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Background Isolated antibodies to DFS70 have been described in healthy individuals and are rarely found in patients with antinuclear antibody-associated autoimmune rheumatic diseases (AARD). However, no data is available on geographic differences in the prevalence of anti-DFS70 antibodies. We aimed to study the prevalence of anti-DFS70 antibodies in blood donor samples from several countries representing various ethnical backgrounds and geographic regions in the world. Methods Sera from apparently healthy blood donors (n≥300 per site) were collected in seven countries (USA, Italy, Spain, Germany, UK, Belgium and Brazil). All samples (n=2628) were tested for anti-DFS70 antibodies by QUANTA Flash DFS70 (Inova Diagnostics, Inc., San Diego, CA, USA). Results The prevalence of anti-DFS70 antibodies varied from 4/321 (1.2%, Italy) to 42/497 (8.5%, USA). Consequently, the prevalence of the antibodies was significantly higher in USA compared to all other countries (p<0.05). In addition, the prevalence in the combined cohort (all sites) was higher in young blood donors (<35 years; 5.0% vs. 2.7%; p=0.0017) and among females (4.5% vs. 3.0%; p=0.0446). However, when cohorts from different countries were corrected for age and gender, no significant difference between the countries were found. Conclusions This is the first study to analyze the prevalence of anti-DFS70 antibodies in different geographic areas using a standardized assay. Our findings show that the antibodies are most prevalent in young females.

Background: Recently, antibodies directed against peptidyl arginine deiminase 3 and 4 (anti-PAD3/PAD4 antibodies), calcium-dependent enzymes that catalyze the conversion from arginine to citrulline, have been described. Furthermore, antibodies that cross-react between PAD3 and PAD4 cause increased PAD4 activity and consequently correlate with joint damage. This study analyzes the correlation of anti-PAD3 antibodies with joint damage., Methods: To validate the novel chemiluminescent immunoassay (CIA) for the detection of anti-PAD3 antibodies, 20 samples were tested by CIA and by immunoprecipitation (IP). Next, 39 RA patients with available joint erosion score (JES), Total Sharp Score (TSS) and Joint Space Narrowing Score (JSNS) were tested for anti-CCP (using different methods) and anti-PAD3 antibodies by CIA., Results: Excellent correlation was observed between the CIA and IP for the detection of anti-PAD3 antibodies (rho=0.85, p<0.0001). The median JES of our 39 patients was 14.1 with a standard deviation of 11.5. Anti-PAD3 antibody levels (rho=0.39, 95% CI=0.1-0.6; p=0.0149) were correlated with JES. No correlation was found with TSS and JSNS. In this cohort, ACPA measured using different anti-CCP assays did not correlate with the JES., Conclusion: In our cohort, anti-PAD3 antibodies correlate with joint erosion score. Therefore, anti-PAD3 antibodies might represent promising markers to predict joint damage in RA patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Ro52 is an interferon-inducible protein of the tripartite motif family. Antibodies against Ro52 have been described in patients with different autoimmune diseases, such as systemic lupus erythematosus and Sjögren's syndrome, that are often associated with anti-Ro60 antibodies. The Ro52 autoantigen is extraordinarily immunogenic, and its autoantibodies are directed against both linear and conformational epitopes. The aim of this study was to evaluate the prevalence of antibodies to the five Ro52 domains, as well as to Ro52 176- to 196-amino acid (aa) and 200-239-aa peptides, in different systemic autoimmune rheumatic diseases (SARDs). We also aimed to verify whether antibodies to a single domain or domain association could increase their diagnostic specificity for any SARD., Methods: Serum samples were obtained from 100 anti-Ro52 antibody-positive patients with SARDs and from 68 controls (50 healthy donors and 18 patients with other autoimmune or allergic diseases). A special line immunoassay was created containing a full-length Ro52 antigen expressed in insect cells using the baculovirus system, five recombinant Ro52 antigen fragments [Ro52-1, Ro52-2, Ro52-3, Ro52-4 (partly overlapping Ro52-1 and Ro52-2), and Ro52-5 (partly overlapping Ro52-2 and Ro52-3)], and two Ro52 peptides (176-196 aa and 200-239 aa), all expressed in Escherichia coli., Results: In patients with SARDs, fragment prevalence rates were as follows: Ro52-1 = 3 %, Ro52-2 = 97 %, Ro52-3 = 0 %, Ro52-4 = 9 %, Ro52-5 = 28 %, Ro52 175-196-aa peptide = 6 %, and Ro52 200-239-aa peptide = 74 %. All control samples were negative for the full-length Ro52 and for the five fragments tested., Conclusions: The main epitope of the Ro52 antigen was localized on fragment 2 (aa 125-267), and the majority (97 %) of SARD sera had antibodies that target this fragment. As most of the samples were positive for fragment 2 and only some for fragments 4 or 5, which partially overlap fragment 2, it seems that the target epitope is localized in the middle of fragment 2 or in the area between fragments 4 and 5. No antibody against a single epitope or a combination of epitopes was linked to any of the single SARDs.

Anti-citrullinated protein antibodies (ACPA) are important serological markers in the diagnosis of rheumatoid arthritis (RA) and are part of the recent disease classification criteria. However, there is a strong need for reliable markers for measuring and predicting joint damage and disease activity. Recently, antibodies directed against carbamylated antigens (anti-CarP antibodies) were identified. A total of 120 RA patients were tested for anti-CCP antibodies using different methods and for anti-CarP antibodies using carbamylated fetal calf serum according to the method described by Shi et al. Additionally, ACPA fine specificities (to three citrullinated peptides) were measured. Disease activity was assessed at baseline using the disease activity score 28 (DAS28) in 80 patients. For 40 RA patients, joint erosion score (JES) was established. The median JES was 14.1 with a standard deviation of 11.5. Anti-CarP antibodies were correlated with joint erosion score (ρ = 0.34, 95% CI 0.03-0.59; p = 0.0332). No correlation between ACPA and joint erosion score was observed. No individual marker correlated with DAS28. When one ACPA peptide was combined with anti-CarP antibodies in a score (ACPA peptide 1 divided by anti-CarP), a statistically relevant correlation was found (p = 0.0264). In this small cohort, the presence of anti-CarP antibodies, but not ACPA correlate with joint erosion score. Anti-CarP antibodies combined with ACPA fine specificities correlated with DAS28. Therefore, anti-CarP antibodies might represent a promising marker to predict joint damage and disease activity in RA patients.

Background: We analyzed the performance of a novel ENA screening chemiluminescent immunoassay (CIA) and the confirmation QUANTA Flash tests., Methods: Sera (n=1079) from patients referred to a rheumatology clinic were screened by QUANTA Flash ENA7 (INOVA Diagnostics). All positive (n=89) and a matched control group (n=90) were reflexed for autoantibodies to the individual antigens. Moreover, sera from patients with systemic lupus erythematosus (SLE, n=252), systemic sclerosis (SSc, n=64), polymyositis/dermatomyositis (PM/DM, n=72), Sjögren's syndrome (SjS, n=39) as well as disease controls (n=605) were tested by ENA7 CIA and by Quanta Lite ENA6 ELISA (INOVA)., Results: 89/1079 (8.3%) samples were ENA7 CIA positive with the following reactivity profile: RNP (36.0%), Sm (13.5%), Scl-70 (9.0%), Jo-1 (0.0%), Ro60 (44.9%), Ro52 (39.3%) and SS-B (24.7%). In the negative group, the reactivity profile was: RNP (1.1%), Sm (1.1%), Scl-70 (2.2%) and 0.0% for Jo-1, Ro60, Ro52 and SS-B. The positive/negative/total agreements (ENA7 CIA vs. confirmation assays) were 95.3%/91.5%/93.3%. The sensitivity of the ENA7 CIA was 62.3% in SLE, 54.7% in SSc, 92.3% in SjS, 50.0% in PM/DM, and 61.8% in the total systemic autoimmune rheumatic disease (SARD) population (specificity 95.0%)., Conclusion: The QUANTA Flash ENA7 CIA is a reliable screening test., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Anti-nucleosome antibodies have a role in the diagnosis and follow-up of systemic lupus erythematosus (SLE) and have a possible correlation with SLE activity and with kidney and hematological involvement. The aim of our study was to detect in 91 patients with rheumatoid arthritis (RA) the positivity of anti-nucleosome antibodies during therapy with three different TNFalpha blocking agents and to underline the possible correlation with the development of antinuclear autoantibodies (ANA) and anti-dsDNA autoantibodies. We detected anti-nucleosome antibodies, ANA, and anti-dsDNA during therapy with three different TNFalpha blocking agents at T-0 and after 12 and 24 weeks of treatment, respectively. Anti-nucleosome antibodies (IgG class) were analyzed by ELISA technique (Orgentec Diagnostika GmbH, Mainz, Germany), ANA both by indirect immunofluorescence (IIF) technique on Hep-2 (Scimedx, USA) and by ELISA (Autoimmune EIA ANA screening test Bio-Rad Laboratories, CA, USA), and anti-dsDNA (IgG and IgM classes) by ELISA (Kallestad, Bio-Rad Laboratories, CA, USA) and confirmed by IIF on Crithidia luciliae (ImmunoConcepts N.A., Sacramento, CA, USA). We observed 19 patients on infliximab treatment at 3 mg/kg every 8 weeks, 43 patients on etanercept treatment at 25 mg twice a week, and 29 patients on adalimumab treatment at 40 mg every other week. At baseline, we observed positivity as follow: in the group of patients treated with infliximab-anti-nucleosome 1/19 (5.26%), ANA 3/19 (15.7%), anti-dsDNA 1/19 (5.26%); in the group treated with etanercept--anti-nucleosome 2/43 (4.65%), ANA 1/43 (2.43%), anti-dsDNA 0/43; and in the group treated with adalimumab--anti-nucleosome 2/29 (6.89%), ANA 1/29 (3.44%), anti-dsDNA 0/29. The results at 12 weeks for the three autoantibodies were: for infliximab--3/19 (15.7%), 10/19 (52.6%), 2/19 (10.5%); for etanercept--3/43 (6.9%), 10/43 (23.2%), 1/43 (2.32%); and for adalimumab--3/29 (10.3%), 4/29 (13.7%), 1/29 (3.4%). At 24 weeks, the results were for infliximab 6/19 (31.5%), 12/19 (63.1%), 2/19 (10.5%); for etanercept 11/43 (25.5%), 22/43 (51.1%), 2/43 (4.65%); and for adalimumab 4/29 (13.7%), 13/29 (44.8%), 1/29 (3.4%). We observed a concordance anti-nucleosome/ANA antibodies of 85.5% (p < 0.001). Our data showed a concordance between anti-nucleosome antibodies and ANA positivity in patients with RA during therapy with TNFalpha blocking agents. The induction of autoantibodies positivity is different for each TNFalpha blocking agent.

