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Tests Used on Biopsy and Cytology Samples to Diagnose and Classify Cancer

When diagnosing cancer, the pathologist will look at the cells in the biopsy or cytology samples under a microscope. This is often all that is needed to determine the type and grade of the cancer, and no additional tests need to be done on the samples.

But sometimes the pathologist will need to do other tests or procedures on the samples to make a diagnosis. Even if the type and grade of the cancer are known, other lab tests might still be done to learn more about the cancer, such as how it might respond to certain treatments. (To learn more about what a pathologist looks for when making a diagnosis, see What Do Doctors Look for in Biopsy and Cytology Samples?)

Here are some of the more common lab tests and procedures done on biopsy or cytology samples. To learn more about the tests that might be done for a specific kind of cancer, see the testing section of our information on that cancer type.

Histochemical stains

These tests use different chemical dyes that are attracted to certain substances found in some types of cancer cells.

For example, the mucicarmine stain is attracted to mucus. Droplets of mucus inside a cell that are exposed to this stain will look pink or red under a microscope. This stain is useful if the pathologist suspects, for example, an adenocarcinoma (a glandular type of cancer) in a lung biopsy. Adenocarcinoma cells can produce mucus, so finding pink or red spots in lung cancer cells exposed to this stain will tell the pathologist that the diagnosis is adenocarcinoma.

Besides being helpful in sorting out different types of tumors, other special stains are used in the lab to identify microorganisms (germs) like bacteria and fungi in tissues. This is important because people with cancer may develop infections as a side effect of treatment, or even because of the cancer itself. It’s also important in cancer diagnosis because some infectious diseases can cause lumps, which might be confused with a cancer. Histochemical stains might show that the lump is from an infection and not cancer.

Immunohistochemical stains

Immunohistochemical (IHC) or immunoperoxidase stains are another useful category of special tests. This method uses immune proteins called antibodies, which will attach to certain substances, called antigens, that are on or in a cell. Each type of antibody only attaches to antigens that fit it exactly.

Certain types of normal cells and cancer cells have unique antigens. If cells have a specific antigen, the antibodies that fit the antigen will bind (stick to) it. To find out if the antibodies have attached to the cells, chemicals are added that make the cells change color only if a certain antibody (and, therefore, the antigen) is present.

Our bodies normally make antibodies that recognize antigens on germs and help protect us against infections. The antibodies used in IHC stains are different. They’re made in the lab to recognize antigens that are linked to cancer and other diseases.

IHC stains are very useful in identifying certain types of cancers. For example, a biopsy of a lymph node might contain cells that clearly look like cancer cells, but the pathologist might not be able to tell whether the cancer started in the lymph node or whether it started elsewhere in the body and then spread to the lymph nodes. This distinction is very important because if the cancer started in the lymph node, the diagnosis would most likely be a lymphoma, whereas if the cancer started in another part of the body and spread to the lymph node, it would be a different type of cancer. The treatment might be very different depending on the type of cancer (as well as some other factors).

There are many antibodies that can be used for IHC tests. Some are very specific, meaning that they react only with one type of cancer. Others may react with a few types of cancer, so several antibodies might be tested to decide what type of cancer it is. By looking at these results, along with how the cancer looks under a microscope, where it is in the body, and other information, it’s often possible to classify the cancer in a way that can help doctors decide the best treatment.

Although IHC stains are used most often to classify cells, they also can be used to find cancer cells. For example, if there are only a few cancer cells in a lymph node, it can be hard for the pathologist to see the cells using only routine stains. But if the pathologist knows what type of cancer they’re looking for, they can choose an IHC stain that uses one or more antibodies known to react with those types of cancer cells, which will help any cancer cells stand out if they are there. IHC stains are sometimes used in sentinel lymph node biopsies, but are generally not used to look at tissue from lymph node dissections, which remove a large number of nodes. (See How Is a Biopsy Done?)

Some IHC stains can help recognize specific substances in cancer cells that might influence a person’s prognosis (outlook) and/or whether they are likely to be helped by certain medicines. For example, IHC is routinely used to check for estrogen and progesterone receptors on breast cancer cells. People whose cells have these receptors are likely to benefit from hormone therapy drugs, which block the production or effects of estrogens. IHC can also help determine the level of HER2 proteins in the cells for some types of cancer, such as breast and stomach cancer. This can help doctors decide if someone is likely to be helped by drugs that target the HER2 protein.

