http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; 24(4): 580–584 © 2014 Japan College of Rheumatology DOI: 10.3109/14397595.2013.844386
ORIGINAL ARTICLE
Diagnostic utility of the Elecsys anti-CCP assay in patients with rheumatoid arthritis Huiming Ye1,2, Falin Chen3, Shuidi Yan2, Yan Zhang2, Zanxi Fang2, Xiaosong Su2, Weicheng Cai4, and Zhongying Zhang2 1Translational Medicine Research Center, School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, P. R. China, 2Department of Clinical Laboratory, Zhongshan Hospital Xiamen University, Xiamen, Fujian, P. R. China, 3Fujian Provincial Center for Clinical Laboratory,
Fuzhou, Fujian, P. R. China, and 4Department of Renal Diseases, Zhongshan Hospital Xiamen University, Xiamen, Fujian, P. R. China Abstract
Keywords
Objective. The automatic anti-cyclic citrullinated peptide (anti-CCP) antibodies assay offered great advantages over traditional methods in terms of improved precision, reliability, technical simplicity, short turnaround time and high-speed throughput. In this study, we evaluated the main technical performance and diagnostic accuracy of the first automatic anti-CCP assay approved in China. Methods. The study comprised 106 rheumatoid arthritis (RA) patients, 203 non-RA rheumatic disease controls and 46 healthy persons. Anti-CCP, rheumatoid factor (RF), α1-acid glycoprotein, C-reactive protein and erythrocyte sedimentation rate were measured and compared. The precision, reference intervals for Chinese population and cut-off value for RA diagnosis, as well as the suitable diluent for anti-CCP were assessed. The positive rate and score of anti-CCP were compared with RF and acute-phase reactants, according to the new RA criteria. Results. Within- and between-run imprecision, expressed as the coefficient of variation, were 0.47–1.36% and 1.15–2.63%, respectively. Upper 95% reference limit of anti-CCP in healthy Chinese was 8.8 U/mL. The area under curve of the receiver operating characteristic (ROC) for anti-CCP and RF were 0.882 (95% CI 0.833–0.930) and 0.844 (95% CI 0.792–0.897), respectively. Based on the cut-off value set by ROC, compared to RF, anti-CCP had higher sensitivity (96.8% vs. 78.3%) and specificity (90.9% vs. 70.7%). With 17 U/mL set as the optimal cut-off for anti-CCP, the total positivity of anti-CCP was comparable to that of RF (76.4% vs. 75.5%), but the high-positivity rate of anti-CCP was significantly higher (74.5% vs. 62.3%, p ⬍ 0.005). Conclusions. Our results confirm anti-CCP as a more sensitive and specific marker than RF for the diagnosis of RA. The diagnostic performance of the Elecsys anti-CCP assay makes it a useful adjunct to clinical practice in the Chinese population.
Anti-cyclic citrullinated peptide antibodies, Diagnostic marker, Electrochemiluminescence immunoassay, Rheumatoid arthritis, Rheumatoid factor
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint swelling, joint tenderness and destruction of synovial joints, leading to severe disability and premature mortality. Its prevalence in mainland China, ranging from 0.2% to 0.37%, is similar to that in most Asian and South American countries. Prevalence in urban and suburban Taiwan, at 0.93%, is closer to that in Caucasians [1]. RA is diagnosed on clinical examination, patient history and serology. The latest classification criteria rank serological testing and measurement of acute-phase markers as important in making a definite diagnosis [2]. In 1998, Schellekens et al. discovered that RA- specific anti-citrullinated peptide antibodies (ACPA) bind to
Huiming Ye and Falin Chen equally contributed to this study. Correspondence to: Zhongying Zhang, Department of Clinical Laboratory, Zhongshan Hospital Xiamen University, 209 Hubin South Road, Xiamen, Fujian Province 361004, P. R. China. Tel: ⫹(86)592-2993328 GMT ⫹ 8h. Fax: ⫹(86)592-2993043. E-mail: [email protected]
History Received 4 February 2013 Accepted 13 July 2013 Published online 31 October 2013
antigenic determinants that contain citrulline, a modified form of arginine produced by the action of peptidyl-arginine deaminase [2]. Anti-cyclic citrullinated peptide antibodies (anti-CCP) are directed against a circular peptide containing an unusual amino acid, citrulline, normally absent from peptides or proteins. AntiCCP appears early in RA: its presence in the blood indicates a 90–95% probability of RA, hence its utility in the diagnosis of unexplained joint inflammation, especially when the traditional rheumatoid factor (RF) test is negative. In the past few years, numerous studies have shown anti-CCP to be a highly sensitive and specific diagnostic marker for RA [3–5]. Additionally, among patients fulfilling the 1987 American College of Rheumatology (ACR) criteria, those who are anti-CCP positive and those who are anti-CCP negative have been shown to differ in pathogenesis, clinical presentation and prognosis [6]. Anti-CCP assays have developed from first generation with low sensitivity (48–68%) to increasingly sensitive second and third generations [7, 8]. Automated immunoassay analyzers provide better performance characteristics [9–11]. The Elecsys anti-CCP assay (Roche Diagnostics GmbH, Mannheim, Germany), the firstautomated anti-CCP assay approved by China’s State Food and
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Drug Administration (SFDA), showed advantages in improvement of turnaround time and increased laboratory efficiency, in a recent study [12]. In the present study, we evaluated the main performance characteristics of this assay, and aimed to investigate its diagnostic accuracy and compare it to RF, according to the newest RA diagnostic criteria [2].
