Reading ANA Patterns Using a HEp-2 or HEp-2000® Substrate

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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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Reading ANA Patterns Using a HEp-2 or HEp-2000® Substrate

The tendency to interpret ANA results (by reading the slides) is to overread and call negative samples positive. Most frequently this is caused by staring too long at the cells, using too high of magnification, and an understandable desire to detect all true positives. With proper training, reading the slides can be greatly simplified.
Follow these three steps:
  1. Look initially at 200x total magnification.
  2. An ANA-positive sample must have a clearly discernible pattern in the nucleus of the interphase cells.
  3. Only use 400x magnification to confirm the pattern seen during the initial screen with 200x.
It is advised that the ANA slide first be viewed using 200x total magnification (20x objective with 10x ocular). During this first assessment, you look for a clearly discernible pattern in the nucleus of the interphase cells. If no discernible pattern is seen, the sample is ANA negative.
Do not stare at the magnified view too long--you could interpret patterns that are not present. Do not switch to a higher magnification to see if you can identify something that isn't visible at 200x. If the sample looks negative at 200x and you go to 400x and start thinking, "I might be able to somewhat see a pattern," the sample is negative. It cannot be overemphasized that to be ANA positive, there must be a clearly discernible pattern in the nucleus of the interphase cells.