Rheumatoid Arthritis: Gilburd B

Sherer Y, Gerli R, Bocci EB, Gilburd B, Vaudo G, Bistoni O, Shoenfeld Y. · Department of Medicine 'B' and Center of Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Israel. · Ann N Y Acad Sci. · Pubmed #17894004 No free full text.

Abstract: The aim of this study was to examine whether heat-shock protein (HSP)-65 autoantibodies are associated with early atherosclerosis in rheumatoid arthritis (RA). Intima-media thickness (IMT) was measured in the carotid arteries of 100 RA patients and 69 control subjects. The IMT was evaluated on both carotid arteries in the common carotid, bifurcation, and internal arteries. Every patient underwent anti-HSP-65 antibody evaluation. Anti-HSP-65 antibodies were not more prevalent among patients compared with controls. Among controls, patients having "positive" anti-HSP-65 tended to have increased carotid artery IMT compared with "negative" patients, whereas among RA patients the opposite association was noted, and positive patients had significantly decreased carotid bifurcation IMT than negative patients without elevated levels of anti-HSP-65. As opposed to the association with cardiovascular diseases and atherosclerosis of anti-HSPs in the general population, among RA patients anti-HSP-65 cannot be regarded as associated with early atherosclerosis.

Sherer Y, Gerli R, Gilburd B, Bartoloni Bocci E, Vaudo G, Mannarino E, Shoenfeld Y. · Department of Medicine B and Center of Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Israel. · Lupus. · Pubmed #17439932 No free full text.

Abstract: Antiphospholipid and anti-oxidized LDL (anti-oxLDL) antibodies are associated with thrombosis and atherosclerosis. Rheumatoid arthritis (RA) is characterized by excess atherosclerosis and cardiovascular diseases. Our aim was to determine whether antiphospholipid and anti-oxLDL antibodies are associated with early atherosclerotic changes in RA. The levels of IgG and IgM anticardiolipin, IgG and IgM anti-beta-2-glycoprotein-I and anti-oxLDL autoantibodies have been evaluated in 82 patients having RA. Carotid artery intima-media thickness (IMT) was measured in the carotid arteries in the common carotid, bifurcation and internal carotid arteries. Elevated levels of IgG anticardiolipin antibodies were detected in 17 of 82 (21%) RA patients, including 7 with medium-to-high levels considered being clinically relevant. These patients had significantly elevated mean carotid and carotid bifurcation IMT compared with RA patients without elevated anticardiolipin. No such association was found regarding other autoantibodies tested. Anticardiolipin antibodies are prevalent in RA and are associated with early atherosclerotic changes, supporting a rational for measuring them in RA, and upon detection treat the patients in order to decrease chances of atherosclerosis progression and thrombosis.

Wu R, Shovman O, Zhang Y, Gilburd B, Zandman-Goddard G, Shoenfeld Y. · Pathway Diagnostics Inc., Malibu, CA, USA. · Clin Rev Allergy Immunol. · Pubmed #17426360 No free full text.

Abstract: To investigate the prevalence of anti-third generation cyclic citrullinated peptide antibodies (anti-CCP3) in patients with systemic connective tissue diseases, we assembled a training set consisting of 115 patients with rheumatoid arthritis (RA), 52 with Calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, 21 with scleroderma, 20 with ankylosing spondylitis, 18 with reactive arthritis, 25 with juvenile rheumatoid arthritis (RA), 51 with osteoarthritis, 26 with mixed connective tissue disease, 23 with primary Sjogren's syndrome, 74 with systemic lupus erythematosus, 49 with Polymyalgia rheumatica, and 39 with polymyositis/dermatomyositis. The commercial enzyme-linked immunosorbent assay (ELISA) was used to detect anti-CCP antibodies, including anti-CCP2 (regular, second generation of CCP antigen) and anti-CCP3 (third generation of CCP antigen) in disease-related specimens and normal controls. These serum samples were also evaluated for anti-centomere antibodies by anti-centromere ELISA kit. The higher frequencies of anti-CCP3 and anti-CCP2 were detected in 75.6 and 70.4% patients with RA, respectively. At the same time, anti-CCP3 (not anti-CCP2) was significantly increased in samples isolated from patients with CREST syndrome. The clinical sensitivity of IgG anti-CCP3 for the patients with CREST syndrome was 29% (15 of 52) and the specificity was 96% (384 of 397), with the exception of the RA group. The anti-centromere antibodies were significantly higher in patients with CREST only. The results of our study suggest that compared to anti-CCP2 assay, the new anti-CCP3 assay can enhance the clinical sensitivity for diagnosis of RA and, as an associate marker combined with anticentromere, can distinguish CREST syndrome from other systemic connective tissue diseases, especially RA. The clinical specificity of anti-CCP3 was lower than anti-CCP2 assay in diagnosis of RA because of the crossreaction to the patients with CREST syndrome.

