Controversies in ANCA testing
ABSTRACTAntineutrophil cytoplasmic antibody (ANCA) detection is a well-known tool for diagnosing small-vessel vasculitis. Its diagnostic utility, however, depends on the methodologic accuracy of the test and the appropriate ordering of testing in the right clinical setting. While ANCA testing is of proven value, the utility of serial ANCA testing is not entirely clear. Correlation of ANCA levels with disease activity and predicted relapse remains unconfirmed. The best gauge of the predictive value of serial testing is to perform long-term serial testing for some individual patients in order to establish a relationship between ANCA level and clinical disease manifestation over time. ANCA-antigen specificity can be used to assess prognosis in patients with ANCA-associated vasculitis. Proteinase 3-ANCA is associated with higher mortality, higher relapse rate, and faster renal deterioration compared with myeloperoxidase-ANCA. Overall, ANCA is an important diagnostic and prognostic marker for small-vessel vasculitis and warrants further investigation.
Antineutrophil cytoplasmic antibody (ANCA) detection is a valuable tool for diagnosing small-vessel vasculitis,1 but measuring and interpreting ANCA levels is an inexact science. There is no single perfect ANCA test, and even a perfect test would not provide definitive clinical answers. The diagnostic utility of ANCA testing depends on the methodologic accuracy of the test and the appropriate ordering of testing in the right clinical setting. This article examines three important questions about this technology:
- What is the best ANCA test methodology?
- What is the prognostic value of serial ANCA testing?
- What is the clinical implication of ANCA type?
WHAT IS THE BEST ANCA TEST METHODOLOGY?
The diagnostic utility of ANCA testing depends on both the methodologic accuracy of the test and the appropriate ordering of tests. Methodologic accuracy comprises the analytic sensitivity and specificity of the test. Analytic sensitivity refers to the accurate identification of the presence of ANCA, and analytic specificity refers to measurement of only the entity in question (ANCA), not confounded by the presence of other entities (antibodies).
Equally as important as analytic accuracy is the appropriate ordering of the tests in the right clinical setting. Using a test that is sensitive to the presence of a specific ANCA type accurately identifies the presence of either proteinase-3 (PR3)- or myeloperoxidase (MPO)-ANCA. Once obtained, test results must be evaluated in terms of their relationship to the diagnosis being considered. If the tests are deemed diagnostically useful based on the results, the data can be used to assess the positive and negative predictive value of the tests.
Immunofluorescence or antigen-specific testing—or both?
A definitive diagnosis is more likely if an immunofluorescence staining pattern of cANCA is paired with the antigen specificity of PR3-ANCA, for example, or a perinuclear immunofluorescence pattern (pANCA) is paired with a positive MPO-ANCA. When positive test pairings have been obtained and the patient’s antigen ANCA reactivity is known, subsequent serial ANCA testing with an antigen-specific assay alone may be indicated, because the ANCA types of patients with vasculitis are unlikely to switch between PR3 and MPO during the course of their disease. If matching pairings are not obtained, the diagnostic utility of the tests remains unconfirmed.
Antigen type (PR3 or MPO) is determined through antigen-specific methods that include solid-phase assays and other methods of bringing the specific antigen in contact with the specific antibody in question. There are two categories of solid-phase assays: the enzyme-linked immunoabsorbent assay (ELISA) and the capture ELISA. In the ELISA methodology, the antigen is directly coated to a plastic plate; in the capture ELISA, an anchor, usually a monoclonal antibody or combination of antibodies, captures the target antigen on the plate. In both ELISA and capture ELISA assays, ANCA contained in the serum sample subjected to testing bind to the immobilized antigen. The amount of ANCA bound to the antigen can then be detected by a secondary antibody that is conjugated with an enzyme that can elicit a color reaction. The intensity of the color reaction is proportional to the amount of ANCA bound to the immobilized antigen.
The ELISA methodology tends to trade off analytic sensitivity for specificity, since the antigen purification process (which allows the ELISA system to increase its specificity) can cause conformational changes to the antigen being bound to the plate. This, in turn, causes a loss of some recognition of the conformationally sensitive ANCA.
In capture ELISA, a specific antibody captures the antigen; this stabilizes the conformation, boosts the analytic sensitivity, and allows a gentler purification process because it only captures the antigen in question and then binds it to the plate. This process decreases false-positive test results caused by residual contaminants in the antigen preparation. Analytic sensitivity issues may come into play if the anchoring monoclonal antibody competes for the epitope on the antigen being recognized by the serum antibody in question (ANCA), causing occasional false-negative results.
Another method now applied to commercial ANCA testing involves bead-based multiplex assays. These assays are based on principles similar to the ELISA or capture ELISA methods. In multiplex microsphere technology, the purified antigen is bound to a polystyrene microsphere instead of a plate. The microsphere is then presented to the antibody in question. The bead is then introduced to a secondary antibody labeled with a fluorescent marker (instead of an enzyme) for detection of the antibody. One advantage of this system is that various beads containing different antigens can be introduced to the same serum sample, and then different color reactions can be measured for each bead. Because only one antigen is bound to each microsphere (eg, PR3-ANCA, MPOANCA or other specific autoantibodies), only specific antibodies will react to each bead of a specific color. If there is no MPO antibody in the sample, there will be no reaction against the MPO antigen bead; however, if PR3-ANCA is present in the sample, it would react with the PR3 antigen beads. Using this methodology, a single serum sample can be tested for a multitude of autoantibodies at the same time (see “Interpreting ANCA results: Accurate tests, appropriate orders,”2–10 above).
