which can induce psychosis resembling schizophrenia. Here, we examined anti-neuronal autoantibodies related to psychosis using both cell- (CBA) and tissue-based assays (TBA) in the cerebrospinal fluid (CSF) of patients with chronic schizophrenia and control participants. First, we screened for the antibodies against leucine-rich glioma-inactivated 1 (LGI1), γ-aminobutyric acid B receptor (GABABR), dipeptidyl aminopeptidase-like protein 6 (DPPX), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR1/R2), and contactin-associated protein-like 2 (CASPR2) in 148 patients with schizophrenia. No antibodies were detected. Next, we performed CBA for NMDAR antibodies in 148 patients with schizophrenia and 151 age- and sex-matched controls. Although we detected relatively weak immunoreactivity for NMDAR in the CSFs of two patients with schizophrenia and three controls, no samples were positive when strict criteria were applied. For TBA in the rat hippocampus and cerebellum, we detected positive signals in the CSFs of 13 patients with schizophrenia and eight controls. Positive samples were analyzed for paraneoplastic syndrome and antinuclear antibodies using immunoblotting. The CSFs of nine patients and six controls were positive for dense fine speckle 70 (DFS70) antibodies. Additionally, antibodies against centromere protein (CENP)-A and CENP-B were detected in patients with schizophrenia. Our results suggest that autoantibodies against NMDAR, LG1, GABABR, DPPX, AMPAR1/R2, and CASPR2 are not associated with the pathogenesis of chronic schizophrenia. Moreover, we emphasize the importance of considering the effect of anti-DFS70 antibodies when analyzing autoantibodies in CSF samples. Conclusively, we obtained no evidence suggesting that the most frequent neuronal autoantibodies in the CSF play a role in the pathogenesis of schizophrenia, even in our sample.

Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging.

Antinuclear antibodies (ANAs) are essential diagnostic markers in systemic autoimmune rheumatic diseases. Among the 30 ANA patterns, homogeneous (AC-1) and dense fine speckled (AC-2) should be focused on owing to their somewhat indistinct presentation in immunofluorescence imaging and distinct correlation with clinical conditions. This study aimed to develop a flowchart to guide discrimination between AC-1 and AC-2 patterns and to re-evaluate ANA samples according to this flowchart to verify its detection ability. We re-evaluated immunofluorescence imaging of 62 ANA blood samples simultaneously subjected to solid-phase assays for autoantibodies against dsDNA, nucleosomes, histones, and DFS70. The results showed statistically significant odd ratios (ORs) of detection of anti-DFS70 using AC-2 after re-evaluation of total samples (OR 101.9, 95% CI 11.7-886.4, p-value < 0.001) and subgroup analysis of patients' samples (OR 53.8, 95% CI 5.9-493.6, p-value < 0.001). The OR of anti-nucleosome/histone/dsDNA detection using AC-1 in re-evaluated data increased to 5.43 (95% CI 1.00-29.61, p-value = 0.05). In the analysis of specific autoantibodies, more than half of the samples with an AC-2 pattern (54.2%) had specific autoantibodies other than anti-DFS70. We conclude that the flowchart for discriminating between AC-1 and AC-2 ANA patterns in this study is a viable practical guide for other laboratories when encountering equivocal ANA results.

Dense fine speckled nuclear immunofluorescence: A mildly reassuring antinuclear antibody pattern meriting consideration.

INTRODUCTION: Antinuclear antibodies (ANAs) are regarded as a hallmark of connective tissue diseases (CTDs) and play a key role in their diagnosis, but the value of some particular antibodies in management of patients and the disease prognosis is controversial. The mechanism underlying the production of ANAs in CTDs, other chronic inflammatory conditions and even in healthy people, is not completely elucidated. Anti-DFS70 antibodies connected with the dense fine speckled autoantigen of 70 kD, known as the lens epithelium-derived growth factor p75, are a subgroup of ANAs. Their presence and coexistence with other antibodies and their clinical significance are the matter of debate. METHODS: Based on literature data, the authors focused on current knowledge explaining the role of anti-DFS70 antibodies in selected CTDs. RESULTS: However, the literature data is ambiguous and does not fully support the validity of the anti-DFS70 assay for a specific CTD diagnosis. Most researchers claim that the presence of anti-DFS70 as the only one usually exclude the diagnosis of CTD. Nevertheless, its coexistence with other ANAs is not an excluding factor but has predictive value due to more favorable course of CTD. Such situations may also suggest an enhanced risk of the development of a CTD in the future. CONCLUSIONS: Although more studies are needed in this field, it seems reasonable to ascertain the presence of anti-DFS70 in routine clinical practice.

