Routine Antinuclear Antibody Research Articles

P-1902. Routine Anti-Nuclear Antibody (ANA) Testing in Patients Experiencing Long COVID: Worth it?

Abstract Background Patients with long COVID are often tested for ANA as part of their routine work-up (Son et al., 2023). However, it is unclear if this testing leads to a rheumatological diagnosis or is actionable. This project assessed the frequency of ANA-positivity in patients experiencing long COVID and the outcomes of rheumatic disease diagnosis and treatment. Methods Electronic medical records (EMR) from January 2022 through May 2023 at the UT Physicians COVID Center of Excellence were reviewed. The population included those experiencing long COVID and presenting with symptoms. Symptoms, personal and family history of rheumatic disease, last known COVID date, ANA test results, referral to rheumatology, diagnoses, and treatment, if any, were recorded. Data was analyzed using Microsoft Excel PivotTables and JavaStat, with right-tailed Fisher Exact Tests. Results The 117-patient sample was 76.92% female, with an average age of 49.21+/-15.09 SD years, with last known COVID between March 2020 and February 2023. 17.95% had a previous rheumatic diagnosis, and 22.22% had a family history of rheumatic disease. The symptoms reported were: fever (6.84%), arthralgia (43.59%), arthritis (22.22%), rash (23.08%), and fatigue (79.49%). 75.21% of patients received an ANA test, of which 43.18% were positive. The mode titer was 1:80, and the most common pattern was nuclear. 48.72% of the patients with a positive test were referred to rheumatology, but only 21.05% received a rheumatological diagnosis. Only 11.97% received any treatment for their symptoms, and most treatments were for symptom control or an anti-inflammatory. On the last visit, only 2.56% had resolution of long COVID symptoms. None of the symptoms were predictive of a positive ANA test. Conclusion The majority of patients experiencing long COVID presenting to our clinic received an ANA test, and almost half had a positive result. Few received any rheumatological diagnosis and most had ongoing symptoms by their latest visit. Although this study is limited by sample size, bias in ANA ordering, and its retrospective nature, these data suggest that routine ANA testing and rheumatology referrals may not be cost-effective in patients experiencing long COVID. Disclosures Luis Ostrosky-Zeichner, MD, FACP, FIDSA, FSHEA, FECMM, CMQ, Cidara: Advisor/Consultant|Enanta: Advisor/Consultant|F2G: Advisor/Consultant|Gilead: Advisor/Consultant|GSK: Advisor/Consultant|Melinta: Advisor/Consultant|Octapharma: Advisor/Consultant|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Pulmocide: Grant/Research Support|Scynexis: Grant/Research Support|Viracor: Advisor/Consultant

Evaluation of Positive Cases for Dense Fine Speckled (DFS) Immunofluorescence Pattern and Anti-DFS70 Antibodies

Objective: The detection of anti-nuclear antibodies (ANA) is crucial in diagnosing systemic autoimmune rheumatic diseases (SARDs). The Dense Fine Speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine ANA screening in patients with various clinical conditions. The aim of this study was to analyze the data of patients who were positive for DFS/antiDFS in our patient population and to show the possible clinical relationship. Methods: In this retrospective study, 7406 patient serum samples sent to our laboratory for routine ANA screening between May 2022-2023 were evaluated for the presence of anti-DFS. Results: In a group of patients referred for routine ANA screening using the indirect immunofluorescence method, the frequency of DFS pattern was found to be 4.55% (337/7406), with ANA positivity detected at a rate of 25.68% (1902/7406). Out of 221 patients with DFS pattern, 181 tested positive for anti-DFS antibodies in both the IIF-ANA and immunoblot (IB) tests. Additionally, 11 of these patients tested positive for other antibodies against different extractable nuclear antigens (anti-ENAs). Conversely, only seven out of the 40 patients who tested negative for anti-DFS antibodies showed positive results for other anti-ENAs. Conclusion: The DFS pattern is often positive in individuals. In patients exhibiting this pattern, anti-DFS70 antibody may be detected alone or in combination with SARD-associated autoantibodies. Therefore, we propose that in this patient cohort, it would be more prudent to screen for additional concomitant autoantibodies with anti-ENA rather than confirming isolated anti-DFS. doi: https://doi.org/10.12669/pjms.41.2.10336 How to cite this: Gurbuz M, Yıldırım BF, Cetinkol Y. Evaluation of Positive Cases for Dense Fine Speckled (DFS) Immunofluorescence Pattern and Anti-DFS70 Antibodies. Pak J Med Sci. 2025;41(2):580-584. doi: https://doi.org/10.12669/pjms.41.2.10336 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recognition of rare antinuclear antibody patterns based on a novel attention-based enhancement framework.