Materials and methods Subjects Study participants were 106 consecutive patients with RA and 203 disease controls, including patients with rheumatic heart disease (RHD) and rheumatic diseases other than RA, presenting between December 2010 and April 2011 in ZhongShan Hospital, XiaMen University. RA patients were diagnosed according to 1987 ACR criteria [13]. They included 79 females, and the mean age was 48 years (range 16–82 years). Eighty-five patients were diagnosed within 2 years. They were mainly treated on methotrexate, leflunomide or sulfasalazine. The disease controls included 55 patients with osteoarthritis (OA) (F:M 43:12; mean age 49 years, range 15–81 years), 29 with ankylosing spondylitis (AS) (F:M 8:21; mean age 33 years, range 18–62 years), 31 with gout (F:M 9:22; mean age 55 years, range 22–81 years), 29 with systemic lupus erythematosus (SLE) (F:M 26:3; mean age 37 years, range 22–75 years), 46 with RHD (F:M 29:17, mean age 49 years, range 32–68 years), and 13 female patients with Sjögren syndrome (SS) (mean age 49 years, range 20–73 years). Normal controls were 46 age- and sex-matched healthy individuals (F:M 35:11; mean age 47 years, range 16–72 years); 120 other healthy individuals (F:M 70:50; mean age 42 years, range 16–72 years) also acted as anti-CCP reference subjects. The study was approved by the local ethics committee. However, only reference subjects were asked for informed consent. They also completed a questionnaire to exclude states that might affect the detection of anti-CCP. Other participants were not asked to provide informed consent since anti-CCP was a routine hospital test. Sample determination All serum anti-CCP antibodies were simultaneously detected by Elecsys assay on an Elecsys 2010 analyzer using the Elecsys antiCCP reagent kit and PreciControl Anti-CCP (Roche Diagnostics). RF, α1-acid glycoprotein (AAG) and CRP were determined by laser nephelometry on a BN™ II analyzer (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) according to the manufacturer’s instructions. Erythrocyte sedimentation rate (ESR) was measured using Ves-matic 30 (Diesse Diagnostica Senese, Siena, Italy). We determined within-run and between-run imprecision according to the Clinical and Laboratory Standards Institute (CLSI) guideline EP15-A2 by assaying two samples containing low and high concentrations in triplicate once daily for 5 days. We also determined reference intervals for the Elecsys anti-CCP assay in Chinese patients according to CLSI guideline C28-A2. As antiCCP is a candidate marker for monitoring clinical response to RA treatment [14, 15], assay linearity is important. Negative serum (the recommended diluent from manufacturer), normal saline, deionized water, phosphate-buffered saline and Elecsys Diluent were selected as diluents for the assay, and their blank values were detected. A high anti-CCP serum sample was twofold diluted by the diluents. Then non-diluted sample and all the 1:2 diluted samples were detected in duplicate. The bias from sample dilution was calculated as Bias ⫽ (Xdet⫺Xanti)/Xanti (Xdet: mean value detected in diluted samples; Xanti: mean value anticipated for diluted samples based on the number of dilutions) and the acceptable bias was set as less than 12.5%. Then we diluted a high anti-CCP serum with
the suitable diluent at threefold, fourfold, fivefold and tenfold. The biases from these diluted samples were also calculated as before. Statistical analysis Sensitivities and specificities were calculated within the manufacturer’s cut-off and the optimal decision threshold that was obtained by receiver operating characteristic (ROC) curve analysis. Statistical analysis was performed using SPSS 13.0 for Windows. Twosided p values ⬍ 0.05 were considered significant throughout. The Standards for Reporting of Diagnostic Accuracy checklist was followed when preparing this report [16].
Results Precision and reference intervals Within- and between-run imprecision for the Elecsys anti-CCP assay in our laboratory, expressed as the coefficient of variation (CV), were 0.47% and 1.15% for 16 U/mL and 1.36% and 2.63% for 84 U/mL, respectively. We set the upper 95% reference limit for anti-CCP as 8.8 U/mL based on 120 Chinese healthy individuals according to the CLSI guideline C28-A2 (Figure 1). Suitable diluent and acceptable dilution In this study, bank values for anti-CCP of normal saline, phosphatebuffered saline, deionized water and Elecsys Diluent were from 7.7 to 15.4 U/mL. Despite compounding bovine serum protein and diluents at albumin concentrations from 10 to 50 g/L, we failed to exorcize the matrix effects. When defining bias from dilution under 12.5% as acceptable, negative serum was the only acceptable diluent in this study and 1:3 was the highest dilution (Table 1). Diagnostic performance of anti-CCP versus RF and acute-phase reactants In the RA group, total positivity was slightly more frequent with anti-CCP than with RF (76.4% vs. 75.5%), but high positivity was significantly higher with anti-CCP (74.5% vs. 62.3%, p ⬍ 0.005). In the 203 disease controls, anti-CCP was positive in only four SLE patients and two RHD patients whereas RF positivity ranged from 7.1% to 33.3%. Acute-phase reactants and ESR showed poor sensitivity and specificity in RA (Table 2 and Figure 2). Areas under the curve (AUC) for anti-CCP and RF in RA versus non-RA were 0.882 (95% CI 0.833–0.930) and 0.844 (95% CI 0.792–0.897), respectively (Figure 3). Detailed diagnostic
Figure 1. Anti-CCP histograms for 120 healthy individuals.
