College of American Pathologists
Immunohistochemical (IHC) Assays: Principles of Analytic Validation

Immunohistochemical (IHC) testing is an essential component of the pathologic evaluation of many specimens and increasingly provides key information that helps determines how patients are treated.

As with any laboratory test, laboratories must validate all IHC assays before they are used to test patient specimens. Unfortunately, recent studies have found significant interlaboratory variation in validation practices and revealed that many laboratories do not follow consistent procedures.

Publication and Educational Materials

The open comment period for the draft recommendations on IHC Assays: Principles of Analytic Validation received over 1000 comments.

The final recommendations will be presented at the CAP ’13 Course: Principles of Analytic Validation in Immunohistochemistry and shortly thereafter published in the Archives of Pathology and Laboratory Medicine.

Scope Questions

  1. When and how should validation assess analytic sensitivity, analytic specificity, accuracy (assay concordance) and precision (inter-run and inter-operator variability)?

  2. What is the minimum number of positive and negative cases that need to be tested to analytically validate an immunohistochemical assay for its intended use(s)? Such uses include identifying cell lineage (non-predictive marker), determining patient treatment (predictive marker), and identifying infectious organisms and rare antigens. Should expression levels be specified for positive cases?

  3. What parameters should be specified for the tissues used in the validation set for the following?
    1. Cytology specimens
    2. Minimum tissue size or minimum quantity of cells
    3. Neoplastic vs nonneoplastic tissues

  4. How do the following preanalytic variables influence analytic validation?
    1. Type of fixative
    2. Type of decalcification solution
    3. Time in decalcification solution
    4. Validation tissues processed in another laboratory

  5. What conditions require assay revalidation?