Due to a significant prevalence of “false negative” ANA results on these newer platforms and an insufficient communication between laboratorians and clinicians, there have been growing concerns about unilateral adoption of these newer screening and high throughput assays. The IIF assay on HEp-2 cells has been replaced in many laboratories since the development of ANA screening assays based on ELISA and automated, high throughput multiplex assays using addressable laser bead and other array technologies for the detection of specific ANA. In the following decades, ANA tests using HEp-2 cells revolutionized the diagnosis of ANA associated rheumatic diseases (AARD) including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SjS), mixed connective tissue disease (MCTD), and idiopathic inflammatory myopathies (IIM). The ANA IIF test initially relied on rodent tissue substrates but contemporary tests use HEp-2 cells, a cell line established in 1952 by Moore and her colleagues from tumors that had been produced in weanling rats exposed to irradiation and corticosteroids injected with epidermoid carcinoma tissue from the larynx of a 56-year-old male. Along with the earlier discovery of the lupus erythematosus (LE) cell and the development of the LE cell test, this ushered in a long and productive age of ANA testing. first described an indirect immunofluorescence (IIF) assay for the detection of anti-nuclear antibodies (ANA). In 1950, Coons and Kaplan described the improvement of an immunofluorescence method for the detection of antigens in tissue cells. 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. Despite all the advances in the field of ANA detection and its contribution to the diagnosis of SARD, significant challenges persist. 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. Due to the subjectivity of this diagnostic platform, automated digital reading systems have been developed during the last decade. Most laboratories use the indirect immunofluorescence (IIF) assay based on HEp-2 cell substrates. Different methods are available for detection of ANA and all bearing their own advantages and limitations.

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).

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