Aims and Objectives: Rheumatoid arthritis is a systemic inflammatory disease whose diagnosis is primarily based on clinical manifestations because of lack of suitable diagnostic tests. As substantial joint damage already occurs by the time patient presents clinically, a validated biomarker for the diagnosis is urgently required. Materials and Methods: Sera from a total of 68 clinically suspected rheumatoid arthritis patients and 68 age-and sex-matched controls were tested for rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α). Results: Anti-CCP and CRP were found to be positive in all patients with positive RF; however, TNF-α was present in only two of them. As regards anti-CCP antibodies, out of the 10 samples that showed positive results, RF, CRP, and TNF-α were also present in 4, 5, and 4 cases, respectively. Conclusion: The recognition of utility of such markers is essential to gain insight into the activity of this disease, which is important for early treatment that may limit functional disability consequent to the disease.
Keywords: Anti-cyclic citrullinated peptide antibodies, C-reactive protein, rheumatoid arthritis, rheumatoid factor, tumor necrosis factor-alpha
How to cite this article:
Kashyap B, Tiwari U, Garg A, Kaur IR. Diagnostic utility of anti-CCP antibodies and rheumatoid factor as inflammatory biomarkers in comparison with C-reactive protein and TNF-α in rheumatoid arthritis. Trop J Med Res 2015;18:5-9
How to cite this URL:
Kashyap B, Tiwari U, Garg A, Kaur IR. Diagnostic utility of anti-CCP antibodies and rheumatoid factor as inflammatory biomarkers in comparison with C-reactive protein and TNF-α in rheumatoid arthritis. Trop J Med Res [serial online] 2015 [cited 2016 Mar 15];18:5-9. Available from: http://www.tjmrjournal.org/text.asp?2015/18/1/5/152534
Rheumatoid arthritis(RA) is a severe, progressive, comorbid systemic inflammatory disease of unknown etiology. Since timely intervention with new and effective treatments can alter the course of the disease, reduce functional impairment, and lengthen life, better biomarkers for diagnosis and prognosis are needed to identify these patients at an early stage in order to fine-tune therapeutic options to the individual patient. 
Rheumatoid factor(RF), an antibody specific for the Fc portion of human IgG, has been historically considered a marker for RA and was one of the diagnostic criteria for RA that was established by the American College of Rheumatology (ACR).  The sensitivity and specificity of RF for the diagnosis of RA has been reported in the range of 50-80% and 70-80%, respectively. , The specificity of the RF test is known to be relatively poor and is often questioned. With around 5% false positivity in the general population, RF is found in many patients with other diseases of infectious or autoimmune origin. Consequently, a search for better diagnostic markers, especially those with improved specificity for RA, ensued. Though due to a low sensitivity and moderate specificity RF has a little diagnostic utility, it has retained its place in practice because of its prognostic capacity and lack of an alternative test.
Citrullination (deimination) of proteins is a chemical reaction which occurs when inflammatory cells release enzymes in local tissues. Citrullination of synovial antigens, especially fibrin, during synovial inflammation probably allows the induction of anti-cyclic citrullinated peptide (anti-CCP) antibody in RA patients through an antigen-conducted activation of B cells. Capacity to form antibodies to citrullinated peptides and not citrullination of peptides per se seems to be unique to RA; the exact significance of antibodies to these peptides, however, remains uncertain. Greater sensitivity and specificity than IgM RF and probable predictability of erosive disease in RA or the eventual development of undifferentiated arthritis into RA makes anti-CCP antibodies potentially important surrogate markers for the diagnosis and prognosis in RA.  The progressive evolution of assays for anti-citrullinated peptide antibody (ACPA) detection has led to a high level of diagnostic accuracy with a specificity of 95-97% and a sensitivity of 67-80%. , The new 2010 RA Classification Criteria, updated to diagnose RA in an earlier phase, include detection of ACPA as a key item for diagnosing the disease.  Anti-CCP guided aggressive treatment at an early stage or correlation of anti-CCP levels with various therapeutic interventions are, however, important areas for research.
Another potential marker for increased risk of RA and disease activity may be C-reactive protein (CRP), since CRP is a sensitive marker of systemic inflammation and is elevated in patients with RA.  The acute-phase reactants like CRP are a class of serum proteins whose concentration in the blood increases after various stimuli such as trauma or inflammation. The magnitude of the acute-phase protein response is roughly proportional to the severity of the stimulus and, therefore, measurements of these proteins can be used to monitor the progress of an inflammatory disorder. Though CRP is a part of ACR core data set for measuring disease activity in RA, the quantitative usefulness has been evaluated in many studies with no clear consensus.
