Diagnostic Performances Of Rheumatoid Factor And Anti-Cyclic Citrullinated Peptide In Rheumatic Patients

ABSTRACT

BACKGROUND: Rheumatoid arthritis is a chronic autoimmune systemic inflammatory disease affecting firstly the small articular structure that characterized by periodic flaring-up ending with irreversible joint damage. Our objective was to test the hypothesis that anti-citrullinated protein antigens have greater diagnostic performance than rheumatic factors in Rheumatoid arthritis.

Methods: A retrospective analysis was conducted for patients who were seen at the rheumatology clinic from Dec 2018 till Mar 2020. The whole cohort was divided into 2 groups: Confirmed rheumatoid arthritis patients (Group I) and Non-Confirmed rheumatoid arthritis patients (Group II). The diagnostic performance was explored by using receiver operator characteristic curves and the area under curves. A Chi-Square, Mann-Whitney-U, and Independent T tests were conducted to compare between the tested groups.

Result: The mean overall age was 58.37±9.96 years, and 155 subjects (47.25%) were male with a male to female ratio of 2.33:1 and 0.44:1 in Group I and Group II, respectively. Rheumatoid factors and anti-citrullinated protein antigens were significantly higher in the radiologically confirmed rheumatoid arthritis cohort than the radiologically non-confirmed rheumatoid arthritis cohort. The best diagnostic cut-off values for rheumatoid factors and anti-citrullinated protein antigens among studied patients were 69.55 IU/ml (Normal range (0-20 IU/ml))  and 89.21 IU/ml (Normal range up to 20 IU/ml) respectively. The area under curve were significantly higher for anti-citrullinated protein antigens than in rheumatoid factors 0.848 (95 CI, 0.801-0.894) and 0.692 (95% CI, 0.624-0.760), respectively.

Conclusion: Anti-citrullinated protein antigens test had a higher diagnostic performance than rheumatoid factors test in rheumatoid arthritis patients’ and so may have important implications for patient’s early diagnosis.

Keywords: Anti-Cyclic Citrullinated Peptide; Rheumatic Factor; Rheumatoid arthritis; diagnostic performance.

INTRODUCTION

 ^[1] RA significantly affects women more than men with interrelated genetic, environmental, and hormonal perpetuating factors. As RA can early cause radiographic-based joint changes, reliable, affordable, diagnostic, and discriminative prognosticators with high performance, specificity, positive predictive value, and accuracy are required to initiate DMARDs as soon as possible before more advanced articular destruction and systemic complications have ensued. ^[2]

The emphasis in the management of RA is early diagnosis and intervention. The hypothesis on which this approach is based is that a possibility of altering the disease process with early intervention. ^[3]Although the 2010 American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) put forward revised classification criteria emphasizing RA characteristics that emerge early in the disease course, including anti-citrullinated protein antigens (ACPAs), a biomarker that predicts aggressive disease, the main purpose of these criteria was to distinguish RA from other forms of arthritis, rather than to identify and diagnose patients with RA in the earlier stages of disease when they might benefit most from intervention ^[4-5].American College of Rheumatology (ACR) criteria for the classification of RA can diagnose an established active RA disease with good sensitivity (~92%) and specificity (89%) after meeting at least 4 criteria from a total 7, it has a poor early diagnostic performance and many pathogeomonic features of arthropathies such as RA, e.g., deformity and nodules, are related to chronicity and are absent at presentation. ^[6-7]

While the diagnosis of RA relies primarily on patient history, radiographic evidence of joint damage, and routine positive acute phase reactants investigations of c-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), RA approved specific laboratory testing of rheumatoid factor (RF) and anti-citrullinated protein antigens (ACPAs) can assist in early diagnosis and differentiating RA from other rheumatic diseases that present with poly-articular arthritis, for establishing an early therapeutic plan before irreversible damage and when treatment is most effective in preserving function.^[8-9]

Although RF has been widely used as a screening and diagnostic test for patients with clinically diagnosed arthritis, it has a poor diagnostic performance with low sensitivity and moderate specificity when used alone. ^[10-11]More recently, a more RA diagnostic and prognosticator specified lab test has been introduced into clinical practice with a sensitivity of 68% and a specificity >97%. ^[12-13] ACPAs test is a highly specific antibody that develops to citrullinated arginine residues in RA patients in which is probably be positive in up to 45% of RF negative patients. ^[11-12]Our objective was to test the hypothesis that ACPAs has greater diagnostic and prognostic performances than RF in RA patients and to determine the optimal cutoff points for both tested prognosticator in our tested RA Jordanian patients.

