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Overview Anti-Nuclear Antibodies (ANA) with a cell line
The term "anti nuclear antibodies" describes a variety of autoantibodies that react with constituents of cell nuclei including DNA, RNA and several proteins and ribonucleoproteins.¹ These autoantibodies occur with high frequency in patients with connective tissue or rheumatic diseases, especially systemic lupus erythematosus. Virtually all SLE patients are ANA positive. This diagnostic sensitivity has led to the incorporation of ANA testing into the 1982 Revised Criteria for the Classification of Systemic Lupus Erythematosus by an American College of Rheumatology subcommittee.² While ANA testing is an excellent screening test for SLE (a negative result virtually rules out active SLE³) it is by no means a specific test. Patients with other connective tissue diseases such as rheumatoid arthritis, scleroderma and dermatomyositis are frequently positive, and low ANA titers may be observed in other disease states and in the normal population. Positive ANA results can occur following severe burns or viral infection and have been reported in some normal, healthy people, especially in older populations. Because of this lack of specificity, it is recommended that all ANA positive samples be titered to endpoint and that more specific testing for autoantibodies to double stranded DNA (dsDNA) and extractable nuclear antigen (ENA) autoantibodies be performed.

Indirect immunofluorescence is the reference method for ANA testing. Common substrates include thin sections of rodent organs or various types of cell lines. It is generally agreed that cell line substrates are preferable to organ sections since these rapidly dividing cells have higher levels of certain clinically relevant antigens, including centromere, SS A(Ro), Scl 70 and PCNA/Cyclin.

Besides the type of substrate, three other factors are critical to the performance of an ANA test: 1) the fixative used in preparing the slide, 2) the fluorescein to protein (F/P) ratio and 3) the immunoglobulin subclass specificity of the conjugate. Some fixatives or combinations thereof are known to destroy certain nuclear antigens and their use should be avoided. The sensitivity and non specific background staining of a conjugate is determined by the F/P ratio while the disease specificity of a conjugate is determined by the immunoglobulin subclass reactivity. Virtually all clinically significant autoantibodies exhibit IgG subclass specificity even in the presence of IgM and IgA specific ANA.⁴ In contrast, ANA found in healthy blood donors are generally of the IgM and IgA subclass only.⁵ Because of this, conjugates specific for IgG are more disease specific.

1. Tan EM: Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Advances in Immunology 33: 167 239, 1982.

2. Tan EM, et al.: The 1982 Revised criteria for the classification of systemic lupus erythematosus. Arthritis and Rheumatism 25: 1271 1277, 1982.

3. Casalo SP, Friou GJ and Myers LL: Significance of antibodies to DNA in systemic lupus erythematosus. Arthritis and Rheumatism 7: 379 390, 1964.

4. Gonzalez E and Rothfield N: Immunoglobulin class and pattern of nuclear fluorescence in systemic lupus erythematosus. The New England Journal of Medicine 274: 1333 1338, 1966.

5. Wiik A: Antinuclear factors in sera from healthy blood donors. Acta Path Microbiology Scand. 84: 215 220, 1976.