Immunocytochemistry and immunohistochemistry
Immunocytochemistry (ICC) and immunohistochemistry (IHC) are laboratory techniques used to detect and visualize specific antigens in cells and tissue sections, respectively. ICC focuses on individual cells, while IHC is applied to tissues, making these techniques essential for diagnosing and classifying various cancers, including lymphomas, leukemias, and breast cancer. By identifying specific antigens, these methods can help determine the origin of undifferentiated tumors and even subclassify tumors that may appear histologically similar but have different prognostic implications.
The procedures involve several steps, including sample collection, immunostaining with antibodies, and diagnostic interpretation. Antigens are typically localized using primary antibodies, often linked to enzymes or fluorescent molecules, which generate detectable signals. For accurate results, it is crucial to include positive and negative controls during the staining process. The techniques pose minimal risks to patients and are generally performed in conjunction with other surgical procedures or biopsy methods. Ultimately, the information gleaned from ICC and IHC plays a significant role in guiding treatment decisions and patient management in oncology.
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Subject Terms
Immunocytochemistry and immunohistochemistry
ALSO KNOWN AS: Immunostain, ICC, IHC
DEFINITION: Immunocytochemistry (ICC) refers to using antibody-based protocols on cells to identify and localize visually specific molecules (antigens). Immunohistochemistry (IHC) refers to similar procedures carried out on tissue sections.
Cancers diagnosed: Undifferentiated or metastatic cancers, leukemias, lymphomas, breast cancers
Why performed: IHC and ICC identify specific antigens on or in tissues or cells. These procedures are valuable in several aspects of the evaluation of human cancers. In undifferentiated malignant tumors, the presence of antigens (for example, tyrosinase) can confirm the origin of the cells, identifying the tumor (in this example, as a melanoma). In metastatic tumors of unknown origin, the presence of antigens with highly restricted specificity can identify the primary site, as in the case of prostate-specific (PSA) expression by a skeletal lesion. Occasionally, subclassification of histologically similar tumors has prognostic implications, as in the case of embryonal carcinomas, distinguished from seminomas by keratin.
Leukemias and lymphomas are especially amenable to IHC-assisted diagnosis and classification because white blood cells' abundant and diverse surface antigens. However, flow cytometry is sometimes favored in this class of malignancies. Several antigens of therapeutic importance can be detected by IHC, including estrogen and progesterone receptors in breast cancer. Finally, IHC can identify antigens associated with tumor cells’ growth rate, such as Ki-67. While primarily performed after the sample collection procedure, immunostaining can also be performed rapidly in intraoperative consultations.
Patient 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.
Steps of the procedure: The pathologist's first task is to construct a differential diagnosis and decide whether immunostaining is necessary. If so, the antigen or antigens to be interrogated must be selected based on specific hypotheses. 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 suits many antigen-antibody interactions. 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 requires exposure of the sample to a specific (primary) antibody under conditions that allow antigen-antibody binding to occur. Hundreds of primary antibodies against tumor-specific molecules are commercially available. The primary antibody is sometimes conjugated to an enzyme called horseradish peroxidase (HRP). This format is rapid, but this direct conjugate-labeled method has low sensitivity and requires high concentrations of antibodies. A more typical procedure involves a different (secondary) antibody that binds to the primary antibody's constant (Fc) region 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 localizing 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, 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 incubating with a color-producing (chromogenic) substrate system that produces insoluble precipitates locally. 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 popular since many immunostains identify cell-surface antigens. Appropriate positive and negative controls should always be stained and evaluated in parallel with the patient sample.
After the procedure: Samples collected as part of larger surgical procedures require standard postoperative care. Simpler biopsy collection procedures require that the biopsy site be kept clean and dry until fully healed. Patients and caregivers are instructed to monitor for signs of infections.
Risks: Immunostaining itself poses no additional risks to the patient.
Results: Meaningful results from the patient sample are only possible with appropriate staining of parallel positive and negative control samples. The most important attribute of an adequate positive control is the heterogeneous distribution of the stain within a cell and among groups of cells. Results for patient samples are usually given semiquantitatively and include estimates of staining intensity, cellular distribution (membranous, cytoplasmic, or nuclear), and abundance of positively staining cells. The report consists of an interpretation that is either favored or ruled out by the observed staining pattern.
Bibliography
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