JIIMC March 2016.cdr ORIGINAL ARTICLE ABSTRACT Objective: To determine the usefulness of anti-Cyclic Citrullinated Peptide and anti-Mutated Citrullinated Vimentin antibody in the diagnosis of seronegative rheumatoid arthritis. Study Design: Descriptive Cross-sectional. Place and Duration of Study: This study was conducted over a period of one year from January, 2010 to December, 2010. Subjects were recruited from Fatima Memorial Hospital, Rheumatology Outpatient Department, Lahore. The research work was conducted at the Department of Physiology and Cell Biology of University of Health Sciences, Lahore. Materials and Methods: A total of 58 known patients of rheumatoid arthritis fulfilling the American College of Rheumatology (ACR) Criteria were included in the study. After selection of subjects, written informed consent was obtained. The venous blood sample was taken and secured in vacutainers. Serum was extracted by centrifugation and stored at -20°C till analysis. Sera of all study subjects were tested by ELISA for presence of rheumatoid factor, anti-MCV and anti-CCP antibodies. The data obtained was analyzed by using SPSS version 17. The diagnostic significance of anti-CCP and anti-MCV antibody for the diagnosis of sero-negative rheumatoid arthritis patients was determined. Results: Serum aCCP antibody was positive in 9 out of 28 RF-ive patients. So the sensitivity of serum aCCP in RF- ive group (n=28) was 32.1%. Serum aMCV antibody was present in 11 out of 28 RF-ive patients. The sensitivity of serum aMCV in RF-ive group was 39.2%. Conclusion: Anti-CCP and Anti-MCV had a higher sensitivity for the diagnosis of seronegative RA. KeyWords: Rheumatoid Arthritis (RA), Rheumatoid Factor (RF), Anti-Mutated CitrullinatedVimentin Antibody (anti-MCV), Anti-Cyclic Citrullinated Peptide Antibody (anti-CCP). progressive disease that produces significant morbidity and premature mortality in many 1 patients. Many studies have shown that the disease progresses rapidly within first two years of onset and 2 can lead to irreversible erosive joint destruction. The diagnosis of RA depends primarily on history and clinical findings. The gold standard for the classification of RA is the American College of 3 Rheumatology criteria (Arnett, et al., 1988). This criterion was not designed for diagnosing RA, but rather to harmonize research in population and family studies for epidemiologic purposes. But they are ubiquitously used as a diagnostic aid. Patient must satisfy four out of seven criteria to be classified as RA. ACR criteria includes: 1)Morning stiffness of more than one hour for at least six weeks 2)Arthritis and soft tissue swelling of more than 3 of 14 joints/ joint groups,present for at least six weeks. 3) Arthritis of hand joints and wrist, present for at least 6 weeks. 4) Symmetric arthritis, present for atleast 6 weeks. 5) Subcutaneous nodules 6) Rheumatoid factor at a Introduction Rheumatoid arthritis (RA) is a common systemic autoimmune disease of multifactorial etiology characterized by chronic inflammation of synovial joints that often leads to joint destruction. Rheumatoid arthritis typically produces symmetrical swelling of peripheral joints of hand and feet, but may affect the large joints as well. Rheumatoid arthritis has a worldwide prevalence of 0.5-3%, being 2-3 times more in women than in men, most frequent during fourth and fifth decade of life. Once established, rheumatoid arthritis is a lifelong Diagnostic Usefulness of Anti-Cyclic Citrullinated Peptide and Anti-Mutated Citrullinated Vimentin Antibodies in the Diagnosis of Seronegative Rheumatoid Arthritis Patients 1 2 3 4 Bushra Gohar Shah , Asma Saeed , Mohammad Faisal Khan , Hamid Javaid Quershi JIIMC 2016 Vol. 11, No.1 Correspondence: Dr. Bushra Gohar Shah Assistant Professor, Physiology Avicenna Medical College, Lahore E-mail: drbushragoharshah@gmail.com 1 2 3 Department of Physiology /Pharmacology / Biochemistry Avicenna Medical College, Lahore 4 Department of Physiology Akhtar Saeed Medical & Dental College, Lahore Funding Source: HEC ; Conflict of Interest: NIL Received: November 10, 2015; Accepted: March 06, 2016 aCCP and aMCV Antibodies in Seronegative RA 3 level above the 95th percentile. 