Frozen section procedure (2023)


Frozen section procedure

Author:Jessica Wallace, P.A., A.S.C.P.

Last author update: 1 October 2011

Last staff update: 3 June 2021

Copyright: 2002-2023,, Inc.

PubMed Search: Frozen section methodology

Page views in 2022: 2,991

Page views in 2023 to date: 2,555

Table of Contents

Definition / general | Indications | Tissue type | Freezing tissue | Cryosectioning | Staining slides | Troubleshooting | Microscopic (histologic) images | Videos | Additional references

Cite this page: Wallace J. Frozen section procedure. website. Accessed July 3rd, 2023.

Definition / general

  • A frozen section (cryosection) is a pathological laboratory technique used for rapid microscopic analysis / diagnosis of a specimen / disease
  • Usually used with oncologic surgery
  • Rapid diagnosis can guide intra-operative patient management


  • Use frozen section to
    • Provide rapid gross or microscopic diagnosis to identify an unknown pathologic process, identify extent of disease / evaluate margins, identify metastases or simply identify a tissue
    • Process tissue to provide appropriate and accurate diagnosis, prognosis and to adhere to research and special study protocols
    • Confirm that pathological tissue is present for diagnosis on permanent sections
  • Do not use if
    • Frozen section diagnosis has no immediate implications for decision making
    • Tissue is needed for permanent processing (is unique or small or requires extensive study for diagnosis)
  • Consider not freezing tissue if
    • Frozen section is known to produce severe artifacts that hinder proper interpretation
    • Tissue is heavily ossified / calcified
    • Risk of serious infection (HIV, TB, hepatitis B or C)
    • Tissue is fatty

Tissue type

  • Tissue should be received fresh, otherwise it will not stay on slide
  • At time of receipt of tissue, decide whether to obtain smears or touch preps and whether to freeze all or part of it
    • Touch preps and smears are often performed on lymph nodes suspicious for lymphoma
    • Some primary small lesions should not be entirely submitted for frozen section
    • There is debate on whether sentinel nodes should be entirely or representatively submitted for frozen section
  • Fixed tissue:
    • There are special slides to keep tissue affixed to slide
    • To freeze fixed tissue, make sure it has been preserved in formalin and not alcoholic fixatives like Carnoy's, because tissue fixed in alcohol is harder to freeze
    • Avoid freezing tissue fixed with heavy metal salts such as B5 and Helly's (Zenker’s formal solution), which can denature proteins and shrink the tissue
    • Avoid hard tissues like bone and cartilage that require decalcification
    • Avoid tissues with a lot of fat
    • Avoid tissues from patients with known TB or other infection (if absolutely necessary, wear appropriate protection)
    • Avoid freezing tissue that will be needed to make a permanent diagnosis

Freezing tissue

  • OCT (optimal cutting temperature) or similar embedding media like TBS or Cryogel should be placed on an appropriate sized chuck that has been precooled in a cryostat
  • The chuck should be clean
  • A toothbrush is useful to remove tissue and OCT
  • Dipping the chuck in methanol removes ice crystals
  • Place the chuck into a -20 to -15 degree (optimal) cryostat; note that the OCT media should not be frozen completely
  • It is better to have a semisolid consistency; this will alleviate tissue artifact
  • Tissue size should be no greater than 3mm - 5mm in greatest dimension (thinner specimens have shorter freezing time and minimal ice crystal artifact formation)
  • The smaller the tissue, the more even and thorough the freeze
  • Place the tissue on the semisolid chuck and add more media rapidly over the tissue, covering it entirely but avoiding overflow
  • Place chuck quickly back into the cryostat
  • Apply heat sink or CO2 aerosol (optional) to rapidly freeze or use "quick freeze" option on cryostat
    • Histobath: being phased out
    • Cryowells: useful in keeping all tissue on an even plane; also helpful in eliminating loss of smaller tissues that are frozen with larger ones, although recommended to not freeze different sizes together
    • Aerosol sprays: often canned CO2 (but may aerosolize infectious diseases)
    • Liquid nitrogen
    • Isopentane based workflow (Virchows Arch 2008;452:305)


  1. Once the chuck is in position, there should be a manual or an automatic advance option to move the block close to the cutting blade

    Frozen section procedure (1)

    Tissue embedded within OCT

  2. Fully face the tissue by using a trim setting on your cryostat; if you do not have this setting, then an advance button should be available, which should be pressed each time before one full revolution of the instrument's wheel
  3. If wells are used to freeze the blocks, then the tissue should be on an even plane and the tissue will be faced faster
  4. To polish the tissue, avoid advancing the cryostat or deselect the trim setting on the cryostat and turn 10 - 15 times
  5. As you cut the tissue, anchor the tissue to prevent folding or curling; this can be done with an anti roll bar (a plastic plate attached to cryostat) or by using a precooled paintbrush with stiff bristles and a wide gripping surface
  6. The brush should be held like a pen with your left hand at an angle
  7. You can rest your fifth finger on the stage for stabilization
  8. Cutting the brushes' bristles at an angle can aid in the brush meeting the tissue flat over its length because you will hold it at an angle
  9. Turn the wheel with your right hand in a continuous motion without stopping; avoid speeding up or slowing down
  10. Avoid stopping the wheel at the beginning of the section, slowly grabbing the tissue and then resuming wheel revolutions; this can cause artifacts such as variation in section thickness and tissue folding
  11. Move the brush as the chuck moves towards the blade; your brush should move down in pace with the chuck