Anti-dense fine speckled 70 (DFS70) autoantibodies: correlates and increasing prevalence in the United States.

Objective: Recent studies report high-titer anti-dense fine speckled 70 (DFS70) autoantibodies in persons with inflammatory conditions, but the clinical significance remains unclear. Our goals were to estimate anti-DFS70 autoantibody prevalence, identify correlates, and assess time trends. Methods: Serum antinuclear antibodies (ANA) were measured by indirect immunofluorescence assay on HEp-2 cells in 13,519 participants ≥12 years old from three time periods (1988-1991, 1999-2004, 2011-2012) of the National Health and Nutrition Examination Survey. ANA-positive participants with dense fine speckled staining were evaluated for anti-DFS70 antibodies by enzyme-linked immunosorbent assay. We used logistic models adjusted for survey-design variables to estimate period-specific anti-DFS70 antibody prevalence in the US, and we further adjusted for sex, age, and race/ethnicity to identify correlates and assess time trends. Results: Women were more likely than men (odds ratio (OR)=2.97), black persons were less likely than white persons (OR=0.60), and active smokers were less likely than nonsmokers (OR=0.28) to have anti-DFS70 antibodies. The prevalence of anti-DFS70 antibodies increased from 1.6% in 1988-1991 to 2.5% in 1999-2004 to 4.0% in 2011-2012, which corresponds to 3.2 million, 5.8 million, and 10.4 million seropositive individuals, respectively. This increasing time trend in the US population (P<0.0001) was modified in some subgroups and was not explained by concurrent changes in tobacco smoke exposure. Some, but not all, anti-DFS70 antibody correlates and time trends resembled those reported for total ANA. Conclusion: More research is needed to elucidate anti-DFS70 antibody triggers, their pathologic or potentially protective influences on disease, and their possible clinical implications.

A novel magnetic bead-based immunoprecipitation method for anti-dense fine speckled 70 antibodies.

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.

The significance of dense fine speckled pattern in antinuclear antibody-associated rheumatic disease and coexisting autoantibodies: A propensity score-matched cohort study.

AIM: To investigate the relationship between the prevalence of antinuclear antibody (ANA) -associated rheumatic diseases (AARD) and the presence of dense fine speckled (DFS) and homogeneous patterns in ANA tests. METHODS: This retrospective study enrolled adult patients with either a DFS or homogeneous pattern in their ANA test. A mixed pattern was defined as the presence of more than one pattern reported in the test. The presence of anti-DFS70 antibodies and other common autoantibodies were detected using EUROLINE ANA Profile 23. A 1:2 propensity score matching was applied to control for demographic and other interfering factors. RESULTS: A total of 59 patients with a DFS pattern were enrolled and compared with a matched homogeneous group. The DFS group had a significantly lower prevalence of AARD (3.4% vs. 16.9%, p = .008) and the subgroup with anti-DFS70 antibodies showed an even lower prevalence (2% vs. 20%, p = .002). Among the 33 patients with monospecific anti-DFS70 antibodies, five had a mixed pattern, and all patients with common autoantibodies had an isolated DFS pattern. CONCLUSIONS: The findings of this study suggest that patients with a DFS pattern in their ANA test may have a lower prevalence of AARD compared with those with a homogeneous pattern. However, an isolated DFS pattern in ANA testing does not necessarily indicate the presence of monospecific anti-DFS70 antibodies or AARD. Confirmatory testing for the monospecific anti-DFS70 antibody is mandatory to exclude AARD.

An Investigation of the Relationship Between Isolated Anti-dense Fine-Speckled 70 Autoantibodies and Allergen-Specific Immunoglobulin E.

Background Although the mechanisms of the formation of anti-dense fine-speckled 70 (anti-DFS70) antibodies are not fully known, there is evidence in the literature that allergic reactions may play a role in their formation. Immunoglobulin E (IgE)-mediated immunopathological mechanisms are increasingly being elucidated in diseases such as atopic dermatitis and urticaria-related diseases. We aimed to reveal its relationship with anti-DFS70 in allergen-sensitive patients with positive specific IgE (sIgE) levels. Methodology The study included samples of 758 patients who underwent antinuclear antibody (ANA) screening and allergen-sIgE testing between January 2019 and January 2022. Patients' clinical diagnoses were retrospectively obtained from the hospital information management system. ANA was tested according to the instructions of the manufacturer by the indirect immunofluorescent antibody method using HEp-2 cell substrates (Euroimmun Luebeck, Germany). Allergen-sIgE was determined by chemiluminescence on the Immulite 2000 XPI system (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) according to the instructions of the manufacturer. Results ANA pattern was detected in 74 samples included in the study. ANA-positive patients were divided into DFS70 (+) and DFS70 (-) groups. A statistically significant increase in the DFS70 pattern was observed in patients with a positive allergen-sIgE test (p < 0.0001). Both allergen-sIgE and DFS70 positivity were statistically significant in younger age groups (p < 0.05). The most common diagnosis was urticaria-related conditions in 23 (31%) patients with a positive allergy test. Conclusions Our study shows that the positivity of the DFS70 pattern is increased in allergen-sensitive patients. Therefore, the allergen-sIgE-mediated allergic disease should be considered in patients with isolated anti-DSF70. Studies with related disease groups are needed to determine whether there is a relationship between anti-DFS70 and allergy-related disease in these patients. If an immunopathological mechanism is not found, these false-positive results can be considered clinically insignificant, and unnecessary consultations can be avoided.

The Nuclear Dense Fine Speckled (DFS) Immunofluorescence Pattern: Not All Roads Lead to DFS70/LEDGFp75.

The monospecific dense fine speckled (DFS) immunofluorescence assay (IFA) pattern is considered a potential marker to aid in exclusion of antinuclear antibody (ANA)-associated rheumatic diseases (AARD). This pattern is typically produced by autoantibodies against transcription co-activator DFS70/LEDGFp75, which are frequently found in healthy individuals and patients with miscellaneous inflammatory conditions. In AARD patients, these antibodies usually co-exist with disease-associated ANAs. Previous studies reported the occurrence of monospecific autoantibodies that generate a DFS-like or pseudo-DFS IFA pattern but do not react with DFS70/LEDGFp75. We characterized this pattern using confocal microscopy and immunoblotting. The target antigen associated with this pattern partially co-localized with DFS70/LEDGFp75 and its interacting partners H3K36me2, an active chromatin marker, and MLL, a transcription factor, in HEp-2 cells, suggesting a role in transcription. Immunoblotting did not reveal a common protein band immunoreactive with antibodies producing the pseudo-DFS pattern, suggesting they may recognize diverse proteins or conformational epitopes. Given the subjectivity of the HEp-2 IFA test, the awareness of pseudo-DFS autoantibodies reinforces recommendations for confirmatory testing when reporting patient antibodies producing a putative DFS pattern in a clinical setting. Future studies should focus on defining the potential diagnostic utility of the pseudo-DFS pattern and its associated antigen(s).

Investigation of dense fine speckled pattern and anti-dense fine speckled 70 antibody by a single step assay.

The indirect immunofluorescence (IIF) method is the gold standard for identifying anti-nuclear antibodies (ANAs). It is recommended that ANA, including the dense fine speckled (DFS) pattern, should be verified with a highly specific confirmatory test after a sensitive screening test. Although methods such as ELISA and LIA are often used to confirm the presence of anti-DFS70 antibodies, new IIF methods have been developed in recent years to prevent the difficulties in the recognition of the DFS pattern and to carry out the confirmatory test in a single step. In this study, we evaluated CytoBead (Generic Assays, Germany) test, which contained both HEp-2 cell substrate and beads coated with DFS70 antigen in one well, in comparison to the routine two-step test strategy. Five hundred forty-one samples were studied by conventional IIF assay, LIA, and CytoBead assay; 264 samples were studied by ELISA. The Bead component of the CytoBead test was found to be reliable as a confirmational test when compared with ELISA and LIA (total agreement values were 85.6% and 87.6%, respectively). The CytoBead ANA DFS70 might be a promising test in the future, allowing both screening and confirmation in a single step, saving time and being easier than two-step testing.

