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Immunohistology


Immunohistology is a branch of immunology that deals with the application of immunologic methods to histology.

The rapid development of immunohistochemistry, a morphology-based technique, has come about through refinements in detection systems and an increasing range of sensitive and specific antibodies that have allowed application of the technique to formalin-fixed, paraffin-embedded tissues. The introduction of heatinduced antigen retrieval has been a significant milestone to compliment these developments so that the immunohistochemistry is firmly entrenched as an indispensable adjunct to morphologic diagnosis. Although this ancillary stain was initially used in a qualitative manner, problems surrounding the many variables that influence antigen preservation in formalin-fixed, paraffin-embedded tissues were not a major issue and laboratories strived to optimize their staining protocols to the material they accessioned and processed. The advent of personalized medicine and targeted cancer treatment has imposed the need to quantitate the stain reaction product and has resulted in calls to standardize the process of immunostaining. A closer examination of the variables that influence the ability to show antigens in formalin-fixed, paraffin-embedded tissues revealed many important variables, particularly in the preanalytical phase of the assay, that are beyond the control of the accessioning laboratory. Although analytical factors have the potential to be standardized, the actions of many pivotal procedures including fixation and antigen retrieval are not completely understood. Postanalytical processes including threshold and cut-off values require consensus and standardization and it is clear that some of these goals can be achieved through the direction of national and international organizations associated with cancer diagnosis and treatment. With the ability to serve as a surrogate marker of many genetic abnormalities, immunohistochemistry enters a new era and the need to better understand some of the mechanisms fundamental to the technique become more pressing and the development of true quantitative assays is imperative. There is also an increasing appreciation that the technique highlights patterns of staining that reflect exquisite localization to organelles and tissue structures that are not appreciable in routine stains, adding a further dimension to morphologic diagnosis.

Immunhistochemistry, when first introduced some 70 years ago, was used in a limited context in diagnostic pathology but subsequent refinements in the procedure and the concomitant development of an ever-increasing range of antibodies resulted in enhanced sensitivity that allowed immunohistochemical staining to be applied to routinely prepared formalin-fixed, paraffin-embedded tissues. The ability to work with formalin-fixed, paraffin-embedded tissues and the sensitivity of the technique has enabled exquisite localization of staining to specific cell structures and organelles. As such, "immunohistology"1 and "immunomorphology" have been proposed as more appropriate terms to emphasize this fundamental attribute of what is primarily a morphology-based investigation. The technique has served as an immensely powerful adjunct to histologic diagnosis and has paved inroads into research, with contributions that compliment and even surpass those of other more established forms of morphologic investigations including histochemistry and electron microscopy.

Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, by colorimetric or fluorescent methods. The immunohistochemist has a number of standard tools to optimize the detection of a particulate analyte. The direct method uses a labeled reagent, such as a fluorescent tag or an enzyme labeled antibody, which can be visualized without further antibody interaction. Indirect methods increase the number of assay steps, but enable the user to amplify their assay for improved sensitivity. For greatest sensitivity, a biotin-streptavidin system should be used.

Another related technique, the ELISpot(Enzyme Linked ImmunoSpot) assay also provides a means of detecting antigens or antibodies in single cells on a solid support. All of the techniques mentioned above rely on a labeled antibody or conjugate as the signaling molecule to detect the protein initially bound to the solid phase. The enzyme labeled reagents are detected with the appropriate chromogenic substrate, which is converted into a colored product. The intensity of signal produced is proportional to the amount of measured antigen bound to the surface.