MATERIALS AND METHODS

A retrospective study was conducted on Royal Rehabilitation Center at the Royal Medical Services Hospital for patients who were seen at the rheumatology clinic from Dec 2018 till Mar 2020 whose clinical symptoms and signs indicated RA. After the baseline demographics, diagnostics, radiographics, and the required lab chemistries were obtained from our institutional electronic health record (Hakeem), the retrospective collected data were divided into 2 groups: Radiologically Confirmed RA patients (Group I) and Radiologically Non-Confirmed RA patients (Group II). The study was reviewed and approved by the standing committee for coordination of health and medical research at the Royal Medical Services (Ref # 41_06/2021).

All RA symptomatic patients met at least 4 out 7 of ACR criteria and all were naïve to disease-modifying anti-rheumatic drugs (DMARDs). Any patient who had missing data was excluded from our study. The patient's Functional assessment was measured using the Health Assessment Questionnaire (HAQ). ACPAs were measured using a commercially available enzyme-linked immunosorbent assay (ELISA), while RFs were quantified based on immunoenzymatically determination of IgM rheumatoid factors which is also based on the ELISA technique. X-rays of affected joints were taken at baseline, 4-6, and 8-12 months and reported by our institutional radiologists.

All RA patient’s collected data were expressed as Mean± SD, Median (Range), or as numbers with percentages by using the Independent Samples T-Test and Independent Whitney U Test or χ² Test for parametric and non-parametric data, respectively. Tested variables analyses were compared for the two compared groups (Radiologically Confirmed RA patients and Radiologically Non-Confirmed RA patients). Of important comparative variables, including ACPAs, RFs, acute phase reactants of CRP and ESR, hemoglobin levels, and symptomatic durations. The diagnostic and prognostic performance of RF vs ACPAs will be explored by a sensitivity analysis using receiver operating characteristic (ROC) curve interpretation to determine the area under the ROC curves (AUROCs), Youden’s indices, sensitivities, specificities, positive and negative predictive values, and the optimal cut-off values for the two tested prognosticators. Statistical analyses were performed using IBM SPSS ver. 25 (IBM Corp., Armonk, NY, USA) and P-values ≤0.05 were considered statistically significant.

Conflict of interest and financial disclosure statement: None declared.

RESULT

Three hundred and twenty-eight patients were included in the study. 176 symptomatic patients (53.66%) according to ACR classification criteria had a confirmed radiological diagnosis of RA at follow-up for at least 1 year. The mean age of the whole study cohort was 58.37±9.96 years, and the RA patients in Radiologically Confirmed RA Group (Group I) were insignificantly older than the RA patients in Radiologically Non-Confirmed RA Group (Group II) [58.55±9.948 years versus 58.09±10.053 years, respectively, p-Value=0.917). Significantly, males were distributed in the study in approximately 1.12: 1 ratio compared to female [155 (47.25%) versus 173 (52.74%), respectively, p-Value=0.003] in which 27.27% (48 RA affected men) and 63.64% (112 RA affected women) were belonged to the Group I compared to 70.39% (107 RA affected men) and 29.61% (45 RA affected women) were belonged to the Group II.CRP, ESR, RF, and ACPAs were significantly higher in radiologically confirmed RA cohort than radiologically non-confirmed RA cohort with Mean±SD of 27.69±2.54 mg/dl, 48.42±6.12 mm/hr, 101.36±18.91 IU/ml, and 167.34±21.23 IU/ml versus 8.29±3.77 mg/dl, 23.09±4.87 mm/hr, 67.53±12.75 IU/ml, and 48.81±17.41 IU/ml, respectively. In contrast, both hemoglobin levels and duration of symptoms in months were significantly lower in Group I compared with Group II with Mean ±SD of 12.55±2.09 g/dl and 13.21±8.95 months versus 13.89±3.12 g/dl and 14.11±7.89 months.

