The diagnostic value of anti-cyclic citrullinated peptide antibodies and rheumatoid factor in patients with rheumatoid arthritis

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1 Formosan Journal of Rheumatology 2008;22:68-73 Original Article The diagnostic value of anti-cyclic citrullinated peptide antibodies and rheumatoid factor in patients with rheumatoid arthritis Hung-Ke Lin 1, Joung-Liang Lan 1,2,3, Der-Yuan Chen 1,2,3, Yi-Hsing Chen 1,2, Wen-Nan Huang 1,4, Tsu-Yi Hsieh 1,3, Chia-Wei Hsieh 1,4, Hsin-Hua Chen 1,2 1 Division of Allergy, Immunology, and Rheumatology, Taichung Veterans General Hospital, Taiwan 2 National Yang-Ming University, Taipei, Taiwan 3 Institute of Medical Technology, National Chung-Hsing University, Taichung, Taiwan 4 Chung-Shan Medical University, Taichung, Taiwan Objective: To identify the diagnostic value of anti-cyclic citrullinated peptide (anti-ccp) antibodies and rheumatoid factor (RF) in patients with rheumatoid arthritis (RA). Methods: Serum levels of anti-ccp antibodies were determined by enzyme-linked immunosorbent assay, and levels of RF were determined by nephelometry in 145 patients with RA and 75 patients with non-ra rheumatic diseases. Results: Among the 145 patients with RA, 119 patients (82.1%) tested positive for anti-ccp antibodies, and 116 patients (80.0%) tested positive for RF. The sensitivity, specificity, positive predictive value, and negative predictive value of anti-ccp antibodies for diagnosing RA were 82.1%, 88.0%, 93.0%, and 71.7% respectively. Those for RF were 80.0%, 62.7%, 81.1%, and 61.0% respectively. The presence of either anti-ccp antibodies or RF increased sensitivity to 88.3%, and when they both were present, the specificity increased to 94.7%. The positive rates for anti-ccp antibodies in the RF-positive RA, RFnegative RA, and non-ra patients were 93.1%, 37.9%, and 12.0% respectively. Conclusion: With its high sensitivity and specificity, the anti-ccp antibodies assay is a useful test for diagnosing RA. The use of anti-ccp antibodies and RF in combination further increases the diagnostic value for RA. Key words: Rheumatoid arthritis, anti-ccp antibodies, rheumatoid factor, diagnostic value Introduction The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical manifestations and serologic tests [1]. Conventionally, the serology test routinely Corresponding author: Der-Yuan Chen, M.D., Ph.D. Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital. No. 160, Sec. 3, Taichung Harbor Road, Taichung 40705, Taiwan. TEL: ext 3354, FAX: dychen@vghtc.gov.tw Received: July 01, 2008 Revised: August 26, 2008 Accepted: September 17, 2008 used in RA is the determination of serum rheumatoid factor (RF). However, it has little predictive value in the general population, since the overall disease prevalence is relatively low. The more specific autoantibodies for the diagnosis of RA, anti-cyclic citrullinated peptide (anti-ccp) antibodies, were discovered in 1964 [2]. Accumulating evidence shows that anti-ccp antibodies are very useful in the diagnosis of RA [3]. They may be present very early in the disease course [4] and are also considered as a prognostic factor for articular destruction [5]. Several studies have shown that anti-ccp antibodies are moderately sensitive but highly specific for the 68

