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The page below is a sample from the LabCE course Autoimmune Diseases and Antinuclear Antibody Testing: Methods and Staining Patterns. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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The Antinuclear Antibody Test: Background, History, and Overview

Starting in the 1950s, the ANA test was designed as a slide-based test performed on blood, using cell substrate. The slide-based test continues to be the gold standard method. In 1957, Holborow et al first demonstrated ANA using indirect immunofluorescence. This was the first indication that processes affecting the cell nucleus were responsible for SLE. In 1959, it was discovered that serum from individuals with SLE contained antibodies that precipitated with saline extracts of nuclei, known as extractable nuclear antigens (ENAs). This led to the characterization of ENA antigens and their respective antibodies. The ANA test essentially determines whether or not antibodies are present in the blood that react to various parts of the nucleus of cells--thus, the term anti-"nuclear" antibody. Because fluorescence techniques are frequently used to actually detect the antibodies in the cells, ANA testing is sometimes referred to as fluorescent antinuclear antibody test (FANA).
In the early days of ANA testing, rodent tissue (stomach, liver, and/or kidney) was commonly used as the substrate. Rodent tissue, however, had several drawbacks. Therefore, in the 1980s, cultured cell lines were examined for utility as an ANA substrate, and the human epithelial-like cell line HEp-2 gained popularity. HEp-2s advantages over rodent tissue are:
  • A large nucleus
  • Better antigen expression
  • Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly)
More recently, a cell line called HEp-2000® (Immuno Concepts, Sacramento, CA) has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This result is a substrate with all of the original advantages of HEp-2, plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.
It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process. Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant, there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero. The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. The guidelines indicate a "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.
The two common tests used for detecting and quantifying ANAs are indirect immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA).