Clin Rheumatol DOI 10.

1007/s10067-011-1861-8

BRIEF REPORT

What is the ability of anti-cyclic citrullinated peptide antibodies determination in synovial fluid in discriminating rheumatoid arthritis from non-rheumatoid arthritis patients? A Tunisian cross-sectional study
Dalila Mrabet & Lilia Laadhar & Hla Sahli & Bchir Zouari & Slim Haouet & Houria Lahmar & Sondes Makni & Slaheddine Sellami

Received: 23 March 2011 / Revised: 9 September 2011 / Accepted: 18 September 2011 # Clinical Rheumatology 2011

Abstract Anti-cyclic citrullinated peptide antibodies (ACPA) seem to be produced locally at the site of joints inflammation in the first stage of rheumatoid arthritis (RA). A strong correlation between serum ACPA and ACPA in the synovial fluid (SF-ACPA) is now suggested. A casecontrol study was conducted to evaluate the usefulness of ACPA determination in SF of patients with RA. A total of 53 patients with a knee-joint effusion (26 RA, 18 peripheral spondyloarthropathies (SPA), and 9 osteoarthritis (OA)) were included in our study. SF samples were obtained by performing therapeutic arthrosynthesis. IgG serum ACPA and SF-ACPA levels were determined by the enzymelinked immunosorbent assay (ELISA). We have also determined IgG levels in serum and SF by nephelometry. Higher levels of IgG ACPA antibodies in SF (p=0.045) and serum (p=0.045) were found in patients with RA with respect to SPA and OA patients. The Spearman correlation analysis showed a significant and positive correlation between ACPA in serum and SF (rho=0.516; p=0.007)
D. Mrabet (*) : H. Sahli : S. Sellami Rheumatology Department, La Rabta Hospital, Tunis 1007, Tunisia e-mail: mrabetdalila@yahoo.fr L. Laadhar : H. Lahmar : S. Makni Immunology Department, La Rabta Hospital, Tunis 1007, Tunisia B. Zouari Epidemiology Department, Faculty of Medicine, Tunis 1007, Tunisia S. Haouet Pathology Department, La Rabta Hospital, Tunis 1007, Tunisia

not only in the RA group but also in patients with SPA. Serum ACPA discriminated RA from non-RA at a cut-off value of 2.7 U/ml (sensitivity, 69%; specificity, 78%; and area under the curve (AUC), 0.72), whereas SF-ACPA discriminated RA from non-RA at a higher cut-off value of 4.95 U/ml (sensitivity, 73%; specificity, 61%; and AUC, 0.71). Our study suggests that the determination of SFACPA give complement information to serum ACPA in patients with RA. Keywords Ankylosing spondylitis . Anti-cyclic citrullinated peptide antibodies . Rheumatoid arthritis . Synovial fluid

Introduction Rheumatoid arthritis (RA) is an inflammatory disease that may lead to joint destruction and bone erosions [1]. Genetic factors including class II major histocompatibility complex (MHC) and non-MHC alleles, environmental factors, and autoimmune processes are highly involved in the pathogenesis of RA [2]. Early diagnosis and effective therapy are crucial in order to prevent joint deterioration, functional disability, and unfavorable disease outcome [1, 3]. During joint inflammation, citrullination of synovial antigens is an active process in which several synovial proteins will be involved [4]. Serum antibodies directed toward citrullinated proteins have been described in 60 80% of patients with RA [4]. Anti-cyclic citrullinated peptide antibodies (ACPA) are produced locally at the site of joints inflammation in the first stage of RA development [4, 5]. This antibody is secreted from B cells which are

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present in the bone marrow as well as in the synovial compartment of ACPA-positive patients with RA [5, 6]. ACPA levels in the synovial fluid (SF-ACPA) are strongly correlated with serum ACPA [7, 8]. Citrullinated antibodies were found not only in the SFs of RA patients but also in other arthropathies [911]. Therefore, presence of ACPA in the SF of patients with arthritis is not specific for RA, but it is an inflammatory-related process [9, 12]. However, in a number of studies, the levels of these antibodies in the SF of RA patients were shown to be significantly higher than in other rheumatic disease [7, 8, 13]. Since production of ACPA occurs prior to onset of arthritis [14], its possible diagnostic benefits are expected to be evident in the SF earlier than in serum. For this reason, a study was conducted to determine the diagnostic usefulness of SFACPA for RA as well as its ability in discriminating RA from non-RA arthritis compared with serum ACPA.