2 Lin et al diagnosis of RA, and their specificity is higher than that of RF [7-10]. Therefore, we conducted a retrospective study to identify the diagnostic value of anti-ccp antibodies and RF in patients with RA. Materials and Methods Materials Two hundred and twenty patients (163 females, 57 males; mean age ± SD, 45.2 ± 16.0 years) randomized selected from 2219 patients with rheumatic diseases who visited the Division of Allergy, Immunology, and Rheumatology, Taichung Veterans General Hospital from 2001 to 2005 were enrolled in this study. Of the 220 patients, 145 (65.9%) patients (106 females, 39 males; mean age ± SD, 48.2 ± 14.9 years) who fulfilled 1987 American College of Rheumatology (ACR) Criteria for RA [1] were included in the RA group. The other 75 patients were classified as non-ra group (57 females, 18 males; mean age ± SD, 39.4 ± 16.3 years). The non-ra group included patients with systemic lupus erythematosus (SLE) (n=19), primary Sjögren's syndrome (SS) (n=33), spondyloarthropathy (n=10), Behcet s disease (n=3), mixed connective tissue disease (n=3), osteoarthritis (n=4), adult onset Still s disease (n=1), antiphospholipid syndrome (n=1), and gouty arthritis (n=1). The age, gender, clinical characteristics, titers of RF and anti-ccp antibodies of each patient were recorded. All serum samples were obtained and stored at 20 C until assayed. The study was approved by the Ethics committee of Taichung Veterans General Hospital. Methods Serum levels of anti-ccp antibodies were determined by the second-generation enzyme-linked immunosorbent assay (INOVA diagnostics, San Diego, CA, USA). The serum samples were evaluated in duplicate, with the upper normal limit of 20 IU/mL being assumed in accordance with the manufacturer's recommendations (cut-off level 20 IU/mL). RF-IgM was determined by nephelometry (Dade Behring, Marburg, GembH, USA) (cut-off level 15 IU/mL). The inter- and intra-assay variabilities of anti-ccp antibodies and RF were both less than 9%. Statistical analysis Continuous variables are expressed as the median ± interquartile range (IQR). The subgroups were compared using the Mann-Whitney U test. Categorical variables, expressed as percentages, were analyzed by Fisher s exact test or Yate s correction of contingency. Comparisons of sensitivity and specificity were made using chi-square test. The most appropriate cut-off values for anti-ccp antibodies and RF were determined by receiver operating characteristic (ROC). We also identified the diagnostic sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and area under curve (AUC) in both tests. All statistical tests were 2-sided and were assessed at the 0.05 significance level. Results Frequencies of anti-ccp antibodies and RF in patients with rheumatic diseases The demographic data and clinical features are summarized in Table 1. Significantly higher values of anti-ccp antibodies were found in the RA patients than in non-ra patients (117.4 ± IU/mL vs. 7.7 ± 5.9 IU/mL, p<0.001). Similarly, higher levels of the RF in the RA patients than non-ra patients was found (72.0 ± IU/mL vs ± 21.9 IU/mL, p<0.001). Anti-CCP antibodies was tested positive in 119 of 145 patients (82.1%) with RA and 9 of 75 patients (12.0%) with non-ra rheumatic diseases, including 8 of 19 patients with SLE (42.1%), and 1 of 33 patients with primary SS (3.0%). Serum RF was detected in 116 Table 1. Demographic data and clinical characteristics of patients with RA and patients with non-ra rheumatic diseases RA (n=145) non-ra (n=75) p Male, n (%) 39 (26.9 %) 18 (24%) Female, n (%) 106 (73.1 %) 57 (76 %) Age at visit (years) (Median ± IQR) 51.0 ± ± 25.0 < Disease duration (mons)(median ± IQR) 18.5 ± ± 23.0 < Values of anti-ccp (Median ± IQR) ± ± 5.9 < Values of RF (Median ± IQR) 72.0 ± ± 21.9 < Abbreviations: RA = rheumatoid arthritis, IQR = interquartile range, anti-ccp = anti-cyclic citrullinated peptide antibody, RF = rheumatoid factor 69