There were insignificant differences between the two tested groups regarding anthropometrics and Health Assessment Questionnaires. Comparative patients ‘data results are fully summarized in Table 1

The optimal cut-off point, sensitivity (TPR), specificity (TNR), Youden’s index (YI), positive and negative predictive values (PPV and NPV), negative likelihood ratio (NLR), and accuracy index (AI) of the two tested prognosticators among the two stratified compared groups are fully illustrated in Table II. The best diagnostic cut-off

values for RF and ACPAs in our study among studied RA patients were 69.55 IU/ml RF (Normal range (0-20 IU/ml)) and 89.21 IU/ml (Normal range up to 20 IU/ml). The AUROCs of ACPAs were significantly higher in our studied RA patients than RF with AUC of 0.848 (95 CI, 0.801-0.894) and 0.692 (95% CI, 0.624-0.760), respectively. The ROC curve analyses of the two tested prognosticators are fully shown in Fig 1.

DISCUSSION

 The present study included RA symptomatic patients with chronic symptoms for at least 12 months who were seen at our rheumatology clinic from Dec 2018 till Mar 2020. To the best of our knowledge, this is the first study in our country that compares two common diagnosticators among two radiologically stratified Jordanian RA groups based on diagnosis confirmation. In the context of ever, dearth of resources, early assessment and triaging with affordable and cost-effective discriminative predictive tools are critically wanted in this chronic progressive affected patient.

Both CRP and ESR are positive acute-phase reactants that are adjunctively used in the diagnosis and following-up RA patients but with low diagnostic performance for both if they are solely used due to falsely elevated in other rheumatology and non-rheumatology inflammatory conditions. ^[13] In contrast, more rheumatology-specific indicators of RF and ACPAs are widely used as the primary biochemical diagnostic parameter. ^[14-16] While RF is a more widely used laboratory marker than ACPAs in RA patient's diagnoses, an early combination of both prognosticators may help establish a diagnosis in the early stages. ^[17-18]

While ACPAs is primarily used for diagnostic purposes, it is also useful for prognostic and diseases activity progression prediction at 3-10 years after onset. In most studies, ACPAs are significantly more correlated with radiological and functional poor RA outcomes than RF. In most studies, ACPAs and RF show comparable sensitivities for the prediction of radiological and functional progression at 5 years, but ACPAs showed greater specificity compared with RA. Also, it is mostly that RF+/ACPAs + patients had poorer progression than RF+/anti-CCP– and RF–/CCP– patients at 5 years. ^[19-21]

In a prospective cohort study of 43 patients with RA not responding to DMARDS treated with infliximab in combination with methotrexate in which the serum samples collected and tested for ACPAs and RF at baseline and after 24 weeks, serum titers of ACPAs and RF decreased significantly after 24 weeks of treatment (ACPAs -14%; RF-20%). Significant decreases in serum ACPAs and RF were observed only in patients with clinical improvement but only changes in ACPAs levels were significantly positively correlated with changes in various clinical measures of RA. ^[22-24]

While the anti-CCP test is a specific marker for the diagnosis of RA. However, this antibody also can be detected in patients with other rheumatic diseases, such as Sjogren's syndrome, and even in healthy people. ^[25-27] In a study of 405 patients originally diagnosed with primary Sjogren’s syndrome, 23 patients (5.6%) progressed to RA after a mean follow-up of 60 months. ^[28] Matsui et al reported a relatively high frequency of anti-CCP in patients with non-RA connective tissue diseases such as SLE (15%), Sjogren's syndrome (14%), polymyositis/dermatomyositis (23%), and scleroderma (16%). ^[29]

