Validation and optimization; both are important processes in histology, but they are not the same thing and can not be used interchangeably. Optimization, thought of simply, is finding the best result. The process of optimization is a technical one, that involves adjusting various conditions like time/temperature, chemical concentration of reagents, to get the greatest amount of specific staining with the least amount of non-specific staining; i.e. the best possible result. This is done on a small number of specimens.
Validation, on the other hand, is a clinical process of confirming that the optimized protocol is applicable on a larger scale. Validation is essentially to confirm that the reactivity observed is appropriate for everything the assay is intended to do, and that you’re seeing consistent, accurate, reproducible, results on a number of different specimens containing varying degrees of target-antigen expression. Validation is required for putting a permanent protocol in place that is documented and what everyone follows in future use of the process.
So, when is something considered validated? It may seem subjective, but something is validated basically just when it is producing consistent results, and the evaluator has signed off on these results being acceptable. The pathologist should be involved in the validation process and selection of control samples. It is important to keep in mind however, that there is only going to be so much of an antigen to detect and you can’t manipulate the results to accommodate personal preferences. Staining should be biologically correct, meaning that if there is low antigen expression, you’re going to have some poorly differentiated staining. You don’t want to manipulate conditions to produce well differentiated staining in every scenario, as you would be getting false results.
Even after something is validated, it is necessary to revalidate if anything in the process, including equipment, reagents, etc. changes.
Learn more in this webinar, Optimization and Validation of Immunohistochemistry Protocols, available on NSH’s Online Learning Center as part of the 2020 Virtual Convention IHC Package or as a solo webinar.