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Table 1. Elecsys anti-CCP assay diluents and dilutions.* Anti-CCP for non-diluted Anti-CCP in 1:2 diluted Bias from 1:2 serum (U/mL) serum (U/mL) Anti-CCP in diluted serum A. Diluents diluents (U/mL) Detection 1 Detection 2 Detection 1 Detection 2 (%) Normal saline 14.3 330.5 332.9 281.5 278.6 69.1 Deionized water 15.4 330.5 332.9 281.2 285.6 71.1 PBS 13.9 330.5 332.9 272.7 272.2 64.5 Elecsys Diluent 7.7 330.5 332.9 240.4 237 44.1 Negative serum ⬍ 7.0 330.5 332.9 157.2 162.4 3.5 Anti-CCP in undiluted serum Anti-CCP in diluted sample (U/mL) (U/mL) B. Dilutions in Bias from diluted negative serum Detection 1 Detection 2 Detection 1 Detection 2 serum (%) 1:3 349.9 351.9 105.7 105.1 9.9 1:4 349.9 351.9 75.5 74 14.8 1:5 349.9 351.9 48.7 49.3 30.1 1:10 349.9 351.9 19.9 21.1 41.6 Anti-CCP, anti-cyclic citrullinated peptide antibodies; PBS, phosphate-buffered saline. *Section A for the evaluation of suitable diluent and section B for the acceptable dilution fold. The bias from sample dilution was calculated as Bias ⫽ (Xdet–Xanti)/Xanti (Xdet: mean value detected in diluted samples; Xanti: mean value anticipated for diluted samples based on the number of dilutions), and the acceptable bias was less than 12.5%. Table 2. RA-specific autoantibody and acute-phase reactant positivity rates in RA patients and disease controls. Patients Disease (n) RA 106 OA 55 AS 29 Gout 31 SLE 29 RHD 46 SS 13 Normal 46
Sex (Female: Male) 79:27 43:12 8:21 9:22 26:3 29:17 13:0 35:11
Age (years, mean, median and range) 48, 48 (16–82) 49, 48 (15–81) 33, 29 (18–62) 55, 59 (22–81) 37, 32 (22–75) 49, 48 (32–68) 49, 51 (20–73) 47, 46 (16–72)
Anti-CCP (ULN 8.8 U/mL) Low positive High positive (%) (%) 1.9 (2/106) 74.5 (79/106) 0 0 0 0 0 0 0 13.8 (4/29) 4.3 (2/46) 0 0 0 2.2 (1/46) 0
RF (ULN 15 IU/mL) Low positive High positive (%) (%) 13.2 (14/106) 62.3 (66/106) 7.3 (4/55) 7.3 (4/55) 7.1 (2/28) 0 3.2 (1/31) 6.4 (2/31) 10.7 (3/28) 10.7 (3/28) 19.6 (9/46) 4.3 (2/46) 33.3 (4/12) 0 6.5 (3/46) 0
AAG Positive (%) 34.4 (30/87) 9.4 (5/53) 33.3 (9/27) 45.2 (14/31) 36.0 (9/25) 10.9 (5/46) 25.0 (3/12) 6.5 (3/46)
CRP Positive (%) 47.4 (45/95) 18.8 (10/53) 44.8 (13/29) 61.3 (19/31) 38.5 (10/26) 23.9 (11/46) 25.0 (3/12) 6.5 (3/46)
ESR Positive (%) 65.3 (49/75) 35.1 (13/37) 41.7 (10/24) 68.2 (15/22) 65.0 (13/20) 52.2 (12/23) 77.8 (7/9) 6.5 (3/46)
AAG, α1-acid glycoprotein; AS, ankylosing spondylitis; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; OA, osteoarthritis; RA, rheumatoid arthritis; RHD, rheumatic heart disease; SLE, systemic lupus erythematosus; SS, Sjögren syndrome. Low positive: higher than upper limit of normal (ULN) but ⱕ 3 ⫻ ULN; high positive: ⬎ 3 ⫻ ULN. Cut-offs for AAG, CRP and ESR: 1200 mg/L, 5 mg/mL and 15 mm/h (male) and 20 mm/h (females).
parameters of anti-CCP and RF are shown in Table 3A/B. At manufacturer cut-offs diagnostic sensitivity was 75.5% in both cases, and specificity 98.4% and 82.8%, respectively. Based on ROC curve analysis, the optimal cut-offs for anti-CCP and RF were set at 7.2 and 25 IU/mL. With these modified cut-offs, sensitivity and specificity were 78.3% and 96.8% for anti-CCP, and 70.7% and 90.9% for RF. Table 3C shows the anti-CCP and RF positivity rates in RA based on our laboratory’s reference interval.
Discussion ACPA has emerged as one of the most important biomarkers in RA, and the CCP test has greatly facilitated its detection [17]. Since the anti-CCP enzyme-linked immunosorbent assay (ELISA) was Figure 2. Scatter plots of anti-CCP and RF level according to disease category.
introduced in 2000, it has been increasingly used as a diagnostic test for RA, and there have been many studies of its performance in the last decade. Increasing data show that the latest generation anti-CCP test is a good diagnostic marker for RA, especially at the early stage [18]. Therefore, new ACR/EULAR (the european league against rheumatism) were proposed in 2010 to include antiCCP testing [2]. The clinical applications of anti-CCP detection, associated with the excellent diagnostic accuracy of the test, have led many manufacturers to produce their own methods, including novel anti-CCP assays using automated platforms that offer great advantages over traditional ELISA in terms of improved precision, reliability, technical simplicity, short turnaround time and highspeed throughput [8]. In China, the SFDA has approved more than ten anti-CCP assays. The Elecsys anti-CCP was the first automated
anti-CCP, anti-cyclic citrullinated peptide antibody; AUC, area under the curve; HP, high positivity; LP, low positivity; Neg, negative (values ⱕ ULN); NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive predictive value; RF, rheumatoid factor; Sens, sensitivity; Spec, specificity; TP, total positivity.
2.8% (3/106) 3.8% (4/106) 5.7% (6/106) 13.2% 62.3% 11.3% (14/106) (66/106) (12/106) C. Comparison of positivity based on local laboratory reference interval 76.4% (81/106) 1.9% 74.5% 75.5% (2/106) (79/106) (80/106)
Anti-CCP Neg and RF HP RF HP RF LP
RF LP and anti-CCP RF Neg and anti-CCP Anti-CCP LP and RF HP or anti-CCP Neg and RF LP HP or RF Neg and HP anti-CCP LP
51.8 18.9 80.8 0.33 87.8 75.0 90.9
7.1
2.8 87.6 0.22 91.2 91.2 96.8
24.5
High positive (%) Low positive (%) Accuracy (%) PPV (%) NPV (%) Spec %
AUC (95% CI) Cut-off Sens (%) B. Diagnostic efficiency based on receiver operating characteristic curve analysis Anti-CCP 7.2 U/mL 78.3 0.882 (0.833–0.930) RF 25 IU/mL 70.7 0.844 (0.792–0.897) Anti-CCP TP (cut-off: Anti-CCP LP Anti-CCP HP RF TP (cut-off: 15 8.8 U/mL) IU/mL)
90.4 88.8
47.0 4.37 95.2 65.0 98.4 82.8
PLR
NLR
68.9 62.3 6.6 13.2 87.0 79.2 0.25 0.30
High positive (%) Low positive (%) Accuracy (%) PPV (%) NPV (%) PLR Spec (%)
Cut-off Sens (%) A. Diagnostic efficiency based on manufacturer’s cut-off Anti-CCP 17 U/mL 75.5 RF 15 IU/mL 75.5
Table 3. Comparing the diagnostic accuracy of anti-CCP and RF in RA patients.