Use of the presence of either RF or anti-CCP had comparable diagnostic utility as anti-CCP alone, but these two tests were complementary in that they increased the sensitivity.^[30-31]Our study suggests that ACPAs had a significantly higher sensitivity (84% vs 75%), specificity (97% vs 72.7%), Youden’s index (81.4% vs 47.7%), positive and negative predictive values (94.79% and 90.58% vs 63.98% and 81.81%, respectively), and accuracy index (92.05% vs 73.60%). In particular, when both tested diagnosticators present, the specificity may increase to >95%.

CONCLUSION

In conclusion, using ACPAs (Normal range up to 20 IU/ml) in RA patients would appear to pick RF (Normal range (0-20 IU/ml)) seronegative early-stage RA patients with significantly higher diagnostic performance and probably higher prognosis predictive capability and so may have important implications for RA patient’s early diagnosis and following-up. In conjunction with the referenced studies, these findings may have a significant impact on the overall RA patients. This study is limited by its retrospective design, confounder effect, and using single-center data. A larger, multisite, and prospective study is needed to control for multiple confounders.

REFERENCES

1. Quinn MA, Conaghan PG, Emery P. The therapeutic approach of early intervention for rheumatoid arthritis: what is the evidence? Rheumatology 2001;40:1211–20.

2. Fuchs HA, Kaye JJ, Callahan LF, et al. Evidence of significant radiographic damage in rheumatoid arthritis within the first 2 years of disease. J Rheumatol . 1989;16:585-591.

3. Quinn MA, Emery P. Window of opportunity in early rheumatoid arthritis: possibility of altering the disease process with early intervention. Clin Exp Rheumatol 2003;21(Suppl. 31):S154–7.

4. Aletaha D,  Neogi T,  Silman AJ, et al. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative, Arthritis Rheum, 2010, vol. 62 (pg. 2569-81)
5. Funovits J,  Aletaha D,  Bykerk V, et al. The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for rheumatoid arthritis: methodological report phase I, Ann Rheum Dis, 2010, vol. 69 (pg. 1589-95)

6. Harrison BJ, Symmons DP, Barrett EM, Silman AJ. The performance of the 1987 ARA classification criteria for rheumatoid arthritis in a population based cohort of patients with early inflammatory polyarthritis. American Rheumatism Association. J Rheumatol 1998;25:2324-2330.

7. Green MJ M-OH, McGonagle et al. Persistence of mild early inflammatory arthritis: the importance of disease duration, rheumatoid factor and the shared epitope. Arthritis Rheum 1999;42:2184–8.

8. Emery P. The optimal management of early rheumatoid disease: the key to preventing disability. Br J Rheumatol 1994;33:765–8.

9. van Zeben D, Hazes JM, Zwinderman AH, Cats A, van der Voort EA, Breedveld FC. Clinical significance of rheumatoid factors in early rheumatoid arthritis: results of a follow up study. Ann Rheum Dis 1992;51:1029–35.

10. Schellekens GA, Visser H, de Jong BAW et al. The diagnostic properties of rheumatoid arthritis antibodies recognising a cyclic citrullinated peptide. Arthritis Rheum 2000;43:155–63.

11. Bizzaro N, Mazzanti G, Tonutti E, Villalta D, Tozzoli R. Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem 2001;47:1089–93.

12. Meyer O, Labarre C, Dougados M et al. Anticitrullinated protein/ peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage. Ann Rheum Dis 2003;62:120–6.

13. van Jaarsveld CH, ter Borg EJ, Jacobs JW et al. The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis. Clin Exp Rheum 1999;17:689–97.