Shovman O, Gilburd B, Zandman-Goddard G, Sherer Y, Orbach H, Gerli R, Shoenfeld Y. · Center for Autoimmune Diseases, Department of Medicine B, Sheba Medical Center, Israel. · Clin Dev Immunol. · Pubmed #16295525 links to  free full text

Abstract: OBJECTIVE: To compare the diagnostic utility of laboratory variables, including matrix metalloproteinase-3 (MMP-3), anticyclic citrullinated peptide (CCP) antibodies, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) in patients with erosive and non-erosive rheumatoid arthritis (RA). METHODS: We assembled a training set, consisting of 60 patients with RA, all fulfilling the revised criteria of the American College of Rheumatology. A commercial enzyme linked immunosorbent assay (ELISA) was used both to test for anti-CCP antibodies (second generation ELISA kit) and MMP; RF were detected by latex-enhanced immunonephelometric assay. CRP was measured by latex turbidimetric immunoassay. RESULTS: The levels of anti-CCP antibody titers and ESR were significantly higher in patients with erosive disease than those in non-erosive RA patients (p < 0.001 and 0.0341) respectively. Moreover, a higher frequency of elevated titers of anti-CCP antibodies was found in RA patients with erosions compared to patients with non-erosive RA (78.3% vs. 43.2% respectively). The ROC curves of anti-CCP passed closer to the upper left corner than those other markers and area under the curve (AUC) of anti-CCP was significantly larger than AUC of other markers (0.755 for anti-CCP, 0.660 for ESR, 0.611 for CRP, 0.577 for RF, and 0.484 for MMP-3 female). A positive predictive value was higher for anti-CCP antibodies in comparison to other markers. We did not find significant statistical correlation between anti-CCP antibody titers and inflammatory markers such as ESR or CRP. However, we confirmed the correlation of elevated titers of anti-CCP antibodies and RF in both groups of patients whereas the degree of correlation was more significant in non-erosive patients. CONCLUSION: The results of our study suggest that the presence of elevated anti-CCP antibody titers have better diagnostic performance than MMP-3, RF, CRP and ESR in patients with erosive RA.

Harel M, Gilburd B, Schiffenbauer YS, Shoenfeld Y. · Center for Autoimmune Diseases, Department of Medicine B, Chaim Sheba Medical Center, Israel. · Clin Dev Immunol. · Pubmed #16295524 links to  free full text

Abstract: The CellScan apparatus is a laser scanning cytometer enabling repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. The CellScan may serve as a tool for diagnosis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) as well as other autoimmune diseases by monitoring FP changes in peripheral blood lymphocytes (PBLs) following exposure to autoantigenic stimuli. Changes in FI and FP in atherosclerotic patients' PBLs following exposure to various stimuli have established the role of the immune system in atherosclerotic disease. The CellScan has been evaluated as a diagnostic tool for drug-allergy, based on FP reduction in PBLs following incubation with allergenic drugs. FI and FP changes in cancer cells have been found to be well correlated with the cytotoxic effect of anti-neoplastic drugs. In conclusion, the CellScan has a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Sherer Y, Gerli R, Vaudo G, Schillaci G, Gilburd B, Giordano A, Bocci EB, Allegrucci R, Marchesi S, Mannarino E, Shoenfeld Y. · Department of Medicine B and Center of Autoimmune Diseases, Sheba Medical Center, Tel Hashomer 52621, Israel. · Ann N Y Acad Sci. · Pubmed #16126971 No free full text.