ACPA for diagnosing RA. The optimal cut-off levels for serum and SF-ACPA that best distinguished RA from non-RA was specified at the maximum value of Youden's index, which was calculated by sensitivity + specificity1. The area under the curve (AUC) was used to estimate the overall accuracy. Diagnostic characteristics of serum ACPA and SFACPA were determined by comparison of RA patients with non-RA controls that is SPA and OA. Diagnostic accuracies of serum ACPA and SF-ACPA were compared by AUC values. Levels of serum ACPA and SF-ACPA were compared between the three groups using one-way analysis of variance (ANOVA) and between RA patients and non-RA controls using Student'st test. Levels of SF-ACPA were presented as median (25th75th percentile). ANOVA test was used to compare the levels of serum ACPA and SFACPA in different groups. Correlations between values were studied by Spearman's rank correlation coefficient; p values less than 0.05 were considered statistically significant. SPSS software version 11.5 was used for statistical analysis.

Materials and methods Patients' characteristics Fifty-three patients with a knee-joint effusion, consisting of 26 patients with RA, 18 with peripheral spondyloarthropathies (SPA), and 9 with osteoarthritis (OA), were included in our study. After informed consent, patients underwent clinical, radiological, and laboratory investigations. We did not test serum or SF-ACPA in normal healthy population. Diagnosis of RA was confirmed by the ACR/EULAR revised criteria [15] and that of SPA was made according to European Spondyloarthropathy Study Group Criteria [16]. Rheumatoid factor (RF) was determined by latex technique. Moreover, SF samples were obtained from all patients by performing therapeutic arthrosynthesis. Paired serum and SF samples were stored at 80C until analysis. IgG serum ACPA and SF-ACPA levels were determined by the second generation of enzyme-linked immunosorbent assay (ELISA) (Euroimmun, Germany). Moreover, we have also determined IgG levels (grams per liter) in serum and SF by nephelometry (The Binding Site, UK). As it is well known that there is a local synthesis of immunoglobulins (IgG) and ACPA antibodies by the rheumatoid synovium, we have performed corrections of ACPA levels in serum and in SF by the determination of ACPA IgG/IgG ratios (median/ 25th75th percentile) in serum and SF in RA and control groups. Statistical analysis Receiver operating characteristics (ROC curves) analysis was constructed by plotting sensitivity against 1specificity to determine the best cut-off values of serum ACPA and SFResults Patient's characteristics Twenty-six patients with RA (21 women, 5 men), with a median age of 46 years (2171 years) and mean disease duration of 13.5 months (326 months), entered this study. The control group was composed of 18 SPA patients (3 women, 15 men) with a median age of 35 years (1976 years) and of 9 OA patients (6 women, 6 men) with a median age of 56.5 years (4378 years). Rheumatoid arthritis cut-off value Eighty percent (21/26) of RA patient's sera were positive for RF. Serum ACPA discriminated RA from non-RA at a cutoff value of 2.49 U/ml. At this level, the sensitivity was 70.4%, the specificity was 70.4%, and there was a high overall accuracy with AUC of 0.716 (95% CI=0.564 0.868; p=0.009). ACPA levels in the serum and synovial fluid of patients The IgG ACPA concentrations in serum and in SF for the different study groups are shown in Table 1. Higher levels of IgG ACPA antibodies in SF (p=0.045) and in serum (p=0.045) were found in patients with RA with respect to SPA and OA patients (Fig. 1). The correction of ACPA concentration in serum as IgG ACPA/total IgG revealed higher levels (median/25th75th percentile) in patients with RA compared with those with SPA or OA (p=0.029).

Clin Rheumatol Table 1 Values of ACPA in serum and synovial fluid of patients with rheumatoid arthritis, spondyloarthropathy, and osteoarthritis Characteristic Serum ACPA (units) (median/25th75th percentile) Synovial fluid ACPA (median/25th75th percentile) Serum ACPA IgG/serum IgG (median/25th-75th percentile) SF ACPA IgG/SF IgG (median/25th-75th percentile) Rheumatoid arthritis 4.33 11.85 0.29 1.24 (0.382.0) (4.08192.53) (0.024.49) (0.4920.45) Spondyloarthropathy 1.29 (03.075) 4.6 (2.5510.69) 0.075 (00.2) 0.054 (0.0280.98) Osteoarthritis 0.94 (0.182.0) 2.50 (1.355.15) 0.044 (0.0170.12) 0.39 (0.260.83)