TNF-alpha role in RA is confirmed by the improvement on joint inflammation and physical function and by the slowing of radiographic damage induced by TNF-alpha blockers, that also reduce Rheumatoid Factor (RF) and anti-CC-P titres. (1) To evaluate the effects of 3 TNF-alpha blockers on (a) disability (HAQ), disease activity (DAS28), acute phase reactants (ERS, CRP); (b) autoantibodies: IgM RF, anti-CCP abs. (2) To evaluate if anti-CCP abs could be useful for testing the efficacy of anti-TNF-alpha agents in the follow-up of RA patients. 34 patients with RA (25 F, 9 M; mean age: 59.1 +/- 12.1 years, mean disease duration: 9.6 +/- 2.3 years; 23/34 positive for anti-CCP abs, 19/34 for IgM RF) were enrolled: 18 received Etanercept (25 mg twice weekly subcutaneously), 8 received Infliximab (3 mg/kg intravenously every 8 weeks) and 8 Adalimumab (40 mg every 14 days). All the patients were evaluated for the above mentioned parameters at baseline (t0) and after 6 months of therapy (t6). Anti-TNF-alpha agents differently reduced HAQ and DAS28, ERS and CRP, RF and anti-CCP ab titres RA patients whose RF (14) and/or anti-CCP abs (17) titres were significantly lowered at the end of the study, at t6 presented a significant reduction in respect to t0 of VES, PCR, DAS28 and HAQ values (p < 0.05 for all comparison). This effect was not shown in patients in whose RF (5) and/or anti-CCP abs (6) titres were not reduced in respect to baseline. In RA, anti-TNF-alpha agents, especially Etanercept, reduce disability, disease activity and acute phase reactants expecially in patients showing reduction of RF and anti CC-P titres.

