Immunoglobulin A antibodies to oxidized collagen type II as a potential biomarker for the stratification of spondyloarthritis from rheumatoid arthritis.

OBJECTIVES: The discovery of diseased tissue-specific neoantigens offers the opportunity to develop important disease tissue-specific biomarkers that can help in the prediction, diagnosis, and stratification of diseases. This opportunity is specifically significant for autoimmune diseases where diagnostic biomarkers are not available. Inflammatory autoimmune diseases are commonly associated with local generation of large amounts of reactive oxidants. We have previously identified oxidative post-translationally modified (oxPTM) tissue-specific neoantigens in rheumatoid arthritis (RA) and type 1 diabetes that elicit an immune response. In the current study, we studied the presence and clinical significance of antibodies to oxPTM collagen type II (CII) in patients with spondyloarthritis (SpA). METHOD: Levels of antibodies specific to native CII and oxPTM-CII were assessed by enzyme-linked immunosorbent assay. RESULTS: Immunoglobulin G (IgG) binding to oxPTM-CII was observed in 52%, 83%, and 28% of serum samples from patients with axial spondyloarthritis (axSpA), RA, and psoriatic arthritis (PsA), respectively. Importantly, while strong IgA anti-oxPTM-CII responses were detected in axSpA and PsA patients, with 47% and 84% respective binders, no IgA anti-oxPTM-CII was detected in RA patients. IgA anti-oxPTM-CII reactivity in axSpA patients treated with biologics was higher and more frequent, with 85% binders compared to 9% binders in patients treated with synthetic disease-modifying anti-rheumatic drugs. CONCLUSION: Our data imply that SpA and PsA are associated with the presence of antibodies to oxPTM-CII, suggesting that there may be a humoral component that may distinguish patients with SpA from RA. Our approach could be adapted to other diseases, particularly to inflammatory autoimmune diseases.

Combining immunofluorescence with immunoblot assay improves the specificity of autoantibody testing for myositis.

OBJECTIVE: Immunoblot (IB) methods are widely used to detect myositis-specific autoantibodies (MSAs); however, false-positive results are common. In this study, we aimed to determine whether associating the anti-nuclear antibody (ANA) IIF pattern may help to improve the specificity of MSA detection by IB in patients with idiopathic inflammatory myositis (IIM). METHODS: Serum samples from 104 patients presenting with muscle weakness/myalgia and positive to at least one MSA by IB (MYOS12 Diver and MIOS7 Diver, D-tek) were tested for ANAs on HEp-2000 cells (Immuno Concepts). The chi-square test was used to analyse the concordance of the MSA result and its corresponding pattern by ANA testing between patients with and without IIM. RESULTS: Eighty-three of the 104 patients had a diagnosis of definite IIM, while in 21 cases, patients were affected by other autoimmune diseases or various non-systemic diseases. Forty nine of 83 (59%) patients in the IIM group and 4/21 (19%) in the non-IIM group showed a concordance between ANA pattern and MSAs by IB (P < 0.001). MSA monopositivity was significantly associated with IIM (91.6%) compared with 61.9% in the non-IIM group (P = 0.0005). CONCLUSIONS: Considering both the MSA result and its corresponding pattern by ANA testing may help to improve the specificity of MSA detection by IB and to confirm the diagnosis of MSA-associated IIM. The monopositivity of MSAs is an important additional tool to validate IB results.

Autoantibody profile and clinical patterns in 619 Italian patients with cutaneous lupus erythematosus.

