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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 4  |  Page : 207-211

Comparative analysis of rheumatoid factor levels by immune turbidimetry and latex agglutination assays among anti-cyclic citrullinated peptide-positive rheumatoid arthritis patients


Department of Microbiology, Guru Teg Bahadur Hospital, University College of Medical Sciences, Delhi, India

Date of Submission25-Aug-2020
Date of Acceptance27-Sep-2020
Date of Web Publication26-Nov-2020

Correspondence Address:
Dr. Bineeta Kashyap
Department of Microbiology, Guru Teg Bahadur Hospital, University College of Medical Sciences, Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/INJMS.INJMS_104_20

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  Abstract 


Background: Rheumatoid arthritis (RA) is one of the most common contributors to the burden of chronic and debilitating polyarthritis. Materials and Methods: Over a period of 3 months, 264 symptomatic patients were tested for the presence of anti-cyclic citrullinated peptide (CCP) antibodies, and the first 50 patients with RA whose serum samples tested positive for anti-CCP (>10 AU/ml) were included in the study. Immune turbidimetric assay was employed to determine the titers of rheumatoid factor (RF) in patients of RA who have tested positive for anti-CCP and its performance was compared with latex agglutination testing modality. Results: Majority (82%) of the study population were female with a male-to-female ratio of 1:4.6. Latex agglutination test for RF using RHELAX-RF test kit yielded RF values ≥10 IU/ml in 31 (62%) patients, while the Immunoturbidimetric test: SPECTRUM RF Test Kit imparted RF titers ranging between 2.4 and 53.76 IU/ml, with a median RF titer of 22.22 IU/ml. The percentage agreement between the latex agglutination and immune turbidimetry assay for analyzing RF was 88%, with a strong positive correlation between latex agglutination and immune turbidimetry assay for RF (Spearman coefficient: r = 0.8097). Conclusion: Since the updated American College of Rheumatology/European League Against Rheumatism classification of RA underscores the precise titers of RF and anti-CCP in scoring RA, absolute titers of RF conferred by the immunoturbidimetry could be a welcome relief to slash inter-observer variations and hand out titers to better classify and treat patients of RA.

Keywords: Anti-cyclic citrullinated peptide, immune turbidimetry, rheumatoid arthritis, rheumatoid factor


How to cite this article:
Kashyap B, Saha R, Sarkar K, Singh NP. Comparative analysis of rheumatoid factor levels by immune turbidimetry and latex agglutination assays among anti-cyclic citrullinated peptide-positive rheumatoid arthritis patients. Indian J Med Spec 2020;11:207-11

How to cite this URL:
Kashyap B, Saha R, Sarkar K, Singh NP. Comparative analysis of rheumatoid factor levels by immune turbidimetry and latex agglutination assays among anti-cyclic citrullinated peptide-positive rheumatoid arthritis patients. Indian J Med Spec [serial online] 2020 [cited 2021 Feb 28];11:207-11. Available from: http://www.ijms.in/text.asp?2020/11/4/207/301652




  Introduction Top


The chronic systemic immune insult of rheumatoid arthritis (RA) has been known for decades, adding to morbidity, disability, socioeconomic burden, and even premature death among sizable populations worldwide across cultures, ethnicities, and diverse geographical settings. Characterized by progressive inflammation of the synovial lining with symmetrical involvement of joints and several hallmark clinical deformities, RA is known to predominantly afflict the elderly and female population. Hence, early diagnosis and prompt intervention forms the cornerstone of management to prevent disability. Since several laboratory parameters and characteristic radiological signs present much later in the course of disease, early markers for RA and timely intervention with disease-modifying antirheumatic drugs (DMRDs) are key index in determining the course of disease. In fact, several studies have established an optimal therapeutic window of 12 weeks commencing from onset of symptoms to favorably reduce articular damage, functional disability, and DMRD-free remissions.[1],[2],[3]