3 Anti-CCP antibody and RF in RA Table 2. Frequencies of anti-ccp antibodies and RF in RA patients and non-ra patients Diseases (n) anti-ccp (%) RF (%) RA (145) 119 (82.1 %) 116 (80.0%) SS (33) 1 (3.0%) 16 (48.5%) SLE (19) 8 (42.1%) 6 (31.6%) Spondyloarthropathy (10) 0 3 (30%) Mixed connective tissue disease (3) 0 2 (66.7%) Osteoarthritis (4) 0 0 Behcet s disease (3) 0 0 Adult onset Still s disease (1) 0 0 Antiphospholipid syndrome (1) 0 0 Gouty arthritis (1) 0 0 Abbreviations: anti-ccp = anti-cyclic citrullinated peptide antibody, RF = rheumatoid factor, RA = rheumatoid arthritis, SS = primary Sjogren s syndrome, SLE = systemic lupus erytyhematosus of 145 patients (80%) with RA and 27of 75 patients (23.1%) with non-ra rheumatic diseases. These patients with rheumatic diseases other than RA had positive RF, including 16 out of 33 patients (48.5%) with SS, 6 out of 19 patients (31.6%) with SLE, 3 out of 10 patients (30%) with spondyloarthropathy, and 2 out of 3 patients (66.7%) with mixed connective tissue disease (Table 2). Among RF-positive RA patients, 108 of 116 patients (93.1%) were anti-ccp antibodies-positive. Among RF-negative RA patients, 11 of 29 patients (37.9%) were anti-ccp antibodies-positive. Diagnostic value of anti-ccp antibodies and RF in patients with RA Based on the cut-off value suggested by the manufacturer, the anti-ccp antibodies had sensitivity, specificity, PPV, and NPV for a diagnosis of RA of 82.1%, 88.0%, 93.0%, and 71.7% respectively. Those for RF were 80.0%, 62.7%, 81.1%, and 61.0% respectively. For anti-ccp antibodies in combination with RF, they were 74.5%, 94.7%, 96.4%, and 65.7% respectively. If Table 3. Diagnostic performance of anti-ccp antibodies and RF in RA Anti-CCP RF Anti-CCP and RF Anti-CCP or RF Sensitivity 82.1% 80.0% 74.5% 88.3% Specificity 88.0% 62.7% 94.7% 57.3% PPV 93.0% 81.1% 96.4% 80.5% NPV 71.7% 61.0% 65.7% 70.5% Abbreviations: anti-ccp = anti-cyclic citrullinated peptide antibody, RF = rheumatoid factor, RA = rheumatoid arthritis, PPV = positive predictive values, NPV = negative predictive values Figure 1. Receiver operating characteristic (ROC) curve and area under curve (AUC) of anti-ccp antibodies and RF. either one was present, they were 88.3%, 57.3%, 80.5%, and 70.5% respectively (Table 3). As shown in Figure 1, the AUC for anti-ccp antibodies was (95% confidence interval (CI) of to 0.921), whereas the AUC for RF was (95% CI of to 0.837). The most appropriate cut-off value of anti-ccp antibodies for diagnosing RA was 34.7 IU/ ml and that of RF was 21.3 IU/mL. Discussion In the present study, the sensitivity and specificity of anti-ccp antibodies for diagnosing RA were 82.1% and 88.0% respectively. The high sensitivity and specificity of our study were consistent with the results of previous studies [10-11]. The frequency of anti-ccp antibodies in RF-negative RA patients was 37.9%, similar to that of previous report (40%) [12], suggesting that the determination of anti-ccp antibodies is more beneficial in the diagnosis of RA in RF-negative patients. Furthermore, the AUC for anti-ccp antibodies was 0.881, which was better than that for RF (0.784). The higher value of AUC made anti-ccp antibodies a better diagnostic tool than RF in the diagnosis of RA. However, there was crossover between the ROC curves of anti-ccp antibodies and RF, which means that anti-ccp antibodies were not superior to RF in all circumstances in the study. In our study, the sensitivity and the specificity of RF for diagnosing RA were 80.0% and 62.7% respectively, which were similar to the results of previous studies [6,13-16]. The PPV of RF for RA (81.1%) was higher 70