    Frozen section procedure (2)

    Riding the block: as the block descends
    toward the brush, the brush keeps pace
    with the block by gently resting on the
    bottom 2 - 3 mm of the block

  12. You can rest your brush softly on the very bottom of your chuck avoiding tissue contact
  13. Pull the brush away easily as the chuck meets the blade

    Frozen section procedure (3)Frozen section procedure (4)

    Catching the curl: as the block meets the blade and
    the section begins its curl, the brush leaves the block
    while catching the curling edge of the section;
    then the brush jumps off the block with the curl

  14. The downward motion of the brush allows you to keep a continuous motion as you take your section

    Frozen section procedure (5)

    Pull over the blanket: the brush holding the curl
    pulls the section horizontally over the stage,
    like pulling the blanket over yourself,
    without pressing the tissue to the stage

  15. A glass slide is gently laid upon the tissue section

    Frozen section procedure (6)Frozen section procedure (7)Frozen section procedure (8)

    Gently touch the section to the slide; avoid stretching or folding the section by keeping a steady hand, and keep the transverse axis of the slide parallel to the section

  16. The tissue section should melt onto the slide
  17. Prepared slides should immediately go into formal alcohol, 95% alcohol (methanol/ethanol) or formalin while awaiting the stain line; if you delay this step, drying artifact will occur
  18. You can take a deeper level after approximately 20 turns (multiple levels may be needed for breast or prostate biopsies)
  19. Optimal cutting thickness is 4 - 7 microns for sectioning and 20 - 40 microns for trimming

Staining slides

  • Keep all stains and solutions fresh and well maintained
  • Dip slide in reagents in this order for H&E staining:
    • After obtaining frozen section, IMMEDIATELY fix in 95% ethanol (even 15 seconds of delay can cause significant artifact)
    • Formal alcohol, formalin or 95% alcohol: 45 - 60 seconds
    • Water: 5 - 7 seconds
    • Hematoxylin: 60 seconds
    • Lithium carbonate or 0.2 % aqueous ammonia (Bluing): 15 - 20 seconds
    • Eosin: 20 - 60 seconds
    • 95% alcohol: 10 seconds
    • 100% alcohol: 10 seconds
    • Xylene, toluene, limonene derivatives and Clearite: 10 seconds
    • Then add mounting media for cover slipping


  • Ice crystal artifacts
    • Due to slow freezing of tissue
    • Solution: Freeze fast (flash / snap); the faster the freeze, the smaller the ice crystals, the less tissue damage (best freezing method is arguably liquid nitrogen)
    • Smaller tissues yield less artifact - optimally tissue should be 0.5 x 0.5 x 0.3 cm or less
    • Never freeze fragments larger than the diameter of the chuck
    • Avoid freezing fat around tissue
    • Blot the outer surface of the tissue dry with gauze before making your block
  • Knife artifact
    • A nicked cutting blade will produce a split / tear in your section
    • Solution: change your blade every few cases; some institutions use a new blade for each case
  • Overfreezing
    • Can cause section to have holes
    • Solution: polish block with a couple extra turns of the blade to create friction and warm up block by pressing on it with your finger (5 - 10 seconds)
  • Underfreezing
    • Underfreezing can be troublesome for fatty tissue
    • Solution: add heat sink to block or select rapid freeze setting on your cryostat (if available)
  • Staining issues
    • Dirty "stain line" can cause floaters (extraneous foreign tissue) to adhere to slides; overly diluted stains and alcohols can diminish slide quality
    • Poor staining hinders frozen section diagnoses, as nuclear detail is compromised
    • Solutions: (a) maintain a clean stain line by frequent solution changes; (b) follow recommended staining times; (c) don't rush
    • Note: brain tissue may stain best in eosin for 60+ seconds
    • Water: should be changed after each frozen section
    • Alcohols and stains: change at least weekly, alcohols may need to be changed more frequently depending on work load
  • Fatty tissue
    • Includes lymph nodes, breast, skin; may be too soft to cut
    • Solution: maintain an extremely cold cutting temperature (-20C)
    • Firm lymph nodes, spleen, brain and liver cut better at -10C; tissue may shatter if sectioning is performed at lower temps
  • Air bubbles
    • May be trapped under cover slips, which can cause the underlying tissue to dry out
    • Solution: make sure an appropriate amount of resin (2 drops) is applied; gently move air bubbles off the slide with finger or tweezers; do not press on the slide too hard or it will break
  • Overly thick sections
    • May cause tissue to fall off slide
    • Solution: reduce the cryostat's sectioning thickness

Microscopic (histologic) images

Images hosted on other servers:

Frozen section procedure (9)Frozen section procedure (10)

Left: ice crystals in edematous stroma by frozen section, right: H&E

Frozen section procedure (11)Frozen section procedure (12)

Nuclear ice crystals (particularly a problem with thinner sections): left - lung adenocarcinoma, right - uterine sarcoma

Frozen section procedure (13)Frozen section procedure (14)Frozen section procedure (15)



Brush technique

Embedding small specimens

Speed embedding

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Frozen section procedure? ›

The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection. The microtome device that cold cuts thin blocks of frozen tissue is called a cryotome.

