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

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

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

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

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.