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There are standard procedures and methods that are used with nearly all types of biopsy or cytology samples. Some types of samples need additional processing.

Routine biopsy processing

After a biopsy sample (sometimes called a specimen) is removed, it is usually put in a container with a mixture of water and formaldehyde (formalin) or some other fluid to preserve it. Sometimes part of the sample might be sent fresh (without any preservative) to the pathology department, so that certain tests that require fresh tissue (like cytogenetics – see below) can be done.

The container is labeled with the patient’s name and other information (such as hospital number and birth date) and the site of biopsy (exactly where on the body it was taken from).

Gross examination

In the pathology lab, the pathologist (a doctor with special training) or another trained pathology professional first looks at the specimen without a microscope. This is called a gross examination (as opposed to a microscopic exam). During the gross examination, the tissue sample’s size, color, consistency, and other characteristics are recorded. The lab staff may even take a picture of the sample as part of the record.

The gross examination is important because the pathologist may see features that suggest a particular diagnosis, such as cancer. It also helps the pathologist decide which parts of a large biopsy sample are the most critical to look at under a microscope. For smaller types of biopsies, such as a punch biopsy or a core needle biopsy, the entire specimen is usually looked at under a microscope.

Processing the specimen for microscopic examination

The biopsy tissue is put into small containers called cassettes, which hold the tissue securely while it’s processed. After processing (which is usually done overnight), the tissue sample is put into a mold with hot paraffin wax. The wax cools to form a solid block that protects the tissue.

This paraffin wax block with the embedded tissue is then cut into very thin slices using an instrument called a microtome. These thin slices of the specimen are placed on glass slides and are dipped into a series of stains or dyes to change the color of the tissue. The color makes different parts of the cells easier to see under a microscope.

For most biopsy specimens, this routine processing is all that’s needed. At this point (usually the day after the biopsy was done), the pathologist looks at the tissue under a microscope. Looking at the solid specimens in this way is called histology, which is the study of the structures of cells and tissues.

Frozen section (intra-operative consultation)

Sometimes during surgery, the surgeon might need information about a tissue sample to help make important decisions, such as how much of an organ to remove. In such situations, the surgeon will ask for an intra-operative (during surgery) pathology consult. This is often called a frozen section exam.

How is a frozen section done?

When a frozen section exam is done, fresh biopsy tissue is sent from the operating room right to the pathologist. Because the patient is often still under general anesthesia (kept in a deep sleep so they don’t feel pain), it’s important that the tissue is looked at quickly (usually within 10 to 20 minutes).

The pathologist will do a gross examination of the fresh tissue to decide which part of it should be looked at under a microscope. Instead of processing the tissue in wax blocks, the tissue is quickly frozen in a special solution that forms what looks like an ice cube around the tissue. It’s then sliced into very thin sections on a special machine, quickly stained (dipped in a series of dyes), and then looked at under a microscope.

The frozen sections typically do not show features of the tissue quite as well as sections of tissue embedded in wax, but they are usually good enough to help the surgeon make decisions during the operation.

Why might a frozen section be done?

Frozen sections are typically done during an operation, when the surgeon needs information right away that can help guide what type of operation should be done. There might be different reasons for this.

To find out if a tumor is cancer: Sometimes the type of operation the surgeon needs to do depends on whether the tumor is cancer (malignant). For instance, just removing a tumor might be enough if a frozen section shows that it’s not cancer (benign). But the surgeon might need to remove a wider margin of tissue around the tumor, as well as nearby lymph nodes, if the tumor is cancer. In a case like this, a frozen section exam often can help the surgeon decide which type of operation is best.

Sometimes, though, the frozen section might not give a definite answer, so the biopsy tissue will still need to go through routine or even special processing. When this happens, the surgeon usually stops the operation and closes the surgical incision (cut). Once the results are back, another operation may be needed.

