ANA-associated Rheumatic Diseases Research Articles

Longitudinal antinuclear antibody titers in systemic lupus erythematosus and other rheumatic diseases.

Antinuclear antibodies (ANAs) are a key feature of systemic lupus erythematosus (SLE) and marker of subclinical autoimmunity. Little is known about longitudinal ANA titers in individuals from the general population or in predicting clinical disease course in persons with rheumatic diseases. We performed an exploratory analysis from an academic health system between 1999 and 2020 to assess intra-individual variation in ANAs longitudinally in persons with SLE, other ANA-associated rheumatic diseases, and ANA+ controls without rheumatic disease. Persons with SLE had a higher odds of positive ANA compared to those with other ANA-associated rheumatic diseases [OR 2.10, 95% CI (1.82, 2.43)] controlling for time and demographics (age, sex, race, ethnicity). Compared to ANA+ controls, the ANA titer strength was significantly higher for both the ANA-associated rheumatic disease (0.33 log units higher) and SLE groups (0.42 log units higher) controlling for demographics and time (p < 0.001 for both). Over time from the first positive ANA, titer strength significantly decreased for all three groups, with average monthly decreases ranging between 0.001 to 0.004 log titer units (p ≤ 0.001 for all). Based on this analysis of electronic health data spanning two decades, ANA titers may be more dynamic than previously accepted in patients with SLE and ANA-associated rheumatic diseases, with average titers tending to be higher in early disease and decreasing over time.

Analysis of Antinuclear Antibody Test Referral Patterns in a Tertiary Care Hospital over Three Years: A Retrospective Observational Study

Introduction: Antinuclear Antibodies (ANA) detection by Human Epithelial Substrate (Hep2) is the recommended screening test for the diagnosis of ANA-Associated Rheumatic Diseases (AARD). ANA is ordered by various specialists in a tertiary care hospital, and a positive ANA result is followed by testing for specific autoantibodies. High pretest probability, pattern type with intensity, and referral departments are key factors determining the autoimmune diagnosis. Aim: To analyse the ANA referral patterns among the various departments and also to estimate the prevalence and clinical significance of rare ANA patterns. Materials and Methods: A retrospective observational study was conducted at Nizam's Institute of Medical Sciences, Hyderabad, a tertiary care hospital, in Hyderabad, Telangana, India where ANA test reports (n=16,994) from various departments over three years (November 2017 to October 2020) were evaluated between November 2022 to December 2022. ANA tests were performed on Hep2 substrate at a 1:100 dilution, and ANA patterns were reported according to the International Consensus on ANA Patterns (ICAP) nomenclature. Statistical analysis of department- wise ANA positivity and fluorescence intensity was conducted, and the final diagnoses of patients with rare ANA patterns (&lt;1%) were noted from clinical records. Fischer's-exact test was used for comparing categorical variables, considering p-value &lt;0.05 as statistically significant. Results: The majority of ANA requests were from the Rheumatology department (5859; 34.5%), followed by nephrology (3132; 18.4%), neurology (1940; 11.4%), general medicine (1646; 9.7%), haematology (1106; 6.5%), and casualty (878; 5.2%), accounting for 85.6% of total referrals. The highest percentage of positivity among ANA referrals was observed in Rheumatology (333; 5.7%), with 58% of positive ANA showing 4+ intensity. No positives were observed from many surgical departments. Rare ANA patterns with a prevalence of less than 1% were observed in 22 patients with mitotic patterns accounting for the majority of rare patterns seen in 11 out of 22 (50%) cases followed by nuclear envelope, rods rings, and nuclear dense fine speckled patterns were observed in six, four, and one patient(s), respectively. The majority of rare ANA patterns had 2+ fluorescence intensity without any associated autoimmune diagnosis. Conclusion: The highest and lowest positivity among ANA referrals were observed in the rheumatology and surgical departments, respectively. Considering the pretest probability of AARD before ordering an ANA test would lead to the optimum utilisation of laboratory services. Mitotic patterns constituted the majority of rare ANA patterns and need to be clinically correlated with antibody titers.

Clinical Performance of the Line Immunoassay, Digital Liquid Chip Method, and Chemiluminescent Immunoassay for Detecting Specific Antinuclear Antibodies.

