How are ANAs measured?
The preferred method is the indirect immunofluorescence (IIF) technique. Permeabilized cells are fixed to a microscope slide and incubated with the patient’s serum, allowing ANAs to bind to the cell nuclei. After washing, a fluorescein-labeled secondary antibody is added, which binds to the patient’s antibodies (which are bound to the nucleus). Cells are visualized through a fluorescence microscope to detect nuclear fluorescence. The amount of ANAs in a patient’s serum is determined by diluting the patient’s serum prior to adding the serum to the fixed cells—the greater the dilution (titer) at which nuclear fluorescence is detected, the greater the amount of ANAs present in the patient’s serum. Most laboratories use human epithelial type 2 (HEp-2) cells (a proliferating cell line derived from a human epithelial tumor cell) for the substrate to detect ANAs. This is because rapidly growing and dividing cells contain a larger array and higher concentration of nuclear antigens (such as Sjögren’s syndrome-related antigen A [SS-A] and centromere antigens). Older testing methods for IIF used rat or mouse liver and kidney tissue as the substrate, which lowered the sensitivity for the detection of antigens such as SS-A.
Enzyme-linked immunoassay (ELISA) methods and multiplex bead assays are now commonly used by many laboratories to detect ANAs as they are cheaper and easier to perform. These platforms generally include an array of 8 to 10 autoantigens compared with >100 autoantigens in HEp-2 cells. Data has shown that ELISA and multiplex bead assays may be less sensitive than IIF methods. Furthermore, ELISA methods can potentially denature the purified target antigen in the solid phase assay raising the potential for false-positive results as well. As such, IIF remains the “gold-standard” according to the American College of Rheumatology. Medicare cost for the ANA screening test is $14.92.