BACKGROUND: Anti-nuclear antibodies (ANA), anti-extractable nuclear antigens (ENA) and anti-dsDNA antibodies are often associated with cutaneous lupus erythematosus (CLE), with variable frequency depending on skin subtype. However, specific data based on large case-series on the pathogenetic, diagnostic and prognostic meaning of such autoantibodies are still lacking. OBJECTIVE: To characterize the correlations between CLE subtypes as well as LE-non-specific skin lesions and their autoantibody pattern. METHODS: Epidemiological, clinical and immunopathological data of 619 Italian patients with CLE and LE-non-specific skin lesions were analysed. Differences in age, sex, clinical features and autoantibody profile were evaluated in each LE subgroup. RESULTS: Anti-nuclear antibodies (P < 0.0001), anti-dsDNA (P < 0.0001), ENA (P = 0.001), anti-Sm (P = 0.001), anti-RNP (P = 0.004) and anti-histone (P = 0.005) antibodies were associated with SLE. A strong association between ANA (P < 0.0001) and anti-dsDNA (P < 0.0001) and female gender was also found: positive ANA and positive anti-dsDNA had a higher prevalence among females. Chronic CLE resulted to be negatively associated with ENA (OR = 0.51, P < 0.0001), anti-Ro/SSA (OR = 0.49, P < 0.0001) and anti-dsDNA (OR = 0.37, P < 0.0001). Intermittent CLE resulted to be negatively associated with ENA (OR = 0.50, P = 0.007) and ANA (OR = 0.61, P = 0.025). Subacute CLE resulted to be associated with ENA (OR = 5.19, P < 0.0001), anti-Ro/SSA (OR = 3.83, P < 0.0001), anti-Smith (OR = 2.95, P = 0.004) and anti-RNP (OR = 3.18, P = 0.007). Acute CLE resulted to be strongly associated with anti-dsDNA (OR = 6.0, P < 0.0001) and ANA (OR = 18.1, P < 0.0001). LE-non-specific skin lesions resulted to be significantly associated with systemic involvement. Livedo reticularis was significantly associated with ENA (P = 0.007) and anti-Ro/SSA (P = 0.036). Palpable purpura and periungual telangiectasia were significantly associated with ANA. CONCLUSION: According to our findings, some well-known associations between CLE subtypes and autoantibody profile were confirmed; moreover, specific association between autoantibodies and LE-non-specific skin lesions was highlighted. A strict association between anti-ENA and anti-Ro/SSA antibodies and livedo reticularis, ANA and palpable purpura, and ANA and periungual telangiectasia was evidenced.

A better definition of the anti-DFS70 antibody screening by IIF methods.

BACKGROUND: Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. METHODS: Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). RESULTS: The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). CONCLUSION: The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

Analytical variability in the determination of anti-double-stranded DNA antibodies: the strong need of a better definition of the old and new tests.

Anti-dsDNA antibodies are a heterogeneous group of antibodies, quite specific for SLE. Their variability is related to the assay used, the immunoglobulin class secondary antibody, and the dsDNA source. The standardization of measuring anti-dsDNA antibodies is still poor and different methods yield different results. Several novel technologies were developed during the last decades that represent viable alternatives to the traditional methods such as the chemiluminescent immunoassay (CIA) and multiplex flow immunoassay (MFI). Additionally, positive results for anti-dsDNA antibodies can be detected in patients with inflammatory arthritis (IA) treated with different biologics reducing its clinical specificity for SLE. Anti-dsDNA antibody levels were evaluated in 246 patient samples: 70 SLE and 176 disease control (including 96 IA during treatment with different biologics), using three enzyme immunoassays (indirect enzyme immunoassay, Bio-Rad Laboratories; chemiluminescent immunoassay, Inova Diagnostics; multiplex flow immunoassay, Bio-Rad Laboratories) and three Crithidia luciliae immunofluorescence tests (CLIFT) (Euroimmun AG, Bio-Rad Laboratories, INOVA Diagnostics). Diagnostic performances were assessed both including and excluding the IA patients. Agreements, measured by the Cohen's Kappa between all methods, ranged from moderate to substantial (0.47-0.68). The clinical sensitivities for the anti-dsDNA antibody tests varied from 5.7% by CLIFT A up to 33.3% provided by EIA while the clinical specificities varied from 89.8% by MFI to 98.9% provided by CLIFT B and C. Newer technologies, such as MFI and CIA, showed great potential as a diagnostic application. Significant variations among anti-dsDNA antibody assays were observed confirming the lack of standardization.

The impact of biological treatments on the anti-dsDNA and anti-nucleosome tests.