Antinuclear antibodies (ANAs) against certain antigens are useful for identifying autoimmune disorders. Although new solid phase-based immunoassays have been developed for evaluating ANAs, the conventional line immunoassay (LIA) is commonly used in clinical practice. To compare the clinical performance of 2 newly developed methods for detecting specific ANAs with LIA. Six hundred ninety-six serum samples were collected from 559 patients with autoimmune disease (AID) and 137 controls. The samples were screened by using the LIA, digital liquid chip method (DLCM), and chemiluminescent immunoassay (CLIA) for specific ANAs. The agreement across assays and the clinical performance of each assay in diagnosing ANA-associated rheumatic diseases (AARDs) were evaluated. Almost perfect agreement was observed among all assays for anti-centromere protein B (κ = 0.85-0.97), anti-ribosome P (κ = 0.85-0.88), anti-SSA 52 (κ = 0.86-0.89), and anti-SSA 60 (κ = 0.89-0.91); moderate to substantial agreement was detected for the autoantibodies against Sm, Jo-1, ribonucleoprotein, Scl-70, and SSB (κ = 0.55-0.80). LIA exhibited better sensitivity for diagnosing AARDs, while DLCM and CLIA demonstrated higher specificity. In the subset of AIDs, especially systemic lupus erythematosus, antibody positive percentages varied greatly between assays. The 3 assays showed comparable qualitative agreement; however, the standardization of testing for ANAs remains challenging owing to intermanufacturer variations. Moreover, DLCM and CLIA exhibited better specificity in distinguishing non-AID individuals, whereas LIA was more sensitive in diagnosing AARDs.

Utility of repeat extractable nuclear antigen antibody testing: a retrospective audit.

Autoantibodies to ENA are frequently ordered during the workup of suspected autoimmune connective tissue diseases. There are no current guidelines for repeat test ordering. The objective of this study was to assess the utility of repeat ENA testing after an initial negative result. A retrospective study was conducted in a single, multicentre tertiary health network in Melbourne, Australia. Results of all ENA tests were extracted from the hospital laboratory information system. For patients who had a change in ENA result from negative to positive, clinical information was obtained from the hospital records regarding new diagnosis of an ANA-associated rheumatic disease (AARD). A total of 23 438 ENA tests were performed in 19 603 patients from 29 July 2013 to 28 September 2020. In total, 20 918 (89.2%) were negative with 215 (0.9%) being equivocal. Of the 2305 positive tests, the most common ENA auto-antibody specificity detected was anti-Ro52 (1185, 51.4%). A total of 2636 of 19 603 patients (13.4%) had more than one ENA test performed during the study period. Of these, most (2523, 95.7%) had stable ENA results with no change compared with the first test. Only 53 patients (2.2%) had an ENA result that changed from negative to positive. Excluding patients with pre-existing rheumatic conditions and those under 18, there were five new AARDs found in the remaining 34 patients. Repeat ENA test results rarely change or result in a new diagnosis of an AARD, with repeated testing only warranted if there is a change in clinical manifestations.

Clinical Performance of Two Automated Immunoassays, EliA CTD Screen and QUANTA Flash CTD Screen Plus, for Antinuclear Antibody Screening.

BackgroundRecently, two fully automated immunoassays for antinuclear antibody (ANA) screening were introduced EliA CTD Screen (Thermo Fisher Scientific, Freiburg, Germany) and QUANTA Flash CTD Screen Plus (Inova Diagnostics, San Diego, USA). We evaluated their clinical performance in comparison with the indirect immunofluorescence assay (IIFA) and analyzed samples with discrepant results.MethodsIn total, 406 serum samples (206 from patients undergoing routine checkups and 200 from rheumatology clinic patients) were assayed using EliA, QUANTA Flash, and IIFA. We evaluated assay concordance and agreement and confirmed the presence of anti-extractable nuclear antigen (ENA) antibodies in samples with discrepant automated immunoassay and IIFA results. Additionally, we compared the clinical performance of each assay in diagnosing ANA-associated rheumatic disease (AARD) and adjusted the cut-off values.ResultsIn rheumatology clinic samples, the concordance and agreement were 91.5% and strong between EliA and QUANTA Flash, 79.0% and weak between EliA and IIFA, and 80.5% and moderate between QUANTA Flash and IIFA, respectively. In automated immunoassay-positive, IIFA-negative samples (N=15), all anti-ENA antibodies detected (6/15) were anti-Sjögren’s syndrome antigen A/Ro (Ro60) antibodies. The automated immunoassays and IIFA showed high accuracy for diagnosing AARD, and adjusted cut-off values improved their sensitivities (EliA with 0.56 ratio, 82.9% sensitivity; QUANTA Flash with 9.7 chemiluminescent units, 87.8% sensitivity).ConclusionsThe two automated immunoassays showed reliable performance compared with IIFA and can be efficiently used with the IIFA in clinical immunology laboratories. Clinical cut-off values can be adjusted according to the workflow in each laboratory.