quantity assay approved in China. In a recent comparative study in Chinese population, the consistent rate and Kappa coefficients among the Elecsys anti-CCP and two ELISA anti-CCP assays were both more than 0.97, and under the optimal cut-off values their diagnostic accurate rates for RA diagnosis were 78.47%, 77.08% and 76.39%, respectively. This study indicated that the assay’s diagnostic performance was satisfactory and comparable to that of classical ELISA assay in Chinese population [19]. In our study, we only evaluated this assay’s precision, reference intervals and suitable diluent that were important in clinical utility. And we aimed to investigate the automatic Elecsys anti-CCP assay’s diagnostic accuracy and compare it to the conventional RA’s autoantibody, rheumatoid factor, according to the 2010 ACR/EULAR RA classification criteria. Within- and between-run CVs in our laboratory were 0.47–1.36% and 1.15–2.63%, thus outperforming the ELISA anti-CCP assay used in previous studies [7,20,21]. Determination of reference values is helpful when implementing the anti-CCP test in terms of the 2010 diagnostic criteria [2]. We identified the 95% upper limit of normal (ULN) for the Elecsys anti-CCP assay in healthy Chinese (n ⫽ 120) as 8.8 U/ml according to CLSI C28-A2. Negative serum was the recommended diluent, but with biohazard. We attempted to identify a suitable diluent for the Elecsys anti-CCP assay. But normal saline, deionized water, phosphate-buffered saline, Elecsys Diluent, and their bovine serum protein compounding solution all had matrix effects. In this study we validated negative serum as the only suitable diluent for the Elecsys anti-CCP assay and the highest acceptable dilution being threefold. ROC curve analysis indicated an optimal cut-off of anti-CCP for RA as 7.2 U/mL, sensitivity and specificity at this value were 78.3% and 96.8%, respectively. The upper 98% reference limit for anti-CCP based on the 120 Chinese healthy individuals was 16.7 U/mL equal to the manufacturer’s cut-off of 17 U/mL, and under this value diagnostic sensitivity was 75.5% and specificity 98.4%, respectively. As diagnostic performance under these two cut-offs was similar, and the lower limit of detection of the Elecsys antiCCP assay is 7.0 U/mL, a cut-off near this value may have poor precision and matrix effects. We recommend both the manufacturer’s cut-off and the 98% upper reference limit in healthy individuals of 17 U/mL as the optimal cut-off for anti-CCP in Chinese RA patients. Diagnostic performance in our study is similar to that in a recent North American population [12]. In addition, assay results are available within 30 min, which is 2 h faster than ELISA, representing a substantial saving in time as well as improved precision. At the same specificity, the sensitivity of Elecsys anti-CCP was higher than that of ELISA anti-CCP in earlier studies (68%, range
NLR
Figure 3. Receiver operating characteristics curves of anti-CCP and RF in RA patients compared with non-RA patients.
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39%–93%) [22, 23]. All our RA patients were diagnosed using 1987 ACR criteria [13], that is without reference to anti-CCP status. Our study therefore differed from the recent Korean study that included anti-CCP status as a diagnostic criterion [8, 24] of its RA population. Although this made sensitivity lower in our study, it better reflects current practice in China. Our results provide further confirmation that anti-CCP is more sensitive and more specific than RF in diagnosing RA. According to the newest RA diagnostic criteria [2], a high-positive anti-CCP or RF scores 3, a low-positive scores 2, and a negative scores 0. In our study 5.6% of the RA population showed RF-negative but anti-CCP positivity, and over 10% of the RA population showed low-positive RF but high-positive anti-CCP, meaning that about 16% of the RA population achieved one or three more scores for a diagnosis of RA with anti-CCP than with RF, while only 6.6% scored more with RF than with anti-CCP. Such differences have practical importance for clinical decision-making in early disease. The Elecsys anti-CCP assay is also effective in differentiating RA from other rheumatic diseases in cases where RF fails as a discriminating tool. We observed low anti-CCP positivity in other rheumatic diseases, including SLE, gout, SS, AS, OA and RHD. Only six of 203 patients with other rheumatic diseases, including four patients with SLE and two with RHD, were positive for antiCCP. A recent study identified CRP and AAG as the most useful acute-phase proteins for assessing disease activity in RA [25]. Positivity rates for AAG, CRP and ESR in RA patients with antiCCP and RF bi-negative were 25% (5/20), 20% (4/20) and 20% (4/20), respectively. Acute-phase reactant testing is therefore recommended for classifying RA. Our results further confirm anti-CCP as a more sensitive and specific marker than RF for the diagnosis of RA. The diagnostic performance of the Elecsys anti-CCP assay makes it a useful adjunct to clinical practice in the Chinese population.
Mod Rheumatol, 2014; 24(4): 580–584
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Acknowledgements
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The authors thank professor Chi-Meng Tzeng in Translational Medicine Research Center, School of Pharmaceutical Sciences, Xiamen University for providing innovative suggestion and for help with the manuscript.
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Funding
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This study was supported partly by National Natural Science Foundation of China (Grant No. 8110331). 19.
Conflict of interest None. 20.