With the diagnostic criteria being updated and re-revised over decades, the quest for precise and consistent diagnostic tools has long eluded the physicians. With a clearer picture of the molecular pathogenesis of RA emerging overtime, several biomarkers have been associated and are currently utilized for classifying and diagnosing RA. Rheumatoid factors (RFs) are autoantibodies to epitopes within the Fc portion of human immunoglobulin G (IgG), IgA, and IgM. However, approximately 50% of the RFs detectable in RA patients belong to IgM subclass, while the clinical significance of RF belonging to other classes is much disputed.[4]

Since the advent of Rose-Waaler test relying on the agglutination of sheep red blood cells opsonized with rabbit IgG (rIG), the detection of RF has come a long way. Several ELISAs, nephelometry, turbidimetry, and latex agglutination formats have been devised for the detection of RF in patient sera. However, with a positivity of 70%, RF in isolation has its own limitations in diagnosing RA. Considering the seropositivity of RF in numerous other rheumatic diseases such as systemic lupus erythematosus, Sjogren's syndrome, primary cryoglobulinemia as well as viral infections or tumors, the specificity of RF in RA appears to be low.[5]

The issue was addressed to an extent with the advent of anti-cyclic citrullinated peptide (anti-CCP) antibodies for RA. Anti-citrullinated protein antibody tests are based on the principle of citrullination catalyzed by the calcium-dependent enzyme peptidylarginine deiminase, which in turn changes positively charged arginine to neutral citrulline as a result of posttranslational modification. Anti-CCP has been detected in sera of 67% of the RA cases and is instrumental in ascertaining early RA disease activity, excluding undifferentiated arthritis as well as classification of RA.[6],[7] Considering the fact that anti-CCP-positive RA endures a more severe clinical course in addition to lower responsive to methotrexate or rituximab treatments, the early diagnosis, monitoring, and aggressive intervention strategies are indispensable in this group of RA patients.[8],[9],[10]

At present, the diagnostic criteria for RA employs RF, anti-CCP antibodies, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR), with varying individual considerations of all these biomarkers. Thus, in the present study, we have tried to elucidate the utility of an immune turbidimetric assay to determine the titers of RF in patients of RA who already have tested positive for anti-CCP (anti-CCP-positive RA) as well as compare the performance of the new test with latex agglutination testing modality.


  Materials and Methods Top


A prospective study was conducted at the Immunology Laboratory, Department of Microbiology, UCMS and GTB Hospital, over a period of 3 months to ascertain the adequacy of an immune turbidimetric assay for determining the levels of RF in serum samples of RA patients. Over a period of 3 months, serum samples from 264 symptomatic patients were tested for the presence of anti-CCP antibodies. Blood samples obtained by venipuncture and transported in nonanticoagulated vacutainers were centrifuged at 2000 rpm for 10 min to separate serum. All grossly hemolyzed and lipemic serum samples were excluded from the study.

ELISA For anti-cyclic citrullinated peptide

The levels of anti-CCP were quantitated in all 264 samples with the help of anti-CP IgG ELISA (Diametra S. r. l., Italy) as per manufacturer's instructions. The first fifty patients with RA whose serum samples tested positive for anti-CCP (>10 AU/ml) were included in the study and processed further.

Latex agglutination test for rheumatoid arthritis

The first fifty patients who tested positive for rheumatoid arthritis by ELISA for anti-CCP were included. “Rheumatoid Factor” (RF) of class IgM, which is specific to the Fc portion of IgG, was semi-quantitatively estimated with the help of RHELAX-RF (Tulip Diagnostic, Goa, India) as per manufacturer's instructions in these fifty samples. The sensitivity of the RHELAX-RF (Tulip Diagnostic, Goa, India) latex agglutination test was ≥10 IU/ml RF; positive as well as negative controls were put up for each batch of tests.

Immunoturbidimetric estimation of rheumatoid factor

SPECTRUM RF Test Kit (Spectrum Medical Industries, Delhi, India) in conjunction with the SPECTRUM Automatic Analyzer (Spectrum Medical Industries, Delhi, India) was used to determine the titer of RF in the first fifty patients' sera that tested positive for anti-CCP by ELISA.