ANA Pozitif Bireylerde ve ANA İlişkili Romatizmal Hastalıklarda Anti-DFS70 Antikorlarının ve DFS paterninin Değerlendirilmesi

Aim: In this study we aimed to find the frequency of anti-DFS70 antibodies and DFS pattern in ANA positive individuals and ANA associated rheumatic diseases (AARDs).Methods: In this study, 337 subjects who were evaluated in a rheumatology clinic with prediagnosis of rheumatic diseases with positive ANA test and had concurrent anti-extractable nuclear antigen (anti-ENA) antibodies results were retrospectively analyzed. Clinical diagnosis of patients and demographic characteristics were obtained from the patients' medical records.Results: A total of 337 subjects (305 women, 32 men) were included in this study. The mean age was 49.8 ± 14.2 years. Of the 337 participants, 111 (32.9%) had an IIF-DFS pattern and 226 (67.1%) had a non-DFS pattern. Anti-DFS70 antibodies were positive in 20.1% of individuals. Sixty eight individuals had AARDs. An IIF-DFS pattern was observed in 22.1% and a non-DFS pattern was observed in 77.9% of individuals with AARDs (p &amp;lt;0.05). Anti-DFS70 antibodies were positive in 13.2% of patients with AARDs. The frequency of AARDs was significantly lower in individuals with anti-DFS70 antibodies compared to individuals with other anti-ENAs antibodies (p &amp;lt;0.05).Conclusion: Anti-DFS70 antibodies may be present in patients with AARDs but AARDs are less prevalent in patients who had anti-DFS70 antibodies, compared with patients who had other anti-ENAs.

Anti-DFS70 Antibodies for Differentiating Systemic Autoimmune Rheumatic Disease in Patients with Positive ANA Tests: A Systematic Review and Meta-Analysis.

Anti-DFS70 antibodies have been proposed as a marker to exclude systemic autoimmune rheumatic disease (SARD). We conducted this systematic diagnostic test accuracy review and meta-analysis to determine the performance of anti-DFS70 antibodies in patients with a positive anti-nuclear antibody (ANA) test result to exclude SARD. We searched PubMed, Embase, Web of Science, Scopus and the Cochrane Library up to 22 February 2021, and included studies examining the diagnostic accuracy of anti-DFS70 antibodies in patients with a positive ANA test result. The results were pooled using a hierarchical bivariate model and plotted in summary receiver operating characteristic curves. R software and Stata Statistical Software were used for the statistical analysis. Eight studies with 4168 patients were included. The summary sensitivity was 0.19 (95% confidence interval: 0.12–0.28) and the specificity was 0.93 (95% confidence interval: 0.88–0.96). The area under the curve was 0.69 (95% confidence interval: 0.64–0.72). The meta-regression analysis showed that targeting only ANA-associated rheumatic disease was associated with higher specificity. In addition, the studies with a non-SARD prevalence of <80% and using a chemiluminescence assay were associated with higher specificity. Anti-DFS70 antibodies have high specificity for the exclusion of SARD among patients presenting with a positive ANA test, but the sensitivity is low.

Comparison of antinuclear antibody profiles obtained using line immunoassay and fluorescence enzyme immunoassay.

