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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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A similar procedure that is also widely used is called Colorzyme®. This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope.

The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed. After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody reagent conjugated to horseradish peroxidase.

When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme-specific substrate. The reaction between the enzyme-labeled antibody and enzyme-specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a pattern characteristic of the particular nuclear antigen distribution within the cells.

If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker staining.

The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, speckled, centromere, and nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)