Anti-DFS70 antibodies in systemic lupus erythematosus: Prevalence in a large Chinese cohort and an unexpected association with anti-dsDNA antibodies by a long-term follow-up.

Objective: Monospecific autoantibodies to dense fine speckles 70 (DFS70) antigen are purported to aid in excluding systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE). However, the non-isolated anti-DFS70 still has a certain prevalence in SLE patients, and the clinical significance remains unclear. We aimed to investigate the prevalence, clinical relevance, and value of long-term monitoring of anti-DFS70 antibodies in SLE patients. Methods: Anti-DFS70 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in 851 SLE patients, 211 healthy individuals, and 194 patients with other SARD (except SLE). Demographic, serological, and clinical associations of anti-DFS70 antibodies were analyzed by a stepwise multivariable logistic regression model. The correlation of anti-DFS70 with anti-dsDNA, anti-C1q, and SLE Disease Activity Index 2000 (SLEDAI-2K) was analyzed. Sixty-one SLE patients with follow-up time ranging from 2 to 57 months were measured anti-DFS70 antibodies using both ELISA and line immunoassay. The dynamic variations of anti-DFS70 antibodies were evaluated with anti-dsDNA, anti-C1q, and SLEDAI-2K during the follow-up. Results: The prevalence of anti-DFS70 was significantly higher in SLE (20.7% (176/851)) than in healthy individuals (9.5% (20/211), p = 0.0002) and other SARD (10.8% (21/194), p = 0.002). Multivariable analysis revealed that anti-DFS70-positive SLE patients were associated with younger age (odds ratio (OR) = 0.982; 95% confidence interval (CI) = 0.969, 0.995), higher frequencies of anti-dsDNA (OR 1.598; 95% CI 1.107, 2.306) and anti-PCNA (OR 6.101; 95% CI 2.534, 14.688), and higher levels of serum IgG (OR 1.097; 95% CI 1.067, 1.129) and were more likely to be accompanied by mucosal ulcers (OR 5.921; 95% CI 1.652, 21.215). The O.D. value of anti-DFS70 positively correlated with levels of anti-dsDNA (r = 0.183, p < 0.0001) and anti-C1q (r = 0.181, p < 0.0001), respectively, but not with SLEDAI-2K (p = 0.920). During the follow-up, 49 (42 negative and 7 positive) patients remained stable with anti-DFS70 levels. The other 12 patients experienced significant changes in anti-DFS70, and 83.3% (10/12) of them showed similar trends between anti-DFS70 and anti-dsDNA by evaluation of dynamic variations. Conclusion: Anti-DFS70 antibodies seem to be prevalent in Chinese SLE patients. The positive association of anti-DFS70 with anti-dsDNA and consistent dynamic variation between anti-DFS70 and anti-dsDNA during the follow-up suggested a potential relationship between anti-DFS70 and anti-dsDNA in patients with SLE.

Analysis of Ana/Dfs70 Pattern in a Large Cohort of Autoimmune/Autoinflammatory Diseases Compared with First Degree Relatives and Healthy Controls Evaluated from Colombia.

BACKGROUND: The presence of Antinuclear antibodies/Dense Fine Speckled 70 (ANA/DFS70) has been proposed as a negative biomarker in the process of exclusion of systemic autoimmune/autoinflammatory rheumatic diseases (SARD). The purpose was to evaluate and characterize ANA/DFS70 patients in a large Colombian population with SARD; rheumatoid arthritis (RA), Psoriasis (PsO), Undifferentiated connective tissue disease (UCTD), first-degree relatives of (FDR), and healthy controls (HC). METHODS: ANA determination was performed using indirect immunofluorescence. Samples with positive dense fine granular staining in the nucleoplasm of the interphase cell (AC2) fluorescence were confirmed with CytoBead/ANA and ANA/modified (Knocked out for the PSPI1 gen). RESULTS: 530 mestizo Colombian participants were included. ANA/DFS70 antibody positivity in the whole group was 2.3%, and 0.8% in SARD; no RA patients were positive. ANA/DFS70 positives in UCTD were three women; the average time of evolution of the disease was 9.4 years. The most frequent clinical findings were arthralgias, non-erosive arthritis, and Raynaud's phenomenon. The PsO positive was a woman with C-reactive protein (CRP) positivity and a negative erythrocyte sedimentation rate (ESR) without any other positive autoantibody or extracutaneous manifestation. FDR and HC positives were 7/8 women. All were negative for other autoantibodies. CONCLUSIONS: ANA/DFS70 autoantibodies were present in Colombian patients with SARD at a shallow frequency, they were more prevalent in healthy individuals.

