Immunohistochemistry

Mesothelioma Immunohistochemistry

Immunohistochemistry, or IHC, is a laboratory test that locates proteins in the cells of a tissue sample by using antibodies that typically bind to the protein being sought out. An antibody is a natural substance the immune system sends out to inhibit a foreign invader. This process is extremely common in the diagnosis of cancer cells.

How Did this Technique Become Accepted in the Diagnosis of Mesothelioma?

Mesothelioma was always difficult to distinguish from adenocarcinoma. Then in the late 1980s, immunohistochemistry became a part of routine pathology. At first there weren’t many biomarkers that were specific to mesothelioma; however, over the years new markers were introduced such as antibodies against:

• Wilms tumor protein 1(1995)
• Calretinin (1998)
• Podoplanin/D2-40 (2005)

These became accepted because they were specific to mesothelioma.

The introduction of these positive markers that are used in conjunction with negative markers, which are biomarkers that are present when there is no mesothelioma, have improved the accuracy of diagnosis. There is still no one reliable mesothelioma immunohistochemical marker. The World Health Organization and the International Mesothelioma Interest Group recommend that when pathologists are performing an immunohistochemical test, they should use a panel of biomarkers made up of at least two positive and two negative.

How is an Immunohistochemical Test Performed?

The biopsied tissue sample is preserved either by embedding it in paraffin wax, or by immersing it in liquid nitrogen to flash freeze it and then placing it in a cryomold. The reason for flash freezing is to provide an option in instances in which the proteins being analyzed are sensitive to the paraformaldehyde used with the paraffin.

The sample is then sliced into thin layers, and those layers are affixed to slides. If the tissue sample was fixed in paraffin, any remaining wax must be removed from the slide otherwise it interferes with the ability of the antibodies to bind with the proteins.

If paraffin was used, the slide is also exposed to prolonged heat to break down the bonds the proteins formed during the fixation process. This makes it easier for the antibodies to access the proteins. A wash is performed to remove any unwanted substances.

The levels of certain enzymes that are present in tissue samples like biotin and peroxidase interferes with the signal given off by antibodies. That is why pathologists perform a step called a “block” that neutralizes these enzymes.

There are five methods of detecting proteins:

• Direct Method – uses a conjugated primary antibody, which is an antibody that has been chemically bound to certain dyes called flourochromes or chromogens. These substances make it easier to see the antibody. This method provides the lowest antibody signal strength.
• Indirect Method – uses a primary antibody followed by a conjugated secondary antibody, which is used to make it easier to see the primary one. Signal strength is stronger than in the direct method.
• PAP Method – uses a third antibody that is bound to the enzyme peroxidase that is normally present in the tissue sample. This third antibody recognizes the secondary antibody that hasn’t been conjugated. This improves signal strength.
• ABC Method – uses a combination of the protein avidin and peroxidase, which recognizes the secondary antibody that has the enzyme biotin added to it.
• Multiple Staining Method – uses more than one antibody conjugated to different dyes that detect more than one protein.