Guide to the Preparation of Frozen Sections

Workstation: Cutting Board and Basic Supplies

Grossing sections for handling tissues received for IOC evaluation need to be equipped with a large cutting board, surgical instruments, and other supplies necessary for dissecting the specimen. Supplies include disposable gloves, disposable scalpel blades and blade holders, short and long blades and blade handles, forceps, sharp scissors, metal probes, rulers, pens, including broad-tip Sharpie and xylene-proof markers for tissue cassettes, tissue cassettes, specimen containers of various sizes, scales, fine brushes, embedding medium (e.g., Sakura Tissue-Tek O.C.T. Compound, StatLab Medical Products Cryo-STAT), cryogen spray coolant, charged slides (polylysine coated), coverslips, mounting medium, paper towels, gauze sponges, disinfectant/detergent, gloves, N-95 masks, various color inks to mark resection margins or orient specimens, cotton-tip applicators to ink specimens, and containers to dispose of sharps ( Figure 3-1 ). The workstation also needs to include multiple containers with the various reagents needed to perform routine hematoxylin and eosin (H&E), toluidine blue, Diff-Quick and Oil Red O stains ( Table 3-1 ), and various requisitions forms.

Intraoperative consultation procedures need to be performed in well-ventilated rooms, ideally equipped with negative vents, to minimize the possibility of spreading mycobacterial and other infections. In addition, the workstation needs to be located in proximity to a sink with running water and to a refrigerator and/or freezers.

The Cryostat

Frozen sections are performed with cryostats, specialized instruments composed of a tissue microtome (cryotome) installed within a cooling chamber that allows for rapid freezing of tissues and a continuous process of cutting tissue at low temperatures ( Figure 3-2 ). Current cryostats use automated electrical refrigeration systems with accurate thermostats.

The cooling chamber of the cryostat has a “work area” to place chucks when cutting another section. Tissue is ideally cut when the chamber is between -10° to -20° C, depending on the tissue. To ensure the cryostat is cool enough for all tissues, it is best to set the chamber temperature to -20° C. Modern cryostats feature a peltier, composed of an electronically controlled cold surface where metal chucks are kept at a reduced temperature and tissues can be quickly frozen by direct contact ( Figure 3-3 ).

The cryotome consists of an objective to place the chucks containing frozen tissues, a stage to hold sharp cutting blades, and an advancing/retracting mechanism. The stage holding blades usually has manual mechanisms that allow for the installation of disposable metal blades or resharpenable knives in older models. Cryotome stages also have mechanisms to vary the angle of the cutting blade that would allow for optimal tissue sectioning. The advancing/retracting mechanism of a cryotome is composed of electrical motors that are controlled with electronic controls that rapidly approximate the objective and metal chucks holding the tissues to the cutting blade and an external manual handwheel that advances the tissue toward the sharp metal blade in fixed increments, ranging from 1 μm and greater. Some cryostat models have optional antiroll guides that assist with keeping sections flat ( Figure 3-4 ). However, using a chilled fine brush to reduce wrinkling of the section as it is cut is a common practice.

Cryostats perform an automatic defrost cycle to reduce frost in the chamber. The time for setting the cryostat to defrost automatically should be set during the late night or early morning when the need to use the cryostat is minimal. Additionally, the College of American Pathologists (CAP) requires that each cryostat used daily must be defrosted and decontaminated at room temperature on a weekly basis.

Different cryostat models are available in the U.S. market, and it is beyond the scope of this chapter to review their technical specifications and/or recommend certain equipment. Pathologists and technicians cutting the frozen sections need to familiarize themselves with the various controls available in different equipment prior to performing IOC.