ObjectiveLIA-ANA-Profile-17S is a multiplex line immunoassay that simultaneously detects 17 antinuclear antibodies (ANAs) against extractable nuclear antigens (ENAs). We evaluated the utility of LIA-ANA-Profile-17S as a supplement to ANA indirect immunofluorescence (IIF) and EliA ENA (a fluorescence enzyme immunoassay) for diagnosis of ANA-associated rheumatic diseases.MethodsSera were collected from 245 patients referred for an ANA IIF test. LIA-ANA-Profile-17S results were compared with those of EliA ENA. The kappa coefficients, agreement rates, and diagnostic performance of these tests were assessed for systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SjS).ResultsWe observed almost perfect interassay agreement for antibodies against Ro52/Ro60, CENP-B, and Scl-70 (kappa = 0.91, 0.97, and 1.00, respectively); strong agreement for anti-SS-B/La antibody (kappa = 0.81); and relatively low agreement for other antibodies, including those against dsDNA, Sm, RNP, and Jo-1. For SLE diagnosis, LIA-ANA-Profile-17S showed lower sensitivity and similar specificity compared with EliA ENA. The sensitivity and specificity of these two assays were similar for SjS diagnosis.ConclusionsThe specificity of LIA-ANA-Profile-17S was enhanced when combined with ANA IIF and was comparable with that of EliA ENA. LIA-ANA-Profile-17S showed relatively good agreement with EliA ENA. In combination with ANA IIF, these assays showed enhanced diagnostic performance.

Evaluation of a novel particle-based multi-analyte technology for the detection of anti-fibrillarin antibodies

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several anti-nuclear antibodies (ANA), including those in the classification criteria (anti-centromere, anti-topoisomerase I (Scl-70), anti-RNA Pol III). However, the presence of less common antibodies such as anti-fibrillarin (U3-RNP) that generate a clumpy nucleolar pattern by HEp-2 indirect immunofluorescence assay (IFA, ICAP AC-9) are considered disease specific and are with clinical subsets of SSc, therefore playing a role in diagnosis and prognosis. A specific and sensitive anti-fibrillarin assay would be an important addition to serological diagnosis and evaluation of SSc. The goal of this study was to evaluate a new particle-based multi-analyte technology (PMAT) for the measurement of anti-fibrillarin antibodies. A total of 149 patient samples were collected including 47 samples from France (Lyon and Paris, n = 32) and Italy (Careggi Hospital, Florence, n = 15) selected based on AC-9 HEp-2 IFA staining (> 1:640, clumpy nucleolar pattern) and 102 non-SSc controls (inflammatory bowel disease (IBD) n = 20, Sjögren’s syndrome (SjS) n = 20, infectious disease (ID) n = 7, systemic lupus erythematosus (SLE) n = 17, rheumatoid arthritis (RA) n = 17, and healthy individuals (HI) n = 21). All samples were tested on the anti-fibrillarin PMAT assay (research use only, Inova Diagnostics, USA). Additionally, the 47 anti-fibrillarin positive samples were also tested on PMAT assays for detecting other autoantibodies in ANA-associated rheumatic diseases (AARD). Anti-fibrillarin antibody data performed by fluorescence enzyme immunoassay (FEIA, Thermo Fisher, Germany) was available for 34 samples. The anti-fibrillarin PMAT assay was positive in 31/32 (96.9%, France) and 12/15 (80.0%, Italy) of samples preselected based on the AC-9 IIF pattern (difference p = 0.09). Collectively, the PMAT assay showed 91.5% (95% confidence interval (CI): 80.1–96.6%) sensitivity with 100.0% (95% CI: 96.4–100.0%) specificity in non-SSc controls. Strong agreement was found between PMAT and FEIA with 100.0% positive qualitative agreement (34/34) and quantitative agreement (Spearman’s rho = 0.89, 95% CI: 0.77.9–0.95%, p < 0.0001). Although most anti-fibrillarin positive samples were mono-specific (69.8%), some expressed additional antibodies (namely Scl-70, centromere, dsDNA, Ro52, Ro60, SS-B, Ribo-P, DFS70, and EJ). In conclusion, this first study on anti-fibrillarin antibodies measured using a novel PMAT assay shows promising results where the new PMAT assay had high level of agreement to FEIA for the detection of anti-fibrillarin antibodies. The availability of novel AFA assays such as PMAT might facilitate the clinical deployment, additional studies, standardization efforts, and potentially consideration of AFA for next generations of the classification criteria.

