What are anti-nuclear antibodies (ANA) and why are they measured?
Anti-nuclear antibodies (ANA) are non-organ-specific autoantibodies that target self-antigens. Screening for them is a key element in diagnosing systemic autoimmune diseases and is either part of the diagnostic criteria for these conditions, such as with systemic lupus erythematosus and mixed connective tissue disease, or it supports their diagnosis, such as with scleroderma, Sjögren’s syndrome, polymyositis, and dermatomyositis.
How does the indirect immunofluorescence assay (IFA) screen for ANA?
The IFA on the human epithelial type 2 (HEp-2) cell substrate is the current gold standard for detecting ANA, according to the American College of Rheumatology. The HEp-2 substrate contains hundreds of native antigens to which autoantibodies present in patient sera will bind, forming distinct staining patterns visible under a fluorescent microscope. These staining patterns are associated with particular autoantibodies indicative of specific autoimmune diseases. IFA test results inform the clinician and direct which confirmatory second-tier tests should be ordered.
In addition to the pattern, a titer is also reported. The titer is the ratio of patient sample to diluent at which the ANA is still visible and is directly proportional to the autoantibody concentration. The titer is correlated with disease severity/activity.
What challenges are associated with this assay and how can they be overcome?
IFA screening for ANA is a laborious manual process that requires experienced laboratory personnel. These technologists must possess a high degree of technical skill to prepare the slides and interpret the staining pattern. Adding to the challenge is the fact that interpretation of slides is subjective and maintaining consistency between technologists can be difficult. At our institution, every slide has a positive and negative control, and two technologists separately review each slide. There are now systems that automate slide preparation and pattern interpretation; however, these systems would need to be properly assessed if a lab were interested in implementing them.
The titer at which laboratories report a positive ANA also varies because of a lack of standardization. As an example, some laboratories may report a positive result at a cutoff of 1:40, whereas others use a cutoff of 1:160. This difference is important, as positive ANA have been documented in approximately 30% of healthy subjects at a 1:40 serum dilution, but only in 5% of healthy subjects at a 1:160 serum dilution (Arthritis Rheum 1997;40:1601–11). A lower titer (i.e., 1:40) may result in a more sensitive screen but would come at the cost of a much lower specificity.
Variability in pattern interpretation and reporting is an additional concern, and international guidelines and recommendations addressing these issues have recently been published (Ann Rheum Dis 2014;1:17–23 and Front Immunol 2015;6:412).
What factors should a laboratory consider when evaluating whether to maintain ANA testing by IFA?
For the IFA, determining if laboratory personnel have the time and expertise to prepare the slides and interpret the patterns consistently is of high importance. High-volume laboratories or laboratories that lack the appropriately trained laboratory personnel may benefit from an automated enzyme immunoassay (EIA) platform instead.
Selection of a testing platform should involve input from both laboratorians and clinicians. The laboratory should explain differences in performance between IFA and EIA assays and define the impact of these changes on test result interpretation. Conversely, clinicians can offer insight on the degree to which the titer and pattern assist them in diagnosing and monitoring their patients. In order to successfully implement either method, laboratorians must be aware of the relative benefits and weaknesses of each as well as how this technology will fit into their current workflow. Regardless of platform selection, ANA testing by either IFA or EIA plays a key role in the diagnosis and monitoring of autoimmune diseases.
The author wishes to thank and acknowledge Mark A. Cervinski, PhD, and Robert D. Nerenz, PhD, for their input.
Zahra Shajani-Yi, PhD, is a second-year clinical chemistry fellow at Dartmouth-Hitchcock Medical Center and the Geisel School of Medicine at Dartmouth. +Email: Zahra.S.Yi@hitchcock.org