To help make sure all of the cancer has been removed: Surgery to treat cancer is often a difficult balance between removing enough tissue to get all of the cancer, and leaving as much normal tissue as possible to help limit side effects from the surgery.

If the surgeon is concerned that a cancer hasn’t been removed completely, a slice from the edge of the tissue that was removed (called a margin) can be sent for a frozen section diagnosis.

If there are no cancer cells at the margin (known as a negative margin), typically no more tissue needs to be removed. Having a negative margin doesn’t guarantee that the cancer won’t come back, although it makes it less likely.

If cancer cells are found at the margin (known as a positive margin), it’s likely that at least some cancer cells weren’t removed. If this happens, the surgeon will usually remove more tissue from the area to try to get all the cancer cells and lower the chances of the cancer coming back. If it’s not possible to remove more tissue, there may be other treatment options, such as using radiation to kill the remaining cancer cells.

Process for Mohs surgery (microscopically controlled surgery)

Mohs surgery (also known as Mohs micrographic surgery, or MMS) can be used to treat certain kinds of skin cancer. In this procedure, the surgeon removes a thin layer of the skin that the tumor might have invaded. The sample is then frozen and sectioned, and then checked under a microscope. If cancer cells are seen, another layer is removed and checked. This process is repeated until no cancer cells are found in the skin samples.

This process is slow, often taking several hours, but it means that more normal skin near the tumor can be saved. This can help the area look better after surgery.

Mohs surgery is a highly specialized technique that should only be done by doctors who have been trained to do it.

Processing cytology samples

How cytology specimens are processed depends on the type of sample.

Some samples are smeared on glass microscope slides by the doctor who gets the sample. These slides, which are called smears, are then sent to the cytology lab, where they’re dipped into a series of stains (colored dyes), much like those used for biopsy samples.

In some situations, a portion of the sample might be stained right away for a preliminary interpretation (called rapid on-site evaluation). This might be done, for example, to help determine which lab tests should be done on the specimen.

Other samples, such as body fluids, can’t be smeared on a glass microscope slide easily because they’re too diluted (that is, there are too few cells in a large amount of fluid). These types of samples need to be concentrated before the cells are put on a glass slide and stained.

After processing and staining, the samples are examined under a microscope, typically by a cytotechnologist. The abnormal cells are found and marked with a special pen. A pathologist will then examine the marked cells to make a diagnosis.

How long are pathology specimens kept?

Clinical labs (including pathology labs) are regulated and certified based on a federal law called CLIA (Clinical Laboratory Improvement Amendments). To be CLIA accredited, labs must keep biopsy and cytology samples for a minimum amount of time. For instance, CLIA says that labs must keep:

• Cytology slides for at least 5 years
• Histopathology slides for at least 10 years
• Paraffin blocks for at least 2 years

Some states have their own laws that require labs to keep pathology samples longer than the time specified by CLIA. And some labs have policies for keeping samples even longer than required by federal or state laws.

If you would like to know if the lab you go to is CLIA certified, you can ask the lab staff. Or if you have questions or concerns, you can reach out to your state’s CLIA agency. A list of contact information for state agencies can be found at cms.gov/medicare/quality/clinical-laboratory-improvement-amendments.

What this might mean for you

Some people might want to get a second opinion on the diagnosis made from their biopsy or cytology tissue sample. Getting another doctor to look at your sample to either confirm the diagnosis or suggest another one is called a pathology review.

The processed samples (slides and blocks) are kept for a certain amount of time (although other parts of the sample are typically discarded). Because of this, the samples can often be sent to another doctor or lab.

Sometimes keeping samples for a longer time can be helpful in other ways. For instance, if someone who had cancer develops a new tumor several years after the first one was removed, doctors often want to know if this tumor is the original cancer coming back (a recurrence) or a new cancer.

This can often be figured out by comparing the histopathology slides from the new tumor to the original tumor. In some cases, it might also be helpful to do more tests (such as immunohistochemical stains) using tissue from the original specimen’s paraffin block.