Background: The early reliable detection and quantification of autoantibodies play an important role in autoimmune disease diagnosis and in disease-course monitoring. New technologies, such as the multiplexed determination of autoantibodies, have recently been introduced and are being adopted more frequently. The aim of this study was to evaluate the ability of a new microdot array-based multiparametric assay (ZENIT AMiDot CTD panel, A. Menarini Diagnostics, Firenze, Italy) to correctly classify patients with autoimmune rheumatic diseases (ARDs) and compare it to a fluorescence enzyme immunoassay (FEIA) for the detection of anti-ENAs. Methods: The study included 69 consecutive samples from patients with ARDs that were analyzed using two different methods (FEIA and AMiDot) to detect anti-CENP B and six anti-ENA antibodies: anti-Scl-70, anti-SSB/La, anti-Jo-1, anti-U1-RNP, anti-Ro52, and anti-Ro60. The control group sera came from sixty-eight blood donors. Tests were run on the automated slide processor ZENIT FLOW, and then the slides were imaged and analyzed using ZENIT fast. Results: Since the samples were selected for at least one antibody positivity with an ARD diagnosis, we did not calculate clinical sensitivity but only specificity, which was 98.53%, ranging from 90% for anti-SSB/La antibodies to 100% for anti-CENP B ones. Mean agreement among the methods assessed by Cohen's kappa was 0.816 ± 0.240. Conclusions: The assay demonstrated good clinical performance and may be considered a valuable aid in detecting ARD patients, offering an alternative to methods such as FEIA which are largely in use today. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by the presence of autoantibodies that are used for classification of the disease. Though routine diagnostics is commonly restricted to measuring rheumatoid factor (RF) and anti-citrullinated protein antibodies, detection of RF IgM, IgG and IgA isotypes, may increase the power of RA serodiagnosis by reducing the number of seronegative patients as well as provide prognostic information. The agglutination-based RF assays, such as nephelometry or turbidimetry, are unable to differentiate isotypes. We compared three different immunoassays used in current laboratory practice to detect RF isotypes. We tested 117 consecutive serum samples that were positive for total RF at nephelometry, from 55 RA and 62 non-RA subjects. IgA, IgG, and IgM isotypes of RF were tested by immunoenzymatic (ELISA, Technogenetics), fluoroenzymatic (FEIA, ThermoFisher) and chemiluminescence (CLIA, YHLO Biotech Co.) immunoassays. Diagnostic performance differed considerably between the assays, especially with regard to RF IgG isotype. Agreement among methods by Cohen's kappa ranged from 0.05 (RF IgG CLIA vs. FEIA) to 0.846 (RF IgM CLIA vs. FEIA). The poor agreement observed in this study indicates substantial lack of comparability among assays for RF isotypes. Harmonization of these tests requires further efforts before their measurement can be used in clinical practice. [ABSTRACT FROM AUTHOR]

Background: The dense fine speckled (DFS) is one of the most common patterns that can be observed as a result of the anti-nuclear antibodies (ANA) test on HEp-2 cells and is mostly caused by antibodies to DFS70 as the main antigenic target. As was recently demonstrated, isolated anti-DFS70 positivity can be used as an aid in the exclusion of ANA associated rheumatic diseases (AARD) due to the opportunity to better interpret unexplained positive IIF ANA results. Methods: Our study included 333 subjects with AARD, 51 undifferentiated connective tissue disease (UCTD) patients, 235 disease controls and 149 healthy blood donors from an Italian cohort. All samples were tested for anti-DFS70 and anti-ENA antibodies using QUANTA Flash assays (Inova Diagnostics, San Diego, CA, USA). Results: No differences in the prevalence of anti-DFS70 antibodies were seen among AARD, non-AARD and UCTD (2.1% [7/333] vs. 2.3% [9/384] vs. 5.9% [3/51], respectively; p-value = 0.188). AARD patients positive for anti-DFS70 antibodies showed in all cases an accompanying anti-ENA specificity. In contrast, monospecific anti-DFS70 antibodies showed a significantly different distribution with a clear trend across the main groups (AARD vs. non-AARD vs. UCTD: 0% [0/7] vs. 22% [2/9] vs. 100% [3/3], p = 0.007). Anti-DFS70 antibody levels among AARD, non-AARD and UCTD patients were not significantly different (p = 0.094). Within the anti-DFS70 antibody positive cases, AARD cohort showed a higher variability (median [min–max]: 3.2 [3.2–450.8] CU) compared to non-AARD (median [min–max]: 3.2 [3.2–75.7] CU) and UCTD patients (median [min–max]: 3.2 [3.2–59.0] CU). Conclusions: Our preliminary data showed a similar frequency of anti-DFS70 antibodies in AARD, UCTD and non-AARD cohorts. Monospecificity of anti-DFS70 antibodies but not their mere presence is the key element in the diagnostic algorithm. Mono-specific anti-DFS70 antibodies might be a helpful biomarker to discriminate individuals with AARD from non-AARD presenting with a positive ANA. [ABSTRACT FROM AUTHOR]

