Standardization of Diagnostic Immunohistochemistry: Literature Review and Geisinger Experience

Abstract

This review article delineates some critical points in preanalytic, analytic, and postanalytic phases; reiterates some important questions, which may or may not have a consensus at this time; and updates the newly proposed guidelines on antibody validation from the College of American Pathologists Pathology and Laboratory Quality Center. Additionally, the article intends to share Geisinger's experience with (1) testing/optimizing a new antibody and troubleshooting; (2) interpreting and reporting immunohistochemistry assay results; (3) improving and implementing a total immunohistochemistry quality management program; and (4) developing best practices in immunohistochemistry.

The mutational spectrum of FOXA2 in endometrioid endometrial cancer points to a tumor suppressor role

“…Using IHC we saw variable FOXA2 expression in EEC, both between tumors, and within tumor samples (Supplemental Fig.4E-I). Quantitative IHC on archived old tissue microarrays was unreliable likely due to epitope stability and/or reduced epitope retrieval for older samples stored in paraffin blocks 25 . Thus mRNA qRT-PCR and protein western blot assays were utilized for evaluation and comparisons of FOXA2 expression in EEC.…”

An Introduction to the Performance of Immunohistochemistry

“…The use of IHC has recently further expanded to assess predictive and prognostic biomarkers in many malignancies including those of the breast, gastrointestinal tract, lung, hematolymphoid and central nervous systems (9, 10). Guidelines for the standardization and analytic validation of immunohistochemical tests have been established by the College of American Pathologists (1, 11). …”

“…The primary antibody, which can be monoclonal or polyclonal, is titrated to optimize contrast between positively staining tissue and nonspecific background staining, with the highest primary antibody dilution to prevent waste (1, 7). In general, monoclonal antibodies, which target a single epitope, tend to be more specific while polyclonal antibodies, which can bind many different epitopes, tend be more sensitive (2).…”

“…This may be combined with various combinations of dilutions of the secondary antibody in the setting of the particular AR method and chromogen to produce optimum staining (7). For the initial titration, an antibody concentration of 1 to 5 μg/mL is usually recommended (1). …”

“…A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (1) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (2, 3) and automated methods for high volume processing with reproducibility (4). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor’s site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining.…”