LECTURE 8.2: Frozen Section in Histotechnology

FROZEN SECTION

Is a rapid diagnostic tool used during surgical procedures.

FROZEN SECTION

It helps the surgeon decide on the next course of action.

FROZEN SECTION

Recommended for examining lipids and nervous tissue elements.

FROZEN SECTION

Commonly used for muscle and nerve biopsies and surgically removed tumors.

• Rapid processing time

• Less equipment required

• Minimal ventilation needs

ADVANTAGES OF FROZEN SECTION:

• Lower morphological detail compared to paraffin sections

• Potential ice crystal artifacts

• Uneven freezing with liquid nitrogen

DISADVANTAGES 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.

APPLICATIONS OF FROZEN SECTION:

1. Liquid Nitrogen

2. Isopentane cooled by liquid nitrogen

3. Carbon dioxide gas

4. Aerosol sprays

FREEZING METHODS:

LIQUID NITROGEN

Rapid but can cause cracking.

(FREEZING METHOD)

ISOPENTANE COOLED BY LIQUID NITROGEN

Ideal for muscle tissue.

(FREEZING METHOD)

CARBON DIOXIDE GAS

Used in freezing microtomes.

(FREEZING METHOD)

AEROSOL SPRAYS

Suitable for small tissue pieces.

(FREEZING METHOD)

1. Cold Knife Procedure

2. Cryostat Procedure

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

FROZEN SECTION TECHNIQUES:

COLD KNIFE PROCEDURE

Uses a freezing microtome.

(FROZEN SECTION TECHNIQUES)

CRYOSTAT PROCEDURE

Uses a cryostat (-10°C to -20°C).

(FROZEN SECTION TECHNIQUES)

-10°C to -20°C

Cryostat temperature:

(FROZEN SECTION TECHNIQUES)

COLD KNIFE PROCEDURE

Is used for cutting frozen tissue sections

COLD KNIFE PROCEDURE

Almost any microtome can be used, provided that freezing and maintaining low temperatures are ensured.

COLD KNIFE PROCEDURE

Carbon dioxide (CO₂) technique is commonly used for freezing.

Carbon dioxide (CO2)

This technique is commonly used for freezing in Cold Knife Procedure.

(COLD KNIFE PROCEDURE)

1. Freezing attachment

2. Rubber hose connecting the freezing platform to the gas

3. CO2 gas cylinder

4. TIssue specimen

5. Freezing attachment emitting CO2 gas

6. Gas control knob

7. Handle

8. Knife holder

9. Microtome knife/razor

10. Freezing attachment/stage

11. Clamp

12. Knife adjustment knob

PARTS OF COLD KNIFE PROCEDURE:

1. Preparation

2. Freezing the Tissue

3. Trimming and Dew Line

4. Section Cutting

5. Transfer and Staining

COLD KNIFE PROCEDURE STEPS:

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.

(COLD KNIFE PROCEDURE)

FREEZING THE TISSUE

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

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

• Freeze just enough to be firm for sectioning.

(COLD KNIFE PROCEDURE)

3-5 mm thick ; 5-seconds

2. Freezing the Tissue

   • Tissue block (________) is oriented with intermittent CO₂ bursts.

   • Bursts last 1-2 seconds each, with _____ intervals.

   • Freeze just enough to be firm for sectioning.

(COLD KNIFE PROCEDURE)

TRIMMING AND DEW LINE

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

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

(COLD KNIFE PROCEDURE)

DEW LINE

The ideal cutting point

(COLD KNIFE PROCEDURE)

SECTION CUTTING

• Sections are cut at 10 microns thickness.

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

(COLD KNIFE PROCEDURE)

10 microns

4. Section Cutting

   • Sections are cut at ___ thickness.

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

(COLD KNIFE PROCEDURE)

TRANSFER AND STAINING

• Sections are placed in a dish of distilled water to separate.

• They are then mounted and stained for microscopic examination.

(COLD KNIFE PROCEDURE)

Solution: Warm with finger to soften

Problem: Too hard frozen (tissue chips)

(COLD KNIFE PROCEDURE: Troubleshooting and Adjustments)

Solution: Apply more CO2 bursts to refreeze

Problem: Not frozen enough (thick/crumbling sections)

(COLD KNIFE PROCEDURE: Troubleshooting and Adjustments)

Solution: Ensure the knife is colder than the tissue

Problem: Knife too warm

(COLD KNIFE PROCEDURE: Troubleshooting and Adjustments)

-40° to -60°C

Optimal temperature range for knife

(COLD KNIFE PROCEDURE: Optimal Temperature Conditions)

-5° to -10°C

Optimal temperature range for tissue

(COLD KNIFE PROCEDURE: Optimal Temperature Conditions)

0° to -10°C

Optimal temperature range for environment

(COLD KNIFE PROCEDURE: Optimal Temperature Conditions)

cold room with controlled humidity

In cold knife procedure, best results are achieved in a?