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ORIGINAL ARTICLE
Diagnostic utility of the Elecsys anti-CCP assay in patients with rheumatoid arthritis Huiming Ye1,2, Falin Chen3, Shuidi Yan2, Yan Zhang2, Zanxi Fang2, Xiaosong Su2, Weicheng Cai4, and Zhongying Zhang2 1Translational Medicine Research Center, School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, P. R. China, 2Department of Clinical Laboratory, Zhongshan Hospital Xiamen University, Xiamen, Fujian, P. R. China, 3Fujian Provincial Center for Clinical Laboratory,
Fuzhou, Fujian, P. R. China, and 4Department of Renal Diseases, Zhongshan Hospital Xiamen University, Xiamen, Fujian, P. R. China Abstract
Keywords
Objective. The automatic anti-cyclic citrullinated peptide (anti-CCP) antibodies assay offered great advantages over traditional methods in terms of improved precision, reliability, technical simplicity, short turnaround time and high-speed throughput. In this study, we evaluated the main technical performance and diagnostic accuracy of the first automatic anti-CCP assay approved in China. Methods. The study comprised 106 rheumatoid arthritis (RA) patients, 203 non-RA rheumatic disease controls and 46 healthy persons. Anti-CCP, rheumatoid factor (RF), α1-acid glycoprotein, C-reactive protein and erythrocyte sedimentation rate were measured and compared. The precision, reference intervals for Chinese population and cut-off value for RA diagnosis, as well as the suitable diluent for anti-CCP were assessed. The positive rate and score of anti-CCP were compared with RF and acute-phase reactants, according to the new RA criteria. Results. Within- and between-run imprecision, expressed as the coefficient of variation, were 0.47–1.36% and 1.15–2.63%, respectively. Upper 95% reference limit of anti-CCP in healthy Chinese was 8.8 U/mL. The area under curve of the receiver operating characteristic (ROC) for anti-CCP and RF were 0.882 (95% CI 0.833–0.930) and 0.844 (95% CI 0.792–0.897), respectively. Based on the cut-off value set by ROC, compared to RF, anti-CCP had higher sensitivity (96.8% vs. 78.3%) and specificity (90.9% vs. 70.7%). With 17 U/mL set as the optimal cut-off for anti-CCP, the total positivity of anti-CCP was comparable to that of RF (76.4% vs. 75.5%), but the high-positivity rate of anti-CCP was significantly higher (74.5% vs. 62.3%, p ⬍ 0.005). Conclusions. Our results confirm anti-CCP as a more sensitive and specific marker than RF for the diagnosis of RA. The diagnostic performance of the Elecsys anti-CCP assay makes it a useful adjunct to clinical practice in the Chinese population.
Anti-cyclic citrullinated peptide antibodies, Diagnostic marker, Electrochemiluminescence immunoassay, Rheumatoid arthritis, Rheumatoid factor
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by joint swelling, joint tenderness and destruction of synovial joints, leading to severe disability and premature mortality. Its prevalence in mainland China, ranging from 0.2% to 0.37%, is similar to that in most Asian and South American countries. Prevalence in urban and suburban Taiwan, at 0.93%, is closer to that in Caucasians [1]. RA is diagnosed on clinical examination, patient history and serology. The latest classification criteria rank serological testing and measurement of acute-phase markers as important in making a definite diagnosis [2]. In 1998, Schellekens et al. discovered that RA- specific anti-citrullinated peptide antibodies (ACPA) bind to
Huiming Ye and Falin Chen equally contributed to this study. Correspondence to: Zhongying Zhang, Department of Clinical Laboratory, Zhongshan Hospital Xiamen University, 209 Hubin South Road, Xiamen, Fujian Province 361004, P. R. China. Tel: ⫹(86)592-2993328 GMT ⫹ 8h. Fax: ⫹(86)592-2993043. E-mail: [email protected]
History Received 4 February 2013 Accepted 13 July 2013 Published online 31 October 2013
antigenic determinants that contain citrulline, a modified form of arginine produced by the action of peptidyl-arginine deaminase [2]. Anti-cyclic citrullinated peptide antibodies (anti-CCP) are directed against a circular peptide containing an unusual amino acid, citrulline, normally absent from peptides or proteins. AntiCCP appears early in RA: its presence in the blood indicates a 90–95% probability of RA, hence its utility in the diagnosis of unexplained joint inflammation, especially when the traditional rheumatoid factor (RF) test is negative. In the past few years, numerous studies have shown anti-CCP to be a highly sensitive and specific diagnostic marker for RA [3–5]. Additionally, among patients fulfilling the 1987 American College of Rheumatology (ACR) criteria, those who are anti-CCP positive and those who are anti-CCP negative have been shown to differ in pathogenesis, clinical presentation and prognosis [6]. Anti-CCP assays have developed from first generation with low sensitivity (48–68%) to increasingly sensitive second and third generations [7, 8]. Automated immunoassay analyzers provide better performance characteristics [9–11]. The Elecsys anti-CCP assay (Roche Diagnostics GmbH, Mannheim, Germany), the firstautomated anti-CCP assay approved by China’s State Food and
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Drug Administration (SFDA), showed advantages in improvement of turnaround time and increased laboratory efficiency, in a recent study [12]. In the present study, we evaluated the main performance characteristics of this assay, and aimed to investigate its diagnostic accuracy and compare it to RF, according to the newest RA diagnostic criteria [2].