Statistical analysis

The percentage agreement between the two testing modalities was calculated, and the correlation between the results of latex agglutination and immune turbidimetric analysis was ascertained by Spearman coefficient. The SPSS 20.0 software (SPSS 20.0, Armonk, NY: IBM Corp) was used for all statistical analysis.


  Results Top


Serum samples from a total of 264 patients presenting with varying degrees of joint pain at various departments of the tertiary care hospital were subjected to ELISA for the detection of anti-CCP antibodies. Among these, 64 were found to harbor detectable levels of anti-CCP, confirming RA in 24% of the tested patients. The first fifty patients of RA, confirmed by ELISA for anti-CCP, were included in the study.

Female patients constituted 82% of the study group, while only 18% comprised male patients, resulting in a male-to-female ratio of 1:4.6. Majority of the study participants hailed from the Inpatient and Outpatient Departments of Medicine and Orthopedics. The ward distribution profile of the study population is depicted in [Figure 1]. The age of the study population ranged from 21 to 70 years, with a median age of 45 years (mean 45.28 ± 12.34 years).
Figure 1: Ward distribution profile

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Latex agglutination test for RF using RHELAX-RF test kit yielded RF values ≥10 IU/ml in 31 (62%) patients, while 19 (38%) patients exhibited titers <10 IU/ml. On the other hand, the SPECTRUM RF Test Kit imparted RF titers ranging between 2.4 and 53.76 IU/ml, with a median RF titer of 22.22 IU/ml. The mean RF titer estimated in the study population was 22.35, with SD ± 15.13 IU/ml. A comparative illustration of RF analysis of study specimens by immunoturbidimetry and latex agglutination test is depicted in [Figure 2] and [Figure 3]. The SPECTRUM RF Test Kit established high RF titers (>20 IU/ml) in 28 (56%) patients, whereas 22 (44%) patients' sera displayed RF titers within the normal range.
Figure 2: Age group-specific depiction of positive results by latex and turbidimetry

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Figure 3: Age group-specific depiction of negative results by latex and turbidimetry

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Discordance in RF titers was observed in five study samples, yielding positive tests by latex agglutination and normal RF titers by immune turbidimetry. The percentage agreement between the latex agglutination and immune turbidimetry assay for analyzing RF was 88%. Upon data analysis, a strong positive correlation was observed between latex agglutination and immune turbidimetry assay for RF, with a Spearman coefficient of r = 0.8097.


  Discussion Top


RA is one of the most common contributors to the burden of chronic and debilitating polyarthritis. With a global prevalence of 0.5%-1%, the incidence and prevalence of RA shows wide variations with respect to geographical regions and ethnicity of populations within the same regions.[11],[12] Several studies have established a higher prevalence of RA in the Western countries in comparison to Asia; however, the role of genetics or environmental factors prompting these fluctuation demands illustrious deliberations.[13] The prevalence of RA in India varies from 0.28% to 0.7%.[14]

The diagnosis of RA has conventionally relied upon a holistic evaluation of clinical, laboratory, and radiological findings. By the end of 2010, the American College of Rheumatology (ACR) criteria had been revised in collaboration with the European League Against Rheumatism (EULAR) to “classification criteria” from “diagnostic criteria.” The new ACR-EULAR criteria attribute greater specificity to positive serum anti-CCP antibodies than a positive RF test and exclude late manifestations of RA such as subcutaneous rheumatoid nodules and radiological findings in addition to imparting due weightage to disease duration and acute-phase reactants such as CRP and ESR. With scores ranging from 0 to 10, a score ≥6 helps define RA and distinguishes patients with a higher likelihood of progressing to chronicity and articular damage. In addition to aiding diagnosis, biomarkers like RF and anti-CCP antibodies also help prognosticate disease outcomes.

A multitude of principles have been employed for determining the levels of these biomarkers to facilitate diagnostic and treatment outcomes. Ranging from conventional ELISA, latex agglutination formats, nephelometry to immune turbidimetry assays, several techniques have emerged over time in quest for precise, simple, cost-efficient testing.[15] Considering the high specificity (98%) of anti-CCP antibodies for RA, it can be demonstrated in the early stages of the disease well before the rise of other serological and clinical findings.[16] The present study determines the efficiency of an immune turbidimetric RF assay in comparison to latex agglutination among patients of RA who have tested positive for anti-CCP antibodies.