Abstract: Antiphospholipid antibodies characterize the antiphospholipid syndrome (APS), but they can also be found in various autoimmune, infectious, and malignant conditions. This study's objectives were to detect the prevalence of antiphospholipid and antioxidized low-density lipoprotein (anti-oxLDL) antibodies among patients with rheumatoid arthritis (RA) who did not have clinical manifestations of APS. Using a standard enzyme-linked immunosorbent assay (ELISA), we evaluated the levels of immunoglobulin G (IgG) and IgM anticardiolipin, IgG, and IgM anti-beta-2-glycoprotein-I (anti-beta(2)GPI), and anti-oxLDL autoantibodies in 82 patients with RA. The cutoff levels for detecting these autoantibodies were 15 IgG phospholipid units (GPL), 15 IgM phospholipid units (MPL), and 25 ELISA units (EU)/mL, respectively. Elevated levels of IgG anticardiolipin antibodies were detected in 17 of 82 (21%) RA patients, including 10 with low levels of IgG anticardiolipin and 7 with medium to high levels of anticardiolipin autoantibodies. IgM anticardiolipin was found in only 1 (1%) patient, and both IgG and IgM anti-beta(2)GPI were found in 3 (4%) patients with RA. Elevated levels of anti-oxLDL antibodies were found in 8 (10%) patients, 4 of whom also had elevated levels of IgG anticardiolipin. We conclude that IgG anticardiolipin autoantibodies can be found in about one-fifth of RA patients who do not have clinical manifestations of APS. Whether the presence of anticardiolipin signifies increased risk for thrombosis and atherosclerosis in these patients should be studied further.

Gerli R, Sherer Y, Vaudo G, Schillaci G, Gilburd B, Giordano A, Bocci EB, Allegrucci R, Marchesi S, Mannarino E, Shoenfeld Y. · Section of Internal Medicine and Oncological Sciences, Center for the Study of Rheumatic Diseases, University of Perugia, Perugia, Italy. · Ann N Y Acad Sci. · Pubmed #16126969 No free full text.

Abstract: This study was designed to compare intima media thickness (IMT) of the carotid arteries among rheumatoid arthritis (RA) patients and controls and to determine whether disease-associated characteristics, smoking, and other classic risk factors for atherosclerosis are associated with IMT values. IMT was measured in the carotid arteries of 101 RA patients and 75 control subjects. The IMT was evaluated in the common carotid (CC), carotid bifurcation (BI), and internal carotid (IC). Eight IMT values were calculated including four mean and four maximal values of CC, BI, IC, and carotid artery (C). The following data were obtained for every patient: age, sex, body mass index (BMI), presence of erosions, extra-articular manifestations, rheumatoid factor, medications, hypertension, hypercholesterolemia, diabetes mellitus, smoking status, daily number of cigarettes, number of smoking years, family history of cardiovascular diseases (CVD), and erythrocyte sedimentation rate (ESR) levels. RA patients had significantly higher mean-BI IMT than controls (1.02 mm vs. 0.89 mm; P < 0.01), higher incidence of increased mean-BI IMT and max-BI IMT, but lower incidence of increased max-IC IMT than controls. Factors significantly associated with IMT in the controls were age, BMI, and hypertension, whereas factors significantly associated with IMT in RA patients were age and smoking status. Mean carotid IMT was associated with all characteristics related to smoking in RA patients. Current smokers had higher mean carotid IMT and internal carotid artery IMT than former smokers. RA is associated with higher carotid artery bifurcation IMT. The profile of factors associated with IMT values is different between RA patients and controls. Smoking is an important factor augmenting early atherosclerosis in RA patients.

Shovman O, Gilburd B, Zandman-Goddard G, Yehiely A, Langevitz P, Shoenfeld Y. · Department of Medicine B and Center for Autoimmune Diseases, Sackler Faculty of Medicine Sheba Medical Center Tel-Aviv University Tel-Hashomer Tel-Aviv Israel. · Autoimmunity. · Pubmed #15804711 No free full text.

Abstract: BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.

Gilburd B, Abu-Shakra M, Shoenfeld Y, Giordano A, Bocci EB, delle Monache F, Gerli R. · Department of Medicine B, Center for Autoimmune Diseases, Sheba Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Hashomer, Israel. · Clin Dev Immunol. · Pubmed #15154612 links to  free full text

