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Journal of the New Zealand Medical Association, 08-October-2004, Vol 117 No 1203

Anti-cyclic citrullinated antibodies: complementary to IgM rheumatoid factor in the early diagnosis of rheumatoid arthritis

Kamal Solanki, Myfanwy Spellerberg, Peter Chapman, Peter Moller, John O’Donnell

Abstract

Aims To compare the diagnostic sensitivity of anti-cyclic citrullinated (CCP) antibodies and rheumatoid factor (RF) in rheumatoid arthritis (RA) within a general hospital setting.

Method Using the American College of Rheumatology (ACR) classification criteria as a gold standard, the frequency of RF and anti-CCP antibody positivity was compared between two groups of RA patients: those with disease duration less than 2 years (early RA, ERA) and those with disease duration more than 2 years (late RA, LRA).

Results In ERA, the diagnostic sensitivity of RF and anti-CCP antibodies was 57% and 79% respectively. In LRA, it was 81% and 84% respectively.

Conclusion Anti-CCP antibodies are 20% more sensitivity than RF in the diagnosis of early RA.

Rheumatoid arthritis affects 1% of the population and is one of the most common serious inflammatory arithritides.1 It is characterised by chronic inflammation of the synovial membrane of diarthrodial joints. This inflammation results in joint damage leading to morbidity and premature mortality .2,3 In many persons, erosion and damage occur within the first 2 years of disease onset.2 Suppressing the inflammatory response early in the course of disease is a major goal of therapy, hence early diagnosis is important.

The American College of Rheumatology (ACR) classification criteria (1987) emphasise features of chronicity and, as such, have limited use in early diagnosis.4 However they are still used as a ‘gold standard’ for research purposes.

IgM rheumatoid factor (RF), which is one of the ACR criterion, is the best known serological marker—however it has a relatively low diagnostic sensitivity (50 to 90%) particularly in early disease and relatively low specificity (87%).5,6 Nevertheless in the appropriate clinical context, rheumatoid positivity is an important finding giving support to the diagnosis of RA.

Newer serological markers have included antibodies to cyclic citrullinated peptide (CCP), Sa protein, heavy chain binding protein (Bi P), and glucose-6-phosphatase isomerase.2 Of these, anti-CCP antibodies seem promising as a diagnostic5,6 and possible prognostic marker in RA.7–10

Citrullination (conversion of arginine residues to citrulline by the enzyme peptidyl arginine deaminase) of filagrin induces an autoantigen that was recognised previously by anti-keratin auto-antibodies. Profilagrin, present in the keratohyaline granules of human keratinocytes, is proteolytically cleaved to filagrin subunits, which form the target autoantigen. Unfortunately these subunits are not stable enough to be used as a commercial substrate in diagnostic tests.

In their study, Schellekens and colleagues reported that 76% of rheumatoid arthritis sera contained autoantibodies reactive with linear synthetic peptides containing citrulline. These antibodies were 96% specific for rheumatoid arthritis5,6

An ELISA based on cyclic-citrullinated peptide (CCP), a more stable compound,6 was shown to efficiently measure these antibodies in the sera of patients with rheumatoid arthritis.

Using ACR criteria as a ‘gold standard’, we assessed the diagnostic sensitivity of anti-CCP antibodies in early and late rheumatoid arthritis within a general hospital setting.

Patients and methods

Christchurch Hospital is both a secondary and tertiary referral centre in the South Island of New Zealand. It has an immediate catchment population of approximately 500,000. Canterbury Health Laboratories provides laboratory support to the hospital. The immunology section of this laboratory routinely stores sera for a minimum of 5 years. These stored samples allowed for the retrospective analysis of anti-CCP antibodies in some patients prior to and following the diagnosis of RA.

Patients referred by their general practitioner to the rheumatology clinic at Christchurch Hospital and subsequently diagnosed with RA formed the study subjects. All patients were given a diagnosis of RA once their caring physician considered that they fulfilled the ACR classification (1987) criteria for RA. Patients were divided into two groups as follows:

Group 1: patients diagnosed with RA of less than 2 years duration who had stored serum available from the time of the initial diagnosis. These patients formed the early rheumatoid arthritis group (ERA)
Group 2: patients with RA who had been diagnosed ≥2 years previously who had a contemporary serum sample available. These patients formed the established rheumatoid arthritis group (LRA)

RF was measured by rate nephelometry (Beckman array) with a cut-off at 40 IU/L. Anti-CCP antibodies were measured by the Quantalite CCP ELISA assay (INOVA Diagnostics, San Diego, CA, USA) with a cut-off at 20 units as specified by the manufacturer. All sera were tested in duplicate in the ELISA and a 5-point standard curve was derived for each microtitre plate.

Statistical Analysis

The McNemars Chi-square test was used for statistical comparison between diagnostic antibody tests within each group.