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist's performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice.

Harmonization of ANA testing challenge: quantification strategy to accurately predict end-point titers avoiding serial dilution.

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results.

B-131 The Prevalence and Clinical Relevance of the DFS Immunofluorescent Staining Pattern in a Large ANA-positive Cohort

Abstract Background The dense fine-speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine antinuclear antibody (ANA) screening. Although various researchers have studied the DFS pattern in recent years, it presents a clinical puzzle since its significance remains elusive. Anti-DFS-70 antibodies occur in healthy individuals with different medical conditions. Unlike the other ANA patterns, they are not associated with systemic autoimmune disease. The present study examined the association of a DFS ANA pattern with the medical conditions of patients who presented to a tertiary care medical center. Methods Retrospective chart review of all laboratory values (January 2020 through June 2022) with ANA- and DFS-positive patterns in patients with ANA tests ordered by various clinicians and compared to the patient diagnosis reported. UMass Human IRB approves this study. Results Of 8885 ANA samples analyzed, 67.8% (6022) were reported negative. Of total positive ANA (2863) samples, 78.6% (2257) had nuclear patterns, 9.1% (260) had mitotic patterns, and 12.1% (346) had cytoplasmic patterns. Of the positive ANA samples, 14.6% (417) had DFS patterns. For samples with DFS pattern, titers ranged from 1:40 to 1:1280; 62.5% were between1:40, and 1:80. Of samples positive for ANA DFS patterns, 76% (317) were from female patients, and 90% (375) were from patients >21 years of age. Among patients with DFS patterns, the most commonly reported diseases were neurological, followed by respiratory, digestive, circulatory, and musculoskeletal (Table 1). Conclusion Clinicians should use a positive ANA with a DFS pattern to indicate pathological phenomena other than autoimmune diseases. Only 3.1% of the patients with DFS patterns have a systemic rheumatic disorder. Clinicians should pay close attention to ANA Patterns, especially DFS staining when using ANA testing.

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.

Prevalence of Anti-Dense Fine Speckled 70 Antibodies in Systemic Autoimmune Rheumatic Diseases and Healthy Controls: A Cross-sectional Study

Introduction: Recognition of Dense Fine Speckled (DFS) Antinuclear Antibody (ANA) pattern is challenging as it is frequently confused with speckled or homogeneous patterns. Identification and confirmation of DFS pattern is important as it is more often seen in healthy individuals, routine laboratory referrals with relatively lesser prevalence in Systemic Autoimmune Rheumatic Diseases (SARD). As there is limited data on the prevalence of DFS pattern in Indian population, present study determined the prevalence and clinical significance of DFS pattern followed by anti-DFS 70 Enzyme Linked Immunosorbent Assay (ELISA) for confirmation. Aim: To estimate the prevalence of anti-DFS 70 antibodies in ANA screening tests, SARD, and healthy individuals by Indirect Immunofluorescence (IIF) screening and confirmation by anti- DFS 70 ELISA. Materials and Methods: A cross-sectional study was conducted over a period of 15 months from January 2017 to April 2018 at Nizams Institute of Medical Sciences, a tertiary care institute in Hyderabad, Telangana, India. A total of 7,550 serum samples were tested by IIF during the study duration. Sera displaying DFS pattern was tested for anti-DFS antibodies and anti-Extractable Nuclear Antigen (ENA) antibodies by ELISA. A total of 49 patients from rheumatology department satisfying SARD criteria and 184 healthy patients were also tested for anti-DFS antibodies. Results: Anti-DFS 70 antibodies were seen in 0.47%, 16.3%, 39.6% among routine ANA screening referrals, SARD patients and healthy individuals, respectively. DFS 70 antibodies were positive in 60% of DFS IIF positive samples with none of them showing anti-ENA positivity or clinical evidence of SARD. Anti- DFS 70 positivity was more commonly seen in younger patients. Anti-DFS antibodies were significantly more common in healthy individuals compared to SARD patients (p -value=0.0022). Conclusion: It is important to confirm DFS IIF pattern with a specific ELISA as isolated DFS antibodies are frequently associated with non SARD conditions.