(COLD KNIFE PROCEDURE: Optimal Temperature Conditions)

Thin sections (<6µm)

In cold knife procedure, ______ are difficult to obtain.

(COLD KNIFE PROCEDURE: Optimal Temperature Conditions)

FREEZING MICROTOME

Specialized apparatus for cutting frozen sections.

(COLD KNIFE PROCEDURE)

FREEZING MICROTOME

Similar to a regular microtome with added features:

• CO₂ gas control and an automatic feed mechanism.

(COLD KNIFE PROCEDURE)

FREEZING MICROTOME

Incorporates:

• Microtome knife control

• CO₂ gas introduction system

• Automatic feed mechanism (adjustable from 5-40 µm)

(COLD KNIFE PROCEDURE)

FREEZING MICROTOME

Features:

• Clamped onto a desk or table.

• CO₂ gas cylinder connected via a rubber hose.

• Insulated rubber plate prevents heat conduction.

• Adjustable knife holder for optimal cutting angle.

(COLD KNIFE PROCEDURE)

CRYOSTAT PROCEDURE

Uses a cryostat, a specialized apparatus for fresh tissue microtomy.

fresh tissue microtomy

In cryostat procedure, it uses a cryostat, a specialized apparatus for ______.

insulated microtome in an electrically driven refrigerated chamber

The cryostat houses an?

CRYOSTAT PROCEDURE

Maintains isothermic conditions at -18 to -20°C for precise sectioning.

isothermic ; -18 to -20°C

Cryostat procedure maintains ____ conditions at ___ for precise sectioning

-18 to -20°C.

Optimum working temperature for cryostat procedure:

(COLD KNIFE PROCEDURE: Temperature Control)

CRYOSTAT PROCEDURE

Fast freezing is critical to prevent artifacts.

(COLD KNIFE PROCEDURE: Temperature Control)

Fast freezing

In cryostat procedure, _____ is critical to prevent artifacts.

(COLD KNIFE PROCEDURE: Temperature Control)

CRYOSTAT PROCEDURE

Slow freezing causes distortion due to ice formation.

(COLD KNIFE PROCEDURE: Temperature Control)

SLOW FREEZING

In cryostat procedure, _____ causes distortion due to ice formation.

(COLD KNIFE PROCEDURE: Temperature Control)

1. Rotary microtome

2. Tissue shelf

3. Knife holder & blade

COMPONENTS OF THE CRYOSTAT:

ROTARY MICROTOME

Moves the tissue via a rotary wheel.

(COMPONENTS OF THE CRYOSTAT)

TISSUE SHELF

Keeps tissue at a lower temperature for freezing

(COMPONENTS OF THE CRYOSTAT)

KNIFE HOLDER & BLADE

Uses low- or high-profile disposable blades.

(COMPONENTS OF THE CRYOSTAT)

KNIFE HOLDER & BLADE

Alternative: Profile C steel blade.

(COMPONENTS OF THE CRYOSTAT)

Profile C steel blade

COMPONENTS OF THE CRYOSTAT

Knife holder & blade:

• Uses low- or high-profile disposable blades.

• Alternative: ______.

• Blade angle: 5°-7°.

(COMPONENTS OF THE CRYOSTAT)

KNIFE HOLDER & BLADE

Blade angle: 5°-7°.

(COMPONENTS OF THE CRYOSTAT)

5°-7°

COMPONENTS OF THE CRYOSTAT

Knife holder & blade:

• Uses low- or high-profile disposable blades.

• Alternative: Profile C steel blade.

• Blade angle: ____.

(COMPONENTS OF THE CRYOSTAT)

ANTIROLL PLATE & TISSUE HANDLING

Prevents curling of cut tissue.

(CRYOSTAT PROCEDURE)

ANTIROLL PLATE & TISSUE HANDLING

Made of glass within a metal frame.

(CRYOSTAT PROCEDURE)

ANTIROLL PLATE & TISSUE HANDLING

Positioned with a gap between the plate and the knife.

(CRYOSTAT PROCEDURE)

ANTIROLL PLATE & TISSUE HANDLING

Alternative: Cool sable hair brush can be used.

(CRYOSTAT PROCEDURE)

COOL SABLE HAIR BRUSH

CRYOSTAT PROCEDURE

Antiroll Plate & Tissue Handling:

• Prevents curling of cut tissue.

• Made of glass within a metal frame.

• Positioned with a gap between the plate and the knife.

• Alternative: ______ can be used.