Improved performance of confirmatory assays for detecting dense fine speckled (DFS) 70 antibodies.

The presence of monospecific dense fine speckled 70 (DFS70) pattern in indirect immunofluorescence assay (IFA) without concomitant systemic autoimmune related diseases (SARD)-associated antibodies could be an exclusion biomarker for SARD. Since interpretation of DFS pattern on IFA is subjective, an assay for confirming the presence of anti-DFS70 is required. We evaluated the performance of two commercial assays [fluorescence enzyme immunoassay (FEIA) and line immunoassay (LIA)] for detecting anti-DFS70. Sera with monospecific DFS (n=176) and without DFS (n=179) pattern from referred patients for ANA testing, in two independent laboratories and healthy donors, were investigated for anti-DFS70 by FEIA (Phadia EliA) and LIA (Euroimmun). The assay performance including sensitivity and specificity at different cut-offs was evaluated, and optimal cut-offs were determined. The newly established optimal cut-offs (2.7 U/mL on EliA, band intensity of 28 on LIA) showed significantly better assay performance in detecting anti-DFS70 and confirming monospecific DFS pattern. A relative sensitivity of 93.7% with relative specificity of 100% on EliA and a relative sensitivity of 96.6% with relative specificity of 95.3% on LIA were achieved. Superior Cohen's Kappa agreements with IFA were also achieved for both assays (0.936 for EliA and 0.906 for LIA). Application of an optimal cut-off can significantly increase the assay performance for anti-DFS70 and enhance the accuracy in reporting DFS pattern by IFA.

Low Prevalence of Anti-DFS70 Antibodies in Children With ANA-Associated Autoimmune Disease.

Introduction: Anti-DFS70 antibodies occur in healthy individuals with various medical conditions. Unlike other anti-nuclear autoantibodies (ANA), they are not associated with systemic autoimmune disease in adult patients. To date, only a few studies have addressed the prevalence and/or clinical relevance of anti-DFS70 autoantibodies in children with and without autoimmune disease. Methods: Included in this retrospective cross-sectional mono-centric study were 308 pediatric patients with suspected or known autoimmune conditions who had a positive ANA in indirect immune fluorescence (IIF) screening and who were screened for anti-DFS70 antibodies by extractable nuclear antigen antibodies (ENA) immunoblot. Patients were assigned to four different diagnostic categories according to their diagnosis in the corresponding medical record: (a) absence of autoimmune or rheumatic disease (noARD, n = 116); (b) suspected autoimmunity without definitive diagnosis (sAI, n = 48); (c) other rheumatic disease (ORD) (n = 115); and (d) ANA-associated autoimmune disease (AARD, n = 29). Results: The prevalence of anti-DFS70 antibodies in the overall cohort was 33.8%. Among children without ARD (46.6%, 54/116), prevalence was significantly higher than among children with ORD (23.7%, 27/115, p = 0.0003) or AARD (17.2%, 5/29, p = 0.0054). Among all of the anti-DFS70 positive patients with AARD, other autoantibodies were found in the ENA immunoblot. In contrast, among anti-DFS70 positive patients with ORD (11.5%, 4/27), sAI (33.3%, 6/18) and noARD (16.7%, 9/54), other autoantibodies infrequently were detected (p = 0.0005). Patients with uveitis rarely were positive for anti-DFS70 antibodies (7.7%, 1/13). No association was found between anti-DFS70 antibodies and a history of allergic conditions (p = 0.51). The concordance between a typical DFS pattern in IIF and the detection of anti-DFS70 antibodies by immunoblot was 59.3%. Conclusion: As with adults, the higher prevalence of anti-DFS70 among children without autoimmune disease confirms the mutual exclusion for this autoantibody in the pathogenesis of ARD. Among ANA-positive children, monospecific anti-DFS70 antibodies may help to discriminate between AARD and not-AARD-related conditions.

