Frequency of Positive Anti-CCP Antibodies in Rheumatoid Arthritis Patients with Negative Rheumatoid Factor

Objective: Rheumatoid arthritis (RA) is a polyarticular autoimmune disease that affects about 1% of the adult population. The disease is characterized by synoviocyte hyperplasia, mainly synovial fibroblasts, resulting in bone and joint destruction. Recent studies have shown that cytokines and other systemic inflammation mediators have a key role in the development of rheumatoid arthritis. Recently, Anti-Citrullinated Protein Antibodies (anti-CCP) have come into use for the diagnosis of rheumatoid arthritis (RA). It has been reported that anti-CCP has quite a high specificity for RA (98%), together with sensitivity similar to that for rheumatoid factor (RF). The study aimed to determine the frequency of positive anti-CCP antibodies in rheumatoid arthritis patients with negative rheumatoid factor.
Methodology:91 patients (both male and females) aged 40-70years with negative RF clinically diagnosed with rheumatoid arthritis were included. Patients with positive rheumatoid factor and joint injuries were disclaimed. The consultant pathologist used a second-generation enzyme-linked immunosorbent assay (ELISA) to assess the serum levels of anti-CCP antibodies. Anti-CCP level >20IU/ ml was considered positive, p-value of 0.05 was considered statistically significant.
Results:In the study, the age range was 40 to 70 years, averaging 53.44 ± 7.16 years. 59 (64.84%) of the patients were between the ages of 40 and 55. Of 91 patients, 56 (61.54%) were women, and 35 (38.46%) were men with a ratio of 1.6:1 man to women. In patients with negative rheumatoid factors, 27 (40.66%) patients were found to have high levels of positive anti-CCP antibodies.
Conclusion: Patients with negative rheumatoid factor showed the high frequency of positive anti-CCP.
Key words:: Antibodies, Rheumatoid arthritis, Rheumatoid factor.

Cytokines and other inflammatory mediators have been shown to play important roles in the production of articular rheumatism and other articular diseases.1 Synovitis is one form of joint disease with symptoms of pain and swelling of the joints.2 RA exists in all racial and ethnic groups, but is most prominent in some demographic groups (for example, Black individuals in the Caribbean region). RA affects the general population. The family members of people with RA are 2 to 3 times more likely to have it than any other person. Monozygotic twins display a disease concordance of up to 20%. Since RA is considered a universal disease, it is suspected that a constant infectious agent is at work.3The pathway of creation of RA is not very well understood. It can potentially cause various symptoms in people with such genetic conditions (e.g., cigarette smoking, infection or trauma). There are pre-inflammation events that lead to tissue changes leading to inflammation or uncontrolled inflammation. Genetically, defects in the immune system may result in the creation of certain illnesses.⁴ Rheumatoid arthritis (RA) is a chronic auto-immune disease that affects many organs and structures in the body.⁵This situation includes inflammation of the synovial joints. Serological testing is widely used in RA to determine the autotherapy, Serum and joint fluids from RA patients contain unique autoantibodies. Rheumatoid factor (RF) is antibodies against the Fc component of IgG autoantibodies.⁶ Rheumatoid factor is a well-established diagnostic method for the diagnosis of rheumatoid arthritis.⁷ Recently, cyclic citrullinated peptide (CCP) antibodies have been found useful in diagnosing rheumatoid arthritis. Anti-CCP patients have got confirmed with RA and RF sensitivity (98% and 97% respectively). Test findings have shown that antibodies against the CCP are also believed to be correlated with active and erosive disease.8 More than 37% of patients with RF negative rheumatoid arthritis had anti-CCP antibodies in their blood supply. ⁹ This study revealed that among 100 people with RA, 92% were with positive Anti-CCP antibodies and 89% with negative rheumatoid factors. We will obtain different results from foreign studies due to our ethnic, geographical and genetic heterogeneity. Through this study, we will analyse and determine how to effectively fix dysfunctional character in our culture. The aim of this study was to determine the frequency of positive anti-CCP antibodies in rheumatoid arthritis patients with negative rheumatoid factor.

