Laboratory Testing for Diagnosis, Management of Patients with Rheumatic Disease

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Table 4: RF & Anti-CCP in Rheumatic & Other Diseases

Antibodies to Citrullinated Proteins

There has been considerable interest in developing a better test for the diagnosis of RA, which has greater sensitivity and specificity than the tests that detect RFs. Within the last decade, as an outgrowth of determining the molecular specificity of antifilaggrin, antikeratin and antiperinuclear antibodies, it was recognized that many patients with RA have antibodies to citrullinated proteins. Proteins that are citrullinated have had an arginine replaced by citrulline, a minor amino acid. This posttranslational modification is mediated by the enzyme peptidyl arginine deiminase 4 (PAD4).

PAD4 is the form of PAD associated with RA. It has been found in bloodstream granulocytes and is localized in the cell nucleus. Its only known activator is a component in cigarette smoke, but its activation is also associated with inflammation. Much more work needs to be done to determine what specifically in RA activates (and downregulates) PAD. Because increased activity of PAD4 has been demonstrated in synovial fluid and synovium of patients with RA and other inflammatory arthritides, but generally not OA, it appears to be associated with inflammation. Citrullination of proteins has also been in noted in multiple sclerosis. The primary physiologic role for PAD enzymes (and the conversion of arginine to citrulline) appears to be related to regulation of nucleosome, that is, histone function. However, the development of antibodies to citrullinated proteins is relatively specific to RA.

To facilitate analysis of antibodies to citrullinated proteins, a number of peptide antigens containing citrulline were created, although these have had a variable degree of sensitivity for recognizing patients with RA. However, a cyclic peptide containing citrulline was found to have a high sensitivity and was subsequently utilized to develop a commercial assay. This test, named anti-CCP (for cyclic-citrullinated peptide) has now been studied extensively, especially in Europe, and it has better sensitivity and specificity for the diagnosis of RA than tests that detect RF; the (positive) predictive value for the diagnosis of RA was 83% for anti-CCP(2), 77% for RF and 83% for the combination of RF and CCP. A number of commercial firms have developed different assays for the detection of these antibodies to CCP, and the assays have been called CCP1, CCP2, CCP3, etc.

There have been numerous claims regarding the superiority of one assay compared with another, and this requires further investigation. In addition, a number of groups have assayed antibodies to other citrullinated proteins—especially fibrinogen and vimentin—and compared these assays to anti-CCP, with variable results. These latter assays are not, at present, commercially available. Most studies to date on examining the frequency, sensitivity, specificity, predictive value of anti-CCP in RA relate to CCP2—which is the assay I employ in my laboratory.