Immunohistochemistry

Immunohistochemistry (IHC) is a procedure done on tissue samples that allows unequivocal identification and localization of specific antigens that may help in the diagnosis of disease. It is especially useful in microscopic analysis of cancer tissue, where the presence or absence of particular molecules may confirm or refute a diagnosis, or guide therapy decisions.

Other Names

When carried out on individual cells (as opposed to intact samples taken from a block of tissue), the procedure is known as immunocytochemistry (ICC). The procedure is also known as immunostaining.

Description

A breast cancer sample where immunostaining shows expression of the estrogen receptor. Source:Cancer.gov

Why Immunohistochemistry Is Done

IHC and ICC are performed to identify specific antigens on or in tissues or cells. There are several cases where the test is especially useful. In cases of undifferentiated malignant tumors, the presence of antigens (such as tyrosinase) can confirm the origin of the cells, identifying the tumor (in this example, as a melanoma). Immunostains are also used to subclassify leukemias and lymphomas. They are also used to analyze poorly differentiated or diagnostically challenging cancers, such as sarcomas,^[1] gynecological malignancies such as ovarian carcinomas^[2] and neural tumors.^[3]

Preparation

Samples taken as part of larger surgeries (such as mastectomy) require general anesthesia preceded by an overnight fast on the part of the patient. Simpler biopsy procedures (such as bone marrow biopsy) may require only local anesthesia with or without sedation.

How Immunohistochemistry Is Done

The first step is taken by the surgeon or the pathologist, who decides that an immunostain is necessary based on the anticipated treatment or the microscopic appearance of the tumor. Then, the antigen(s) to be interrogated must be determined. Usually this is straightforward, since many samples routinely require immunostains for specific antigens—breast cancer samples, for example, are routinely stained for estrogen receptors. Sample acquisition, immunostaining, and diagnostic interpretation are the next steps. Individual cells for ICC can be scraped or brushed from surfaces, aspirated from cavities or compartments via a fine needle, or concentrated from body fluids by centrifugation. For IHC, tissue samples are usually obtained by standard biopsy or open procedures. Routine tissue processing involving formalin fixation, paraffin embedding, and sectioning is suitable for many antigen-antibody interactions, but in some cases the interaction requires special sample handling such as frozen sectioning (to preserve the antigen) or heating (to increase exposure or "retrieve" the antigen). Immunostaining itself requires exposure of the sample to a specific (primary) antibody under conditions that allow antigen-antibody binding to take place. Hundreds of primary antibodies against tumor-specific molecules are commercially available. In some cases, the primary antibody is conjugated to an enzyme such as horseradish peroxidase (HRP). This format is rapid, but this direct conjugate-labeled method has low sensitivity and requires high concentrations of antibody. A more typical procedure involves a different (secondary) antibody that binds to the constant (Fc) region of the primary antibody and is linked to HRP or another enzyme. Since multiple secondary antibodies can attach to a single primary antibody, sensitivity is improved. Other methods are possible, but all result in localization of a detectable "tag" molecule near the antigen(s) of interest. Typically used tags include enzymes such as HRP, alkaline phosphatase, glucose oxidase, and beta-galactosidase. Antibody-conjugated fluorescent molecules are sometimes used as tags, but visualization requires specialized light sources and filters, and fluorescent tags degrade quickly. If higher magnification is needed, then antibody-conjugated gold particles can also serve as tags and are visualized with the electron microscope. Gold particle sizes can be tightly controlled, facilitating double labeling in individual sections.

Signal generation on samples with attached enzyme tags is achieved by incubation with a color-producing (chromogenic) substrate system that results in local production of insoluble precipitates. Diaminobenzidine or aminoethylcarbazole are popular substrates for HRP because the catalysis products are stable and easily identified in the microscope as brown or red deposits, respectively. Glucose oxidase and its substrate tetrazolium are popular for double-labeling techniques, since the blue reaction product is easily distinguished from products of HRP. The final processing step is counterstaining and mounting slides. Counterstaining is necessary to visualize cells and structures around the immunostain. Hematoxylin, which stains nuclei blue, is a popular choice, since many immunostains identify cell surface antigens. Appropriate positive and negative controls should always be stained and evaluated in parallel with the patient sample.^[4]

Risks

The procedure poses no special risk to the patient. The reagents themselves pose no special risk to the laboratory's technical staff. .

References

1. ↑ Gupta R, Gupta S, Aggarwal D, Singh S. Primary pleomorphic undifferentiated sarcoma of the kidney: a rare renal tumor. Indian J Pathol Microbiol. 2008 Oct-Dec;51(4):573-6. Citation | PDF
2. ↑ Prat J. Ovarian carcinomas, including secondary tumors: diagnostically challenging areas. Mod Pathol. 2005 Feb;18 Suppl 2:S99-111. Abstract | Full Text | PDF
3. ↑ Dedeurwaerdere F, Giannini C, Sciot R, et al. Primary peripheral PNET/Ewing's sarcoma of the dura: a clinicopathologic entity distinct from central PNET. Mod Pathol. 2002 Jun;15(6):673-8. Abstract | Full Text | PDF
4. ↑ Ramos-Vara JA. Technical aspects of immunohistochemistry. Vet Pathol. 2005 Jul;42(4):405-26. Abstract | Full Text | PDF

External Links

Immunoquery is a web-based service that compiles lists of antibodies that have been used to interrogate a wide range of malignancies.