Current issues of standardization of antinuclear antibodies’ detection by indirect immunofluorescence on HEp-2 cells

Background. Antinuclear antibodies (ANA) are a group of antibodies that target nuclear and cytoplasmic antigens. Testing for ANA using an immunofluorescence assay (IFA) on HЕp-2 cell (IFA-HEp-2) is a screening test for the diagnosis of systemic autoimmune rheumatic diseases, druginduced lupus erythematosus, autoimmune liver diseases, juvenile idiopathic arthritis. In routine ANA testing final report forms bears important, but unstandardized variables (e. g., screening and final titers of ANA, coding of glow types, the procedure for performing confirmatory tests). Aim of the study. Develop a standard report form for IFA-HEp-2 testing results.Material and methods. Survey of 10 immunological and clinical diagnostic laboratories using the ICAP questionnaire adapted by the Working Group on Standardization of the definition of ANA by the IFA-HEp-2 method (Committee on Immunology of the Association ‘Federation of Laboratory Medicine’).Results. According to the results of a survey: most of the participants use a screening dilution of serum 1:160; indicates the maximum end titer of antinuclear factor (ANF), cytoplasmic staining; identifies staining types (including AC encoding); considers it mandatory to make confirmatory tests to detect ANA to individual nuclear antigens in ANF-positive patients, but does not provide a list of this tests in the results form; describes ANF titer for each of the detected glows separately; designates ANF titers using a colon and does not give a clinical interpretation of the results. Based on interlaboratory consensus, ICAP recommendations and the requirements of ISO 15189, the standard report form for IFA-HEp-2 testing results was developed.Conclusions. To achieve high quality of interaction between clinical diagnostic/immunological laboratories and clinical departments of health care facilities, it’s necessary to introduce into practice a standard form for IFA-HEp-2 testing results, developed relying on modern clinical recommendations and interlaboratory consensus.

The choice of anti-LEDGF/DFS70 assay matters: a comparative study of six assays

Lens-epithelial derived growth factor (LEDGF/DFS70) autoantibodies result in the commonly observed dense fine speckled (DFS) pattern by anti-nuclear antibody (ANA) assay. However, there is no consensus approach for confirmation of this autoantibody specificity. To evaluate current approaches, we examined inter-assay agreement between six anti-LEDGF/DFS70 assays. A total of 395 consecutive sera samples from routine ANA diagnostics were obtained, tested by routine ANA, anti-ENA line immunoblot assay (LIA) and anti-dsDNA assay and with six anti-DFS/LEDGF assays: the EuroLine-LIA (Euro-LIA), Medical and Biological Laboratories ELISA (MBL-ELISA), Phadia-EliA (EliA), QUANTA Flash CLIA, EuroImmun ELISA (Euro-ELISA) and Immco-Diagnostics HEp-2 ELITE/DFS-Knockout (HEp-2KO). Of 395 sera, 108 tested positive by at least one assay. Despite general good concordance between all assays across the cohort (Gwet's AC1=0.89), within the target DFS-ANA pattern group inter-assay agreement was poor (AC1=0.59). Euro-LIA, CLIA and MBL-ELISA assays were most concordant, but CLIA and Euro-LIA were also most likely to identify discordant positive results. EliA and Euro-ELISA had poorer agreement, which could be attributable to ill-matched cut-offs between assays. HEp-2KO was frequently discordant with all other assays tested. Euro-LIA, CLIA and MBL-ELISA were most concordant at manufacturer's specifications and are suited for use in clinical laboratories. Modified assay thresholds are required to ensure comparative results for Euro-ELISA and EliA. HEp-2KO assay is frequently discordant with all other assays, making it less suited for routine diagnostics. The study highlights the importance of considering inter-assay variability when developing a diagnostic strategy for anti-LEDGF/DFS70 autoantibodies in clinical laboratories.