Anti-DFS70 Antibodies Are Associated With Proliferative Lupus Nephritis and Renal Pathological Activity.

Objective: The significance of anti-dense fine speckles 70 (DFS70) antibodies in systemic lupus erythematosus (SLE) is still unclear, especially in lupus nephritis (LN) patients. We investigated the prevalence, clinical and pathological relevance of anti-DFS70 antibodies in LN patients. Methods: Anti-DFS70 antibodies were measured using enzyme-linked immunosorbent assays in 377 biopsy-proven LN patients, 268 non-LN SLE patients, 232 chronic kidney disease (CKD) patients, and 78 healthy individuals (HI). Demographic, clinical, and pathological parameters were compared between LN patients with and without anti-DFS70 antibodies. Stepwise multivariable logistic regression was performed to identify covariates associated with anti-DFS70 antibodies. Results: The prevalence of anti-DFS70 antibodies in LN (19.6%) was comparable to non-LN SLE patients (19.8%, P=0.9630), but was significantly higher than CKD patients (13.4%, P=0.0468) and HI (9.0%, P=0.0252). Using multivariable logistic regression analysis, the titer of anti-double-stranded DNA (dsDNA) antibodies (adjusted odds ratio=1.002, 95% confidence interval 1.001-1.003, P=0.004) was associated with positive anti-DFS70 antibodies in LN patients. In addition, anti-DFS70 antibodies were more prevalent in proliferative LN (22.0%, 68/309) compared to membrane LN patients (10.2%, 6/59, P=0.0376). Furthermore, LN patients with positive anti-DFS70 antibodies had significantly higher activity index (AI) compared to patients who were negative (8.0 vs 6.0, P=0.0131). However, the chronicity index was similar between the groups (3.0 vs 3.0, P=0.8412). Conclusion: Anti-DFS70 antibodies were not associated with LN development in SLE patients but were associated with anti-dsDNA antibodies, proliferative LN, and renal AI. This suggests their potential to serve as a non-histological biomarker for LN subclass and activity status.

Anti-dense fine speckled (DFS) antibody: its staining pattern in indirect immunofluorescence and its clinical relevance.

Background: Dense fine speckled (DFS) pattern is defined by very intense, heterogeneous speckled staining of nucleoplasms of interphase HEp-2 cells and chromosomal areas of metaphase cells. The association of Anti-DFS70 and rheumatologic signs, symptoms, and diagnosis were evaluated.Methods: One-hundred-eight anti-DFS70 positives who got consecutively admitted to the Rheumatology clinic between January and June 2020 were analyzed. The clinical and laboratory findings of positives for anti-DFS70 antibody were compared with those with DFS pattern ANA IFA staining rates. Also, anti-DFS70 positivity rates and their correlation with the DFS staining pattern were analyzed retrospectively in 1016 CTD patients.Results: The most common complaint was joint pain seen in 77 (71.3%) and the most common laboratory abnormality was RF-positivity observed in 10/108 (9.3%) who had anti-DFS70 positivity. The most common ANA staining pattern was DFS (72/108; 66.7%); one-third had other than DFS. No statistical significance was found for the association of any of the rheumatological complaints and laboratory findings with the DFS staining pattern. ANA analysis was performed in a total of 964/1016 (94.88%) CTD patients and 44 (4.56%) of these positive for anti-DFS70. The correlation coefficient showed good correlations between the DFS pattern staining and anti-DFS70 antibody positivity (r=+0.773, p<0.001).Conclusions: Anti-DFS70-positives have a low rate of CTD. A low anti-DFS70 positivity rate was observed in patients with CTD. As such, it can be considered that anti-DFS70 does not predict CTD or even excludes it.

Clinical utility of anti-DFS70 for identifying antinuclear antibody-positive patients without systemic autoimmune rheumatic disease.

