What is a Frozen Section?
A frozen section is a term referring to a section of tissue that has been rapidly cooled using cryostat. It is an important feature that is needed in hospitals to assist with the diagnoses of lesions and the extent of the lesion during surgery. The cryostat is an instrument used to freeze the human tissue samples and cut it for microscopic section. It is used to aid in the immediate diagnosis of lesions to help medical professionals plan the management for the relevant patient. Frozen sections are also helpful for immunofluorescence and enzyme immunochemistry studies. Another useful indication would be to stain certain carbohydrates and lipids present in the tissue.
The Principle of Frozen Section
When the tissue sample goes through rapid freezing, it converts water into ice which acts as an embedding media allowing the tissue to be sectioned. The tissue can become firmer if the temperature of the tissue sample is lowered while increasing the temperature softens the tissue. Some important factors to note are:
The temperature range in the cryostat machine usually ranges from 0⁰C to -35⁰C. The majority of the tissue can be appropriately sectioned between -15⁰C to -25⁰C. Tissue samples that contain water can be sectioned at a higher temperature while tissues that contain more fat will need to be cut at a lower temperature.
The knife inside the rotary microtome is fixed, and the tissue can be moved with the help of the rotary wheel.
A tissue shelf in one side of the microtome can be used to keep the tissue. This helps to keep the samples at a freezing temperature as the temperature in the tissue shelf is usually lower compared to the cabinet temperature.
There is a small place to place the knife and brush holder in front of the microtome machine.
To obtain an even pressure during sectioning of the tissue samples throughout the whole length, the blade should be fixed to the holder with the knife angle kept between 5⁰ to 7⁰.
An antiroll plate is available in front of the knife to prevent the rolling of the tissue during the cutting process. The antiroll plate is usually glass within a metal frame.
A cool sable hair brush is also available to obtain unrolled tissue.
Depending on the manufacturer, the specimen holder can be available in various shapes and sizes.
The optimal cutting temperature (OCT) compound such as resin and water-soluble glycols will be used as an embedding medium to hold the tissue.
Cryostat Sectioning
The tissue sample and requisition form should first be identified.
Look at the clinical information available as it can help achieve the possible differential diagnoses.
Observe the gross appearance of the tissue in terms of color, consistency, texture, and presence of sutures used to mark the anatomical position of the sample.
Identify the resection margins and planes. Use different ink colors for identification of medial and lateral margins.
When cutting the tissue, ensure that the tissue is fresh, preferably dry, and free from any gauze, sutures, or staples. The tissue is then cut into multiple small pieces to assist freezing. Multiple sections of the tissue should be obtained to help minimize error and understand the primary pathology. Use a sharp blade to cut the most crucial area using gentle pressure.
To embed tissue in the mold, keep a small piece in the center of the mold and pour OCT in excess over it. A tissue holder is then placed over the tissue.
Place the tissue in the frozen section chamber. To hasten the process, cold spray can be used.
Load the cutting knife at the proper alignment.
Once the tissue is frozen, it will appear whitish. Place the in frozen tissue the holder of the microtome and trim to remove the excess OCT. This prepares the tissue surface for sectioning.
Cut the tissue gently and spread it over the antiroll plate using a cooled brush.
Press a glass slide firmly over the section and fix immediately in methanol for 1 minute. 95 percent ethanol can also be used for tissue fixation (for a few seconds). Rapid fixation is a must as a delay results in swollen cells and hazy cell margins.
Staining
Staining is usually done using hematoxylin and eosin (H&E) and toluidine blue stain. The slide is first rinsed in tap water and put in hematoxylin for a minute. It is then rinsed in tap water for 5 seconds followed by another rinse in Scott's tap water for another 5 seconds. The slide is then dipped in eosin for 20 seconds and rapidly rinsed in tap water after.
Conclusion
The frozen section refers to the process where there is rapid tissue section cooled with a cryostat to provide an immediate report of the tissue sample. The cryostat freezes the tissue allowing it to be cut for a microscopic section. The conversion of water into ice acts as the embedding media for cutting the tissue. It is a technique that is mostly used for the rapid diagnosis of lesions during intraoperative management. This helps to determine the extent of the lesion, allows for immunofluorescence and immunohistochemistry study, and staining of specific carbohydrate and lipid in the tissue. This article has described the principle, techniques, indications, and instructions on how to make a good quality frozen tissue section.
References
Dey P. (2018) Frozen Section: Principle and Procedure. In: Basic and Advanced Laboratory Techniques in Histopathology and Cytology. Springer, Singapore.