7) Radiological changes suggestive of joint erosion/ and or periarticular osteopenia. By the time clinical diagnosis of RA is made, irreversible joint erosions usually have occurred. Ongoing research has shown that early therapeutic intervention results in earlier disease control and 2 consequently less joint damage. There is no single test or finding that can diagnose rheumatoid arthritis. Rheumatoid factor is the only serological test included in the ACR criteria. However, this auto-antibody lacks specificity. It may be found in patients with other autoimmune diseases and infectious disorders. It may also be present in sera of apparently healthy elderly individuals. Upto 25% of patients with rheumatoid arthritis have negative rheumatoid factor test 4 (seronegative). Therefore, disease-specific auto antibodies are needed for early diagnosis. Currently available data suggest that the diagnosis of RA can be made by testing antibodies to citrulline- containing peptides such as anti-perinuclear factor(APF), anti-keratin antibody (AKA), anti- fillagrin antibody and anti-cyclic citrullinated peptides (anti-CCP) antibody. These all belong to the family of anti-citrullinated protein/ peptide antibody 5 (ACPA). All these antibodies recognize the antigenic 6 epitope containing citrulline, which is generated by post-translational modification of naturally occurring amino acid arginine by the activity of 7 enzyme peptidyl arginine deiminase (PAD). Citrullinated peptides have been synthesized as 6 antigens for diagnostic immunoassays. Several assays for detecting anti-citrullinated peptide antibodies (ACPA´s) have been developed employing filaggrin derived peptides (CCP-assay), viral citrullinated peptides (VCP-assay), mutated 8 citrullinatedvimentin (MCV-assay). The Anti-MCV assay (ELISA) for the detection of antibodies against citrullinatedvimentin uses an antigen with a genetically modified sequence, which is most 8 abundant in patients with rheumatoid arthritis. Positivity of these markers in the rheumatoid factor negative RA patients would suggest their additional benefit in the early diagnosis of this subgroup of patients, which are often diagnosed and treated late. It is expected that the results of the present study will help the clinicians in early diagnosis and timely management of this debilitating disease. The aim of this study was to investigate the diagnostic value of antibodies against mutated citrullinatedvimentin (anti-MCV) and antibodies to cyclic citrullinated peptides (anti-CCP) in the diagnosis of seronegative rheumatoid arthritis patients. Materials and Methods This descriptive cross sectional study was conducted over a period of one year from January, 2010 to December, 2010. Fifty-eight subjects were recruited from Fatima Memorial Hospital, Rheumatology Outpatient Department, Lahore, by convenient sampling technique. The research work was conducted at the Department of Physiology and Cell Biology of University of Health Sciences, Lahore. A total of 58 patients attending the Rheumatology outpatient department of Fatima Memorial Hospital, Lahore were recruited in the study. All the patients fulfilled the American College of Rheumatology criteria for RA and were diagnosed by the rheumatologist. The study was approved by the Ethical and Review Committee of University of Health Sciences, Lahore. Informed written consent was taken from each study participant. A purposefully designed proforma was used to record data of the subjects including age, gender, disease duration, clinical characteristics and medication used. The venous blood samples were taken and secured in vacutainers. Serum was extracted by centrifugation and stored at -20°C till titer of anti- CCP and anti-MCV antibodies. Rheumatoid factor titers were determined by ELISA (Highton, et al., 1986) using commercially available ImmuLisa anti-RF antibody IgM ELISA kit (Immco Diagnostics, USA), with an automated EIA analyzer [Coda, Bio-Rad Laboratories, Hercules, CA, USA].Results were interpreted as follows:RF-IgM value of less than 7 IU/ml was considered as negative. RF-IgM value of 7-9 IU/ml was considered as borderline. RF-IgM value of more than 9 IU/ml was considered as positive. Serum anti-MCV antibody levels were determined by 9 ELISA using ELISA kit (Cusabio Biotech Co., Ltd, China), with an automated EIA analyzer [Coda, Bio- Rad Laboratories, Hercules, CA, USA]. Serum anti- 10 CCP antibody levels were determined by ELISA using commercially available ELISA kit (Immco JIIMC 2016 Vol. 11, No.1 4 aCCP and aMCV Antibodies in Seronegative RA Diagnostics, USA), with an automated EIA analyzer [Coda, Bio-Rad Laboratories, Hercules, CA, USA]. 