Cryotomy
The History of Freezing Techniques
Frozen section techniques were developed in an attempt to eliminate artifacts in tissue specimens caused by exposure to normal processing procedures. Boyle, who sectioned frozen eyes, accomplished the first recorded frozen section in 1663. The initial freezing techniques were accomplished by outdoor freezing in cold weather or with volatile chemicals such as ether and rigolene. As the techniques advanced, freezing was accomplished by using carbon dioxide, Freon, isopentane, liquid nitrogen slush and other cryogens. Thermoelectric cooling is now also an accepted method for routine cryotomy for light microscopic evaluation.
Factors Affecting Frozen Sectioning
Specimens are usually mounted on a specimen holder by means of a compound, such as Cryo-STAT (CAT# Cryo-STAT) that is formulated to freeze at a specific temperature and optimized to have sectioning characteristics similar to frozen tissue specimens. The compound can be used to either serve as a glue to attach the specimen to the holder, leaving the specimen exposed and reducing rolling during sectioning, or to surround the specimen and act as a support medium during the sectioning process. When the compound surrounds the specimen it becomes a thermal insulator so care must be taken during the freezing process to prevent freezing artifacts (ice crystal damage). The compound can also be used to provide a protective covering to prevent desiccation (drying out) of the specimen during storage. Proper freezing techniques are extremely important and must be selected according to the requirements and resources available in individual laboratories.
Sectioning specimens at the proper temperature is critical. If the specimen is too warm, sections will compress and bunch up on the knife edge. If the specimen is too cold, it breaks and shatters under the pressure of the blade. Each type of biological specimen and industrial material will have its own optimum cutting temperature range. It is common for a specimen to have constituents that have very different characteristics for adequate sectioning and, when this occurs, it is preferable to freeze to the temperature of the constituent with the lowest optimum temperature.
If the previously frozen specimen, knife or anti-roll plate becomes too warm, they can be cooled by spraying with an aerosol cryogen, such as the environmentally friendly Statfreeze 3 ™ Freezespray (CAT# SL202).
Routine Frozen Section Staining
Frozen sections for intraoperative diagnosis require a rapid, high quality stain. The general preference seems to be a stain with results resembling the Hematoxylin and Eosin stain on routine permanent sections. The Hematoxylin used for staining can be either progressive or regressive. A progressive hematoxylin with a high stain concentration accelerates the staining process. The eosin can be used alone or in a combination of stains to provide additional contrast and intensity.
Cryo-STAT (CAT# Cryo-STAT) is a freezing compound that sections smoothly and evenly with a firm and consistent quality. It dissolves readily, without residue, when it is exposed to immersion in water or other aqueous media. It is available in 4 oz plastic bottles.
Statfreeze 3™ Freezespray (CAT# SL202) does not contain any ingredients presently known to harm the environment. It is available in 10 oz. cans that are available individually or 12/case.
Product information on Harris Hematoxylin (CAT #SL90), Gill’s 1, 2 and 3 Hematoxylin (CAT# SL94, SL95 and SL97 respectively), Eosin (CAT#SL98) and Treosin (CAT# DL93) can be found in the H&E section of this manual.
