Tests used on biopsy and cytology specimens to diagnose cancer

The type and grade of a cancer is usually clear when the cells are seen under a microscope after routine processing and staining, but this is not always the case. Sometimes the pathologist needs to use other procedures to make a diagnosis.

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-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. Adenocarcinomas can produce mucus, so finding pink-red spots in lung cancer cells will tell the pathologist that the diagnosis is adenocarcinoma.

Besides being helpful in sorting out different kinds of tumors, other types of 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 cause lumps to form which might be confused with a cancer until histochemical stains prove that the patient has an infection and not cancer.

Immunohistochemical stains

Immunohistochemical (IHC) or immunoperoxidase stains are another very useful category of special tests. The basic principle of this method is that an immune protein called an antibody will attach itself to certain substances, called antigens, that are on or in the cell. Each type of antibody recognizes and attaches to antigens that fit it exactly. Certain types of normal cells and cancer cells have unique antigens. If cells have a specific antigen, they will attract the antibody that fits the antigen. To find out if the antibodies have been attracted 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 routinely processed biopsy of a lymph node may contain cells that clearly look like cancer, but the pathologist may not be able to tell whether the cancer started in the lymph node or whether it started elsewhere in the body and has spread to the lymph nodes. If the cancer started in the lymph node, the diagnosis would be lymphoma. If the cancer started in another part of the body and spread to the lymph node, it might be metastatic cancer. This distinction is very important because treatment depends on the type of cancer (as well as some other factors, too).

There are hundreds of antibodies used for IHC tests. Some are quite specific, meaning that they react only with one type of cancer. Others may react with a few types of cancer, so several antibodies may be tested to decide what type of cancer it is. By looking at these results along with the cancer’s appearance after the biopsy specimen is processed, its location, and other information about the patient (age, gender, etc.), it’s often possible to classify the cancer in a way that can help select the best treatment.

Although IHC stains are used most often to classify cells, they also can be used to detect or recognize cancer cells. When a large number of cancer cells have spread to a nearby lymph node, these cells are usually recognized easily when the pathologist looks at the lymph tissue under the microscope using routine stains. But if there are only a few cancer cells in the node, it can be hard to recognize the cells using only routine stains. This is where IHC stains can help. Once the pathologist knows the kind of cancer to look for, he or she can choose one or more antibodies known to react with those cells. More chemicals are added so that the cancer cells will change color and clearly stand out from the normal cells around them. IHC stains are generally not used to look at tissue from lymph node dissections (which remove a large number of nodes), but they are sometimes used in sentinel lymph node biopsies. (See Sentinel lymph node mapping and biopsy in our review of biopsy types.)

Another specialized use of these stains is to help distinguish lymph nodes that contain lymphoma from those that are swollen from increased numbers of normal white blood cells (usually as a response to infection). Certain antigens are present on the surface of white blood cells called lymphocytes. Benign (non-cancerous) lymph node tissue contains many different types of lymphocytes with a variety of antigens on their surface. In contrast, cancers like lymphoma start with a single abnormal cell, so the cancer cells that grow from that cell typically share the chemical features of the first abnormal cell. This is especially useful in diagnosing lymphoma. If most of the cells in a lymph node biopsy have the same antigens on their surface, this result supports a diagnosis of lymphoma.

Some IHC stains can help recognize specific substances in cancer cells that influence a patient’s prognosis and/or whether they are likely to benefit from certain drugs. For example, IHC is routinely used to check for estrogen receptors on breast cancer cells. Patients 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 which women with breast cancer are likely to benefit from drugs that block the growth-promoting effects of abnormally high levels of HER2 protein.

Electron microscopy

The typical medical lab microscope uses a beam of ordinary light to look at specimens. A larger, much more complex instrument called an electron microscope 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 in deciding whether a cell is cancer. But it sometimes helps find very tiny details of a cancer cell’s structure that provide clues to the exact type of the cancer.

For instance, some cases of melanoma, a highly aggressive skin cancer, may look like other types of cancer under the 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 establish the type of cancer and helps in choosing the best treatment plan.

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 also helps tell lymphomas from non-cancer diseases in the lymph nodes.

A sample of cells from a biopsy, cytology specimen, or blood specimen is treated with special antibodies. Each antibody sticks only to certain types of cells that have the antigens that fit with it. The cells are then passed in front of a laser beam. If the cells now have those antibodies, the laser will make them give off light that’s then measured and analyzed by a computer.

Analyzing cases of suspected leukemia or lymphoma by flow cytometry uses the same principles explained in the section on immunohistochemistry:

  • Finding the same substances on the surface of most cells in the sample suggests that they came from a single abnormal cell and are likely to be cancer.
  • Finding several different cell types with a variety of antigens means that the sample is less likely to contain 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 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.