What are the steps of frozen section? ›

1) Close up of a flat embedded block showing slight retraction of medium around the tissue. 2) Place a drop of embedding medium on the chuck face. 3) Press the block face to a flat freezing surface such as cryostat stage or any of the freezing apparatus. 4) Remove the chuck with a tap of the over chuck freezing block.

What are the disadvantages of frozen section? ›

These are: sampling error, ice crystal artifacts, lack of special studies, and lack of consultation. I would like to discuss what I consider the limitations in frozen section.

What is the difference between biopsy and frozen section? ›

Frozen section examinations are a specific biopsy procedure. They allow surgeons to rapidly diagnose a suspicious mass during a surgical procedure. Doctors commonly call the procedure cryosection. During cryosection, the surgeon removes part of the tissue.

How do you perform a frozen section biopsy? ›

What is a Frozen Section Biopsy?
  1. The anesthetist will anesthetize the patient.
  2. The surgeon will make an incision exposing the tumor.
  3. The surgeon will take a sample of the tumor and give it to a pathologist.
  4. The pathologist quickly freezes the sample in a cryostat and takes a thin slice of the sample.
Mar 31, 2020

How long does a frozen section take? ›

A frozen section usually takes between 30 and 45 minutes. Sometimes the frozen section approach is non-diagnostic or inconclusive because only a limited number of sections can be taken and examined by the pathologist in the short time available.

Is frozen section a surgical procedure? ›

During the frozen section procedure, the surgeon removes a portion of the tissue mass. This biopsy is then given to a pathologist (a doctor who examines tissues and uses laboratory tests to make a diagnosis).

Can frozen section be wrong? ›

Tissue Types with Concordance and Discordance. Serial no. Diagnostic accuracy of frozen section was 95.5% (191/200 cases) when compared with permanent section. Discordance was found in nine cases with false negative diagnoses in 7 cases and false positive diagnosis in 2 cases.

How much does a frozen section cost? ›

The estimated cost of intraoperative frozen section averaged as much as $3,123 per patient, with a cost-benefit ratio of 20:1. Conclusions: Intraoperative frozen section margins are accurate, but they are costly and cannot reliably eradicate positive final margins.

What is the reason for frozen section? ›

The frozen section is mainly used for rapid diagnosis of the lesion for intraoperative management, to know the extent of the lesion, to do enzyme immunocytochemistry and immunofluorescence study and also to stain lipid and certain carbohydrate in the tissue.

How long does it take to get a frozen biopsy result? ›

A razor-thin slice of tissue is extracted from the frozen section, prepared on a slide and placed under the microscope for review. In many other medical centers, this process takes at least 24 hours to complete. After reviewing the sample, the pathologist conveys the test results to the surgeon in the operating room.

How thick is tissue in frozen section? ›

Cryosectioning at temperatures lower than −35°C requires the use of a cryogen such as liquid nitrogen. section thickness range from 1–100 µm or higher.

Is frozen section the same as Mohs? ›

It is different from Mohs surgery, because larger areas around the tumor are taken during the excision, the microscopic evaluation technique of the tumor is slightly different, and any surgeon (including a plastic surgeon) can perform the excision.

What are the limitations of frozen section biopsy? ›

Nevertheless, frozen biopsy has several limitations, such as cytological and architectural distortion, artifacts, and its low performance in some specific neoplasms, which explain why frozen section biopsy is a tool for patient care that does not replace the conventional biopsy.

What are the three stages of freezing? ›

The freezing process is divided into three phases: initial freezing, water-ice phase transition, and deep freezing. The melting process is also divided into three phases: initial melting, ice-water phase transition, and deep melting.

What is a frozen section quizlet? ›

Frozen section. thin piece of tissue cut from a frozen specimen for rapid examination under a microscope. Unfixed tissue. best frozen section. Formol-Calcium.

How is a frozen section sent to the lab quizlet? ›

How is a frozen section sent to the laboratory? (D) Frozen section specimens are not placed in solution because they can react with tissue and affect the pathologist's diagnosis. A frozen section is the cutting of a thin piece of tissue from a frozen specimen. This permits examination under a microscope.

What is freezing procedures? ›

Freeze food quickly and at as low a temperature as possible. Zero degrees F or lower is recommended. If you are freezing a large quantity of food at one time, set the freezer temperature 10 degrees lower than normal until the food is frozen. To avoid fluctuating temperatures do not open the freezer more than necessary.


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