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

E-consults: an effective way to decrease clinic wait times in rheumatology

BackgroundTo evaluate the effect of E-consults on wait times and resource utilization for positive antinuclear antibody (ANA) referrals in outpatient rheumatology.MethodsWe conducted a pre-post study of E-consult implementation for positive ANA referrals. We retrospectively reviewed “positive ANA” referrals from 1/2015–3/2017. A statistical process control chart was created to display monthly average wait times for in-person clinic visits and to identify special cause variation. Final diagnoses, wait times and resource utilization were recorded and compared between E-consults and in-person referrals.ResultsThere were 139 referrals for positive ANA with 126 occurring after E-consult implementation in August 2015. Forty-four percent (55/126) of referrals were E-consults; 76% did not have an in-person visit after initial electronic rheumatology recommendation. A control chart demonstrated special cause variation in the form of a shift from June 2016 – January 2017, suggesting a temporal association between decreased wait times and the implementation of E-consults. Eleven patients were diagnosed with ANA-associated rheumatic disease; the majority of patients (73%, 86/139) did not have a rheumatologic diagnosis. Overall E-consults utilized more labs than in-person visits, but this was not statistically significant. In-person visits utilized more imaging studies, which was statistically significant.ConclusionE-consults are an effective way to address positive ANA consults without significant increase in resource utilization and were temporally associated with decreased wait times for in-person visits.

Monospecific anti-DFS70 antibodies are moderately helpful in excluding ANA-associated rheumatic disease in patients presenting with a dense fine speckled pattern – A systematic review and meta-analysis of diagnostic test accuracy

Monospecific anti-DFS70 antibodies are moderately helpful in excluding ANA-associated rheumatic disease in patients presenting with a dense fine speckled pattern – A systematic review and meta-analysis of diagnostic test accuracy

P92 Referral with a positive antinuclear antibody? Lessons from prospective triage and diagnosis in a single-centre service evaluation of 1,547 new rheumatology referrals

Abstract Background Blood tests assessing for the presence of antinuclear antibodies (ANA) are frequently used as a screening method for ANA-associated rheumatic disease (AARD), including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), undifferentiated connective tissue disease (UCTD), and Sjögren's syndrome (SjS). Past research suggests a positive ANA makes up between 4 and 17% of new rheumatology referrals, however little is known about how more detailed referral information can be used to triage these referrals. This service evaluation explores how ANA and clinical referral data relate to the final diagnoses. Methods New out-patient referrals to a single consultant rheumatologist at one UK centre were prospectively coded, over a 40-month period (04/16-08/19), prior to clinical assessment. Data collected included anonymised baseline demographics, referral source, summarised by ‘key referral features’ [ANA testing; number of AARD flags e.g. Raynaud’s (RP), serositis, photosensitivity, inflammatory markers], and diagnosis after assessment. Referral text was coded using up to four 4 ‘key-features’ in line with established clinical reasoning theory (e.g. polyarthralgia, fatigue, synovitis, raised inflammatory markers). The assessment diagnosis was coded against established rheumatological diagnostic categories, with a separate code for awaiting investigations. The data was anonymised, password protected, and analysed for the service evaluation. Results Of the 1,748 referrals, 177 (10.1%) were excluded because of incomplete data, leaving 1547 for analysis. Of these patients 76(5%) were ANA+, with no prior diagnosis of a AARD. The ANA+ 76 were middle aged (mean 56years; SD 14.6years), and predominantly female (60/76). The main referrers were primary care (56/76) and haematology (8/76), the remainder from eight secondary care specialities and extended-scope-physiotherapy. The majority (58/78,76%) had non-inflammatory diagnosis (e.g. osteoarthritis); 9 (12%) were diagnosed AARD (SLE,3; SjS,2; SSc,3; UCTD,1); 9 had other inflammatory disease (gout,3; PMR,1; reactive,1; undifferentiated inflammatory arthritis,4). The AARD flags prospectively coded from the initial referral, prior to assessment, in ANA+ patients were as follows: 0 flags (e.g. ANA+, fatigue, arthralgia), 1/43 patients had AARD (2.3%); 1 flag, 5/29 AARD (17.2%); 2 flags 1/3 AARD (33%); 3 flags 1/1 AARD (100%). Of the 1,471/1,546 referrals with no ANA test, 3 were diagnosed as a AARD (UCTD,1 ; SLE,2). Conclusion This service evaluation shows ANA positive referrals account for 5% of new referrals to our centre, and is the first to our knowledge to evaluate ANA positive referrals based on prospective AARD flags from the referral to evaluate the likelihood of a CTD. Many referrals from the cohort diagnosed with a new CTD had prior ANA testing (75%). These data can potentially help to train and educate specialists in ANA requesting when referring to rheumatology, and assist other departments implementing AARD flags to triage referrals. Disclosures N. Cleaton None. J. Bateman None.

