Frozen Section Principle And Procedure

The frozen section is the rapid tissue section by cooling the tissue with the help of cryostat to give an immediate report of the tissue sample. This is especially needed in a large hospital to diagnose the lesion or extent of the lesion at the time of operation.

The cryostat is the instrument that has the arrangement to freeze the tissue and also to cut the frozen tissue for microscopic sections.

Indications Of Frozen Sections

The frozen section is used mainly for immediate diagnosis of the lesion for management and to know the extent of the lesion. It is also helpful to do enzyme immunochemistry and immunofluorescence study.

At times, frozen section tissue is used for the demonstration of fat and carbohydrate in the tissue sample.

The Principle of Frozen Section

The rapid freezing of the tissue sample converts the water into ice. The firm ice within the tissue acts as embedding media to cut the tissue.  Lowering the temperature makes the tissue more firm, whereas increasing temperature makes the tissue softer.

Cryostat Machine Proper:

The temperature range in the machine: The cryostat machine has the usual temperature range from 0 °C to −35 °C. Most of the tissue is sectioned properly between −15  °C and  −25 °C. The water-containing tissues can be sectioned at a higher temperature, and fat-containing tissue needs a much lower temperature to cut. 

Rotary microtome: Rotary microtome is placed inside the cabinet of the cryostat.  Here the knife is fixed and the tissue is moved with the help of a rotary wheel. 

Tissue shelf: Just on one side of the microtome, there is a tissue shelf to keep the tissue. In this place, the samples are kept for freezing.

Usually, the temperature of the tissue shelf is lower than the overall cabinet temperature. Place to keep the brush and knife holder: Just in front of the microtome machine, there remains a small place to keep the brush and knife holder. 

Knife or blade: Nowadays, low- or high-profile disposable blades are used. The blade should be properly fixed to the holder to get an even pressure in the whole length. Alternatively, a Profile  C steel blade is also used. The angle of the knife is kept between 5° and 7°.

Antiroll plate: Just in front of the knife, there is an antiroll plate that prevents the rolling of the cut tissue. It is usually a glass plate within a metal frame. The undersurface of the plate has free space, and there is a gap between the knife and the plate. 

Alternating to the antiroll plate, a cool sable hair brush can be used to get unrolled tissue.  Specimen holder: The specimen holder or chuck is supplied by the manufacturers in different sizes and shapes. Usually, these are round metal structures. 

Embedding medium: This medium is used to hold the tissue over the chuck. Presently optimum cutting temperature (OCT) compound is used as embedding medium. The OCT is made of water-soluble glycols and resin.

Cryostat Sectioning

The process of the cryostat sectioning needs the following steps:

1. Grossing and cutting the specimen:  The cutting surface of the tissue should be smooth. The following steps in grossing of the  tissue are mandatory for accurate reporting:

• Identify the tissue sample of the patient and  the requisition form: This is the first and  foremost part of the frozen tissue grossing 

• Salient clinical information: The essential clinical information is very helpful as it guides the pathologist to reach a possible differential diagnosis.

• Tissue appearance: The gross appearance of the tissue such as color, texture, consistency, and any suture to mark the anatomical position.  

• Resection margin: It is very important to identify the resection margins of the tumor. The resection planes and margins should be inked thoroughly. The different colors of ink can be used for medial and lateral margin identification.

Cutting the tissue: The tissue should be fresh without any fixative. The tissue should be preferably dry, and it should not be wrapped in a gauze piece. Any suture, staple, or sharp hard structure should be removed from the tissue sample.

Now the tissue is cut into small pieces as it facilitates freezing. Take multiple sections of the tissue to understand the main pathology and to minimize the error.

Use a new sharp scalpel blade, and first cut the most important area that needs microscopic examination. It is preferable to use the gentle stroke of the scalpel rather than too much pressure. 

Cytology of the tissue: At times the imprint of the tissue on the slide provides good morphological details such as lymphoma of the lymph node.  Similarly crushing of tissue also provides excellent morphological details such as in the case of tissue of the brain tumor.

1. Tissue embedding in the mold: A small piece of the tissue is kept in the center of the mold, and then the OCT is poured over it in excess. Then the tissue holder or chuck is firmly placed over the tissue with overflown OCT.

2. Tissue loading in the frozen section chamber: The tissue is now placed in the frozen section chamber and cold spray can be used to make the process faster.

3. Loading the blade: The cutting knife or blade is now loaded and the proper alignment is done.

4. Trimming the tissue: The loss of normal or natural color to whitish color indicates that the tissue is frozen. The frozen tissue in the tissue holder is now placed in the holder of the microtome. The block is trimmed to remove the excess OCT and to get the smooth tissue surface for sectioning.

5. Sectioning: The tissue is now cut gently and is spread over the antiroll plate with the help of a brush. The brush should be cooled. The tip of the tissue is guided by the brush.

6. Section lifting: The glass slide of normal room temperature is pressed firmly over the tissue section, and normally the tissue sticks immediately.

7. Fixation: The tissue should be immediately fixed in methanol for 1 min or 95% ethanol for a few seconds. Rapid fixation within a few seconds is mandatory. In case of delayed fixation, the cells are swollen, and the cytoplasmic margin may be ruptured giving a hazy appearance of the margin of the cells.