Introduction/objectives: Accurate interpretation of DFS70 (dense fine speckled 70) and mixed antinuclear antibodies (ANAs) patterns can be challenging using conventional HEp-2 immunofluorescence (IIF) method. We evaluated a novel HEp-2 IIF substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of engineered HEp-2 devoid of the DFS70 autoantigen and conventional HEp-2 cells. The study assessed the utility of the new substrate in ANA screening and its advantages. Method: One thousand and five consecutive routine samples sent for ANA screening were tested on both standard HEp-2 and the HEp-2 ELITE DFS70 KO substrates (ImmuGlo ANA HEp-2 and HEp-2 ELITE/DFS70-KO, Trinity Biotech, Buffalo, NY). Anti-DFS70 antibody specificity was additionally determined by immunoblot (IB). Clinical and serological data were included in the analysis of the overall impact of the novel HEp-2 substrate on DFS pattern interpretation. Results: Of the 22 cases suspected as positive for DFS pattern alone or in combination with homogeneous or speckled patterns on conventional HEp-2 cells, 17 were interpreted with a higher accuracy using the new HEp-2 ELITE method as positive for DFS70 (monospecific DFS70 (10), mixed DFS70 (7)), speckled (3), and DFS (2) patterns. Conclusions: The new substrate was not only useful in deciphering unclear mixed ANA patterns but also highly sensitive in detecting DFS70 pattern in comparison to the DFS70 positivity obtained using IB. [ABSTRACT FROM AUTHOR]

Objective. The main objective was to determine the prevalence of anti-dense fine speckled (DFS70) antibodies in a stable population of undifferentiated connective tissue disease (UCTD) to better define their potential role. Methods. Immunological and clinical records of 91 long-standing UCTD patients were studied. DFS pattern was determined using the IIF ANA test on HEp-2 cells and anti-DFS70 antibodies were tested by chemiluminescence assay and by DFS70 line immunoassay. Results. Twelve (13.2%) of 91 serum samples were positive for anti-DFS70 antibodies by chemiluminescence assay and line immunoassay. There was no statistical significance between the prevalence of anti-ENA and anti- DNA autoantibodies in patients with and without anti-DFS70 antibodies. No differences were found in the clinical characteristics of both groups. The presence of the anti-DFS70 antibodies was related to the younger age class. Conclusion. The high prevalence of anti-DFS70 antibodies in the UCTD patients suggested the potential role of these autoantibodies as a marker in the evolution of UCTD to CTD. [ABSTRACT FROM AUTHOR]

Introduction. The objective of the present study was to compare QUANTA Flash dsDNA, a chemiluminescent immunoassay (CIA) on the BIO-FLASH, a rapid-response chemiluminescent analyzer, to three other anti-dsDNA antibody assays and to Crithidia luciliae indirect immunofluorescence test (CLIFT). Methods. In the first part of the study, 161 samples, 61 from patients suffering from systemic lupus erythematosus (SLE) and 100 from a disease control group, were tested by QUANTA Flash dsDNA CIA, QUANTA Lite dsDNA SC ELISA, BioPlex 2200 multiplex flow immunoassay (MFI), ImmuLisa dsDNA ELISA, and NOVA Lite CLIFT. A second cohort of 69 SLE patients was then tested by QUANTA Flash dsDNA and CLIFT to expand the study. Results. The overall qualitative agreements varied between 77.0% (NOVA Lite CLIFT versus QUANTA Lite) and 89.4% (ImmuLisa versus NOVA Lite CLIFT). The clinical sensitivities for the anti-dsDNA antibody tests varied from 8.2% (NOVA Lite CLIFT) to 54.1% (QUANTA Lite), while the clinical specificities varied from 88.0% (BioPlex 2200) to 100.0% (NOVA Lite CLIFT). Good correlation was found between QUANTA Flash dsDNA and NOVA Lite CLIFT. Conclusion. Significant variations among dsDNA methods were observed. QUANTA Flash dsDNA provides a good combination of sensitivity and specificity for the diagnosis of SLE and good agreement to CLIFT. [ABSTRACT FROM AUTHOR]