Tumor necrosis factor-alpha (TNF-α), yet another marker of disease activity, is one of the key cytokine molecules that causes inflammation in RA and plays a dominant role in rheumatoid synovitis.  TNF-α is now recognized as a mediator of a wide variety of effector functions which are recognized components of the RA disease spectrum, including endothelial cell activation and chemokine amplification leading to leukocyte accumulation; osteoclast and chondrocyte activation promoting articular destruction; nociceptor sensitization; impaired cognitive function and metabolic syndrome.  All these have led to the potential role of TNF-α inhibitors to induce a rapid and sustained attenuation of disease activity in patients with RA.
The best characterized predictors for rapid progression are the number of swollen joints and the levels of acute phase reactants, as swollen joints indicate synovitis and the acute phase response acts as a biomarker of pro-inflammatory cytokine production. The objective of the study was to compare the diagnostic utility of the two most widely used serological markers of RA, i.e, anti-CCP antibodies and RF, and correlate their potential as inflammatory biomarkers with important markers of disease activity like CRP and TNF-α.
Materials and Methods
This case-control study was conducted in the immunology section of the Department of Microbiology, University College of Medical Sciences and Guru Tegh Bahadur Hospital, Delhi. Sixty-eight patients with RA as per ACR criteria were enrolled in the study. At inclusion, the patients had symptom duration of at least 6 weeks, but less than 6 months, and were not receiving any glucocorticoid or immunosuppressant drug. Each enrolled patient gave written consent prior to being included in the study. Healthy hospital personnel (n = 68) without any history of inflammatory diseases served as controls. Serum samples were obtained from both patients and controls, and were aliquoted and stored at 80°C until assayed.
Detection of RF, anti-CCP antibodies, CRP, and TNF-α was done with the help of commercially available kits following the manufacturers’ instructions. RF was detected by RHELAX-RF (Tulip Diagnostic, Goa, India) which is a latex agglutination slide test for the detection of RFs of the IgM class with a sensitivity of 10 IU/ml. Anti-CCP antibodies were analyzed with a commercial enzyme-linked immunosorbent assay (ELISA) IMTEC-CCP-Antibodies (IMTEC Human, Weisbaden, Germany) which is a test system for measuring IgG class autoantibodies against cyclic citrullinated peptides in human serum or plasma. The interpretation of the results was possible by correlating the absorbance of the reference control and the samples. CRP was assayed using RHELAX-CRP (Tulip Diagnostic, India) which is a slide test for detection of CRP based on the principle of latex agglutination with a sensitivity of 0.6 mg/dl. TNF-α was assayed by a commercial solid-phase sandwich ELISA (Diaclone, Besancon Cedex, France) and the sensitivity minimum detectable dose was found to be 8 pg/ml.
In our study, 50% of patients were between 21 and 40 years of age and 20% were between 41 and 50 years of age. The extremes of age, i.e, less than 20 years and more than 50 years, constituted 30% of the enrolled patients. Fifty-one out of 68 patients, i.e, 75% of patients, enrolled in the study were females. Male:Female ratio in our study was 1:3 [Figure 1]. The frequency distribution of clinical wards from where the patients were admitted is depicted in [Figure 2]. Out of a total of 68 patients, 29 patients were from orthopedics ward and 23 from medicine ward. Four patients were from neurology, three from dermatology, two from surgery, and one patient was from pediatrics ward.
Figure 1: Age and sex distribution of the patients enrolled in the study (N = 68)
Figure 2: Ward-wise distribution of cases (N = 68)
Out of 68 patients who were clinically suspected cases of RA, only 4 patients had positive RF while 10 patients had positive anti-CCP. CRP was positive in 9 cases and TNF-α was positive in 14 of the total cases. None of the controls were positive for any of the markers except anti-CCP antibodies which were present in two females who were 22 and 25 years old, respectively [Figure 3].
Figure 3: Comparative positivity of inflammatory markers among the cases (N = 68)
[Table 1] shows the comparative evaluation of RF and anti-CCP antibodies in correlation with CRP and TNF-α. Anti-CCP and CRP were found to be positive in all patients with positive RF; however, TNF-α was present in only two of them. As regards anti-CCP antibodies, out of the 10 samples that showed positive results, RF, CRP, and TNF-α were also present in 4, 5, and 4 cases, respectively. All four markers were simultaneously present in only two cases, whereas combinations of anti-CCP/RF/CRP, anti-CCP/RF/TNF-α, anti-CCP/CRP/TNF-α, and RF/CRP/TNF-α were present in four, two, three, and two cases, respectively.