Abstract: BACKGROUND: Flow-based, multiplex bead arrays (MBA) have been developed for a variety of applications including the detection of antibodies to extractable nuclear antigens (ENA). It offers a rapid and sensitive method to assess multiple analyses in a single tube/well. PURPOSE: To evaluate the Athena Multi-Lyte ANA Test System utilizes Luminex Corporation's MBA technology for the detection of antinuclear antibodies (ANA) and ENA antibodies in the sera of patients with Sjogren's syndrome (SS). METHODS: MBA assay was used to detect ANA and ENA antibodies in the sera of 37 patients with SS and 96 sera from healthy subjects. RESULTS: All patients were women. Their mean age was 48.7 years and the mean disease duration was 7.27 years. ANA was found in 3 (3%) sera of healthy subjects by the AtheNA system and in 2 (2%) sera by the ELISA kit. A 99% concordance between the 2 assays was found. A 94.6% concordance between the 2 assays was found by testing the sera of patients with SS for ANA. By the AtheNA system, none of the sera of 37 patients with SS had autoantibodies reacting with Sm, Jo-1, dsDNA or histones. Anti-RNP antibody was found in 5.4% of the sera and 2.7% of the sera reacted with Scl-70 and histones. Anti-SS/A and anti-SS/B were identified in 84 and 76% of the sera, respectively. CONCLUSION: The AtheNa Multi-Lyte ANA Test System offers a sensitive and specific result for the detection of ANA and ENA antibodies in the sera of patients with SS.

Marai I, Gilburd B, Blank M, Shoenfeld Y. · Department of Medicine B, Center for Autoimmune Diseases, Sheba Medical Center, Tel- Hashomer, Israel. · Hum Antibodies. · Pubmed #14646033 No free full text.

Abstract: BACKGROUND: Antiphospholipid antibodies are a family of autoantibodies that exhibit a broad range of target specificities and affinities, all recognizing various combinations of phospholipids, phospholipid binding proteins or both. OBJECTIVE: To evaluate the frequency of anticardiolipin (aCL) or anti-beta-2 glycoprotein I (anti-beta2GPI) antibodies in a cohort of patients with primary (PAPS) and secondary (SAPS) antiphospholipid syndrome and other rheumatic and infectious conditions. METHODS: Sera were drawn from 226 patients with PAPS (n= 66), SAPS (n= 60), rheumatoid arthritis (RA) (n= 30), scleroderma (SSc) (n= 30) or syphilis (n= 30). IgG, IgM, IgA aCL and anti-beta2GPI antibodies were determined for all patients. We employed Varelisa diagnostic kits (Pharmacia Diagnostics GmbH & Co.KG, Germany). RESULTS: In APS patients: aCL and anti-betaGP1 antibodies were detected in 81.8% and 70% in PAPS and SAPS patients, respectively. In PAPS, aCL were detected in 71.2%, and anti-beta2GP1 in 50% of patients. In SAPS, aCL were detected in 63.3%, and anti-beta2GP1 in 53.3% of patients. In syphilis, aCL and anti-beta2GP1 antibodies were detected in 46.7% of patients in low levels: aCL in 36.7% and anti-beta2GP1 in 20% of patients. aCL antibodies were detected in 10%, 13.3%, and 0% of RA patients, SSc patients, and healthy persons, respectively. Anti-beta2GP1 antibodies were not detected in RA patients, SSc patients, and healthy persons. CONCLUSIONS: aCL and anti-beta2GP1 antibodies are more frequently found in sera of APS patients, and can be found in low levels in syphilis. Anti-beta2GP1 assay found to be more specific than aCL in antiphospholipid antibodies determination.

Orbach H, Gilburd B, Brickman CM, Gerli R, Shoenfeld Y. · Department of Medicine and Rheumatology Unit, Bikur Cholim Hospital, Jerusalem, Israel. · Isr Med Assoc J. · Pubmed #12455175 links to  free full text

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Steiner G, Shovman O, Skriner K, Gilburd B, Langevitz P, Miholits M, Hoet R, Levy Y, Zandman-Goddard G, Hoefler E, Smolen JS, Shoenfeld Y. · Department of Internal Medicine III, University of Vienna, Austria. · Clin Exp Rheumatol. · Pubmed #12180437 No free full text.

