ABSTRACT
The development of molecular diagnostics in cancer includes careful considerations around the molecular endpoint, the assay specificity, the assay sensitivity, robustness and standardisation. Within the present chapter, the molecular endpoint is a protein and the diagnostic assay includes immunohistochemistry (IHC) as the main technology. However, even if a protein is the molecular endpoint the activation state of the protein has to be decided upon before designing the test assay. Activated proteins may be a stronger predictive endpoint as compared to non-activated proteins. To differentiate between proteins and activation stage it is extremely important to ensure the optimal specificity of the assay. In respect of an IHC assay, the specificity is very much dependent on the specificity of the primary antibody, which can be assured by several technical tools. In contrast, the IHC assay sensitivity is more dependent on pre-analytical issues as well as the analytical visualisation process. New visualisation techniques will in a short time enable “single molecule detection” which will supersede all previous IHC sensitivity levels and revolutionise IHC to become a strong quantitative molecular diagnostic technique. The pre-analytical parameter of tissue fixation is still a challenge when it comes to standardisation of the IHC assay, but the future inclusion of detecting 2housekeeping proteins (internal tissue controls) will indeed increase the standardisation and robustness levels and thereby optimise the IHC assay quality and patient care.
