Pathologists often perform tests on the tissue sample sent for pathological examination and you may read the results of these tests in your pathology report. While some tests are only performed in certain labs, many tests are so common that they are used almost everywhere. This page will review some of the most commonly used tests that are performed on tissue submitted for pathological examination.
All tissue submitted to a lab for pathological examination is first fixed in formalin to preserve the tissue, embedded in paraffin wax to make it strong, cut into very thin sections, and then placed on a slide so that it can be viewed under a microscope.
Hematoxylin and eosin (H&E)
Most tissues are transparent when cut very thinly which means they cannot be seen under a microscope until a dye is added to give the tissue colour. To accomplish this, most labs start by staining at least one section from each piece of tissue with a combination of dyes called hematoxylin and eosin (typically just called H&E). This stain is in fact so common that some reports will just call it the ‘routine stain’.
The H&E stain works because hematoxylin, a blue dye, preferentially stains the nucleus while eosin, a pink dye, preferentially stains the components of the cell body. This stain allows pathologists to identify both normal and abnormal cells and to assess the overall architecture of the tissue. In many cases a diagnosis can be made off of the H&E stain alone without the need for any further tests.
Immunohistochemistry (IHC) is a commonly used test that allows pathologists to identify cells based on the specific proteins they produce. This test allows pathologists to better understand both the function and origin of the cell.
The test works by attaching a probe (an antibody) to the protein of interest (the antigen). Importantly, the probe will only stick to cells that contain the protein of interest. A second probe is then added to the tissue which makes the cells containing the initial protein change colour.
When the tests works well, only the cells which contain the protein of interest should change colour. When viewed under a microscope the target cells stand out in sharp contrast to the unstained cells in the background.
There are literally thousands of probes available to identify thousands of different proteins. This variety allows pathologists to distinguish between cells that otherwise look identical. Immunohistochemistry also allows pathologists to identify specific proteins that can help predict the behavior of a disease or the response to drugs such as chemotherapy.
When an immunohistochemistry test identifies a protein in your tissue, the result is called ‘positive’ or ‘reactive’. When no protein is identified, the result will be described as ‘negative’ or ‘non-reactive’.
A special stain is a chemical that reacts with tissue to produce specific a colour in the tissue. The tissue can then be viewed under a microscope by your pathologist who will examine the amount of colour produced and the location of the colour within the tissue. The results of this examination will be described in your pathology report.
The colour produced in the tissue depends on the special stain used and pathologists have hundreds of different special stains to choose from. The choice of which special stain to use depends on the question your pathologist is trying to answer. For example, some special stains are used to highlight infectious organisms such as fungus and bacteria by turning them black or red. This makes them easier to see under a microscope.
Other special stains are used to highlight specific kinds of cells. For example, some special stains turn cells that contain a chemical called mucin blue or red. This type of special stain is very useful when the cell of interest is very small or when there are only a small number of them in a large population of other cell types.
Some of the most common special stains include:
Fluorescence in situ hybridization (FISH)
Most of the genetic material inside a cell is stored on chromosomes (DNA). Some tumours contain changes that alter the location or amount of genetic material in a predictable manner. Pathologists use their knowledge of these changes to identify specific types of tumours.
Fluorescence in situ hybridization (FISH) uses a fluorescent of probe which has been designed to stick to a unique segment of DNA (the target). Unlike the probes used in immunohistochemistry, FISH probes only produce a colour when they are exposed to certain types of light. As a result, pathologists need to use a special type of microscope which can produce specific types of light and detect the colour coming from the probe.
The results are captured in an image on a computer for further analysis. When the probe shows a change in the location of the target DNA, the result is called a translocation. When the probe shows a change in the total amount of target DNA, the result is called an amplification or deletion depending on the type of change identified.