Frozen Section Lecture

By Dr. Jerome A. Tan, RMT, MLS (ASCPi), MD

Page 1: Objectives

1. Describe the basic techniques of frozen section processing.

2. Explain the principle involved.

3. Enumerate the advantages and disadvantages.

Page 2: Introduction to Frozen Section

• 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

Page 3: Applications of Frozen Section

• 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.

Freezing Methods

1. Liquid Nitrogen – Rapid but can cause cracking.

2. Isopentane cooled by liquid nitrogen – Ideal for muscle tissue.

3. Carbon dioxide gas – Used in freezing microtomes.

4. Aerosol sprays – Suitable for small tissue pieces.

Page 4: Frozen Section Techniques

1. Cold Knife Procedure

   • Uses a freezing microtome.

2. Cryostat Procedure

   • Uses a cryostat (-10°C to -20°C).

3. Tissue must be fresh and frozen quickly to avoid artifacts.

Page 5-6: Cold Knife Procedure Steps

1. Preparation

• 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.

2. Freezing the Tissue

• Tissue block (3-5 mm thick) is oriented with intermittent CO₂ bursts.

• Bursts last 1-2 seconds, with 5-second intervals.

3. Trimming and Dew Line

• Tissue is manually lifted to the knife and trimmed until flat.

• Warm the surface slightly to reveal the dew line (ideal cutting point).

4. Section Cutting

• Sections are cut at 10 microns thickness.

• Sections stick to the knife and are removed with a moistened brush or finger.

5. Transfer and Staining

• Sections are placed in distilled water to separate.

• Mounted and stained for microscopic examination.

Page 7-8: Freezing Microtome

• 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.

Page 9: Cryostat Components & Temperature Control

• 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°.

Temperature Control

• Optimum working temperature: -18°C to -20°C.

• Fast freezing prevents ice crystal artifacts.

Page 10-11: Specimen Handling in Cryostat

Antiroll Plate & Tissue Handling

• Prevents curling of cut tissue.

• Made of glass within a metal frame.

• Alternative: Cool sable hair brush.

Specimen Holder & Embedding Medium

• 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.

Page 12: Effects of Incorrect Freezing

• Freezing artifacts appear as holes in tissue.

• Most problematic in skeletal muscle & delicate tissues.

Cold Knife vs. Cryostat Procedure Summary

Page 13-14: Frozen Section Processing

1. 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.

2. Mounting

   • Free-floating sections are transferred onto albuminized slides.

   • Mounted sections are placed directly on a glass slide.

3. Staining

   • Temporary Stain: Toluidine Blue (rapid)

   • Permanent Stain: Hematoxylin & Eosin (H&E)

Page 15-16: Factors Affecting Cryostat Sectioning

• Tissue & knife temperature must be optimal.

• Fatty/mucin-rich tissues require lower temperatures.

Freezing Previously Fixed Tissue

• Cryostat sections of fresh tissue adhere well.

• Formalin-fixed tissue requires special coatings (e.g., albumin or chrome-glycerin jelly).

Page 17-18: Freeze-Drying & Freeze-Substitution

Freeze-Drying

• Rapid freezing at -160°C to -180°C with liquid nitrogen.

• Dehydration through sublimation in a vacuum chamber at -30°C to -40°C.

Freeze-Substitution

• Alternative method using acetone at -70°C.

• Sections are embedded in paraffin for long-term preservation.

Applications of Freeze-Drying

• Immunocytochemistry

• Fluorescent antibody studies

• Scanning electron microscopy

Page 19: Storage of Frozen Sections

• Frozen sections should not be left exposed in the cryostat.

• Unprotected sections dehydrate quickly.

• For long-term storage, wrap and freeze at -70°C.