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Immunohistochemistry and Special Stains in the Diagnosis and Classification of Cancer

Nyamweya is a Kenyan scholar who has done many years of research on a diversity of topics


Immunohistochemistry (IHC) involves the use of polyclonal and monoclonal antibodies to detect specific antigens existing in tissue sections for cancer classification and diagnosis. It is suited for determining the tissue distribution for a specific antigen to aid the diagnostic effort by the surgeon (Sharma et al, 2017). Its application requires the use of biopsies, which undergoes incubation with suitable antibody. The development of IHC involves the use of localized pneumococcal antigens and enzymes such as alkaline phostatase and peroxidease. The aim is to limit the damage to the cell or tissue during the staining process by reducing the number of antibodies.

Fig 1: Epitopes and the Paratopes

The IHC uses specific antigen-antibody reactions as opposed to the enzyme staining techniques for the tissue, proteins and enzymes identification. It is used to predict the tumors’ prognosis by identifying the oncogenes, enzymes, tumor cells, tumor suppressor genes and specific antigens. Physicians use specific tumor markers to diagnose cancer as malignant or benign, identify both stage, grade and the cell type for the tumor. Also, the physician defines the metastasis source to identify the site where the tumor primarily originated (Sharma et al, 2017). IHC targets specific protein markets for accurate tumor diagnosis and classification.

Fig 2. Summary of Immunohistochemistry (IHC) Laboratory Procedure

The procedure involves the application of primary antibodies, the monoclonal and polyclonoal, to bind with the target antigen in the first stage. Next is to bind the primary antibodies with the secondary antibodies through an indirect IHC process. A detection system then builds at the secondary antibodies. Horseradish Peroxidase (HRP) enzyme is utilized in the detection process. Finally, there is the formation of a chromogen, either DAB (brown) or AP (red) visible under a microscope (Yatabe et al, 2019). The formation of the chromogens marks a typical IHC stain with the visibility of brown precipitate indicating the target antigen, Cytokeratin 5. The location, pattern and amount of staining are useful information for diagnosis.

Specific Stains

Specific Stains uses different staining techniques to identify a particular tissue or cell using dye. There are three basic types of staining including Trichome staining, Haematoxylin and Eosin and the Periodic Acid Schiff. The most commonly used Special Stains include those that target connective tissues, Trichrome-Masson. In this case, three dyes are utilized in the analysis, collagen fibres (blue), muscles (red) and nuclei (black). In cancer diagnosis, the three dyes are used to distinguish between collagen present in tumors and the smooth muscles, hence detect an increase in collagen (Yatabe et al, 2019). Minimum dosage and time for proteolysis digestion are recommended to avoid denaturing the epitopes and tissue structure. Excessive heating might also lead to antigen loss from protein denaturalization.

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The Verhoeff-van Gieson’s Elastic Stain is suitable for tumor diagnosis and classification in blood vessels (Shedge et al, 2020).

Fig 3: The Verhoeff-van Gieson’s Elastic Stain Staining Process

During the staining process, it is important to enhance the dehydration process to remove water from the target tissues. Next is to embed the tissue with wax to extract the cellular structures. There is the sectioning of the tissue through the preparation of ribbons to help in the microscopic examination. Lastly, the antigens are retrieved by identifying the markings from the dye. The procedure involves the use of ferric chloride, sodium thiosulfate and van Gieson’s solution (Shedge et al, 2020). The results are as shown below in Fig 3 and Fig 4.

Fig 4. Colageno: rojo Fig 5. Musculo: Amarillo, Fibras elasticas: negro


Sharma, S., Kamala, R., Nair, D., Ragavendra, T. R., Mhatre, S., Sabharwal, R., ... & Rana, V. (2017). Round cell tumors: classification and immunohistochemistry. Indian journal of medical and paediatric oncology: official journal of Indian Society of Medical & Paediatric Oncology, 38(3), 349.

Shedge, S., Roy, P., Shedge, A., & Doshi, M. A. (2020). Periodic acid schiff (PAS) staining: a useful technique for demonstration of carbohydrates. Medico Legal Update, 20(2), 353-357.

Yatabe, Y., Dacic, S., Borczuk, A. C., Warth, A., Russell, P. A., Lantuejoul, S., ... & Moreira, A. L. (2019). Best practices recommendations for diagnostic immunohistochemistry in lung cancer. Journal of Thoracic Oncology, 14(3), 377-407.

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