Clinical associations of serum Golgi apparatus antibodies in an immunology laboratory cohort.

Anti-Golgi antibodies (AGAs) are rare antibodies that are found as distinct, polarised cytoplasmic staining on the HEp-2 substrate. We performed a review of patients that demonstrated this autoantibody in a large laboratory cohort in Australia. Over a 24-month period, all patients that had a sample submitted for routine antinuclear antibodies (ANAs) that had AGA staining were retrospectively identified. Medical records were perused to identify clinical associations. There were 23813 ANAs identified with 34 patients (0.14%) demonstrating AGA staining. AGAs were found in a variety of inflammatory disorders, malignancies and liver diseases. They did not associate with any other significant ANA, and in contrast with previous studies, we did not find any association with systemic autoimmune rheumatic diseases. Anti-Golgi antibodies are rare, non-specific and possibly a bystander phenomenon. Future studies are required to study the origin and longitudinal clinical associations of these autoantibodies.

Prevalence of autoantibodies to cellular cytoplasmic and mitotic antigens in routine antinuclear antibody reporting: Implementation of international consensus on antinuclear antibodies patterns guidelines

Background: The international consensus on antinuclear antibody (ANA) patterns (ICAP) guidelines recommends that non-nuclear patterns should be reported as positive for ANA. Therefore, a prospective study was planned to investigate the frequency of cytoplasmic and mitotic ANA patterns in routine reporting in a high-throughput lab in a tertiary referral center in Northern India. Materials and Methods: Whole blood samples received were centrifuged and serum was separated within 4 hours of collection. Sera were subjected to indirect immunofluorescence (IIF) assay on HEp-2 cells for ANA determination in a dilution of 1:40, per standardized protocol in our laboratory. IIF staining patterns and intensity were evaluated and the results interpreted. Results: Over a 3-month period, of the 3796 serum samples received for ANA testing, 81 (4.9%) samples were positive for cytoplasmic, 7 (0.4%) for mitotic, and 2 (0.1%) for DNA topoisomerase I (Topo I)-like pattern. The most frequent cytoplasmic fluorescence pattern was dense fine speckled (2%) and the most frequent mitotic pattern was NuMA-like (0.2%). The prevalence of total positive ANA in our study population was found out to be 43.6%. Conclusions: Implementation of ICAP guidelines into routine ANA reporting helped us in detecting important cytoplasmic antibodies such as antimitochondrial antibodies, anti-ribosomal P protein antibodies, anti-Jo-1antibodies, anti-Topo I antibodies and allowed us to introduce more specific and confirmatory tests for the same. The new format for ANA reporting also encouraged clinician-laboratory crosstalk.

Evaluation of the clinical utility of disease-specific antinuclear antibody screening tests using chemiluminescence enzyme immunoassay for routine antinuclear antibody testing

Evaluation of the clinical utility of disease-specific antinuclear antibody screening tests using chemiluminescence enzyme immunoassay for routine antinuclear antibody testing

Clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies testing

BackgroundIt is has been proposed that the presence of anti-DFS70 antibodies could be used to eliminate a SARD diagnosis. However, anti-DFS70 antibodies were also observed at relatively high frequency in patients with SARD. The clinical significance of these antibodies is therefore not yet clear. In this study, we assessed the clinical significance of the presence of anti-DFS70 antibodies. MethodsA total of 3432 sera samples were obtained from patients who underwent routinely requested ANA screening in the clinical laboratory of a University Hospital, between June 2017 and June 2019. Antinuclear antibody testing was performed by indirect immunofluorescence (IIF) on Hep-2 cell substrates (Euroimmun, Germany), and anti-ENA were measured by LIA (Euroimmun, Germany). ResultsAmong 3432 serum samples tested for ANA, 57.3% were ANA positive by IIF. Participants had mean age of 46.4 and 74.8% of the participants were female. Only 11.4% of the study population had SARD. The frequency of DFS pattern by IIF was 8.1%. Analysis of the DFS pattern positive samples by LIA revealed the presence of anti-DFS70 autoantibodies in 67.5% of all DFS, AC-2 pattern ANAs. When using the results of the ANA IIF HEp-2 DFS pattern/Anti-DFS70 LIA, likelihood ratio (LR) for SARD was 0.33. When comparing the ANA IIF HEp-2 DFS pattern with anti-DFS70 LIA, the ANA IIF HEp-2 DFS pattern's LR was lower (0.63 vs 0.85). ConclusionsAlthough the DFS pattern cannot exclude the presence of SARD, the likelihood is lower than with other patterns. Therefore, anti-DFS70 antibodies represent an important biomarker that can aid in the interpretation of positive ANA patients and, therefore, should be included in test algorithms for ANA testing. The optimal test algorithm might be laboratory specific being dependent on referral patterns for ANA testing.

