Immunohistochemistry Basics & Diagnosis of Neoplasia

By Angela Ellie, DVM, PhD

Immunohistochemistry has been available for many years, but is becoming much more widely used with advances in automated staining processes, available antibodies, and knowledge. The principle behind the technique is relatively simple: all cells have antigens, and the distribution of antigens is tissue-dependent.

In the case of tumors, certain cell types express unique markers or antigens that can be used to identify their tissue of origin. Antibodies designed to specifically bind to those antigens are used to identify whether those antigens are present in a tissue section. Since antigens and antibodies are not visible on routine histologic sections, the antibody is bound, either directly or indirectly, to a colorimetric agent, or chromagen, which allows the pathologist to visualize the antigen if present.

Although the technique sounds relatively simple, there are many potential pitfalls. Antibodies may bind nonspecifically to other antigens, resulting in false-positive staining. If reagents are not properly washed off between steps, it may appear that the antibody has bound to the antigen when it has not, again resulting in false-positive staining. Antibodies also have to be diluted to an optimum concentration prior to use. Improper dilution can result in over or under staining, resulting in either false positives or false negatives or equivocal results.

Immunohistochemical stains are based on markers expressed by normal cells. Although neoplastic cells typically express these same markers, anaplastic tumors can either lose or gain expression of normal markers.Many of these cell markers are expressed by a wide variety of cells and are used only to narrow down the list of differentials and not to make a specific diagnosis.

Cost of immunohistochemistry is variable and is dependent on the specific antibody or antibodies used. In cases where immunohistochemistry would be helpful, it will be suggested by the pathologist in the biopsy report. Immunohistochemistry is performed on formalin-fixed, paraffin-embedded tissues that may have been archived for several years at the diagnostic laboratories after sample submission. Therefore, it is not usually necessary to submit additional tissues for immunohistochemistry as the originally submitted tissues will still be available for use.

The following are some of the most common immunohistochemical markers that we use:

  • Vimentin: This is the most ubiquitous intermediate filament in the body. It is expressed in mesenchymal cells including fibroblasts, myocytes, melanocytes, endothelium, adipocytes, chondrocytes, lymphocytes, and macrophages. It is typically used to verify that a tumor is of mesenchymal rather than epithelial origin.
  • Cytokeratins: This is actually a group of intermediate filaments of varying molecular weights. They are expressed in epithelial cells. Tumors that express cytokeratin include carcinomas, mesothelioma, chordoma, thymoma, synovial sarcoma, and meningioma. This stain often is used in conjunction with vimentin to differentiate between mesenchymal or epithelial origin in poorly differentiated tumors.
  • Desmin: This is normally expressed in skeletal and smooth muscle, and cardiac muscle. It can also be used to help identify leiomyomas/leiomyosarcomas and rhabdomyosarcomas. Other commonly used antibodies to specifically diagnosis leiomyoma/leiomyosarcoma or rhabdomyosarcoma are smooth muscle actin and skeletal muscle myosin, respectively.
  • S-100: This is a relatively nonspecific marker that stains cells of neural crest origin including glial cells, adipocytes, chondrocytes, and melanocytes. Tumors that are S-100 positive include schwannomas, neurofibromas, astrocytomas, oligodendrogliomas, nerve sheath tumors, chordomas, chondrosarcomas, melanomas, liposarcomas, and synovial sarcomas.
  • Melan-A: This is expressed by melanocytes and is used to identify amelanotic melanomas.
  • CD3: This is a marker for T-lymphocytes.
  • CD79: This is a marker for B-lymphocytes and plasma cells. CD3 and CD79 are typically used together either to verify that a round cell tumor is a lymphoma or to establish that a lymphoma is of B- or T-cell origin. Other antibodies commonly used in immunohistochemistry for detecting B-lymphocytes include CD20 and BLA-36.
  • CD18: This is a panleukocyte marker including lymphocytes and macrophages. It is often used to identify tumors of histiocytic origin but CD3 and CD79 often have to be used concurrently to rule out lymphoma. Substantial inflammation within a tumor can make interpretation difficult since normal inflammatory cells also express CD18.

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