4 Lin et al than that in one report (56%); whereas the NPV (61%) was lower [17]. The reason is that with RF, as with any diagnostic test, the predictive value is affected by the estimated likelihood of disease prior to conducting the test [18]. It has a lower PPV if the test is conducted among patients with non-ra rheumatic diseases (SLE, primary SS, and cryoglobulinemia) or few clinical features of systemic rheumatic disease. The sensitivity, specificity, and PPV in our study were all relatively higher than those in other reports [17]. Our patients who had existing arthritic or rheumatic problems may have contributed to this discrepancy. Detailed history taking and physical examination made anti-ccp antibodies and RF have better diagnostic value for RA. The presence of either anti-ccp antibodies or RF increased the sensitivity of anti-ccp antibodies for diagnosing RA from 82.1% to 88.3%; however when they both were presented, the specificity increased from 88.0% to 94.7%. In view of practical use, it would be helpful to perform both the RF and anti-ccp antibodies assays in patients with suspected RA. In addition, we observed anti-ccp antibodies in patients with other rheumatic diseases including 8 of 19 patients with SLE (42.1%), and 1 of 33 patients with primary SS (3.0%). Three patients in the non-ra group who had high anti-ccp antibodies titers (>60 IU/mL) also had SLE. When we used the appropriate cut-off level value (anti-ccp antibodies 34.7 IU/mL) for diagnosing RA, only 4 (21%) SLE patients tested positive for this antibody. Our data were consistent with those of a recent study in which anti-ccp antibodies were detected in 15 % of patients with SLE, and 14% in those with SS [19]. Besides, 6 of 19 (31.6%) SLE patients and 16 of 33 (48.5%) SS patients had RF. In another study, 104 patients with SLE were evaluated for arthritis. Of the patients with erosive arthritis (n=12), 8 patients tested positive for anti-ccp antibodies (66.7%) and 6 patients also met criteria for RA [20]. However, the 8 SLE patients with positive anti-ccp antibodies in our study either didn t receive further radiographic exams of peripheral joints or lost follow up, and thus, the rate of erosive arthritis was not known. As for primary SS, it has been reported that small number of patients tested positive for anti-ccp antibodies. In these patients, SS was closely associated with synovitis and therefore was a predictor of progression to RA or a more inflammatory process of synovial tissue [21]. The only SS patient with anti-ccp antibodies in our study didn t have erosive arthritis. Further follow-up and evaluation may be needed to determine the disease progression in such cases. In conclusion, anti-ccp antibodies have better diagnostic value than RF in diagnosing RA in Taiwanese patients. Whether we can use anti-ccp antibodies as a substitute for RF in 1987 ACR Criteria for RA Classification is currently under discussion [22]. Detail history taking and physical examination make the use of RF and anti-ccp antibodies more reliable tools. Anti- CCP antibodies and RF in combination further increases the diagnostic value for RA. Acknowledgements The authors thank the Biostatistics Task Force of Taichung Veterans General Hospital, Taichung, Taiwan, ROC, for statistical assistance. References 1. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31: Nienhuis RLF, Mandema E. A new serum factor in patients with rheumatoid arthritis, the antiperinuclear factor. Ann Rheum Dis 1964;23: Russell AS, Devani A, Maksymowych WP. The role of anti-cyclic citrullinated peptide antibodies in predicting progression of palindromic rheumatism to rheumatoid arthritis. J Rheumatol 2006;33: Vincent C, de Keyser F, Masson-Bessie`re C, Sebbag M, Veys EM, Serre G. Anti-perinuclear factor compared with the so called antikeratin antibodies and antibodies to human epidermis filaggrin, in the diagnosis of arthritides. Ann Rheum Dis 1999;58: van der Helm-van Mil AH. Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther 2005;7:R Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM, Breedveld FC, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 2000;43: Goldbach-Mansky R, Lee J, McCoy A, Hoxworth J, Yarboro C, Smolen JS, et al. Rheumatoid arthritis associated autoantibodies in patients with synovitis of recent onset. 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5 Anti-CCP antibody and RF in RA 10. Lee DM, Schur PH. Clinical utility of the anti-ccp assay in patients with rheumatic diseases. Ann Rheum Dis 2003;62: Zeng X, Ai M, Tian X, Gan X, Shi Y, Song Q, et al. Diagnostic value of anti-cyclic citrullinated Peptide antibody in patients with rheumatoid arthritis. J Rheumatol 2003;30: Kastbom A, Strandberg G, Lindroos A, Skogh T. Anti-CCP antibody test predicts the disease course during 3 years in early rheumatoid arthritis (the Swedish TIRA project). Ann Rheum Dis 2004;63: Weinblatt ME, Schur PH. Rheumatoid factor detection by nephelometry. Arthritis Rheum 1980;23: Saraux A, Berthelot JM, Chales G, Le Henaff C, Mary JY, Thorel V, et al. Value of lantibodiesoratory tests in early prediction of rheumatoid arthritis. Arthritis Rheum 2002;47: Bas S, Perneger TV, Kunzle E, Vischer TL. Comparative study of different enzyme immunoassays for measurement of IgM and IgA rheumatoid factors. Ann Rheum Dis 2002;61: Bizzaro N, Mazzanti G, Tonutti E, Villalta D, Tozzoli R. Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem 2001;47: Silveira IG, Burlingame RW, von Muhlen CA, Bender AL, Staub HL. Anti-CCP antibodies have more diagnostic impact than rheumatoid factor (RF) in a population tested for RF. Clin Rheumatol. 2007;26: Shmerling RH, Delbanco TL. The rheumatoid factor: an analysis of clinical utility. Am J Med 1991;91: Matsui T, Shimada K, Ozawa N, Hayakawa H, Hagiwara F, Nakayama H, et al. Diagnostic utility of anti-cyclic citrullinated peptide antibodies for very early rheumatoid arthritis. J Rheumatol 2006;33: Chan MT, Owen P, Dunphy J, Cox B, Carmichael C, Korendowych E, McHugh NJ. Associations of erosive arthritis with anti-cyclic citrullinated peptide antibodies and MHC Class II alleles in systemic lupus erythematosus. J Rheumatol. 2008;35: Fabiola Atzeni, Piercarlo Sarzi-Puttini, Nicola Lama, Eleonora Bonacci, Francesca Bobbio-Pallavicini, Carlomaurizio Montecucco, et al. Anti-cyclic citrullinated peptide antibodies in primary Sjögren syndrome may be associated with nonerosive synovitis. Arthritis Research & Therapy 2008;10:R Liao KP, Batra KL, Chibnik L, Schur PH, Costenbader KH. Anti-CCP revised criteria for the classification of rheumatoid arthritis. Ann Rheum Dis 2008, January 30 as doi: / ard

6 Lin et al 抗環瓜氨酸肽抗體及類風濕因子在類風濕關節炎的診斷價值 1 林泓科 1,2 陳信華 1,2,3 藍忠亮 1,2,3 陳得源 1,2 陳怡行 1,4 黃文男 1,3 謝祖怡 1,4 謝佳偉 1 台中榮民總醫院過敏免疫風濕科 2 國立陽明大學 3 國立中興大學 4 中山醫學大學 目的 : 研究抗環瓜氨酸肽抗體及類風濕因子在類風濕關節炎的診斷價值 方法與材料 : 在 145 個類風濕關節炎病人及 75 個非類風濕關節炎病人身上, 以酵素免疫分析法測定抗環瓜氨酸肽抗體數值, 另外以濁度測定法測量類風濕因子之高低 結果 : 在 145 個類風濕關節炎病人中,119 (82.1%) 個病人有抗環瓜氨酸肽抗體,116 (80%) 個有類風濕因子 抗環瓜氨酸肽抗體的敏感度 專一度 陽性預測率 陰性預測率各別是 82.1% 88.0% 93.0% 71.7%; 而類風濕因子則各別為 80% 62.7% 81.1% 61.0% 若抗環瓜氨酸肽抗體或類風濕因子兩者其一為陽性, 則敏感度有 88.3%; 若兩者皆為陽性, 則專一度高達 94.7% 在類風濕因子陽性 類風濕因子陰性 及非類風濕關節炎這三組中, 抗環瓜氨酸肽抗體的陽性率各為 93.1% 37.9% 12.0% 結論: 抗環瓜氨酸肽抗體因為有高度的敏感性及專一性, 因此是一個診斷類風濕關節炎很好的工具, 若抗環瓜氨酸肽抗體及類風濕因子合併使用, 可更進一步增加診斷類風濕關節炎的能力 關鍵詞 : 類風濕關節炎 抗環瓜氨酸肽抗體 類風濕因子 診斷價值 73