By Dr. Jerome A. Tan, RMT, MLS (ASCPi), MD
Describe the basic techniques of frozen section processing.
Explain the principle involved.
Enumerate the advantages and disadvantages.
Frozen section is a rapid diagnostic tool used during surgical procedures.
It helps the surgeon decide on the next course of action.
Recommended for examining lipids and nervous tissue elements.
Commonly used for muscle and nerve biopsies and surgically removed tumors.
Advantages | Disadvantages |
---|---|
Rapid processing time | Lower morphological detail compared to paraffin |
Less equipment required | Potential ice crystal artifacts |
Minimal ventilation needs | Uneven freezing with liquid nitrogen |
Rapid pathological diagnosis during surgery.
Diagnostic and research enzyme histochemistry.
Demonstration of lipids, carbohydrates, and soluble substances.
Immunofluorescent and immunohistochemical staining.
Specialized silver stains in neuropathology.
Liquid Nitrogen – Rapid but can cause cracking.
Isopentane cooled by liquid nitrogen – Ideal for muscle tissue.
Carbon dioxide gas – Used in freezing microtomes.
Aerosol sprays – Suitable for small tissue pieces.
Cold Knife Procedure
Uses a freezing microtome.
Cryostat Procedure
Uses a cryostat (-10°C to -20°C).
Tissue must be fresh and frozen quickly to avoid artifacts.
Soak filter paper in gum syrup and place it on the microtome stage.
Apply short bursts of CO₂ to freeze the filter paper onto the stage.
Tissue block (3-5 mm thick) is oriented with intermittent CO₂ bursts.
Bursts last 1-2 seconds, with 5-second intervals.
Tissue is manually lifted to the knife and trimmed until flat.
Warm the surface slightly to reveal the dew line (ideal cutting point).
Sections are cut at 10 microns thickness.
Sections stick to the knife and are removed with a moistened brush or finger.
Sections are placed in distilled water to separate.
Mounted and stained for microscopic examination.
Specialized apparatus for cutting frozen sections.
Similar to a regular microtome but with additional features:
CO₂ gas control
Automatic feed mechanism (adjustable from 5-40 μm)
Maintains isothermic conditions at -18°C to -20°C for precise sectioning.
Rotary microtome: Moves the tissue via a rotary wheel.
Tissue shelf: Keeps tissue at a lower temperature for freezing.
Knife holder & blade:
Uses low- or high-profile disposable blades.
Blade angle: 5°-7°.
Optimum working temperature: -18°C to -20°C.
Fast freezing prevents ice crystal artifacts.
Prevents curling of cut tissue.
Made of glass within a metal frame.
Alternative: Cool sable hair brush.
Specimen holder (chuck): Available in different shapes and sizes.
Embedding medium: Optimum Cutting Temperature (OCT) compound.
Made of water-soluble glycols & resin.
Supports the tissue for firm but flexible sectioning.
Freezing artifacts appear as holes in tissue.
Most problematic in skeletal muscle & delicate tissues.
Cold Knife Procedure | Cryostat Procedure |
---|---|
Used for rapid frozen tissue sectioning | Controlled sectioning at -18°C to -20°C |
Requires temperature control | Fast freezing prevents ice crystal formation |
A freezing microtome improves efficiency | Antiroll plate & OCT compound improve sectioning |
Tissue Freezing & Sectioning Steps
Place a drop of water on the block holder to secure tissue.
Hold tissue flat and apply CO₂ bursts.
Move knife slowly through the tissue above the frozen line.
Mounting
Free-floating sections are transferred onto albuminized slides.
Mounted sections are placed directly on a glass slide.
Staining
Temporary Stain: Toluidine Blue (rapid)
Permanent Stain: Hematoxylin & Eosin (H&E)
Tissue & knife temperature must be optimal.
Fatty/mucin-rich tissues require lower temperatures.
Cryostat sections of fresh tissue adhere well.
Formalin-fixed tissue requires special coatings (e.g., albumin or chrome-glycerin jelly).
Rapid freezing at -160°C to -180°C with liquid nitrogen.
Dehydration through sublimation in a vacuum chamber at -30°C to -40°C.
Alternative method using acetone at -70°C.
Sections are embedded in paraffin for long-term preservation.
Immunocytochemistry
Fluorescent antibody studies
Scanning electron microscopy
Frozen sections should not be left exposed in the cryostat.
Unprotected sections dehydrate quickly.
For long-term storage, wrap and freeze at -70°C.