Question

Describe the principles of immunocytochemistry/immunohistochemistry and how the technique can be usefully employed in the diagnosis and classification of tumours

Answer 1

Principles of immunohistochemistry:

The principle of IHC has existed since the 1930s, but it was not until 1941 that the first IHC study was reported.Coons and his colleagues used Fluorescein isothiocyanate (FITC)-labeled antibodies with a fluorescent dye to localize pneumococcal antigens in infected tissues. With the expansion and development of IHC technique, enzyme labels have been introduced, such as peroxidase[8,9] and alkaline phosphatase. Colloidal gold label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy levels. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography. The aim of IHC is to perform most IHC staining by causing least damage on the cell or tissue, and by using least amount of antibody, it finds a way in the tumor typing and tumor markers.

Principles of immunocytochemistry:

There are very many methods for performing immunocytochemistry – comprehensive accounts are available on the internet (eg under Immunocytochemical Methods). One well established and useful method will be described here – the indirect (two-layer) method using a peroxidase polymer-labelled second antibody.

The antigen of interest is preserved in the cells by fixation with alcohol and/or formalin. The primary antibody, usually raised in a rabbit or a mouse, binds to the antigen and excess is washed off with buffer. The secondary antibody is raised (in goat, or other species) to immunoglobulins of the primary antibody host (eg. goat anti-mouse Ig) and binds to the primary antibody at the site where it is attached to its antigen. The secondary antibody is labelled so that the reaction site can be visualised in the microscope. The most efficient label is polymerised horseradish peroxidase which provides a large amount of enzyme label on the second antibody molecule. Following further washing, the peroxidase is reacted with its substrate, hydrogen peroxide, and a chromogen, usually diaminobenzidine (DAB) which gives a dark brown, insoluble precipitate at the site of reaction. The preparation can then be counterstained with haematoxylin, dehydrated in graded alcohols, cleared in a solvent such as xylene and permanently mounted.

Application of immunocytochemistry/immunohistochemistry:

To predict the prognosis of tumors by identification of enzymes, tumor-specific antigens, oncogenes, tumor suppressor genes, and tumor cell proliferation markers. Analysis of tumors by these methods is a significant improvement over the conventional prognostic considerations by clinical staging and histologic grading. IHC is used for disease diagnosis, drug development, and biological research. Using specific tumor markers, physicians use IHC to diagnose a cancer as benign or malignant, determine the stage and grade of a tumor, and identify the cell type and origin of a metastasis to find the site of the primary tumor. IHC is also used in drug development to test drug efficacy by detecting either the activity or the up- or down-regulation of disease targets.Immunocytochemistry involves mainly small cell blue tumors.Tumors that fall in this category include lymphorma,rhabdomyosarcoma,neuroblastoma,nephroblastoma.

Tumors of Uncertain Histogenesis:

IHC methods have brought about a revolution in approach to diagnosis of tumors of uncertain origin, primary as well as metastatic from unknown primary tumor. A panel of antibodies is chosen to resolve such diagnostic problem cases. The selection of antibodies being made is based on clinical history, morphological features, and results of other relevant investigations. Immunohistochemical stains for intermediate filaments are expressed by tumor cells (keratin, desmin, vimentin, neurofilaments, and glial fibrillary acidic proteins).