14. Kroot EJ, de Jong BAW, van Leeuwen MA et al. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum 2000;43:1831–5.

15. Auger I, Sebbag M, Vincent C, et al.: Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen. Arthritis Rheum. 2005, 52:3424-3432. 10.1002/art.21391.
16. Tebo AE, Jaskowski T, Davis KW, et al.: Profiling anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis. Pediatr Rheumatol Online J. 2012, 10:29. 10.1186/1546-0096-10-29.
17. Oberle EJ, Harris JG, Verbsky JW: Polyarticular juvenile idiopathic arthritis - epidemiology and management approaches. Clin Epidemiol. 2014, 6:379-393. 10.2147/CLEP.S53168.
18. Nell-Duxneuner V, Machold K, Stamm T, et al.: Autoantibody profiling in patients with very early rheumatoid arthritis: a follow-up study. Ann Rheum Dis. 2010, 69:169-174.
19. Sieghart D, Platzer A, Studenic P, et al.: Determination of autoantibody isotypes increases the sensitivity of serodiagnostics in rheumatoid arthritis. Front Immunol. 2018, 9:876.10.3389/fimmu.2018.00876
20. Kim SK Bae J Lee H Kim JH Park S-H Choe J-Y . Greater prevalence of seropositivity for anti-cyclic citrullinated peptide antibody in unaffected first-degree relatives in multicase rheumatoid arthritis-affected families. Korean J Intern Med. 2013;28(1):45–53.
21. Scott LJ . Tofacitinib: a review of its use in adult patients with rheumatoid arthritis. Drugs. 2013;73(8):857–874.
22. Nüßlein HG, Alten R, Galeazzi M, Lorenz HM, Nurmohamed MT, Bensen WG, et al. Prognostic factors for abatacept retention in patients who received at least one prior biologic agent: an interim analysis from the observational, prospective ACTION study. BMC Musculoskelet Disord. 2015;16:176.
23. Lv Q, Yin Y, Li X, Shan G, Wu X, Liang D, et al. The status of rheumatoid factor and anti-cyclic citrullinated peptide antibody are not associated with the effect of anti-TNFα agent treatment in patients with rheumatoid arthritis: a meta-analysis. PLoS One. 2014;9:e89442.

24.  Alessandri C, Bombardieri M, Papa N, Cinquini M, Magrini L, Tincani A, Valesini G. Decrease of anti-cyclic citrullinated peptide antibodies and rheumatoid factor following anti-TNFalpha therapy (infliximab) in rheumatoid arthritis is associated with clinical improvement. Ann Rheum Dis. 2004 Oct; 63(10):1218-21.

25.  Sauerland U, Becker H, Seidel M et al. (2005) Clinical utility of the anti-CCP assay: experiences with 700 patients. Ann N Y Acad Sci 1050, 314– 8.

26.  Atzeni F, Sarzi-Puttini P, Lama L et al. (2008) Anti-cyclic citrullinated peptide antibodiesin primary Sjögren syndrome may be associated with nonerosive synovitis. Arthritis Res Ther 10, R51.

27.  Hodkinson B, Meyer PW, Musenge E et al. (2010) The diagnostic utility of the anti-CCP antibody test is no better than rheumatoid factor in South Africans with early rheumatoid arthritis. Clin Rheumatol 29, 615– 8.

28.  Ryu YS, Park SH, Lee J et al. (2013) Follow-up of primary Sjögren's syndrome patients presenting positive anti-cyclic citrullinated peptides antibody. Rheumatol Int 33, 1443– 6.

29.  Matsui T, Shimada K, Ozawa N et al. (2006) Diagnostic utility of anti-cyclic citrullinated peptide antibodies for very early rheumatoid arthritis. J Rheumatol 33, 2390– 7.

30.  Yang X, Cai Y, Xue B, Zhang B. Diagnostic value of anti-cyclic citrullinated peptide antibody combined with rheumatoid factor in rheumatoid arthritis in Asia: a meta-analysis. J Int Med Res. 2021.

31.  Esmat, Mamdouh, Moghazy, Hoda Boghdady, et al. Anti-Cyclic Citrullinated Peptide ( ACCP ) Antibody versus Rheumatoid Factor ( RF ) for Diagnosis of Rheumatoid Arthritis. Egyptian Journal of Medical Microbiology. 2021