Genetic tests


Normal human cells have 46 chromosomes (pieces of DNA and protein that control cell growth and function). Some types of cancer have one or more abnormal chromosomes. Recognizing abnormal chromosomes helps to identify those types of cancer. This is especially useful in diagnosing some lymphomas, leukemias, and sarcomas. Even when the type of cancer is known, cytogenetic tests may help predict the patient’s outlook. Sometimes the tests can even help predict which chemotherapy drugs the cancer is likely to respond to.

Several types of chromosome changes can be found in cancer cells:

  • A translocation means part of one chromosome has broken off and is now located on another chromosome.
  • An inversion means that part of a chromosome is upside down (now in reverse order) but still attached to the right chromosome.
  • A deletion indicates part of a chromosome has been lost.
  • A duplication happens when part of a chromosome has been copied, and too many copies of it are found in the cell.

Sometimes, an entire chromosome might be gained or lost in the cancer cells.

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 about 3 weeks to get results.

Fluorescent in situ hybridization

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 is also important in determining if certain targeted drugs might help patients with some types of cancer. For example, FISH can show when there are too many copies (called amplification) of the HER2 gene, which can help doctors choose the best treatment for some women with breast cancer.

Unlike standard cytogenetic tests, it’s not necessary to grow cells in lab dishes for FISH. This means FISH results are available much sooner, usually within a few days.

Molecular genetic tests

Other tests of DNA and RNA can be used to find most of the translocations found by cytogenetic tests. They can also find some translocations involving parts of chromosomes too small to be seen under a microscope with usual cytogenetic testing. This type of advanced testing can help classify some leukemias and, less often, some sarcomas and carcinomas. These tests are also useful after treatment to find small numbers of remaining leukemia cancer cells that may be missed under a microscope.

Molecular genetic tests can also identify mutations (abnormal changes) in certain areas of DNA that control cell growth. Some of these mutations may make cancers especially likely to grow and spread. In some cases, identifying certain mutations can help doctors choose treatments that are more likely to work.

Certain substances called antigen receptors are on the surface of immune system cells called lymphocytes. Normal lymph node tissue contains lymphocytes with many different antigen receptors, which help the body respond to infection. But some types of lymphoma and leukemia start from a single abnormal lymphocyte. This means all these cancer cells have the same antigen receptor. Lab tests of the DNA of each cell’s antigen receptor genes are a very sensitive way to diagnose and classify these cancers.

Polymerase chain reaction (PCR): This is a very sensitive molecular genetic test for finding specific DNA sequences, such as those occurring in some cancers. Reverse transcriptase PCR (or RT-PCR) is a method used to detect very small amounts of RNA. 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. RT-PCR can be used to find and classify cancer cells.

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, but its value for more common types of cancer is less certain. The disadvantage is that doctors are not always sure whether having a few cancer cells in the bloodstream or a lymph node means that a patient will actually develop distant metastases that will grow enough to cause symptoms or affect survival. In treating patients with most common cancer types, it’s still not clear whether 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 one or even 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 microarrays: These tiny devices are in some ways like computer chips. The advantage of this technology is that relative levels of hundreds or even thousands of different RNAs from one sample can be compared at the same time. The results tell which genes are active in a tumor. This information can sometimes help predict a patient’s prognosis (outlook) or response to certain treatments.

This test is sometimes used when a cancer has spread to several parts of the body but doctors aren’t sure where it started. (These are called cancers of unknown primary.) 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. (To learn more about this type of cancer, see Cancer of Unknown Primary.)

DNA sequencing: For the past couple of decades, DNA sequencing has been used to identify people who have inherited genetic mutations that greatly increase their risk of developing certain types of cancer. In this case, the testing generally uses DNA from blood cells of either patients who already have certain cancers (such as breast cancer or colon cancer) or from the blood of their relatives who do not have any known cancer but may be at increased risk.

Doctors have started using DNA sequencing of some cancers to help predict which targeted drugs are most likely to work in individual patients. This practice is sometimes called “personalized oncology” or “precision oncology.” At first, DNA sequencing was done for only one gene or for a few genes that were known to be most often affected for certain types of cancer. Recent progress has made it possible to sequence many more genes, or even all of the genes from a cancer (although this is still not done routinely). This sequence information sometimes shows unexpected mutations in genes that are affected less often, and may help the doctor choose a drug that otherwise would not have been considered and avoid other drugs that are unlikely to be helpful.

The American Cancer Society medical and editorial content team
Our team is made up of doctors and master’s-prepared nurses with deep knowledge of cancer care as well as journalists, editors, and translators with extensive experience in medical writing.

Last Medical Review: March 30, 2015 Last Revised: July 30, 2015

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