The majority of the suspected RA patients harboring elevated anti-CCP levels in their serum samples were females (82%). The male-to-female ratio of 1:4.6 within the study population aligned with the findings of several other studies depicting a female preponderance.[17] The higher predisposition of women to develop RA has been widely speculated. Several studies have documented the association of breastfeeding, use of oral contraceptives as well as menopause to the risk for developing RA disease, thus implicating a plausible hormonal impact in RA development.[18] The age of the study participants ranged between 21 and 70 years (mean 45.28 ± 12.34 years), which was in alignment with findings of other studies related to RA.[19]

The immune turbidimetric established positive RF results in 56% of the anti-CCP-positive patient sera, while the latex agglutination test depicted a positivity of 62% in the same specimens. Other authors have reported RF positivity in 90%-40% of the anti-CCP-positive patients.[20],[21] The two methods of analyzing RF (turbidimetry and latex agglutination) showed a high positive correlation with a correlation coefficient of 0.8097. Similar findings have been demonstrated by authors comparing latex agglutination and turbidimetry for determining RF among suspected RA patients irrespective of their anti-CCP antibody status.[19] However, five study samples yielded discordant results (positive tests by latex agglutination and normal RF titers by immune turbidimetry), which was much higher than that reported by other studies.[19] Nevertheless, the findings of various authors have reported discrepancies in comparability between RF testing formats such as ELISA, nephelometry, and turbidimetry.[22],[23] These variations may be at least partially attributed to the difference in sensitivities of the testing kits as well as in the disease activity of the study population, considering the fact that the present study group included patients only comprising anti-CCP-positive patients. Nevertheless, similar results depicting higher positivity among agglutination testing in comparison to nephelometry have also been reported in certain studies from around the world.[24]

Since RF testing exhibits seronegativity in 30%-50% of the patients with early-stage RA, clinical significance of isolated readings is of limited value; biomarkers (RF, anti-CCP, and CRP) should be interpreted with caution and invariably in conjunction to other disease parameters.[25] As depicted in [Figure 2] and [Figure 3], the latex agglutination test displayed higher positivity than turbidimetry within the age brackets of 40-69 years, while the immune turbidimetry yielded more negative results within the same age group. With comparable sensitivities of serum anti-CCP with RF in diagnosing RA, the cumulative benefit of simultaneously testing for anti-CCP and RF cannot be discounted. Despite the discrepancies, there is a strong positive correlation (r = 0.8097) between the immune turbidimetry and latex agglutination assay for RF, as re-emphasized by other studies.[15],[19]


  Conclusion Top


It is quite evident from the strong positive correlation between the immune turbidimetry and latex agglutination resulting from the present study and the findings of the above authors that both the testing techniques yield RF positivity which can be interchangeably used depending on resources. The immune turbidimetry, however, ameliorates optimal prognostication and might be better suited to classify RA and monitor prognosis due to its precision. The latex agglutination format of testing on the contrary is merely a semi-quantitative measure yielding titers in dilutions that are visually interpreted. The subjective interpretation and calculations derived from observed value are invariably prone to inter-observer variations, which is nullified by the turbidimetry analyzer that bypasses the possibility of human error during interpretation. The precision and ease of the immune turbidimetric RF analysis not only paves the way for better diagnosis but also facilitates correlate clinical outcomes with serological results in order to formulate a holistic management strategy.

The complex interplay of immune mechanisms that contribute to the pathogenesis of RA calls for strategic implementation and evaluation of diagnostic tests. The updated ACR/EULAR classification of RA reinforces the titers of RF and anti-CCP to ascertain scores. The absolute titer conferred by the SPECTRUM RF Test Kit in conjunction with the analyzer could be a welcome relief to slash inter-observer variations and hand out precise titers, which in turn could facilitate the classification of RA in addition to determining the prognosis of disease.

Financial support and sponsorship

None.

Conflicts of interest

There are no conflicts of interest.



 
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