Clinical significance of anti-DFS70 antibody in antinuclear antibody-positive samples with a dense fine speckled pattern

Abstract Background When the dense fine speckled (DFS) pattern-antinuclear antibodies (ANA) are detected in the indirect immunofluorescence (IIF) assay, the presence of anti-dense fine speckles 70 (DFS70) antibodies has been suggested to facilitate the exclusion of ANA-associated rheumatic diseases (AARD). We evaluated the potential use of anti-DFS70 antibodies for verifying AARD in patients with a positive ANA result of the DFS pattern. Methods A total of 5509 patients who were requested ANA testing were included. The DFS pattern was confirmed using two IIF assays. Semiquantitative DFS70 ELISA (Euroimmun, Germany) was examined in samples with the DFS pattern. Results Among 639 ANA-positive patients, 19.6% displayed the DFS pattern. And 17.6% of patients with the DFS pattern were diagnosed with AARD. The low titer of 1:80 was more prevalent in the non-AARD group than in the AARD group (64.1% vs. 4.5%, p &lt; 0.0001). Anti-DFS70 antibodies were positive in 60.0% of patients with the DFS pattern. The frequency of anti-DFS70 positivity was higher in the non-rheumatic disease (NRD) group (74.2%) than in the other rheumatic disease group (43.2%, p = 0.003) and the AARD group (45.5%, p = 0.019). Conclusions The DFS pattern is present in both AARD and non-AARD cases. In the DFS pattern, a low titer of 1:80 and isolated anti-DFS70 antibodies without AARD-associated antibodies represent a low likelihood of AARD. The presence of anti-DFS70 antibodies cannot exclude AARD and should be analyzed in combination with AARD-associated antibodies in the diagnostic algorithm.

Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies.

Solid phase assays (SPAs) and automated microscope systems are increasingly used to screen for antinuclear antibodies (ANAs). The goal of this study was to evaluate the performance of three automated ANA screening assays; NOVA Lite HEp-2 using NOVA View® (NV, Inova Diagnostics), an automated indirect immunofluorescence method, EliA™ CTD Screen (Fluorescence Enzyme Immunoassay, FEIA; Thermo Fisher) and QUANTA Flash® CTD Screen Plus (Chemiluminescence immunoassay, CIA; Inova Diagnostics). The assays were performed on 480 diagnostic samples from patients with an ANA-associated rheumatic disease (AARD; systemic lupus erythematosus, primary Sjögren's syndrome, systemic sclerosis, inflammatory myopathy, mixed connective tissue disease) and on 767 samples from diseased and healthy controls. Using cut-offs proposed by the manufacturers, the sensitivity was 95%, 80.5% and 86% for NV, FEIA and CIA, respectively. The corresponding specificity was 61% (NV), 97.5% (FEIA) and 88% (CIA). The sensitivity associated with a specificity of ~95% was 79%, 82% and 78% for NV, FEIA, and CIA, respectively. Receiver operating characteristics (ROC) curve analysis revealed no differences in area under the curve (AUC) between the 3 assays when all diseases were grouped. For Sjögren's syndrome, the AUC was higher for SPAs than for NV, whereas for systemic sclerosis, the AUC was higher for NV than for CIA. For all assays, the likelihood ratio for AARD increased with increasing antibody levels and for double positivity of NV with SPA. In conclusion, the performance of automated SPA and IIF was assay- and disease-dependent. Taking into account antibody levels and combining IIF with SPA adds value.

