Anti-citrullinated peptide antibodies and rheumatoid factor isotypes in the diagnosis of rheumatoid arthritis: an assessment of combined tests
Introduction
Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease characterized by chronic joint inflammation (which often leads to the destruction of bone and cartilage) and the presence of autoantibodies, including rheumatoid factor (RF) and highly RA-specific anti-citrullinated protein antibodies (ACPAs usually measured as anti-CCP) [1]. It has been shown that RF and ACPA are present before the appearance of the clinical symptoms of arthritis, thus suggesting that the initial immune dysregulation occurs years before symptomatic disease [2]. It has also been shown that ACPAs are specific prognostic markers, and predict the erosive or non-erosive progression of the disease, thus making them useful for the optimal therapeutic management of RA patients [3].
Anti-keratin antibodies (AKAs) directed against fillagrin and anti-perinuclear factor (APF) have been historically described in RA patients, whereas ACPAs bind to citrullinated filaggrin, an epithelial protein containing citrulline residues as a result of post-translational modification [4]. In 1998, Shellekens et al. reported that AKAs recognize an epitope of citrullinated peptides in the serum of RA patients, and this led to the development of the first-generation ACPA test (designated anti-CCP), which used a mixture of CCP as a coating. However, its sensitivity was no more than 50%, and it was later replaced by second- and third-generation tests (CCP2 and CCP3), which used a mixture of synthetic cyclic peptides as a coating and increased sensitivity to 80% [5], [6]. The currently available ACPA assays use one of two synthetic peptide mixtures: CCP2 (Euro-Diagnostica) or CCP3 (Inova) [7]. The CCP2 peptide sequence was identified by screening highly complex peptide libraries using highly reactive serum taken from RA patients, whereas CCP3 was designed by means of combinatorial peptide engineering and contains multiple citrullinated epitopes in a conformational structure that increases epitope exposure and immunoreactivity, especially in the case of early RA [8]. Because of patent restrictions, most manufactures use the same synthetic peptide mixture as a coating, which means that the differences between them are more due to the method/instrument than the coating itself.
Today, ACPA tests are systematically added to clinical and radiological investigations when diagnosing RA, and the inclusion of ACPA positivity in the new 2010 RA criteria underlines their importance: RF and ACPAs together have a positive predictive value of nearly 100% [9].
The first international reference ACPA preparation has now been developed, an important step in reducing inter-laboratory and inter-method variability [10]. Furthermore, increasing demand has led a number of manufacturers to improve their own methods, which are mainly based on enzyme-linked immunosorbent assays (ELISAs), although a new multiplex flow immunoassay has recently been set up and shown very good concordance with ELISA [11].
The primary aim of this study was to determine the sensitivity and specificity of different ACPA assays and IgA, IgG and IgM isotypes of RF in a cohort of patients with early RA in order to assess the value of combining the tests [12], [13], [14]. The secondary aim was to compare the diagnostic sensitivity and specificity of the newly available ACPA assays with the previous tests.
Section snippets
Serum sampling
Serum samples were obtained from 46 patients with early RA diagnosed on the basis of the 2010 American College of Rheumatology (ACR) criteria (42 females and 4 males; mean age 65.2 ± 7.3 years; disease duration 1.5 [0.5–5.2] months) who attended the Rheumatology Unit of San Giovanni di Dio Hospital, Florence (Italy), between July 2011 and April 2012. Three of the patients were being treated with one or more immunosuppressants (methotrexate, sulfasalazine) and 43 with one of the biological drugs
Results
At the manufacturers' cut-off range values varied from 45.65% to 73.91% for diagnostic sensitivity and from 87% to 95% for diagnostic specificity.
The EliA CCP-Phadia test was the most specific of the APCA assays (95%), and had the best LR + and PPV, although the second best performers (Bio-Rad CCP and Axis-Shield CCP) were also very specific.
The EliA CCP-Phadia already reaches the 95% specificity at manufacturer cut-off, while others' cut-off already needs to be increased.
INOVA 3.1 was the most
Discussion
The recent addition of ACPA to RF testing in the 2010 RA classification criteria has reinforced the role of laboratory tests in RA diagnostics [9]. The later CCP3.1 version detects ACPA IgG + IgA, and is still the only assay approved by the American Food and Drug Administration (FDA) for the early detection of RA (510 k number: K072944).
Over the last few years, many studies have evaluated the diagnostic performance of ACPA assays using a variety of diagnostic platforms [15], [16], [17], [18], [19]
Conclusions
During last years the role of APCAs have been developed by new methods or by different diagnostic platforms such as fluorescence enzyme immunoassay (FEIA) and multiplex flow immunoassay (MFI).
MFI and FEIA technologies provide rapid results and are both totally automated. Their advantage over ELISAs is that it is possible to use a single sample run and gain constant random access, thus increasing efficiency and improving laboratory workflows.
New assays based on citrullinated proteins instead of
Conflicts of interest
The authors declare that they have no conflict of interest.
Authors' contributions
MI, MM, FA and MB contributed equally to the manuscript.
Acknowledgments
We gratefully aknowledge the technical assistance of R. Bambi, G. Ciotta, F. Priami, F. Soldaini, C. Taddei, and M. Valentini. We would like to thank Phadia AB-Upsala for the statistical analyses. All of the companies gifted their immunoassay kits.
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