Materials and methods Subjects Study participants were 106 consecutive patients with RA and 203 disease controls, including patients with rheumatic heart disease (RHD) and rheumatic diseases other than RA, presenting between December 2010 and April 2011 in ZhongShan Hospital, XiaMen University. RA patients were diagnosed according to 1987 ACR criteria [13]. They included 79 females, and the mean age was 48 years (range 16–82 years). Eighty-five patients were diagnosed within 2 years. They were mainly treated on methotrexate, leflunomide or sulfasalazine. The disease controls included 55 patients with osteoarthritis (OA) (F:M 43:12; mean age 49 years, range 15–81 years), 29 with ankylosing spondylitis (AS) (F:M 8:21; mean age 33 years, range 18–62 years), 31 with gout (F:M 9:22; mean age 55 years, range 22–81 years), 29 with systemic lupus erythematosus (SLE) (F:M 26:3; mean age 37 years, range 22–75 years), 46 with RHD (F:M 29:17, mean age 49 years, range 32–68 years), and 13 female patients with Sjögren syndrome (SS) (mean age 49 years, range 20–73 years). Normal controls were 46 age- and sex-matched healthy individuals (F:M 35:11; mean age 47 years, range 16–72 years); 120 other healthy individuals (F:M 70:50; mean age 42 years, range 16–72 years) also acted as anti-CCP reference subjects. The study was approved by the local ethics committee. However, only reference subjects were asked for informed consent. They also completed a questionnaire to exclude states that might affect the detection of anti-CCP. Other participants were not asked to provide informed consent since anti-CCP was a routine hospital test. Sample determination All serum anti-CCP antibodies were simultaneously detected by Elecsys assay on an Elecsys 2010 analyzer using the Elecsys antiCCP reagent kit and PreciControl Anti-CCP (Roche Diagnostics). RF, α1-acid glycoprotein (AAG) and CRP were determined by laser nephelometry on a BN™ II analyzer (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) according to the manufacturer’s instructions. Erythrocyte sedimentation rate (ESR) was measured using Ves-matic 30 (Diesse Diagnostica Senese, Siena, Italy). We determined within-run and between-run imprecision according to the Clinical and Laboratory Standards Institute (CLSI) guideline EP15-A2 by assaying two samples containing low and high concentrations in triplicate once daily for 5 days. We also determined reference intervals for the Elecsys anti-CCP assay in Chinese patients according to CLSI guideline C28-A2. As antiCCP is a candidate marker for monitoring clinical response to RA treatment [14, 15], assay linearity is important. Negative serum (the recommended diluent from manufacturer), normal saline, deionized water, phosphate-buffered saline and Elecsys Diluent were selected as diluents for the assay, and their blank values were detected. A high anti-CCP serum sample was twofold diluted by the diluents. Then non-diluted sample and all the 1:2 diluted samples were detected in duplicate. The bias from sample dilution was calculated as Bias ⫽ (Xdet⫺Xanti)/Xanti (Xdet: mean value detected in diluted samples; Xanti: mean value anticipated for diluted samples based on the number of dilutions) and the acceptable bias was set as less than 12.5%. Then we diluted a high anti-CCP serum with
the suitable diluent at threefold, fourfold, fivefold and tenfold. The biases from these diluted samples were also calculated as before. Statistical analysis Sensitivities and specificities were calculated within the manufacturer’s cut-off and the optimal decision threshold that was obtained by receiver operating characteristic (ROC) curve analysis. Statistical analysis was performed using SPSS 13.0 for Windows. Twosided p values ⬍ 0.05 were considered significant throughout. The Standards for Reporting of Diagnostic Accuracy checklist was followed when preparing this report [16].
Results Precision and reference intervals Within- and between-run imprecision for the Elecsys anti-CCP assay in our laboratory, expressed as the coefficient of variation (CV), were 0.47% and 1.15% for 16 U/mL and 1.36% and 2.63% for 84 U/mL, respectively. We set the upper 95% reference limit for anti-CCP as 8.8 U/mL based on 120 Chinese healthy individuals according to the CLSI guideline C28-A2 (Figure 1). Suitable diluent and acceptable dilution In this study, bank values for anti-CCP of normal saline, phosphatebuffered saline, deionized water and Elecsys Diluent were from 7.7 to 15.4 U/mL. Despite compounding bovine serum protein and diluents at albumin concentrations from 10 to 50 g/L, we failed to exorcize the matrix effects. When defining bias from dilution under 12.5% as acceptable, negative serum was the only acceptable diluent in this study and 1:3 was the highest dilution (Table 1). Diagnostic performance of anti-CCP versus RF and acute-phase reactants In the RA group, total positivity was slightly more frequent with anti-CCP than with RF (76.4% vs. 75.5%), but high positivity was significantly higher with anti-CCP (74.5% vs. 62.3%, p ⬍ 0.005). In the 203 disease controls, anti-CCP was positive in only four SLE patients and two RHD patients whereas RF positivity ranged from 7.1% to 33.3%. Acute-phase reactants and ESR showed poor sensitivity and specificity in RA (Table 2 and Figure 2). Areas under the curve (AUC) for anti-CCP and RF in RA versus non-RA were 0.882 (95% CI 0.833–0.930) and 0.844 (95% CI 0.792–0.897), respectively (Figure 3). Detailed diagnostic
Figure 1. Anti-CCP histograms for 120 healthy individuals.