25U/ml was taken as cut-off value for anti-CCP antibodies. The data was entered into SPSS version 17. Diagnostic sensitivity of anti-CCP and anti-MCV antibodies for the diagnosis of Rheumatoid arthritis in sero-negative patients were calculated by table of 2 x 2. Statistical analysis was done to determine the usefulness of the diagnostic sensitivities of anti-CCP and anti-MCV antibodies. P value of < 0.05 was considered to to be statistically significant. Results The study population (n=58), comprised of 58 rheumatoid arthritis patients, out of which 38 were females and 20 were males. Mean ±SEM age of the RA patients was 44±1.2 years. Median (IQR) disease duration was 5(4-8) years. Median (IQR) anti-CCP antibodies titer (IU/ml) was 10.8(0.00-340.5). Median (IQR) anti-MCV antibodies titer (IU/ml) was 19.7(14.2-30.06). (Table I) Sub-grouping of RA group was done on the basis of presence or absence of RF, aCCPAb, or aMCV Ab in the sera. Subgroups were named RF+ive group (gp), RF-ivegp, aCCP+ivegp, aCCP-ivegp, aMCV+ivegp, aMCV-ivegp. RF testing by ELISA technique, was done in a total of 58 RA patients. Out of the RA patients (n=58), 30 (52%) were RF+ive and 28 (48%) were RF-negative. In the RA group (n=58), 34 (58%) were aCCP+ ive and 24(41.4%) were aCCP –ive. The sensitivity of serum aCCP antibodies for RA was calculated to be 58.6%. Serum aCCP antibody was positive in 9 out of 28 RF- ive patients. So the sensitivity of serum aCCP in RF- ive group (n=28) was 32.1%. In the RA group (n=58), 20(34.5%) patients were aMCV+ and 38(65.5%) were aMCV –ive, at cutoff value of 25U/L (Table I). The sensitivity of serum aMCV antibodies for RA was calculated to be 34.5% at the manufacturer's cutoff value of 25U/L. Serum aMCV antibody was present in 11 out of 28 RF-ive patients. The sensitivity of serum aMCV in RF-ive group was 39.2%. Discussion A close relationship exists between autoimmunity and antibodies; despite this, some patients are p e rs i ste n t l y n e ga t i ve fo r d i s e a s e s p e c i f i c autoantibodies. These conditions have been defined as seronegative autoimmune diseases. Although the prevalence of seronegative autoimmune diseases is low, they may represent a practical problem because they are often difficult and challenging cases for the 11 clinicians/rheumatologist. About 80% of the patients affected by RA are positive for RF, the rest 20-25% being seronegative. A more disease specific marker for RA may help in diagnosing early disease and seronegative RA patients resulting in reduced joint damage. It is therefore important to differentiate between RA and other forms of arthritis early after the onset of symptoms. Therefore, a specific and sensitive serological marker, which is present very early in the disease, is needed so that the rheumatologist are able to target the use of potentially toxic and expensive drugs to those patients, where the benefits clearly outweighs the risk. Keeping in view the need of a more sensitive marker, especially in the seronegative cases, the present study aimed to evaluate the sensitivity of anti-CCP and anti-MCV antibodies in local Pakistani seronegative RA subjects. In seronegative cases of arthritis, the differential diagnosis is not easily established in the early disease course. Especially seronegative patients need the determination of an additional marker for RA besides rheumatoid factor to confirm diagnosis. The high specificity of anti-CCP antibodies has been reported in RF-neative RA patients. In the present study, 9 out of 28 seronegative patients were positive for anti- CCP antibodies. So, the sensitivity of anti-CCP antibodies in the seronegative sub-group was 32.1%. TableI: Serum RF, aCCP and aMCV status in the RA group (n=58) JIIMC 2016 Vol. 11, No.1 5 aCCP and aMCV Antibodies in Seronegative RA In a study, conducted by Alexiou, et al., sensitivity in seronegative group was reported to be 34.9%, which 12 is almost comparable to our results. Similarly, 13 Mobini, et al., found sensitivity in seronegative 14 group to be 33.3%, whereas Vanichapuntu, et al., found sensitivity value of 20% in the seronegative 15 sub-group whereas Serdaroglu, et al., reported sensitivity of 14.3% in seronegative group. Thus, anti-CCP antibody serves as a better diagnostic marker in the diagnosis of RA, especially in the seronegative group. The sensitivity of anti-MCV in the sero-negative RA group was 39.2%, as 11 out of the 28 RF-ive patients were anti-MCV positive. This finding is supported by recent results of other authors, that the higher sensitivity of anti-MCV especially in the sero- negative patients makes it a more valuable marker in 16 17 the diagnosis of RA. Wagner, et al., reported sensitivity of anti-MCV in the sero-negative group of 43% and sensitivity of anti-CCP