IHC stands for Immunohistochemistry, a technique used in pathology to visualize specific molecules within tissue samples. This method combines immunology and chemistry, relying on the highly specific binding reaction between an antibody and its target molecule, known as an antigen. Pathologists use this diagnostic tool to determine the presence, location, and abundance of these target molecules within a biopsy or surgical specimen.
Decoding Immunohistochemistry
The foundational concept of IHC is the specific interaction between an antigen and an antibody, often described as a lock-and-key mechanism. Antigens are the target molecules, typically proteins, present on or within the cells that characterize a specific disease or cell type. Antibodies are specialized immune system proteins that recognize and bind to a unique site, called an epitope, on the target antigen.
To make this binding visible, the antibody is linked to a reporter system. In the most common method, an enzyme, such as horseradish peroxidase (HRP), is attached to the antibody. When the antibody binds to the target antigen, the enzyme is localized precisely at that spot.
A colorless chemical compound, called a chromogen, is then introduced. The enzyme acts as a catalyst, chemically altering the chromogen to produce an insoluble, visible colored precipitate at the site of the antigen-antibody complex. This color change, often brown or red, allows the pathologist to pinpoint where the target protein resides within the tissue’s cellular structure.
The Procedure
The IHC process begins with preparation of the tissue sample, which is typically fixed in formalin and embedded in paraffin wax to preserve cellular architecture. The resulting tissue block is then sliced into very thin sections, often about 4 micrometers thick, using a microtome. These sections are mounted onto glass slides, ready for staining.
A primary step for fixed tissues is antigen retrieval, which uses heat or enzymes to “unmask” binding sites hidden during fixation. The slide is then treated with the primary antibody, which binds directly to the specific antigen of interest. After washing away any unbound primary antibody, a secondary antibody is applied.
This secondary antibody binds to the primary antibody and is often linked to the enzyme that generates the color. Finally, the chromogen substrate is added, reacting with the enzyme to create the colored deposit. A counterstain, such as hematoxylin, is also applied to stain the cell nuclei a contrasting color, providing a background to visualize the tissue structure.
Essential Clinical Applications
IHC is a fundamental tool in the clinical diagnosis of disease, particularly in oncology. A primary application is the differential diagnosis of tumors, where it helps pathologists classify a tumor by identifying specific protein markers. For instance, a panel of IHC stains can differentiate between a carcinoma, a lymphoma, or a melanoma, which may look similar under a standard stain.
IHC is also used to determine the primary site of a metastatic tumor when the original source is unknown. By testing for tissue-specific markers, such as Cytokeratin 7 or Cytokeratin 20, the pathologist can narrow down the potential origins, which guides appropriate treatment. The technique also provides prognostic information about how a tumor is likely to behave. For example, the Ki-67 protein marker, which indicates cell proliferation, is assessed using IHC; high expression suggests a faster-growing, more aggressive tumor.
IHC plays a role in personalized medicine by identifying molecular targets for specific drugs. In breast cancer, for instance, IHC determines the expression of hormone receptors like Estrogen Receptor (ER) and Progesterone Receptor (PR), as well as the HER2 protein. A positive result for ER/PR guides the use of hormone-blocking therapies, while HER2 positivity indicates eligibility for HER2-targeted drugs.
Understanding the Results
A pathologist interprets the final stained slide by examining the tissue under a microscope, looking for the presence, intensity, and location of the colored precipitate. An “IHC positive” result means the targeted antigen was detected, confirmed by the presence of the colored stain. Conversely, an “IHC negative” result means the target protein was not present at a detectable level.
Interpretation goes beyond simple positive or negative findings and includes analyzing the staining pattern. For example, the stain might be localized to the cell nucleus, cytoplasm, or cell membrane, providing information about the protein’s function and cell type. The intensity of the color, ranging from weak to strong, is also evaluated, often using a semi-quantitative scoring system that considers both intensity and the percentage of stained cells. These findings are integrated with other diagnostic information to confirm a diagnosis, predict disease behavior, and select the most effective therapeutic strategy.