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Table 1. Elecsys anti-CCP assay diluents and dilutions.* Anti-CCP for non-diluted Anti-CCP in 1:2 diluted Bias from 1:2 serum (U/mL) serum (U/mL) Anti-CCP in diluted serum A. Diluents diluents (U/mL) Detection 1 Detection 2 Detection 1 Detection 2 (%) Normal saline 14.3 330.5 332.9 281.5 278.6 69.1 Deionized water 15.4 330.5 332.9 281.2 285.6 71.1 PBS 13.9 330.5 332.9 272.7 272.2 64.5 Elecsys Diluent 7.7 330.5 332.9 240.4 237 44.1 Negative serum ⬍ 7.0 330.5 332.9 157.2 162.4 3.5 Anti-CCP in undiluted serum Anti-CCP in diluted sample (U/mL) (U/mL) B. Dilutions in Bias from diluted negative serum Detection 1 Detection 2 Detection 1 Detection 2 serum (%) 1:3 349.9 351.9 105.7 105.1 9.9 1:4 349.9 351.9 75.5 74 14.8 1:5 349.9 351.9 48.7 49.3 30.1 1:10 349.9 351.9 19.9 21.1 41.6 Anti-CCP, anti-cyclic citrullinated peptide antibodies; PBS, phosphate-buffered saline. *Section A for the evaluation of suitable diluent and section B for the acceptable dilution fold. The bias from sample dilution was calculated as Bias ⫽ (Xdet–Xanti)/Xanti (Xdet: mean value detected in diluted samples; Xanti: mean value anticipated for diluted samples based on the number of dilutions), and the acceptable bias was less than 12.5%. Table 2. RA-specific autoantibody and acute-phase reactant positivity rates in RA patients and disease controls. Patients Disease (n) RA 106 OA 55 AS 29 Gout 31 SLE 29 RHD 46 SS 13 Normal 46
Sex (Female: Male) 79:27 43:12 8:21 9:22 26:3 29:17 13:0 35:11
Age (years, mean, median and range) 48, 48 (16–82) 49, 48 (15–81) 33, 29 (18–62) 55, 59 (22–81) 37, 32 (22–75) 49, 48 (32–68) 49, 51 (20–73) 47, 46 (16–72)
Anti-CCP (ULN 8.8 U/mL) Low positive High positive (%) (%) 1.9 (2/106) 74.5 (79/106) 0 0 0 0 0 0 0 13.8 (4/29) 4.3 (2/46) 0 0 0 2.2 (1/46) 0
RF (ULN 15 IU/mL) Low positive High positive (%) (%) 13.2 (14/106) 62.3 (66/106) 7.3 (4/55) 7.3 (4/55) 7.1 (2/28) 0 3.2 (1/31) 6.4 (2/31) 10.7 (3/28) 10.7 (3/28) 19.6 (9/46) 4.3 (2/46) 33.3 (4/12) 0 6.5 (3/46) 0
AAG Positive (%) 34.4 (30/87) 9.4 (5/53) 33.3 (9/27) 45.2 (14/31) 36.0 (9/25) 10.9 (5/46) 25.0 (3/12) 6.5 (3/46)
CRP Positive (%) 47.4 (45/95) 18.8 (10/53) 44.8 (13/29) 61.3 (19/31) 38.5 (10/26) 23.9 (11/46) 25.0 (3/12) 6.5 (3/46)
ESR Positive (%) 65.3 (49/75) 35.1 (13/37) 41.7 (10/24) 68.2 (15/22) 65.0 (13/20) 52.2 (12/23) 77.8 (7/9) 6.5 (3/46)
AAG, α1-acid glycoprotein; AS, ankylosing spondylitis; CRP, C reactive protein; ESR, erythrocyte sedimentation rate; OA, osteoarthritis; RA, rheumatoid arthritis; RHD, rheumatic heart disease; SLE, systemic lupus erythematosus; SS, Sjögren syndrome. Low positive: higher than upper limit of normal (ULN) but ⱕ 3 ⫻ ULN; high positive: ⬎ 3 ⫻ ULN. Cut-offs for AAG, CRP and ESR: 1200 mg/L, 5 mg/mL and 15 mm/h (male) and 20 mm/h (females).
parameters of anti-CCP and RF are shown in Table 3A/B. At manufacturer cut-offs diagnostic sensitivity was 75.5% in both cases, and specificity 98.4% and 82.8%, respectively. Based on ROC curve analysis, the optimal cut-offs for anti-CCP and RF were set at 7.2 and 25 IU/mL. With these modified cut-offs, sensitivity and specificity were 78.3% and 96.8% for anti-CCP, and 70.7% and 90.9% for RF. Table 3C shows the anti-CCP and RF positivity rates in RA based on our laboratory’s reference interval.
Discussion ACPA has emerged as one of the most important biomarkers in RA, and the CCP test has greatly facilitated its detection [17]. Since the anti-CCP enzyme-linked immunosorbent assay (ELISA) was Figure 2. Scatter plots of anti-CCP and RF level according to disease category.
introduced in 2000, it has been increasingly used as a diagnostic test for RA, and there have been many studies of its performance in the last decade. Increasing data show that the latest generation anti-CCP test is a good diagnostic marker for RA, especially at the early stage [18]. Therefore, new ACR/EULAR (the european league against rheumatism) were proposed in 2010 to include antiCCP testing [2]. The clinical applications of anti-CCP detection, associated with the excellent diagnostic accuracy of the test, have led many manufacturers to produce their own methods, including novel anti-CCP assays using automated platforms that offer great advantages over traditional ELISA in terms of improved precision, reliability, technical simplicity, short turnaround time and highspeed throughput [8]. In China, the SFDA has approved more than ten anti-CCP assays. The Elecsys anti-CCP was the first automated
anti-CCP, anti-cyclic citrullinated peptide antibody; AUC, area under the curve; HP, high positivity; LP, low positivity; Neg, negative (values ⱕ ULN); NLR, negative likelihood ratio; NPV, negative predictive value; PLR, positive likelihood ratio; PPV, positive predictive value; RF, rheumatoid factor; Sens, sensitivity; Spec, specificity; TP, total positivity.