Flow cytometry

Flow cytometry is often used to test the cells from bone marrow, lymph nodes, and blood samples. It’s very accurate in finding out the exact type of leukemia or lymphoma a person has. It can also help tell lymphomas from non-cancer diseases in the lymph nodes.

For this test, a sample of cells from a biopsy, cytology, or blood sample is treated with special antibodies. Each antibody sticks only to certain types of cells that have the antigens that fit with it. The antibodies are linked to chemicals that are fluorescent (that is, they give off light of a certain color when exposed to a laser beam).

The cells are put into a stream of liquid that is passed through a laser beam, and an instrument measures the color and brightness of the light each cell gives off. A computer collects and analyzes the data to help doctors recognize and classify cancers.

Along with analyzing suspected cases of leukemia or lymphoma, flow cytometry can also be used to measure the amount of DNA in cancer cells (called ploidy). Instead of using antibodies to detect protein antigens, cells can be treated with special dyes that react with DNA.

  • If there’s a normal amount of DNA, the cells are said to be diploid.
  • If the amount of DNA is abnormal, the cells are described as aneuploid. Aneuploid cancers of most (but not all) organs tend to grow and spread faster than diploid ones.

Another use of flow cytometry is to measure the S-phase fraction, which is the percentage of cells in a sample that are in a certain stage of cell division called the synthesis or S phase. The more cells that are in the S-phase, the faster the tissue is growing and the more aggressive the cancer is likely to be. 

Image cytometry

Like flow cytometry, this test uses dyes that react with DNA. But instead of suspending the cells in a stream of liquid and analyzing them with a laser, image cytometry uses a digital camera and a computer to measure the amount of DNA in cells on a microscope slide. Like flow cytometry, image cytometry also can determine the ploidy of cancer cells.

Electron microscopy

The typical microscope uses a beam of light to look at specimens. An electron microscope is a larger, much more complex instrument that uses beams of electrons. The electron microscope’s magnifying power is about 1,000 times greater than that of an ordinary light microscope. This degree of magnification is rarely needed to determine if a cell is cancer. But it sometimes can help find very tiny details of a cancer cell’s structure that provide clues to the exact type of the cancer.

For instance, some melanoma skin cancers may look like other types of cancer under an ordinary light microscope. Most of the time, these melanomas can be recognized by certain IHC stains. But if those tests don’t give a clear answer, the electron microscope may be used to identify tiny structures inside melanoma cells called melanosomes. This helps diagnose the type of cancer and might help in choosing the best treatment plan.

Genetic and genomic tests

Different types of lab tests might be done on the biopsy or cytology samples to learn more about the changes in genes (or patterns of changes in several genes) in cancer cells. These types of tests might help determine which type of cancer a person has. In some situations, they might also be helpful in determining treatment options.

Cytogenetic tests

In some cancers, the cells have one or more abnormal chromosomes (long strands of DNA that contain our genes, which control cell growth and function). Recognizing abnormal chromosomes can help identify these cancers. This is especially useful in diagnosing cancers such as lymphomas, leukemias, and sarcomas.

Even when the type of cancer is known, cytogenetic tests may help predict a person’s outlook. Sometimes the tests can even help predict which treatments are likely to be helpful.

Several types of chromosome changes might be found in cancer cells. For example:

  • translocation means part of one chromosome has broken off and is now attached to another chromosome.
  • An inversion means that part of a chromosome is now in reverse order but is still attached to the right chromosome.
  • deletion means part of a chromosome has been lost.
  • duplication means there are extra copies of part of the DNA in a chromosome.

Sometimes, an entire chromosome might be gained or lost in the cancer cells. (Normal human cells have 46 chromosomes.)

For cytogenetic testing, cancer cells are grown in lab dishes for about 2 weeks before their chromosomes can be looked at under the microscope. Because of this, it usually takes at least this long to get results.

Fluorescent in situ hybridization (FISH)

Fluorescent in situ hybridization (FISH) is a lot like cytogenetic testing. It can find most chromosome changes that can be seen under a microscope in standard cytogenetic tests. It can also find some changes too small to be seen with usual cytogenetic testing.

FISH uses special fluorescent dyes linked to pieces of DNA that only attach to specific parts of certain chromosomes. FISH can find chromosome changes like translocations, which are important to help classify some kinds of leukemia.

Finding certain chromosome changes can also be important in determining if certain targeted drugs might be helpful in treating some types of cancer. For example, FISH can show when there are too many copies of the HER2 gene (known as HER2 amplification or HER2 overexpression). This can help doctors determine if drugs that target HER2 might be helpful in people with cancers that have too much HER2, such as some breast, stomach, and other cancers.