However, the correction of SF-ACPA by the ratio IgG ACPA/total IgG in SF showed higher values (median/ 25th75th percentile) in RA patients when compared to the control group (SPA and OA) but without a significant difference (p=0.138). In the control group, the serum and SF levels of IgG ACPA were higher in patients with SPA when compared with OA group but without a significant difference (respectively, p=0.425 and p=0.253). The Spearman correlation analysis showed a significant and positive correlation between ACPA in serum and SF (rho=0.516; p=0.007) in the RA study group. A positive and significant correlation was also found between serum ACPA and SF IgG levels (rho=0.480; p=0.013). Concerning the group of patients with SPA, Spearman correlation analysis showed also a significant and positive correlation between ACPA in serum and SF (rho=0.641; p=0.006). However, in the OA group, there was no significant correlation between ACPA in serum and SF (rho=2.67; p=0.488). Sensitivity and specificity of serum and synovial fluid levels of anti-CCP Serum ACPA discriminated RA from non-RA at a cut-off value of 2.7 U/ml. At this level, the sensitivity is 69%, the

specificity is 78%, and there was a high overall accuracy with AUC of 0.72 (95% CI=0.570.86; p=0.006). SF-ACPA discriminated RA from non-RA at a cut-off value of 4.95 U/ml. At this level, the sensitivity is 73%, the specificity is 61%, and there was a high overall accuracy with AUC of 0.71 (95% CI=0.560.85; p=0.009). Low specificity of serum ACPA (78%) and low specificity of SF-ACPA (61%) indicated that about one third of non-RA controls which included patients with SPA or OA had higher than expected levels of ACPA in the serum and in the SF. We have also determined sensitivity and specificity of the combination of these two tests (serum and SF-ACPA). This combination was discriminant (p=0.012), but it did not provide further information because its sensitivity and specificity did not differ from those of each test taken separately (overlap of confidence intervals (CI) with 95% CI=0.3720.76 for the sensitivity and 95% CI=0.5590.9 for the specificity, respectively). Proportion of false positive and false negative in the determination of serum and synovial fluid ACPA levels The proportion of false positive serum ACPA was 22% in nonRA groups. The proportion of false negative serum ACPA was 31% in RA group. The positive predictive value of the serum ACPA test was 75% in RA patients. The positive predictive value of the SF-ACPA test was 65% in the RA group. This high rate of false positive serum ACPA and SF-ACPA tests as observed in our study may indicate that a proportion of our non-RA controls, in particular, those with higher levels of ACPA, may be at earlier stages of RA who have not progressed to typical clinical form of RA. In another stage of our study, we proposed to identify RA patients among negative serum ACPA group, in particular, in serum RF negative patients by determination of SF-ACPA. However, the total number of negative serum RF and negative serum ACPA patients did not exceed 12 patients. That is why it was not possible to evaluate sensitivity and specificity of this test in such reduced population. We also studied the serum and synovial ACPA levels in patients with RA seronegative for IgM-RF. The total number of these patients was five. Among these five patients, three had positive ACPA levels (p=0.28) and two

Fig. 1 Serum and synovial ACPA levels in the different groups

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had positive synovial ACPA levels (p=0.59). That is why we considered that the determination of positive serum and synovial ACPA levels in the group of RA seronegative for RF was not contributive in our study because of the reduced sample size.

Discussion The findings of the present study indicate significantly higher concentration of ACPA in the SF and in serum of patients with RA compared with non-RA patients. SF-ACPA was strongly correlated with serum ACPA, and increased concentration of SF-ACPA was in concordance with increased level of antibody in the serum of RA patients. At present, data concerning diagnostic performance of SF-ACPA are lacking. Although increased level of SF-ACPA in RA was shown in a few previous studies [7, 8, 13], diagnostic utility of this marker as shown in this study was observed only in few previous studies [8, 13]. Assuming that serum anti-ACPA is derived from synovium as a result of diffusion of these antibodies from the SF into the general circulation, therefore, any diagnostic performance of serum ACPA is expected to be yielded by the SF-ACPA as well. This assumption is supported regarding to strong correlation between SF-ACPA and serum ACPA, which was observed in this study as well as other previous studies [7, 8]. Caspi et al. [7] have conducted a study to assess the levels of ACPA in SFs of patients with RA, psoriatic arthritis (PsA), and OA. Mean levels of SF-ACPA were significantly increased in RA joint effusions compared with PsA and OA (p<0.003). No significant difference was noted between OA and PsA. Spadaro et al. [8] have demonstrated, in a case-control study, that lower levels of IgG ACPA antibodies in SF (p<0.01) and serum (p<0.005) were found in patients with PsA when compared with those with RA, but without difference with OA. The correction of ACPA concentration in SF as IgG ACPA/total IgG revealed higher values (p<0.002) in RA patients with respect to those with PsA. The findings of this study [14] indicate that SF-ACPA yields similar diagnostic utility as serum ACPA does. Hence, during approach of patients with early arthritis, determination of SF-ACPA could be suggested. However, accessibility to SF during management of patients with early arthritis provides opportunity for determination of SF-ACPA. This latter may confer additional benefits due to earlier appearance of antibody compared with serum, lower threshold level for RA diagnosis, and more frequent SF-ACPA positivity compared with serum ACPA positivity in seronegative arthritis [14], which could not be raised by serum examination. However, our study suggests that the determination of IgG SF-ACPA in patients with RA did not give complement information to serum ACPA. Moreover, a significant correlation was found between serum

