Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies

Abstract

The detection of autoantibodies that target intracellular antigens, commonly termed anti-nuclear antibodies (ANA), is a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Different methods are available for detection of ANA and all bearing their own advantages and limitations. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. In addition, solid phase immunoassays using well characterized antigens have gained widespread adoption in high throughput laboratories due to their ease of use and open automation. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. This review provides a comprehensive overview of the current status on ANA testing including automated IIF reading systems and solid phase assays and suggests an approach to interpretation of results and discusses meeting the problems of assay standardization and other persistent challenges.

Figure 1

Illustration of pretest and posttest probability. Posttest probability (predictive value) for systemic lupus erythematosus as a function of pretest probability and as a function of indirect immunofluorescence (IIF) and solid phase assay (SPA) (EliA CTD screen, Thermo Fisher) test result. Values for likelihood ratios are from Bossuyt and Fieuws [31], WBC = white blood cell.

Figure 2

Change in referral patterns. Historically, when the ANA HEp-2 test became available in around 1960 exclusively rheumatologist and clinical immunologists ordered the ANA test. With the emerging recognition that many other diseases are associated with ANAs, a broad range of clinical disciplines order the ANA test. With changes in the ANA referral pattern and the associated decrease in the pretest probability, the posttest probability significantly decreases (indicated by the triangle).

Figure 3

Characteristic staining pattern of anti-DFS70 antibodies. The characteristic dense fine speckled (DFS) staining pattern of interphase HEp-2 cells is indicated by the blue arrow and the strong chromatin staining of mitotic cells by the red arrow. (a) Wide field view using 40x magnification, (b) dense fine speckled pattern of an interphase nucleus, and (c) of the metaphase chromatin of a mitotic cell.