Immunohistochemistry (IHC) is a technique which is used to study the tissue distribution of a defined target. It relies on antibodies to detect specific proteins in a sample of tissue which has been paraffin embedded or frozen and can provide a wealth of data regarding protein localization and relative levels of expression. In a clinical laboratory setting this information is used in the diagnosis of various disease conditions, as well as to help predict prognosis and to evaluate a patient’s response to treatment. It can also be used to advance understanding of disease etiology and progression.
IHC for cancer diagnosis
IHC is regularly employed to diagnose cancers since tumor cells often present specific de novo antigens or exhibit irregular levels of protein expression in comparison to healthy tissue samples. It can facilitate the qualification of a cancer as benign or malignant, can allow clinicians to determine the grade of a tumor, and enables identification of the tissue of origin to drive appropriate therapeutic intervention.
A multitude of IHC markers have been identified for clinical oncology, some which are unique to a particular type of cancer and others that demonstrate expression across a range of tumors. It is common for researchers to simultaneously evaluate multiple markers, the detection of which relies on the use of high quality, clinically-validated antibodies to stain biopsies which have been acquired from both healthy and diseased tissue.
Generation and interpretation of clinical IHC data
Once a tissue sample has been obtained for IHC analysis, it is either fixed and then paraffin embedded or is immediately frozen in liquid nitrogen. Although the subsequent downstream staining process differs depending on the method of sample preservation (for example paraffin embedded sections often necessitate antigen retrieval, whereas this step is not usually required for frozen tissue sections where fixation is mild), a typical IHC staining protocol involves permeabilization, blocking, immunostaining and detection. Antibody binding is visualized using detection moieties which include fluorescent dyes, enzymes or colloidal gold. These may be directly conjugated to the primary antibody or attached to an appropriate secondary reagent. As for any experimental protocol, each step of the procedure requires careful optimization.
For the correct interpretation of clinical IHC data, it is essential to understand the biological and physiological relevance of any markers. A literature review can provide information such as the expected distribution pattern and expression level of the antigenic target. Additionally, it can inform the selection of relevant positive and negative control tissue samples.
Antibodies for clinical IHC
It is fundamental that trusted and well-validated antibodies are used for clinical IHC. These specialized reagents undergo rigorous characterization to assess specificity and sensitivity and are frequently compared to other antibodies raised against the same target to ensure accurate and consistent staining.
In addition to staining positive and negative tissue samples, it is good practice to test any antibodies in at least one other non-IHC method. This could, for instance, be immunocytochemical staining of relevant cells fixed within microplate wells, or Western blot analysis of lysates, recombinant proteins or knockout samples. The inclusion of appropriate controls is vital to any clinical IHC experiment, and antibody-based controls should include staining in which the primary antibody or secondary antibody has been omitted, as well as isotype controls. The latter involves incubation of the sample with isotype matched primary antibodies that have no specificity to the antigenic target. The aim of these controls is to identify the source of any background staining which may be observed.