How are the tests to identify ANCAs performed, and how should they be interpreted?
- • ANCAs are antibodies directed against specific proteins in granules of the cytoplasm of neutrophils and lysosomal proteins in monocytes, and are present in the sera of patients with several underlying diseases.
- • ANCAs can be detected using alcohol-fixed neutrophils on a glass slide using the classic indirect immunofluorescence assay (IFA). Test serum is incubated on a slide, during which time the ANCAs bind. After washing, an antihuman IgG antibody labeled with fluorescein is incubated on the slide. After a second washing, the slide is examined using a fluorescence microscope. The highest (most dilute) titer at which fluorescence is detected, and the pattern, is noted. Two patterns are generally important in primary AAV.
- • Cytoplasmic (c)-ANCA pattern is characterized by diffuse staining of the neutrophil cytoplasm. The protein recognized by c-ANCA is nearly always PR3, a serine proteinase present in primary azurophilic granules of neutrophils.
- • Perinuclear (p)-ANCA pattern results in perinuclear cytoplasmic staining. The protein recognized by p-ANCA is often MPO, and less commonly is elastase or other proteins (lactoferrin, cathepsin G, bactericidal/permeability-increasing protein [BPI], catalase, lysozyme, and others) within primary azurophilic or specific granules of neutrophils. Patients with p-ANCA with MPO specificity are those most likely to have AAV.
- • Specific enzyme-linked immunosorbent assays (ELISAs) for antibodies directed against the PR3 and MPO autoantigens should also be obtained in addition to testing for ANCAs via IFA.
- • Data suggests that the sensitivity of ELISA testing for PR3 and MPO antibodies (for the diagnosis of AAV) may exceed that by ANCA testing via IFA with some ELISA testing platforms. As such, ANCA testing by IFA should not be viewed as a screening test for AAV. Owing to improved sensitivity and specificity, testing for ANCAs by IFA in addition to ELISA for PR3 and MPO should all be requested when evaluating a patient for suspected AAV.
- • ANCAs can be seen in conditions outside of AAV (including other rheumatic conditions, ulcerative colitis, following exposure to medications such as hydralazine, etc.; see Questions 41 and 42).
- • In patients with AAV, the presence of anti-PR3 or anti-MPO antibody may carry greater prognostic significance (disease relapse as well as clinical outcomes including response to therapy) than the clinical designation of GPA or MPA.