Analytical and clinical comparison of two fully automated immunoassay systems for the detection of autoantibodies to extractable nuclear antigens

BackgroundDetection of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IIFA) is increasingly substituted by fully automated solid phase immunoassays. This study evaluated the performance of an automated chemiluminescence immunoassay (CIA) and fluorescence enzyme immunoassay (FEIA) and compared their performance to that of IIFA. MethodsThe study included an unselected prospective study population suspected of systemic autoimmune rheumatic disease. ANA were measured by IIFA, while in parallel sera were tested by CIA QUANTA Flash CTD Screen Plus on the BIO-FLASH® and FEIA EliA CTD Screen on the Phadia® 250 system. As validation, retrospective cohorts of patients with ANA-associated rheumatic disease (AARD) and healthy controls were tested. ResultsProspectively, sensitivity of IIFA, CIA and FEIA was 90%, 99% and 92%, respectively. Specificity was 76%, 76% and 84%, respectively. Total percent agreements between the three methods were 75.2% (IIFA vs. CIA), 79.2% (IIFA vs. FEIA) and 85.4% (FEIA vs. CIA). The AUC values were 0.95 for CIA and 0.93 for FEIA and did not significantly differ. Retrospectively in individual AARD cohorts, similar results were obtained comparing both CTD screens. ConclusionsBoth FEIA and CIA CTD screen significantly outperformed IIFA, with a higher specificity for FEIA and higher sensitivity for CIA. Based on ROC analysis, major contributor to the difference between the two solid phase immunoassays was the cut-off.

Antinuclear antibody testing: discordance between commercial laboratories.

Antinuclear antibody (ANA) test results frequently affect the course of patients' evaluations, diagnosis, and treatment, but different laboratory centers may yield conflicting results. This study investigated the degree of agreement between laboratory results in a group of subjects who had ANA testing performed at two commercial laboratories. This was a chart review study, in which all ANA tests ordered by the authors from one commercial laboratory over a 4-year period were queried. Corresponding patient charts were reviewed, and if ANA testing had also been performed at the second commercial laboratory, subjects were entered into the study. The primary measurement was agreement between paired ANA results, and we performed sensitivity analysis using varying criteria defining agreement (criteria A to criteria D [strictest to most lenient definition of agreement]). Other data captured included relevant data obtained through the course of evaluation (e.g., presenting complaints, exam findings, other laboratory data) and final diagnoses. Of 101 paired ANA tests, there was 18% agreement according to the strictest criteria and 42% according to the most lenient. Of the seven subjects with ANA-associated rheumatic disease, none of the paired tests were in agreement according to criteria A (two agreed according to criteria D). Our findings demonstrate poor agreement between paired ANA tests performed at two commercial laboratories. The low level of agreement may have far-reaching clinical implications. Specifically, this finding calls into question the reliability of ANA testing as it is currently performed and suggests that results may in part depend upon the laboratory center to which patients are referred.

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.

The Clinical Utility of a Positive Antinuclear Antibody Test Result

BackgroundThis retrospective study investigated the clinical utility of a positive antinuclear antibody (ANA) test performed outside of the rheumatology setting. Prior studies have investigated the frequency of ANA positivity within the general population. The purpose of this investigation was to evaluate the clinical utility of a positive ANA test result in a real-world setting by reviewing the final diagnoses of patients who were referred to a tertiary rheumatology clinic for evaluation of a positive ANA test result. MethodsWe reviewed the records of patients presenting to the authors between July 2007 and July 2009. Patients were included in the evaluation if they were referred for a positive ANA test result. All relevant descriptive and laboratory data were collated, as were the initial reasons for ordering ANA testing and the ultimate diagnoses reached. Positive predictive values for a “positive ANA test result” were calculated for all antinuclear antibody-associated rheumatic diseases and for lupus specifically. ResultsA total of 232 patients were referred for a positive ANA test result. The positive predictive value of a positive ANA test result in this cohort was 2.1% for lupus and 9.1% for any antinuclear antibody-associated rheumatic disease. No antinuclear antibody-associated rheumatic disease was identified in patients with an ANA<1:160. The most common reason for ordering ANA testing was widespread pain (54/232, 23.2%). ConclusionsIn this retrospective study, more than 90% of patients who were referred to a tertiary rheumatology clinic for a positive ANA test result had no evidence for an ANA-associated rheumatic disease. The poor predictive value of a positive ANA in this cohort was largely attributable to unnecessary testing in patients with low pretest probabilities for ANA-associated rheumatic disease.