Background Anti-double stranded DNA antibodies are a very heterogeneous group of antibodies, quite specific for systemic lupus erythematosus. Newer technologies, such as addressable laser bead immunoassays (ALBIA), show great potential as a diagnostic application. The production of anti-double stranded DNA antibodies is often encountered in inflammatory arthritis; however, literature reports that the actual onset of drug induced lupus in patients treated with biological drugs is a rare event. False positive results for anti-double stranded DNA and anti-nucleosome antibodies detected in patients with inflammatory arthritis treated with different biologics prompted the investigation of full autoantibody profiles to evaluate each biomarker's diagnostic performance in systemic lupus erythematosus. The aim of the study was to compare the diagnostic performance of anti-double stranded DNA antibody and anti-nucleosome antibody methods and to evaluate the value of simultaneously measuring anti-double stranded DNA and anti-nucleosome antibodies, along with other anti-nuclear antibody analytes, as biomarkers for systemic lupus erythematosus, using a more appropriate control cohort including inflammatory arthritis patients with a non-clinical drug induced lupus. Methods Anti-double stranded DNA and anti-nucleosome antibody levels were evaluated in 247 patient samples: 70 systemic lupus erythematosus, 177 disease controls (including 97 inflammatory arthritis during treatment with different biologics) using the Bio-Rad BioPlex® 2200. Results Anti-nucleosome antibodies demonstrated greater clinical sensitivity and specificity than anti-double stranded DNA antibodies. At the manufacturers' cut-off range, considering the two markers as a single or combined test, the "anti-double stranded DNA test or anti-nucleosome antibodies" was the most sensitive combination (0.400) with the best negative likelihood ratio (0.62) and negative predictive value (0.803). Conclusion Anti-nucleosome antibodies are a more sensitive and specific biomarker of systemic lupus erythematosus than anti-double stranded DNA antibodies. Anti-nucleosome antibodies and anti-double stranded DNA antibodies are independent and complementary markers of systemic lupus erythematosus diagnosis and, therefore, are strongly suggested as combined tests (positive predictive value = 0.938). Moreover, the combined use of the two tests may help to overcome the decreased specificity percentage of the anti-double stranded DNA test, when considering an inflammatory arthritis cohort under biological therapies. The ALBIA method for anti-nuclear specificity detection allows a full autoantibody assessment, resulting in a much higher clinical specificity for systemic lupus erythematosus in the presence of ≥3 positive markers and significantly more positive likelihood ratio when ≥2 positive markers are present.

An effective algorithm for the serological diagnosis of idiopathic inflammatory myopathies: The key role of anti-Ro52 antibodies.

BACKGROUND: Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests. METHODS: Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods. RESULTS: Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA. CONCLUSIONS: Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended.

Jaccoud's arthropathy, an unusual manifestation of idiopathic retroperitoneal fibrosis: rapid improvement of symptoms after tocilizumab treatment.

Jaccoud's arthropathy (JA) is a chronic, non erosive, rheumatoid-like deformity associated with rheumatic fever (RF) and systemic lupus erythematosus and with other diseases such as psoriatic arthritis, connective tissue diseases, hypocomplementemic urticarial vasculitis, infections, sarcoidosis and neoplasia. We described a case of JA in a patient with cutaneous psoriasis but with a particular disease evolution associated with idiopathic retropritoneal fibrosis (IRF), evaluated with computed tomography, magnetic resonance and 18F-FDG PET/ CT. The patient, following failure with steroids, methotrexate and etanercept, was treated with tocilizumab (8 mg/kg) once every 4 weeks for 6 months. A rapid improvement of symptoms and disappearance of 18F-FDG uptake was shown. We describe a review of literature of rheumatic manifestations of IRF and the possible role of interleukin-6 in the pathway of JA and IRF.

The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test.

According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight (n = 84), Helios (n = 85) and NOVA View (n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A (k = 0.82) and B (k = 0.72), and almost perfect for C (k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C (k = 0.86; k = 0.82) and substantial agreement for A (k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform.

The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature.

Anti-nuclear antibody (ANA) positivity suggests CTD but can also lead to a diagnosis of UCTD when a patient does not fulfill the CTD diagnostic criteria. An anti-dense fine speckled (DFS) immunofluorescence (IIF) pattern can be observed when using an ANA test on HEp-2 cells and is due to the presence of antibodies to the nuclear DFS70 antigen that has rarely found in CTD. Serological testing for anti-DFS70 antibodies could therefore play a very interesting negative predictive role in stratifying patients on the basis of the evolution of UCTD to CTD. We described two patients ANA and anti-DFS70 positive in which the use of new method allowing the immunoadsorption of anti-DFS70 antibodies has permitted to exclude the incorrect diagnosis of CTD.