Abstract
Anti-CCP assays (antibody to cyclic citrullinated peptides) possess a high specificity for RA (rheumatoid arthritis). In lack of an international reference material the absolute values among different assays strongly diversify. The cut-off showed a variance from 5 U/mL Euroimmun anti-CCP to 25 U/mL Euro-Diagnostica Immunosan RA. The Autoantibody Standardization Committee is currently trying to establish a reference specimen of human polyclonal anti-CCP.
Key Words::
Introduction
The diagnosis of rheumatoid arthritis (RA) often relies on clinical manifestations and laboratory tests as rheumatoid factor and C-reactive protein.
Recently, the identification of citrulline as a target of a whole set of auto-antibodies detected in the sera of RA patients has led to the development of anti-CCP assays (antibody to cyclic citrullinated peptides) which possess a high specificity for RA. The clinical performance of anti-CCP assays has been further improved by the use of multiple citrullinated peptides, resulting in a second generation of anti-CCP assays [Citation1,Citation2]. Regarding nomenclature it is also common to use ACPA (anti-citrullinated protein/peptide antibodies) instead of anti-CCP [Citation3].
Example for quantifying anti-citrullinated auto-antibodies:
The Roche Elecsys anti-CCP assay uses a set of cyclic citrullinated peptides and is therefore a so-called second generation assay. The principle of the test is an IgG-capture format [Citation4].
Traceability: The reference standardization for Elecsys anti-CCP was done by a method comparison of native patient samples using a commercially available second-generation anti-CCP assay. In a second step 6 working calibrators were value assigned on the previous method comparison. These second line calibrators (master calibrators) are used for master curve fitting using the Passin-Bablok regression procedure [Citation6] which is stored in the reagent barcode. These second line calibrators are only in use ‘in house’. They also secure the lot to lot consistency. The third line working calibrators with 2 set points were value assigned with the master curve fitting. The Passing-Bablok regression function in addition to the working calibrators adopt the quantification of assay results to each individual analyzer.
Clinical utility
The anti-CCP assays were optimized by having a high specificity and a good discrimination to other rheumatic and non-rheumatic diseases. For demonstration of the process Roche Elecsys anti-CCP is used. In an external clinical study including 792 confirmed RA patients, 420 asymptomatic healthy individuals and 907 patients with rheumatic and non-rheumatic disorders an optimal cut-off of 17 U/mL was determined. Using this cut-off the sensitivity was calculated to be 67,4% with a specificity of 97,0%. In a next step the Roche Elecsys anti-CCP was compared to other anti-CCP available assays. For example a 97.3% agreement was found in the comparison of Euro-Diagnostica Immunoscan RA assay with the Roche Elecsys anti-CCP assay (N =1,606 samples). The different cut-offs of 17 U/mL Roche and 25 U/mL Euro-Diagnostica were taken into consideration [Citation4].
Comparison of assay results
In lack of an international reference material the absolute values among different assays strongly diversify [Citation3]. The cut-off showed a variance from 5 U/mL Euroimmun anti-CCP; 7 U/mL Phadia ELIAS CCP to 25 U/mL Euro-Diagnostica Immunoscan RA [Citation3].
By comparing different assays it is helpful to use a direct comparison of negative and positive values in a 4 field plot design to visualize the concordance and disconcordance of the two compared assays instead of a conventional method comparison. When the anti-CCP assay in the laboratory is changed the absolute values will differ and the cut-off has to be adopted to the new method.
International standardization efforts
The Autoantibody Standardization Committee (ASC) [Citation5] headed by Pier Luigi Meroni recognized the need for reference human autoimmune sera. The ASC is currently trying to establish a reference specimen of human polyclonal anti-CCP. The work is still in a validation phase. The use of International Units, based on the reactivity of a reference serum, will hopefully harmonize the diagnosis of which serum is anti-CCP positive and which is not [Citation7]. Reference standards for other auto-antibodies as anti-nuclear antibodies (ANA) are still available [Citation5].
A project proposal to the IFCC scientific division for standardization of anti-CCP assays is set by J. Sheldon “Harmonisation of 5 autoantibody tests in the investigation of autoimmune rheumatic and vasculitic diseases” (personal communication PL Meroni to start a working group including anti-CCP in this project)
Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.
References
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