Autoantibodies to mRNA processing pathways (glycine and tryptophan-rich bodies antibodies): prevalence and clinical utility in a South Australian cohort

Autoantibodies to glycine and tryptophan-rich bodies (GWB) can be detected on routine antinuclear antibodies (ANA) testing and might have important disease associations. The aim of this study was to investigate the prevalence of anti-GWB antibodies identified on routine ANA testing, define their antigenic specificities and describe their clinical association. Anti-GWB antibodies were identified by distinct cytoplasmic staining pattern on all samples referred for ANA testing over a 6-month period. All positive anti-GWB samples were further tested on a multiplex addressable bead immunoassay (ALBIA) with known GWB antigens. Extractable nuclear antigens (ENA) were characterised by line immunoblot assay. Clinical details were collected retrospectively by contacting patients and the requesting clinicians. Eleven patients (7 females, 4 males) out of a total of 2136 positive ANAs requested on 11,265 samples had the classical GWB pattern (0.5%). The median age of patients was 66 years (range 39-92). There was no consistent disease association. Ten were confirmed to have distinct antigenic specificity for known GWB antigens. Ge-1/Hedls and RAP55 were the most common antigenic specificity targets [seen in 7 patients (64%) and in 5 patients (45%), respectively]. Ro52 was positive in 5/9 (56%) patients, SSB in 2/9 (22%) patients and Ro60 in 1/9 (11%) patient. The clinical association of anti-GWB antibodies is uncertain but might point towards autoimmune origin of certain non-specific musculoskeletal symptoms. The antigenic specificity of anti-GWB reactivity could point towards specific clinical associations: anti-RAP55 and Ge-1 in non-specific musculoskeletal conditions versus anti-GW182 in neurological diseases.

Autoantibodies in interstitial lung diseases

The role of autoantibody testing for patients with interstitial lung disease is an evolving area. Recent guidelines recommend routine anti-nuclear antibodies, rheumatoid factor, and anti-citrullinated cyclic peptide antibody testing for patients undergoing diagnostic evaluation for interstitial lung disease, with further autoantibody testing reserved for selected cases guided by rheumatological features. Even this approach may miss patients with clinically significant autoantibodies when interstitial lung disease is the dominant or first manifestation of autoimmune disease. We retrospectively performed autoimmune serology in a clinically well characterised cohort of interstitial lung disease patients. Using stored serum, additional testing was performed to ensure all patients had complete autoantibody profiles including anti-nuclear antibodies, extractable nuclear antigen antibodies, double-stranded DNA antibodies, rheumatoid factor, anti-citrullinated cyclic peptide antibodies, anti-neutrophil cytoplasmic antibodies, and myositis antibodies. Eighty patients with interstitial lung disease, and available stored serum, were assessed. Mean age at interstitial lung disease diagnosis was 65.2 years and 42 patients were male. Positive autoimmune serology was found in 56 of 80 (70.0%) patients; the most common positive result was anti-nuclear antibodies (n=34; 42.5%). Myositis antibodies were detected in 13 of 80 (16.2%) patients. Four (5%) patients had elevated anti-citrullinated cyclic peptide antibodies, and two (2.5%) patients had detectable myeloperoxidase antibodies. Eleven (13.7%) patients with negative anti-nuclear antibodies had other significant disease associated autoantibodies. An extended panel of autoantibody testing may detect cases of connective tissue disease associated interstitial lung disease, regardless of clinical or radiological subtype, and prior to extra-pulmonary manifestations of systemic autoimmunity.