This cross-sectional study was conducted over a period of one year from January, 2020 to December, 2020 at blood bank of Pakistan Atomic Energy Commission (PAEC) General Hospital, Islamabad, Pakistan. Ethical approval was obtained from hospital ethical committee prior to the study. A total of 121 patients diagnosed with polycythemia were selected by purposive sampling. Among total of 121 patients, 116 (95.86%) were male and 6 (4.95%) were female with male to female ratio of 19:1. The mean age of the patients at the time of presentation was 45 years, with a range of 19 to 76 years. Patients having Hb > 16.5 g/dl and hematocrit > 48 were selected for the study. Hemoglobin and hematocrit were done on peripheral collected in EDTA. All samples were run on hematology analyzer Sysmex XN1000 (Japan). Phlebotomy was done using 17G needle after informed consent and after obtaining vital signs. Patients were counseled about the procedure and were constantly monitored. Results were entered and analyzed using SPSS version 20.0.

Out of 121 patients, the most common blood group was B positive [47 (39%)], while the least common were O negative and AB negative, with one patient belonging to each blood group (0.8% each), figure 1. The average pre phlebotomy hemoglobin of the patients was 17.45g/dl while post-phlebotomy, hemoglobin was 15.97g/dl. Among all, patients those underwent phlebotomy, 89 (73.5%) had a fall in Hb of greater than or equal to 1g/dl, while in 32 (26.4%) patients, Hb drop was less than 1g/dl. 105 patients out of 121 underwent more than one therapeutic phlebotomies to maintain their hemoglobin and hematocrit within normal range, figure 2. The patients presented with rise in hemoglobin were also suffering from a number of underlying causes including smoking, cardiac disease, renal or liver disease and chronic obstructive pulmonary disease. Smoking was found to be the cause of secondary polycythemia in 27.27% of the patients, and it was followed by unknown cause in 23.9% patients, figure 3.

The World Health Organization (WHO) defines JAK2V617F as a major criterion for the diagnosis of Polycythemia Vera. Other markers include Hb> 16.5 in men and 16 in women, or hematocrit >49% in men and 48% in women, or RBC mass >25% above the mean predicted value.12 Polycythemic patients present with a variety of symptoms, among which headache is quite common.13 Facial plethora is also seen in a large number of patients. Several patients complain of erythromylgia or pruritus. Splenomegaly is seen in a large number of patients with polycythemia vera 14 In the current study, smoking was found to be the most common cause of secondary polycythemia. The studies have also reported that smokers have increased red cell mass and decreased plasma volume, due to increased carboxyhemoglobin levels which result in hypoxia.15 A study conducted on 40 healthy individuals and 40 smokers in Taif, Saudi Arabia reported that smoking causes damage to alveolar tissue, resulting in inflammation, as well as injury to vascular tissue which causes leukocytes to increase. Smoking also adversely affects gaseous exchange in the lungs, resulting in hypoxia and leading to an increase in the red cell mass.¹⁶ In our study, JAK2 mutation status was analyzed to diagnose patients of polycythemia vera, however it was only found positive in 02 patients out of 121. Bases on this finding other patients were evaluated for secondary causes. In 29 patients of study among 121, the cause of polycythemia could not be determined despite conducting all investigations and taking a detailed history. In another study conducted on 231 patients, in 40 patients no cause of polycythemia was detected.¹⁷
Phlebotomy is the mainstay of treatment in polycythemic patients, with an aim to decrease hematocrit to below 45%. Taking in account the hemoglobin, phlebotomy would be considered successful if Hb falls 1g/dl or more after one phlebotomy. Phlebotomy also results in a decrease in blood viscosity that reduces subsequent occurrence of thrombotic events. Studies have also shown that at maintained hematocrit below 45%, the risk of cardiovascular morbidity is decreased. In our study, we recorded the Hemoglobin level of patients before and after phlebotomy. About 73.5% of patients had a fall in Hb of 1g/dl or more, while in 26.4% of patients, a fall in Hb was less than 1g/dl.

A number of factors can lead to an increase in Hb, among them smoking is a very common cause along with other associated illnesses like renal disease, lung disease, cardiac disease and malignancies. Phlebotomy is the basis of treating polycythemia, although in secondary polycythemia the underlying cause requires further diagnosed and treated accordingly.

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6. Öcal R, Arslan Y, Özgür G, Doğan D, Yıldırım M, Öcal N. Do we care enough about the role of smoking in the etiology of polycythemia?. GULHANE MEDICAL JOURNAL. 2019;61(2):46-51. doi: 10.26657 Alkhedaide AQ. Tobacco smoking causes secondary polycythemia and a mild leukocytosis among heavy smokers in Taif City in Saudi Arabia. Saudi journal of biological sciences. 2020;27(1):407-11. doi: 10.1016/j.sjbs.2019.11.001
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