Immunohistochemistry

What is immunohistochemistry (IHC)?

Immunohistochemistry (IHC) is a lab technique pathologists use to look for signs of disease in a tissue sample. A pathologist uses lab tests to diagnose medical conditions.

As part of your diagnosis, a healthcare provider may remove tissue and send it to a lab for testing. For example, they may remove part of a tumor and send it to a lab to test for cancer cells. This is called a biopsy. An IHC is just one method a pathologist may use to study the sample once it arrives at the lab.

IHC is the most common type of immunostaining. Immunostaining involves using antibodies and special markers to “label” parts of a tissue sample so they’re easier for pathologists to identify.

The word “immunohistochemistry” provides clues about what’s involved:

• Immuno means relating to your immune system. Your immune system detects substances in your body (called antigens). It makes antibodies to find and destroy antigens that don’t belong, like pathogens (viruses, bacteria, fungi and parasites) and cancer cells. An IHC uses the antigen-finding properties of antibodies to detect and grab onto antigens in a tissue sample. Antibodies stain the sample so the pathologist can see the antigens they’re attached to when viewed beneath a microscope.
• Histo means tissue. An IHC examines a tissue sample.
• Chemistry studies the tiny building blocks that make up all matter, including human tissue. An IHC uses a microscope to see otherwise invisible antigens that may indicate diseases.

When is immunohistochemistry performed?

An IHC can be used to:

• Diagnose a condition: An IHC allows healthcare providers to diagnose conditions like cancer. It can help providers determine the type of cancer (for example, carcinoma, melanoma or sarcoma). It also allows healthcare providers to pinpoint the origins of cancer that’s spread (metastatic cancer).
• Determine prognosis: An IHC can determine how high-risk, or aggressive, a cancer is. It also can help providers stage and grade cancer. This information can help providers determine the best options for treatment.
• Predict treatment response: An IHC can identify characteristics of tumor tissue that provide clues about how cancer may respond to treatment. For example, pathologists can identify breast and prostate cancers that are likely to grow in the presence of certain hormones, like estrogen and testosterone. These cancers may respond best to treatments that block these hormones (hormone therapy).
• Monitor treatment response: An IHC allows providers to monitor whether treatments are working to rid your body of the disease.

Researchers also perform IHC to develop new drug treatments. IHC helps researchers learn more about how the smallest parts of your body work, like your cells and the molecules inside them. IHC provides insight into how diseases affect these processes and what treatments can help.

What diseases can be diagnosed by immunohistochemistry?

Healthcare providers most commonly use IHC to diagnose cancer, but it can also diagnose other conditions, including Alzheimer’s disease, Parkinson’s disease and muscular dystrophy.

It can identify pathogens that cause infection, too. The first successful IHC stain occurred in 1941, when researchers (Coons, et al.) identified the bacteria associated with pneumonia (pneumococcus) in a tissue sample.

How does the test work?

An IHC uses antibodies to detect a target antigen in a tissue sample. The target antigen is a marker, indicating a specific disease is present. If the antibody recognizes the antigen, it will attach (bind) to it. The binding process is similar to a lock (antigen) and key (antibody). If the antibody binds to the antigen, the tissue sample will stain a certain color when viewed beneath a microscope.

It works like this:

• A pathologist links an antibody to an enzyme that will react if the antibody binds to the target antigen.
• The antibody binds to the target antigen if it’s present.
• The attachment causes the enzyme to react.
• The reaction causes the tissue sample to stain a certain color when viewed beneath a microscope.

For the results to be reliable, pathologists must accurately complete multiple steps.

Preparing the sample

Preparing the sample ensures it stains correctly. If an antigen is present, it stands out in colored segments against the background. To prepare the sample, pathologists:

1. Preserve the tissue. Tissue consists of cells that die over time. Preserving, or “fixing,” the tissue slows the process. Fixation maintains the tissue’s structure, so it stains effectively. One of the most common substances used to fix the tissue is formalin, a formaldehyde solution.
2. Ensure antigens are accessible. The fixation process can sometimes block parts of the antigen so the antibody can’t bind to it. A process called antigen retrieval can re-expose the antigen’s binding points so antibodies can attach.
3. Block similar structures where an antibody may bind. Sometimes, antibodies bind to substances similar in structure to the target antigen — but that aren’t the same. Pathologists block these structures beforehand so the antibody only attaches to the target antigen.

Selecting antibodies

Pathologists select antibodies known to bind to the target antigen. IHC uses either polyclonal antibodies or monoclonal antibodies.

• Polyclonal antibodies:A mix of different antibodies. These antibodies may attach to multiple binding sites on an antigen.
• Monoclonal antibodies: Identical copies of the same antibody. Monoclonal antibodies will only attach to a specific binding site on an antigen.

Detecting the antigen

Pathologists prepare the antibody to stain tissue containing the antigen.

Pathologists:

1. Link the antibodies to an enzyme. Examples of enzymes include horseradish peroxidase and alkaline phosphatase.
2. Add the antibody with the enzyme to the tissue sample.
3. View the sample underneath a microscope.
4. Look for staining that indicates the antigen is present.

The first successful IHC used a similar process. Instead of linking the antibody to an enzyme, the researchers linked it to a fluorophore. A fluorophore absorbs light and reflects it. The fluorophore stains the sample when viewed under a fluorescence microscope. This technique is now considered a different type of immunostaining called immunofluorescence.

What are the limitations of immunohistochemistry?

There aren’t standard guidelines for each step in immunohistochemistry. Different labs use different techniques, which means results may vary.

Also, recent research has shown that not all antibodies available for IHC do what they’re supposed to — that is, detect the target antigen in a sample. If there are problems with the antibodies, a test may give results that are false, including:

• False-positive: An IHC detects an antigen that is not present.
• False-negative: An IHC doesn’t detect an antigen that is present.

Labs must have quality controls in place so that every step preserves the tissue and ensures a high-quality stain. To improve IHC accuracy, pathologists can test antibodies on tissue known to contain the target antigen to ensure it stains before testing an unknown tissue sample.

How accurate is immunohistochemistry?

When performed correctly and with quality controls in place, immunohistochemistry is a reliable method for cancer diagnosis. One study reports that IHC can accurately identify the primary location of metastatic cancer with 70% to 90% accuracy.

Is immunohistochemistry a biopsy?

No, but they’re related. Immunohistochemistry is one type of lab technique a pathologist may perform on a tissue sample collected for a biopsy.

A note from Cleveland Clinic

Immunohistochemistry (IHC) allows pathologists to achieve something remarkable. It enables them to see signs of disease invisible to the naked eye. IHC uses your body’s powerful fighters, antibodies, to expose harmful, microscopic substances that cause disease. It’s just one method that allows scientists to study, diagnose and — most importantly — fight cancer from the lab.