(CRYOSTAT PROCEDURE: Antiroll Plate & Tissue Handling)

SPECIMEN HOLDER (CHUCK)

Available in different shapes and sizes.

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

CHUCK

Specimen holder in cryostat procedure which is available in different shapes and sizes

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

OPTIMUM CUTTING TEMPERATURE (OCT)

CRYOSTAT PROCEDURE

Specimen Holder & Embedding Medium:

• Specimen holder (chuck): Available in different shapes and sizes.

• Embedding medium: ____ compound.

  • Made of water-soluble glycols & resin.

  • Supports the tissue and ensures a firm but flexible sectioning process.

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

OPTIMUM CUTTING TEMPERATURE (OCT)

Embedding medium used in cryostat procedure which is made of water-soluble glycols & resin and supports the tissue and ensures a firm but flexible sectioning process.

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

OPTIMUM CUTTING TEMPERATURE (OCT)

Made of water-soluble glycols & resin.

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

OPTIMUM CUTTING TEMPERATURE (OCT)

Supports the tissue and ensures a firm but flexible sectioning process.

(CRYOSTAT PROCEDURE: Specimen Holder & Embedding Medium)

• Tissue must remain frozen solid during sectioning.

• Prevents compression & distortion during cutting.

• Knife should be chilled to avoid melting the tissue.

IMPORTANCE OF MAINTAING THE FROZEN STATE IN CRYOSTAT PROCEDURE:

Overfreezing

What is the cause?

Issue: Tissue too cold

(CRYOSTAT PROCEDURE)

Becomes brittle, shatters upon cutting

What is the effect?

Issue: Tissue too cold

(CRYOSTAT PROCEDURE)

Underfreezing

What is the cause?

Issue: Tissue too warm

(CRYOSTAT PROCEDURE)

Tissue compresses & distorts

What is the effect?

Issue: Tissue too warm

(CRYOSTAT PROCEDURE)

Ice crystal formation

What is the cause?

Issue: Slow freezing

(CRYOSTAT PROCEDURE)

Freeze-thaw artifacts (holes in tissue)

What is the effect?

Issue: Slow freezing

(CRYOSTAT PROCEDURE)

Tissue too cold

What is the issue?

Cause: Overfreezing

Effect: Becomes brittle, shatters upon cutting

(CRYOSTAT PROCEDURE)

Tissue too warm

What is the issue?

Cause: Underfreezing

Effect: Tissue compresses & distorts

(CRYOSTAT PROCEDURE)

Slow freezing

What is the issue?

Cause: Ice crystal formation

Effect: Freeze-thaw artifacts (holes in tissue)

(CRYOSTAT PROCEDURE)

ICE CRYSTAL ARTIFACTS

Appear as holes in tissue.

(CRYOSTAT PROCEDURE: Freezing Artifacts & Their Impact)

CRYOSTAT PROCEDURE: Freezing Artifacts & Their Impact

Most problematic in skeletal muscle & delicate tissues.

skeletal muscle & delicate tissues

In cryostat procedure, freezing artifacts & their impact are the most problematic in what tissues?

(CRYOSTAT PROCEDURE: Freezing Artifacts & Their Impact)

COLD KNIFE PROCEDURE

Is used for rapid frozen tissue sectioning.

rapid frozen tissue

Cold Knife Procedure is used for?

COLD KNIFE PROCEDURE

Success depends on temperature control and proper technique.

• temperature control

• proper technique

Success in Cold Knife Procedure depends on:

COLD KNIFE PROCEDURE

A freezing microtome improves efficiency and accuracy.

FREEZING MICROTOME

In Cold Knife Procedure this improves efficiency and accuracy.

CRYOSTAT PROCEDURE

Allows controlled frozen sectioning at -18 to -20° C.

-18 to -20° C.

Cryostat Procedure allows controlled frozen section at what temperatures?

CRYOSTAT PROCEDURE

Fast freezing prevents ice crystal formation.

FAST FREEZING

In Cryostat Procedure what prevents ice crystal formation?

CRYOSTAT PROCEDURE

Antiroll plate & OCT compound improve sectioning quality

ANTIROLL PLATE & OCT COMPOUND

In Cryostat Procedure what improves sectioning quality?

CRYOSTAT PROCEDURE

Maintaining the right temperature is crucial

Maintaining the right temperature

What is crucial in Cryostat Procedure?

1. Identify the tissue sample of the patient.

2. Clinical information

3. Tissue appearance

4. Anatomy of the tissue

5. Color the resection planes and margins

6. Section cutting

7. Cytology preparation (if needed)

GROSSING FOR FROZEN SECTION TISSUE:

COLD KNIFE PROCEDURE (Freezing Microtome)

Uses a freezing microtome for sectioning.