Clinical use of anti-DFS70 autoantibodies.

The dense fine speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine anti-nuclear antibody (ANA) screening. There is a negative association between anti-DFS70 status and systemic autoimmune rheumatic disease (SARD), especially in the absence of concomitant SARD-specific autoantibodies. The purpose of this study was to determine the need for confirming anti-DFS70 status when a DFS pattern is observed in IIF-ANA. The frequency of anti-DFS70 detection on Western blot and the positive rate of connective tissue disease (CTD)-related autoantibody screening with a fluorescence-based enzyme immunoassay was evaluated in DFS (n = 182) and non-DFS (n = 359) groups. Specific autoantibodies against 15 autoantigens were identified by line immunoassay. We evaluated the frequency of cases of DFS mistaken for non-DFS and non-DFS cases mistaken for DFS, as well as the clinical impacts of these misinterpretations. Among cases of IIF-ANA with an observable DFS pattern, 68.1% had only anti-DFS70 without CTD-related autoantibodies, 20.3% were false positive for IIF-ANA, and the remaining 11.5% had CTD-related autoantibodies independent of anti-DFS70 status. These results indicated that CTD-related autoantibodies may be present with or without anti-DFS70 even if a DFS pattern is observed in IIF-ANA. Among patients who are ANA negative or have a low probability of SARD, an anti-DFS70 confirmation test has no clinical benefit and cannot replace specific tests for detecting CTD-related autoantibodies. Specific tests to detect CTD-related autoantibodies should be performed instead of anti-DFS70 confirmation tests when a DFS pattern is observed in IIF-ANA.

Prevalence and serological profile of anti-DFS70 positive subjects from a routine ANA cohort

Anti-Dense Fine Speckled 70 (DFS70) antibodies are a common finding in clinical laboratory referrals. High prevalence of DFS70 autoantibodies in healthy population and usual negative association with Antinuclear Antibody (ANA)-associated autoimmune rheumatic diseases (AARD) were reported. The aim of this study was to evaluate the prevalence of DFS70 autoantibodies and their association with other autoantibodies in the context of a routine ANA referral cohort. Consecutive sera submitted for ANA screening were analyzed for anti-DFS70 antibodies by indirect immunofluorescence (IIF) (n = 3175, 1030 men and 2145 women) then confirmed by immunoblotting. Anti-DFS70 positive samples were also assayed for a large spectrum of other circulating autoantibodies. The prevalence of anti-DFS70 antibodies was 1.7% in the whole population and 4.6% in the ANA-positive samples. Comparison between DFS70 IIF and immunoblotting showed an excellent correlation between the two methods. The prevalence of anti-DFS70 positive was significantly higher in females (2.1%, 45/2145) than in males (1.0%, 10/1030). Of note, no concomitant autoantibodies were found in the DFS70-positive male group compared with DFS70-positive females group that showed other serum autoantibodies in the 51% of cases. Anti-DFS70 reactivity in male population may represent an useful biomarker predicting the absence of other autoantibodies. On the contrary, the serological profile of DFS70-positive females required further investigations in order to define the presence of concomitant disease-marker autoantibodies.

Reflex testing of speckled cytoplasmic patterns observed in routine ANA HEp-2 indirect immunofluorescence with a multiplex anti-synthetase dot-blot assay: a multicentric pilot study.