ACPA and SF-ACPA not only in patients with RA but also in SPA group. These findings confirm the hypothesis that citrullinated antibodies were found not only in the SF of RA patients but also in other arthropathies, as it was found by other studies [911]. The major limitation of our study was the small sample size for both RA and non-RA patients, which can be considered as the major cause of insignificant differences between comparison groups. Moreover, another important limitation of our study was the high rate of false positive serum ACPA and SF-ACPA tests. These findings may indicate that a proportion of our non-RA controls, in particular, those with higher levels of ACPA, may be at earlier stages of RA who have not progressed to typical clinical form of RA. Further prospective studies of early arthritis on a higher number of patients are required to determine diagnostic as well as predictive ability of SF-ACPA. In these studies, prospective follow-up of patients with and without ACPA in the SF will clarify diagnostic or prognostic ability of SF-ACPA.

Disclosures None.

References
1. Alamanos Y, Drosos AA (2005) Epidemiology of adult rheumatoid arthritis. Autoimmun Rev 4:130136 2. Van der Helm-van Mil AH, Wesoly JZ, Huizinga TW (2005) Understanding the genetic contribution to rheumatoid arthritis. Curr Opin Rheumatol 17:299304 3. Raza K, Buckley CE, Salmon M, Buckley CD (2006) Treating very early rheumatoid arthritis. Best Pract Res Clin Rheumatol 20:849863 4. Vossenaar ER, Nijenhuis S, Helsen MM et al (2003) Citrullination of synovial proteins in murine models of rheumatoid arthritis. Arthritis Rheum 48:24892500 5. Reparon-Schuijt CC, van Esch WJ, van Kooten C et al (2001) Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis. Arthritis Rheum 44:4147 6. Rosengren S, Wei N, Kalunian KC, Zvaiffer NJ, Kavanaugh A, Boyle DL (2008) Elevated autoantibody content in rheumatoid arthritis synovia with lymphoid aggregates and the effect of rituximab. Arthritis Res Ther 10:R105 7. Caspi D, Anouk M, Golan I et al (2006) Synovial fluid levels of anti-cyclic citrullinated peptide antibodies and IgA rheumatoid factor in rheumatoid arthritis, psoriatic arthritis, and osteoarthritis. Arthritis Rheum 55:5356 8. Spadaro A, Riccieri V, Scrivo R, Alessandr C, Valesini G (2007) Anti-cyclic citrullinated peptide antibody determination in synovial fluid of psoriatic arthritis. Clin Exp Rheumatol 25:599604 9. Van Venrooij WJ, Zendman AJ (2008) Anti-CCP2 antibodies: an overview and perspective of the diagnostic abilities of this serological marker for early rheumatoid arthritis. Clinic Rev Allerg Immunol 34:3639 10. Vossenaar ER, Smeets TJ, Kraan MC, Raats JM, van Venrooij WJ, Tak PP (2004) The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum 50:34853494 11. Fabien N, Olsson NO, Goetz J et al (2008) Prevalence of autoantibodies to cyclic citrullinated peptide in patients with

Clin Rheumatol rheumatic diseases other than rheumatoid arthritis: a French multicenter study. Clin Rev Allergy Immunol 34:4044 12. Ryke D, Nicholas AP, Cantaert T et al (2005) Synovial intracellular citrullinated proteins colonizing with peptidyl arginine deimiase as pathophysiologically relevant antigenic determinants of rheumatoid arthritis-specific humoral autoimmunity. Arthritis Rheum 2:2323 2330 13. Lundberg K, Nijenhuis S, Vossenaar ER et al (2005) Citrullinated proteins have increased immunogenicity and arthrogenicity and their presence in arthritic joints correlate with disease severity. Arthritis Res Ther 7:458467 14. Heidari B, Abedi H, Firouzjahi A, Heidari P (2010) Diagnostic value of synovial fluid anti-cyclic citrullinated peptide antibody for rheumatoid arthritis. Rheumatol Int 30:14651470 15. Rantap-Dahlqvist S, De Jong BA, Berglin E et al (2003) Antibodies against cyclic citrullinated peptide and IgA rheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum 48:27412749