Results

Table 1 summarises the results of both rheumatoid factor and anti CCP antibodies in the two patient groups.

In group 1, the diagnostic sensitivity of RF and anti CCP antibodies was 57% and 79% respectively (p=0.009) while in group 2 it was 81% and 84% respectively (p=1.0).

Six patients within group 1 had more than one serum sample stored from the time of initial presentation. Of these, two patients were negative for both anti-CCP antibodies and RF at initial presentation. The other four patients were anti-CCP positive and RF negative at initial presentation.

Analysis of stored serum samples revealed that the two double negative patients seroconverted for anti-CCP between 6 and 48 months and seroconverted for RF between 15 and over 48 months. In the four patients who were anti-CCP antibody-positive and RF-negative at initial presentation, RF seropositivity developed in three patients between 16 and 48 months and the fourth patient remained RF negative.

Table 1 Diagnostic sensitivity of rheumatoid factor and anti-cyclic citrullinated (CCP) antibodies in early and late rheumatoid arthritis

(Click here to view Table 1)

Discussion

General practitioners and specialist physicians are often faced with evaluating patients with polyarthralgia and polymyalgia.

Typically, such patients eventually fall into four categories.

Patients destined to suffer a self-limiting disorder (most often idiopathic, but occasionally associated with an infection).
Patients suffering from a chronic diffuse pain disorder (such as fibromyalgia).
Patients destined to develop degenerative joint disease.
Patients ultimately destined to develop some form of chronic inflammatory joint disease.

Patients destined to develop rheumatoid arthritis form a large sub-group of the last category. The importance of identifying these RA patients early has been increasingly emphasised as strategies are developed to reduce disease morbidity.2,11

Early intervention with disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate, salazopyrin, and more recently anti-tumour necrosis factor α (anti-TNFα) therapy has been associated with more favourable long–term outcomes.9,11

Anti-CCP antibodies have a high positive predictive value for RA in patients with polyarthralgia, particularly if used in conjunction with clinical findings and rheumatoid factor.12 Thus there is the potential to identify patients for therapy at an early stage. In addition, anti-CCP antibodies also have prognostic value in identifying those patients at greater risk of erosive disease.6,7,13

The results of our study support previous findings from reference and research centres that anti-CCP antibodies tend to appear earlier and are therefore of added value in the in early diagnosis of rheumatoid arthritis. In our study, diagnostic sensitivity was increased by 20% over IgM rheumatoid factor in the early RA group.

Prior to commissioning the anti-CCP ELISA in our laboratory, the specificity of the assay was evaluated using stored serum samples from 54 patients with non-RA rheumatic diseases: SLE (n=23); Wegener’s granulomatosis (n=9); psoriatic arthritis (n=3); mixed connective tissue disease (n=2); and one patient each with giant cell arteritis, CREST syndrome, ankylosing spondylitis, and juvenile chronic arthritis.

Three of the 54 were positive giving a specificity of 94%, a result comparable with published data.4 The three ‘false positive’ sera where from patients with SLE and all had low titre concentrations of anti-CCP antibodies.

In recognition of the benefits of early intervention in RA, there have been calls to establish new prognostic criteria.2 Despite their demonstrated diagnostic and prognostic value9 it has been suggested that anti-CCP antibodies should be excluded from such criteria on the pretext that the test is not widely available.2

Cost is the primary determinant of availability. In New Zealand, the price of tests varies from laboratory to laboratory. Rheumatoid factor measured by nephelometry is about $NZ15.00/test (exclusive of taxes) whereas currently the price of anti-CCP antibodies is about $NZ60.00/test. The cost of assay kits and the cost of labour are the main determinants of price. More general use, automation, and competition will see prices fall.

Anti-CCP antibodies have all the hallmarks of establishing themselves firmly in the diagnostic algorithm of rheumatoid arthritis providing additive sensitivity to rheumatoid factor.

Author information: Kamal K Solanki, Rheumatology Registrar, Christchurch Hospital, Christchurch; Myfanwy B Spellerberg, Section Head Immunology, Canterbury Health Laboratories, Christchurch; Peter T Chapman, Rheumatologist, Christchurch Hospital, Christchurch; Peter Moller, Rheumatologist, Christchurch Hospital, Christchurch; John L O’Donnell, Clinical Immunologist, Canterbury Health Laboratories and Christchurch Hospital, Christchurch

Acknowledgments: We acknowledge the kind and dedicated efforts of the laboratory staff of the immunology section Canterbury Health Laboratories, Mrs Christine Martin for secretarial support ,and Dr Christopher Frampton for advice on statistical analyses.

Correspondence: Dr John O’Donnell, Department of Rheumatology Immunology and Allergy, Canterbury Health Laboratories, PO Box 151, Christchurch. Fax: (03) 364 1241; email: john.odonnell@cdhb.govt.nz

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