Principles of Analytic Validation of Immunohistochemical Assays

All clinical practice guidelines issued by the College of American Pathologists (CAP) Pathology and Laboratory Quality Center for Evidence-based Guidelines (Center) are reviewed every five years to determine if the original guideline remains current. The field of clinical immunohistochemistry (IHC) has evolved significantly since the publication of the original guideline in 2014; as such, the guideline was updated based on a systematic review of the medical literature.

• Recommendation 6 provides validation/verification recommendations for IHC assays that have separate scoring systems employed depending on tumor site and/or clinical indication (eg, human epidermal growth receptor 2 [HER2], which has different scoring systems depending on tumor site, and programmed death receptor-1 [PD-L1], which employs different scoring systems based on tumor site and/or tumor type). The recommendation stipulates that laboratories should separately validate/verify each assay-scoring system combination.
• Since the publication of the original guideline, laboratories have frequently requested more definitive validation guidelines for IHC performed on cytology specimens that are not fixed identically to those tissues used for initial assay validation. Based on this feedback and literature published since the initial guideline, conditional recommendation 9 and statement 10 require that laboratories perform separate validations with a minimum of 10 positive and 10 negative cases for IHC performed on specimens fixed in alternative fixatives. The guideline panel recognize that these new recommendations will impose an added burden to laboratories; however, the literature has shown variable sensitivity of IHC assays performed on specimens collected in fixatives often used in cytology laboratories compared with formalin-fixed, paraffin embedded (FFPE) tissues.
• The original guideline and associated publications recommended varying concordance requirements for estrogen receptor, progesterone receptor, and HER2 IHC performed on breast carcinomas. These variable concordance requirements have been uniformly set to 90% for all IHC assays in this updated guideline (recommendation 2).
• This update provides more explicit verification requirements for unmodified United States Food and Drug Administration (FDA) approved/cleared assays (statement 5).

As with any clinical evidence-based guidelines, laboratories are not required to follow the recommendations. While these recommendations may be incorporated into future versions of the CAP Laboratory Accreditation Program (LAP) Checklist; the major changes to this guideline noted above are not currently required by LAP or any regulatory/accrediting agency. Laboratories are encouraged to adopt these evidence-based recommendations to increase the quality and safety of these clinically important assays.

Although this guideline prospectively applies to new assays used on patient specimens, The Clinical Laboratory Improvement Amendments of 1988 (CLIA), and by extension, CAP LAP, requires laboratories to validate/verify the performance characteristics of all assays before issuing results on patient specimens. Thus, even if an assay has been in clinical use, the lack of documentation of a previous analytic validation may result in a citation by an accrediting agency. We remind the reader that both the CAP and other organizations have promoted validation guidelines in the past, providing timely and relevant guidance to Laboratory Directors prior to this update.

It is NOT the intention of the new statements 9 and 10 to mandate revalidation of all IHC assays that are currently performed on cytology specimens. However, as stipulated in the original guideline, some sort of validation study should have been performed on cytology specimens whose tissues were not fixed in the same manner as the tissues used for original assay validation. If documentation of such a study does not exist, the laboratory may not be in compliance with an accrediting agency.

Assessment of these two variables is not explicitly included in the updated guideline. However, it would be wise for IHC laboratory medical directors to design both a validation plan and a quality management plan that evaluate these two variables. A reasonable strategy might include running the validation set on different instruments over a period of a few days. Each of these runs should be performed by a different laboratory personnel.

Statement 1 provides for many different comparators as models for validation/verification study design. These options are provided as part of this statement because most IHC assays do not have an applicable gold standard suitable for determining the performance characteristics of the new assay. As guidance, the following list is ordered from the most stringent to the least stringent comparator. It is emphasized that an IHC medical director may choose any of these options as the basis for an IHC validation study design:

• Comparing the new assay’s results to IHC results from cell lines that contain known amounts of protein (“calibrators”).
• Comparing the new assay’s results with results of a non-immunohistochemical method, such as flow cytometry or fluorescent in-situ hybridization.
• Comparing the new assay’s results with the results of testing the same tissues in another laboratory using a validated/verified assay.
• Comparing the new assay’s results with the results of prior testing of the same tissues with a validated/verified assay in the same laboratory.
• Comparing the new assay’s results with the results from testing the same tissues in a laboratory that performed testing for a clinical trial.
• Comparing the new assay’s results with the expected architectural and subcellular localization of the antigen.
• Comparing the new assay’s results against percent positive rates documented in published clinical trials.
• Comparing previously graded tissue challenges from a formal proficiency testing program (if available) with the graded responses.