2.8% (3/106) 3.8% (4/106) 5.7% (6/106) 13.2% 62.3% 11.3% (14/106) (66/106) (12/106) C. Comparison of positivity based on local laboratory reference interval 76.4% (81/106) 1.9% 74.5% 75.5% (2/106) (79/106) (80/106)
Anti-CCP Neg and RF HP RF HP RF LP
RF LP and anti-CCP RF Neg and anti-CCP Anti-CCP LP and RF HP or anti-CCP Neg and RF LP HP or RF Neg and HP anti-CCP LP
51.8 18.9 80.8 0.33 87.8 75.0 90.9
7.1
2.8 87.6 0.22 91.2 91.2 96.8
24.5
High positive (%) Low positive (%) Accuracy (%) PPV (%) NPV (%) Spec %
AUC (95% CI) Cut-off Sens (%) B. Diagnostic efficiency based on receiver operating characteristic curve analysis Anti-CCP 7.2 U/mL 78.3 0.882 (0.833–0.930) RF 25 IU/mL 70.7 0.844 (0.792–0.897) Anti-CCP TP (cut-off: Anti-CCP LP Anti-CCP HP RF TP (cut-off: 15 8.8 U/mL) IU/mL)
90.4 88.8
47.0 4.37 95.2 65.0 98.4 82.8
PLR
NLR
68.9 62.3 6.6 13.2 87.0 79.2 0.25 0.30
High positive (%) Low positive (%) Accuracy (%) PPV (%) NPV (%) PLR Spec (%)
Cut-off Sens (%) A. Diagnostic efficiency based on manufacturer’s cut-off Anti-CCP 17 U/mL 75.5 RF 15 IU/mL 75.5
Table 3. Comparing the diagnostic accuracy of anti-CCP and RF in RA patients.
quantity assay approved in China. In a recent comparative study in Chinese population, the consistent rate and Kappa coefficients among the Elecsys anti-CCP and two ELISA anti-CCP assays were both more than 0.97, and under the optimal cut-off values their diagnostic accurate rates for RA diagnosis were 78.47%, 77.08% and 76.39%, respectively. This study indicated that the assay’s diagnostic performance was satisfactory and comparable to that of classical ELISA assay in Chinese population [19]. In our study, we only evaluated this assay’s precision, reference intervals and suitable diluent that were important in clinical utility. And we aimed to investigate the automatic Elecsys anti-CCP assay’s diagnostic accuracy and compare it to the conventional RA’s autoantibody, rheumatoid factor, according to the 2010 ACR/EULAR RA classification criteria. Within- and between-run CVs in our laboratory were 0.47–1.36% and 1.15–2.63%, thus outperforming the ELISA anti-CCP assay used in previous studies [7,20,21]. Determination of reference values is helpful when implementing the anti-CCP test in terms of the 2010 diagnostic criteria [2]. We identified the 95% upper limit of normal (ULN) for the Elecsys anti-CCP assay in healthy Chinese (n ⫽ 120) as 8.8 U/ml according to CLSI C28-A2. Negative serum was the recommended diluent, but with biohazard. We attempted to identify a suitable diluent for the Elecsys anti-CCP assay. But normal saline, deionized water, phosphate-buffered saline, Elecsys Diluent, and their bovine serum protein compounding solution all had matrix effects. In this study we validated negative serum as the only suitable diluent for the Elecsys anti-CCP assay and the highest acceptable dilution being threefold. ROC curve analysis indicated an optimal cut-off of anti-CCP for RA as 7.2 U/mL, sensitivity and specificity at this value were 78.3% and 96.8%, respectively. The upper 98% reference limit for anti-CCP based on the 120 Chinese healthy individuals was 16.7 U/mL equal to the manufacturer’s cut-off of 17 U/mL, and under this value diagnostic sensitivity was 75.5% and specificity 98.4%, respectively. As diagnostic performance under these two cut-offs was similar, and the lower limit of detection of the Elecsys antiCCP assay is 7.0 U/mL, a cut-off near this value may have poor precision and matrix effects. We recommend both the manufacturer’s cut-off and the 98% upper reference limit in healthy individuals of 17 U/mL as the optimal cut-off for anti-CCP in Chinese RA patients. Diagnostic performance in our study is similar to that in a recent North American population [12]. In addition, assay results are available within 30 min, which is 2 h faster than ELISA, representing a substantial saving in time as well as improved precision. At the same specificity, the sensitivity of Elecsys anti-CCP was higher than that of ELISA anti-CCP in earlier studies (68%, range
NLR
Figure 3. Receiver operating characteristics curves of anti-CCP and RF in RA patients compared with non-RA patients.
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39%–93%) [22, 23]. All our RA patients were diagnosed using 1987 ACR criteria [13], that is without reference to anti-CCP status. Our study therefore differed from the recent Korean study that included anti-CCP status as a diagnostic criterion [8, 24] of its RA population. Although this made sensitivity lower in our study, it better reflects current practice in China. Our results provide further confirmation that anti-CCP is more sensitive and more specific than RF in diagnosing RA. According to the newest RA diagnostic criteria [2], a high-positive anti-CCP or RF scores 3, a low-positive scores 2, and a negative scores 0. In our study 5.6% of the RA population showed RF-negative but anti-CCP positivity, and over 10% of the RA population showed low-positive RF but high-positive anti-CCP, meaning that about 16% of the RA population achieved one or three more scores for a diagnosis of RA with anti-CCP than with RF, while only 6.6% scored more with RF than with anti-CCP. Such differences have practical importance for clinical decision-making in early disease. The Elecsys anti-CCP assay is also effective in differentiating RA from other rheumatic diseases in cases where RF fails as a discriminating tool. We observed low anti-CCP positivity in other rheumatic diseases, including SLE, gout, SS, AS, OA and RHD. Only six of 203 patients with other rheumatic diseases, including four patients with SLE and two with RHD, were positive for antiCCP. A recent study identified CRP and AAG as the most useful acute-phase proteins for assessing disease activity in RA [25]. Positivity rates for AAG, CRP and ESR in RA patients with antiCCP and RF bi-negative were 25% (5/20), 20% (4/20) and 20% (4/20), respectively. Acute-phase reactant testing is therefore recommended for classifying RA. Our results further confirm anti-CCP as a more sensitive and specific marker than RF for the diagnosis of RA. The diagnostic performance of the Elecsys anti-CCP assay makes it a useful adjunct to clinical practice in the Chinese population.
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Acknowledgements
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The authors thank professor Chi-Meng Tzeng in Translational Medicine Research Center, School of Pharmaceutical Sciences, Xiamen University for providing innovative suggestion and for help with the manuscript.
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Funding
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This study was supported partly by National Natural Science Foundation of China (Grant No. 8110331). 19.
Conflict of interest None. 20.
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