INTRODUCTION: The antinuclear antibody (ANA) test is a screening test for systemic autoimmune rheumatic disease (SARD). We hypothesised that the presence of anti-DFS70 in ANA-positive samples was associated with a false-positive ANA test and negatively associated with SARD. METHODS: A retrospective analysis of patient samples received for ANA testing from 1 January 2016 to 30 June 2016 was performed. Patient samples underwent ANA testing via indirect immunofluorescence method and anti-DFS70 testing using enzyme-linked immunosorbent assay. RESULTS: Among a total of 645 ANA-positive samples, the majority (41.7%) were positive at a titre of 1:80. The commonest nuclear staining pattern (65.5%) was speckled. Only 9.5% of ANA-positive patients were diagnosed with SARD. Anti-DFS70 was found to be present in 10.0% of ANA-positive patients. The majority (51/59, 86.4%) of patients did not have SARD. Seven patients had positive ANA titre > 1:640, the presence of anti-double stranded DNA and/or anti-Ro60. The presence of anti-DFS70 in ANA-positive patients was not associated with the absence of SARD (Fisher's exact test, p = 0.245). CONCLUSION: The presence of anti-DFS70 was associated with a false-positive ANA test in 8.6% of our patients. Anti-DFS70 was not associated with the absence of SARD.

Frequency of ANA/DFS70 in Relatives of Patients with Rheumatoid Arthritis Compared to Patients with Rheumatoid Arthritis and a Healthy Population, and its Association with Health Status. / Frecuencia de ANA/DFS70 en familiares de pacientes con artritis reumatoide comparados con pacientes con artritis reumatoide y población sana y su asociación con el estado de salud.

INTRODUCTION: DFS70 ANAs have attracted interest due to their frequency in individuals with no clinical evidence of systemic autoimmune rheumatic disease, groups with genetic risk for rheumatoid arthritis (RA) were not assessed. OBJECTIVE: To determine the frequency of ANA and DFS70 ANA in blood relatives (BR) of people with RA compared to patients with early RA (ERA), and control individuals, and its association with health status. METHODOLOGY: A cross-sectional study with an analytical component. Sixty ERA patients, 60 BR and 120 control individuals paired by age and sex were studied. Hep2-ANA and DFS70 ANA were studied. The absolute and relative frequencies and associations were established using logistic regression models, with a significance level of 95%. RESULTS: 43% ANA in ERA, 30% in BR, and 25.8% in control individuals 1:80. The fine dense granular pattern based on conventional Hep2 was found in 12.9% of the positive samples, and 1.66% of the total samples. There was no detection of DFS70 ANAs in patients with ERA. In ERA there was an association between the presence of ANA and inflamed joints (p=.02), CRP (p=.01), DAS28CRP (p=.03) and HAQ (p=.04). There was an association between ANA and elevated CRP (p=.05) in the BR. In the control individuals, there was an association between ANA and painful joints (p=02). In DFS70 ANA individuals we observed an association between a normal ESR p=.032, BR (-), p=.044 and absence of painful joints, p=.039. CONCLUSIONS: The frequency of DFS70 ANA in the groups studied was low, none of the patients with ERA was positive. The presence of DFS70 ANA was only confirmed in systemically healthy individuals.

Infusion of anti-DFS70 antibodies prolonged survival of lupus-prone mice.

BACKGROUND: Systemic-lupus-nephritis is a chronic autoimmune disease characterized by immune complex deposition and a flare of autoantibodies and leading to renal injury. OBJECTIVES: To expose anti-Dense-Fine-Speckled-70 (DFS70)-antibodies to genetically-prone-lupus-mice. METHODS: NZBXW/F1 female mice were monitored for the onset of glomerulonephritis by proteinuria upon infusion of anti-DFS70 (40 µg/mouse), commercial-human-IgG (cIgG) or phosphate-buffered-saline (PBS) as controls. The survival time was detected by mice death. Circulating anti-dsDNA were tested by ELISA. Proteinuria, was defined by a standard semi-quantitative-Bayer-Multistix-dipstick. Kidney histology was analyzed by periodic-acid-Schiff-PAS staining. RESULTS: A significantly higher percentage of anti-DFS70-infused mice exhibited prolonged survival time as compared with cIgG and PBS-subjected mice (p < 0.022). One mouse out of 10 mice injected with anti-DFS70-antibodies died at week 36, whereas, 6 out of 10 mice subjected with PBS found dead at this time. Eighty percent of anti-DFS70 injected mice did not show severe glomerulonephritis by histology. CONCLUSIONS: anti-DFS70 attenuated the progression of glomerulonephritis and prolonged the survival time. Circulating anti-DFS70-autoantibodies may confer a protective role against renal injury in murine-lupus-nephritis. Our data may propose a novel therapy approach for lupus patients.