to be 30.8%. In seronegative RA patients, the sensitivity of anti-MCV 18 was superior over anti-CCP. Soos, et al., reported sensitivity of anti-MCV in the sero-negative group to 19 be 29.4%. Narvaez, et al. documented sensitivity of 23% in their series of sero-negative RA patients. Limitations of the Study Small sample size was the limitation of this study. Further studies with greater number of RA patients are recommended. Conclusion Both anti-CCP and anti-MCV antibodies can be used for the early diagnosis of sero-negative patients of Rheumatoid Arthritis. Moreover anti-MCV antibody has a significantly higher sensitivity as compared to anti-ccp antibodies for the diagnosis of seronegative RA Recommendations Clinicians must be aware of the implications of delayed diagnosis in RA. Keeping in view the cost- effectiveness, this study emphasizes the utility of RF initially for the diagnosis of RA, reserving ACPA's for seronegative RA patients where strong clinical suspicion exists. Acknowledgement This research was funded by the Higher Education Commission of Pakistan. REFERENCES 1. Lee DM, Weinblatt ME. Rheumatoid Arthritis. Lancet 2001; 358: 903-11. 2. Combe B. Progression in early rheumatoid arthritis. Best Pract Res ClinRheumatol 2009; 23: 59-69. 3. Saraux A, Berthelot JM, Chales G, Le Henaff C, Thorel JB, Hoang S, et al. Ability of the American College of Rheumatology 1987 criteria to predict rheumatoid arthritis in patients with early arthritis and classification of these patients two years later. Arthritis Rheum 2001; 44: 2485-91. 4. Marianna, MN. Rheumatoid factors: What do they tell us? J Rheumatol. 2002; 29: 2034-40. 5. Zendman AJ, van Venrooij MJ, Pruijn GJ. Use and significance of anti-CCP autoantibodies in rheumatoid arthritis. Rheumatology. 2006; 45: 20-5. 6. Schellekens GA, de Jong BA, van den Hoogen FH, van de Putte LB, van Venrooij WJ. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis- specific autoantibodies. J. Clin Invest 1998; 101: 273-81. 7. Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. Bioessays 2003; 25: 1106-18. 8. Bang H, Egerer K, Gauliard A, Berg W, Fredenhagen G, Feist E, et al. Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis. Arthritis & Rheumatism. 2007; 56: 2503-11. 9. Coenen D, Verschueren P, Westhovens R, Bossuyt K. Technical and diagnostic performance of 6 assays for the measurement of citrullinated protein/peptide antibodies in the diagnosis of rheumatoid arthritis. Clin Chem. 2007; 53: 498-504. 10. 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. 11. Alessandri C, Conti F, Conigliaro P, Mancini R, Massaro L,Valesini G. Seronegative autoimmune diseases. Ann NY Acad Sci. 2009; 1173: 52-9. 12. Alexiou I, Germenis A, Ziogas A, Theodoridou K, Sakkas LI. Diagnostic value of anti-cyclic citrullinatedpeptide antibodies in Greek patients with rheumatoid arthritis.BMC Musculoskeletal Disorders. 2007; 8: 37. 13. Mobini M, Kashi Z, Mahdavi, MR. The role of rheumatoid factor and anti-cyclic citrullinated peptide antibody in diagnosis of rheumatoid arthritis. IRCMJ. 2010; 12: 100-3. 14. Vanichapuntu M, Phuekfon P, Suwannalai P, Verasertniyom O, Nantiruj K, Janwityanujit S. Are anti-citrulline autoantibodies better serum markers for rheumatoid arthritis than rheumatoid factor in Thai population? Rheumatol Int. 2010; 30: 755-9. 15. Serdaroglu M, Cakirbay H, Deger O, Cengiz S, Kul S. The association of anti-CCP antibodies with disease activity in rheumatoid arthritis.Rheumatol Int. 2008; 28: 965-70. 16. Ladislav S, Walter G, Peter S. Laboratory biomarkers or imaging in the diagnostics of rheumatoid arthritis?BMC Medicine. 2014; 12: 49. 17. Wagner E, Skoumal M, Bayer PM, Klaushofer K. Antibody against mutated citrullinatedvimentin: a new sensitive JIIMC 2016 Vol. 11, No.1 6 aCCP and aMCV Antibodies in Seronegative RA marker in the diagnosis of rheumatoid arthritis. RheumatolInt 2009; 29: 1315-21. 18. Soos L, Szekanecz Z, Szabo Z, Fekete A, Zeher M, Horvath IF, e t a l . C l i n i c a l e v a l u a t i o n o f a n t i - m u t a t e d citrullinatedvimentin by ELISA in rheumatoid arthritis. J Rheumatol. 2007; 34: 1658-63. 19. Narvaez J, Sirvent E, Narvaez JA, Bas J, Gomez-Vaquero C, Reina D, et al. Usefulness of magnetic resonance imaging of the hand versus anticycliccitrullinated peptide antibody testing to confirm the diagnosis of clinically suspected early rheumatoid arthritis in the absence of rheumatoid factor and radiographic erosions.Semin Arthritis Rheum.2007; 38: 101-9. JIIMC 2016 Vol. 11, No.1 7 aCCP and aMCV Antibodies in Seronegative RA