Table 1: Correlation of rheumatoid factor, anti-CCP antibodies, C-reactive protein, and TNF-α among the cases (N=68)
For decades, the diagnosis of RA has been primarily based on clinical manifestations due to lack of reliable alternative tests. Approximately one-third of the RA patients do not fulfill the ACR classification criteria, which makes the diagnosis of this disease difficult in the early stages.  Adding to the problem is the fact that substantial irreversible joint damage occurs within the first 2 years by the time the diagnosis can be confirmed by radiological or laboratory parameters. , Hence, optimization of timely and aggressive disease-modifying anti-rheumatic drug (DMARD) treatment demands prompt and accurate diagnosis and prognostic information. Though RF test has been widely used routinely in the diagnosis of RA, the enhanced specificity and early prediction of joint damage have made the assay for anti-CCP antibodies an attractive option. To better correlate these two markers for the diagnosis of RA and also to evaluate their role in inflammation, this study was planned.
RA, the most common inflammatory arthritis affecting roughly 0.5-1% of the general population worldwide with a male to female ratio of 1:2.5, may appear at any age, but it is most commonly seen among those aged from 40 to 70 years.  In our study, 36.76% of the clinically suspected RA patients belonged to this age group; the maximum (70.59%) belonged to 21-50 years age group with an overall male to female ratio of 1:3.
A recent study reports RF positivity in 90% and 40% of anti-CCP positive and negative patients, respectively, compared to a positivity of 40% and 0% in anti-CCP positive and negative patients, respectively, in our study.  The same study also found a small but significant correlation between RF and anti-CCP, though no significant correlation was found between anti-CCP and CRP as a marker of disease activity.
Another study reported that 50% of the patients were positive in both tests with 78% of the RF-positive and 40% of the RF-negative RA patients being anti-CCP antibody positive, unlike our study where 100% and 9% of RF-positive and -negative patients, respectively, were positive for anti-CCP antibodies.  This study confirms that the diagnostic sensitivity of anti-CCP antibodies in patients with recent-onset RA is the same as that of agglutinating RF and that seropositivity for the two tests correlates significantly. A study reported that 41.4% of patients were positive for RF and 41.7% of patients positive for anti-CCP at baseline, in contrast to 5.9% and 14.7% anti-CCP and RF positivity, respectively, in our study.  Kroot et al., identified 66% positive RA serum samples with CCP-ELISA, which was only 14.6% in our case.  In another study,  the results from the seronegative RA patients demonstrated high prevalence of anti-CCP positivity (60%) in the RF-negative RA patients, which was higher than previously published results where prevalence was reported to be between 20%  and 43%,  3and in our study was as low as 8.8%. Such a variation in the positivity is not clear though generation of ELISA used, population dynamics, or geographic location could be responsible for this. Also, a study on the prediction of disease course of RA by anti-CCP reports that the proportion of anti-CCP antibody positive patients increases with the number of ACR criteria fulfilled.  In another study, both RF and anti-CCP antibody tests were reported to be positive in 40.4% and negative in 28.1% of cases, as compared to our study where they were together positive in 5.9% and negative in 85.3% of RA patients. 
Patients with RA show considerable variability in disease activity that can be difficult to predict at the onset of disease. The characterization of acute phase reactants’ responses in RA is essential to gain insight into the activity of this disease and to assess the degree of inflammation. A study on the association between acute phase reactant response and the disease activity score concluded that CRP was elevated in RA patients as compared to controls, with a significant correlation observed with the disease activity score.  In our study, CRP was positive in 9 out of 68 RA patients and it correlated with RF in 4, anti-CCP in 5, and TNF-α in 4 cases. A previous study observed that CRP was significantly higher in the anti-CCP positive patients than in the anti-CCP negative group and the differences in disease activity measures between IgM RF or IgA RF-positive and-negative patients showed the same tendency as with anti-CCP.  In our study, CRP was positive in 5/10 (50%) anti-CCP positive and 4/58 (6.9%) anti-CCP negative patients and in 4/4 (100%) RF-positive and 0/64 (0%) RF-negative patients. One study, however, did not show a significant and convincing trend, contrary to other studies, regarding the use of CRP in RA patients.  Soluble TNF receptors are found in high concentrations in the synovial fluid and serum of patients with RA.  In our study, TNF-α was positive in 14 out of the 68 suspected cases of RA and the positivity correlated with RF, anti-CCP, and CRP in 2, 4, and 4 cases, respectively. In another study on 242 RA patients, anti-CCP antibodies positively correlated with higher erythrocyte sedimentation rate (ESR), CRP, swollen joint count, and worse physician global assessment ratings. If other possible causes of alteration in these surrogate inflammatory markers values are closely monitored before interpretation, the diagnostic utility of this measure should further improve.
Though RA is a disease defined by well-accepted criteria, the clinical presentation and molecular pathogenesis of this disease are varied and complex due to which prioritizing diagnostic tests or predicting treatment responsiveness is often not so easy. Our study addresses the important issue of the status of serological markers present in RA, which may predict the development of disease or prognosticate the damage that has occurred. The recognition of utility of such markers is important, as early detection of the disease will allow for early treatment, which may limit functional disability consequent to the disease.