Unlike with standard cytogenetic tests, the cancer cells don’t need to be grown in lab dishes for FISH. This means FISH results are available much sooner, usually within a few days.

Molecular tests

Some newer types of sensitive lab tests can also find changes in the DNA, RNA, or proteins in cancer cells. These tests can be used to help determine the type of cancer a person has, as well as if certain treatments are likely to be helpful. Some of these tests can also be used to see if treatment is working, or to look for signs that cancer has come back.  

Testing for changes in a person’s cancer cells to help determine the best care (including treatment) is sometimes referred to as biomarker testing. Using the results of these types of tests to help plan treatment for each person is known as precision or personalized medicine.

Polymerase chain reaction (PCR): This is a very sensitive molecular test for finding specific DNA sequences, such as those occurring in some cancers.

Reverse transcriptase PCR (RT-PCR) is a method used to detect very small amounts of RNA in a sample. RNA is a substance related to DNA that’s needed for cells to make proteins. There are specific RNAs for each protein in our body.

An advantage of RT-PCR is that it can detect very small numbers of cancer cells in blood or tissue samples that would be missed by other tests. RT-PCR is used routinely for detecting certain kinds of leukemia cells that remain after treatment.

It’s less clear, though, if RT-PCR is useful for more common types of cancer. Doctors aren’t always sure if finding a few cancer cells in the blood or in a lymph node means that a person’s cancer will grow and spread enough to cause symptoms or affect their survival. In treating people with most common cancer types, it’s still not clear if finding a few cancer cells with this test should be a factor in choosing treatment options.

RT-PCR can also be used to sub-classify cancer cells. Some RT-PCR tests measure levels of several RNAs at the same time. By comparing the levels of important RNAs, doctors can sometimes predict whether a cancer is likely to be more or less aggressive (likely to grow and spread) than would be expected based on how it looks under the microscope. Sometimes these tests can help predict whether a cancer will respond to certain treatments.

Gene expression profiling: These tests compare the levels of many different RNAs from one sample at the same time. The results tell which genes are active (expressed) in a tumor, rather than just looking for changes in individual genes.

This pattern of gene activity can sometimes help predict a person’s prognosis (outlook), which can help determine further treatment options. For example, for some early-stage breast cancers, this type of testing can show how likely it is the cancer will come back after surgery (and possibly radiation). This can help determine if chemotherapy should be part of further treatment.

This type of test can also be helpful when a cancer is found in different parts of the body, but doctors aren’t sure where it started. (This is called a cancer of unknown primary, or CUP.) The RNA pattern of these cancers can be compared with the patterns of known types of cancer to see if they match. Knowing where the cancer started is helpful in choosing treatment. These tests can help narrow down the cancer type, but they are not always able to tell the exact type of cancer with certainty.

DNA sequencing: Some types of tests can determine the actual sequence of DNA – the order of the chemicals that make up its code – to look for gene mutations (changes) inside cells.

This type of testing has been available for the past couple of decades, where it has been used mainly to identify people who have inherited gene mutations that greatly increase their risk of developing certain types of cancer.

Over time, doctors have learned more about the gene changes in cancer cells, and the technology behind DNA sequencing has improved to make it easier to do. Newer, next generation sequencing (NGS) tests are now used to look for gene changes in some types of cancers that can help predict which treatments, such as targeted drugs, are likely (or not likely) to work for each person.

DNA sequencing might be done to look at only a small number of genes for certain types of cancer. But as more gene changes are discovered in cancer cells (and as medicines are developed to target cells with these gene changes), sequencing is now likely to include more genes, or even all of the genes in a cancer cell (although this is still not done routinely). Sometimes DNA sequencing information might even show an unexpected gene mutation, which might help the doctor choose a drug that otherwise would not have been considered.

Getting the results of molecular tests

Some types of molecular tests might be done in the pathology lab at the center where you’re getting your care, but others might need to be sent out to a central lab for testing. Depending on the test, it might take at least a couple of weeks to get these results.

Once the results are back, a member of your health care team should go over them with you. Ask them to explain the results in a way you can understand, including how they might affect your treatment options and help predict your outlook.

The American Cancer Society medical and editorial content team

Our team is made up of doctors and oncology certified nurses with deep knowledge of cancer care as well as journalists, editors, and translators with extensive experience in medical writing.

Last Revised: August 1, 2023

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