Immunofluorescence on HEp2-cells is the standard diagnostic assay for the detection of anti-nuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding, and are associated with various antibodies, including anti-synthetase antibodies. However, classic ENA testing generally identifies only anti-Jo-1. Moreover, anti-synthetase syndrome is increasingly recognized as a pleomorphic entity, possibly presenting as isolated arthritis or interstitial lung disease. Sera referred for routine ANA testing were selected on the basis of the presence of a fine dense speckled cytoplasmic pattern (254 samples) and compared to control sera with negative cytoplasm (239 samples). All 493 samples were tested with a commercial synthetase profile dot-blot (D TEK - Alphadia-Alifax) including anti-Jo1, anti-PL7, anti-PL12, anti-EJ, anti-OJ, anti-KS, anti-ZO, anti-HA, anti-SRP, and anti-Ribosome P0. Retrospective clinical data was searched for positive patients. Dot-blot identified 18/254 (7.1%) positive sera in the samples with a cytoplasmic fluorescence pattern and 4/239 (1.7%) in the control group (χ2 = 8.4627; p = 0.003625). Blot intensity was more intense in samples with concordant cytoplasmic staining (cytoplasmic negative 27 ± 12.4; cytoplasmic positive 53.9 27 ± 27.7; p = 0.0027). In the positive samples, 8/18 had a highly compatible diagnosis (myositis, interstitial lung disease, arthritis), 7/18 an uncharacterized connective tissue disease, and 3 a diagnosis not associated with the presence of anti-synthetase antibodies. We evaluated the performance of a dot-blot assay for anti-cytoplasmic antibodies in a serologic cohort presenting a cytoplasmic speckled pattern found during routine ANA testing. This algorithm enabled the identification of a significant quota of patients with rare anti-synthetase antibodies and an incomplete or atypical clinical picture. Reflex testing strategies of speckled cytoplasmic patterns with multiplex assays containing cytoplasm-specific antigens, as opposed to standard ENA testing, may yield important data and for this reason should be implemented in routine ANA testing.

Application of anti-DFS70 antibody and specific autoantibody test algorithms to patients with the dense fine speckled pattern on HEp-2 cells

Objectives: Whereas antinuclear antibodies (ANAs) detected by indirect immunofluorescence (IIF) have diagnostic significance, the dense fine speckled (DFS) pattern on HEp-2 cells may be an exclusionary marker for ANA-associated rheumatic disease (AARD). The aim of this study was to evaluate a new algorithm considering anti-DFS70 antibodies for routine ANA testing.Method: From ANA requested sequential 10 528 sera, 181 sera samples showing the DFS pattern were additionally tested for anti-DFS70 antibodies by an enzyme-linked immunosorbent assay (ELISA-DFS70) and for specific-ANAs. Specific-ANAs(+)/IIF-DFS(–) control sera samples (n = 50) were also tested.Results: Of the 181 IIF-DFS-positive sera samples, 82.9% (n = 150) were from non-AARD patients and 112 (61.9%) patients had non-rheumatic diseases (NRD), including the most common clinical feature of dermatitis (18.2%). The ELISA-DFS70 was positive in 109 (60.2%) sera and was negative in all control sera. Specific-ANAs were similarly detected as 25.7% (28/109) and 22.2% (16/72) of ELISA-DFS70(+) and ELISA-DFS70(+) patients, respectively (p > 0.05). The prevalence of non-AARD was 95.1% and 25.1% in the ELISA-DFS70(+)/specific-ANAs(–) and ELISA-DFS70(–)/specific-ANAs (+) groups, respectively.Conclusions: In patients with a HEp-2 DFS pattern, the additional ELISA-DFS70 and specific-ANAs test could improve the efficiency of diagnosing AARD. The detection of anti-DFS70 antibodies should be included in test algorithms for ANA testing.

Association of systemic lupus erythematosus with uveitis.

Systemic lupus erythematosus (SLE) can be associated with uveitis. The reported prevalence of SLE in patients with uveitis varies from 0.1% to 4.8%. Accordingly, the positive predictive value of antinuclear antibody testing in diagnosing SLE in a patient with uveitis varies enormously. An accurate estimate of SLE prevalence in uveitis is needed to establish the value of routine antinuclear antibody testing in patients with uveitis. A literature review using the Medline database was performed to find studies reporting data on uveitis etiology from January 1, 1984, to March 20, 2015. Studies were included where there were sufficient data to draw conclusions on the prevalence of SLE as an etiological factor in uveitis. Data for 53 315 patients were reviewed and 63 studies from 30 countries were included. The prevalence of SLE as a cause of uveitis was estimated to be 0.47% (95% CI, 0.41%-0.53%). The positive predictive value of routine antinuclear antibody testing was 2.9% (95% CI, 2.65%-3.19%). Systemic lupus erythematosus is a rare cause of uveitis. Routine antinuclear antibody testing has a low positive predictive value for SLE. These data suggest such testing should be reserved for patients where there is a higher pretest probability of SLE.