Abstract: OBJECTIVE: Anti-RA33 antibodies occur in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD) and target the A2/B1 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex 4 which forms part of the spliceosome. The aim of the present study was to evaluate the immune response and pathological features induced in mice immunized with anti-RA33 antibodies or patient-derived recombinant single-chain variable fragments (scFv) of anti-RA33 antibodies. METHODS: In the first set of the experiment, two strains of mice (C57BL/6J and BALB/c) were immunized with IgG preparations obtained from two patients with RA and one normal donor. One of the patients had high titer anti-RA33 antibodies; the other one showed weak borderline reactivity. In the second set of the experiment three groups of C57BL/6J mice were immunized, respectively, with affinity-purified (AP) anti-RA33 antibodies, scFv of anti-RA33 antibodies and normal human IgG. The immunological response induced in immunized mice was studied by immunoblotting and line immunoassay (LIA). The presence of arthritis, serositis or myositis was assessed six-months following initial immunization. RESULTS: While anti-RA33 antibodies developed in only two of the mice immunized with different human IgG fractions, anti-RA33 antibodies were clearly detected in 7 sera of 13 mice immunized with AP anti-RA33 antibodies three months after the boost immunization and, moreover, also in 2 sera of 13 mice immunized with scFv of anti-RA33 antibodies. In contrast, mice immunized with normal human IgG did not develop anti-RA33 antibodies. Interestingly, transient autoantibody production against another nuclear autoantigen, U1 snRNP, was observed in 3 C57BL/6J mice immunized with scFv and in 1 mouse immunized with AP autoantibodies. However, these immunological responses were not associated with pathological findings. CONCLUSIONS: Active immunization of naive mice with AP anti-RA33 antibodies and scFv of anti-RA33 antibodies resulted on the one hand in the production of murine anti-RA33 antibodies and led, on the other hand, to transient "autoantibody spread" to snRNP component of the spliceosome and other nuclear autoantigens. This "autoantibody spread" probably reflected disregulation of the idiotypic anti-idiotypic cascade.

Afek A, George J, Gilburd B, Rauova L, Goldberg I, Kopolovic J, Harats D, Shoenfeld Y. · Institute of Pathology, Sheba Medical Center, Faculty of Medicine, Tel Aviv University, Tel Hashomer, Sackler, Israel. · J Autoimmun. · Pubmed #10677242 No free full text.

Abstract: Heat shock proteins are a family of approximately 25 highly conserved proteins upregulated in response to various forms of stress. They play an active role in the development autoimmune diseases in animals, and have been incriminated in human autoimmune diseases (i.e. rheumatoid arthritis, multiple sclerosis). It has been previously shown, that an induced immune response against Heat shock protein 65 (HSP-65) results in atherosclerotic lesions in normocholesterolemic rabbits. We have supported these findings showing that C57BL/6 mice immunized with HSP-65 and fed a high-fat diet develop enhanced fatty streaks. To create a model that will eliminate the need for exogenous supplementation of a high-fat diet, we have immunized LDL receptor deficient (LDL-RD) mice with HSP-65 or with heat-killed Mycobacterium tuberculosis (Mt). Seven groups of LDL-RD mice (n=10), were immunized subcutaneously with different concentrations of HSP-65, Mt or bovine serum albumin (BSA). All mice were fed a normal chow-diet for 3 months. The mice immunized with the higher doses of Mt developed significantly larger fatty streaks when compared with their BSA immunized littermates. The size of the lesions in the aortic sinus were: 31,562+/-5,994 microm(2)in the 10 microg Mt and 52,777+/-5,245 microm(2)in the 100 microg Mt vs. 11, 500+/-3,750 microm(2)in the BSA group (P<0.05). In the HSP-65-immunized mice, only the group injected with the highest dose (5 microg, twice) developed significantly larger fatty streaks when compared with the BSA-immunized group (28,611+/-4,716 microm(2)vs. 11,500+/-3,750 microm(2)respectively, (P<0.05). The HSP-65-but not the Mt- or BSA-immunized mice developed high titers of anti HSP-65 antibodies, beginning 10 days after the immunization, which persisted until they were killed. Immunohistochemical staining showed CD3-positive lymphocytes in the aortic sinus of mice immunized with Mt or HSP-65, but not in the control group. Thus, we established a mouse model of HSP-65 immune mediated atherosclerosis devoid of high fat diet supplementation. This model will enable us to further study the role of the immune system in atherosclerosis, via HSP-65 and raise novel immunomodulatory therapeutic modalities.

Bayer PM, Bauerfeind S, Bienvenu J, Fabien N, Frei PC, Gilburd B, Heide KG, Hoier-Madsen M, Meroni PL, Monier JC, Monneret G, Panzeri P, Shoenfeld Y, Spertini F, Wiik A. · Zentrallabor Wilhelminenspital, Vienna, Austria. · J Autoimmun. · Pubmed #10441172 No free full text.

Abstract: The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sjögren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations.