Processing

Tissue Processing

Course Information

  • Course Code: MLTD1109

  • Semester: Fall 2022

Objectives

  • By studying this material, students will be able to meet the following Learning Outcome for MLTD 1109:

    • Description of the basic principles of fixation, processing, and theoretical principles of histological staining mechanisms.

Learning Objectives

  • List the steps involved in tissue processing.

  • Outline features of processing programs and indicate where each is necessary.

  • Describe the differences between open and closed tissue processors.

  • Define key terms:

    • Dehydration

    • Clearing

    • Infiltration

    • Universal solvents

  • List properties and safety precautions for common dehydrants.

  • List universal solvents.

  • List properties and safety precautions of common clearing agents.

  • List properties of common infiltrating agents.

  • Describe tissue effects of over-processing and under-processing of tissues.

  • Identify quality processes used in tissue processing, including:

    • Maintenance
      a Reagent rotations

    • Reagent QA checks

Readings

  • Reference Text:
    Carson, Chapter 5, pp. 85-96

Goal of Processing

  • The main goal is to prepare fixed tissue to be sliced into very thin sections (typically 4-5 microns) for mounting on slides for staining.

  • The end process results in tissue embedded in a block of paraffin that is stable indefinitely and kept on file for 20 years or longer.

Types of Tissue Processors

Closed System Tissue Processors

  • Most commonly used today.

  • Features:

    • Specimens are kept in an enclosed retort.

    • Processing fluids are pumped in and out sequentially.

    • Programmable settings; some units include 'safe modes' to continue processing after power issues.

    • Fumes are either exhausted to a nearby fume hood or filtered through charcoal.

Open System Processors

  • Older model of processing system.

  • Features:

    • Tissues are held in a basket, reagents are in stationary containers, and the basket moves from station to station.

    • Increased exposure to air leads to higher risk of fumes, evaporation, and humidity absorption.

    • Risks of desiccation or prolonged exposure to reagents in power losses or malfunctions, resulting in over-processing.

Processor Programs

  • Laboratories establish processing protocols based on specimen type and size:

    • Routine Program:

    • For most tissue types, especially large surgical specimens, with a cycle time of approximately 12-14 hours.

    • Biopsy Program:

    • Tailored for smaller tissues with considerably shorter processing times, as over-processing can lead to compromised tissue quality.

    • Extended Program:

    • For more dense or fatty tissues requiring longer processing times at each station to avoid under-processing, which can hinder microtomy and impair staining ability.

Sample Processor Program (Routine)


  • Processing Steps:

    Position

    Reagent

    Time/Temperature

    Function


    1

    FORMALIN

    20 min/ 37°C

    Fixation


    2

    H2O

    2 min/37°C

    Removes formalin


    3

    70% ALCOHOL

    20 min/37°C

    Displaces H2O


    …

    …

    …

    …


    13

    PARAFFIN WAX

    60 min/57°C

    Infiltration

    Processor Maintenance

    • Fluid Checks:

      • Ensure reagents cover specimens adequately.

    • Rotating Reagents:

      • After processing a batch, replace the most contaminated reagent and rotate the remaining reagents.

    • Paraffin Temperatures:

      • Set at 2-4 °C above melting point (usually 60 °C).

    • Reagent Lines:

      • Regular flushing to prevent buildup.

    • Retort Seal:

      • Must be in good condition and free from paraffin buildup.

    Processing Steps in Detail

    Step 1 - Dehydration

    • Goal: Remove free water without affecting water that is molecularly bound within tissue.

    • Attracts water using:

      • Hydrophilic reagents that draw out water.

      • Repeated dilution with dehydrating agents.

    Types of Dehydrating Agents

    1. Alcohols:

      • a. Ethanol

      • b. Methanol

      • c. Isopropanol

      • d. Butanol

    2. Acetone

    1.a Ethanol (Ethyl Alcohol)

    • Best dehydrant, offering reliable results and rapid dehydration.

    • Properties:

      • Clear, colorless, and flammable (store safely).

      • Hydrophilic and easily mixed with water.

      • Strictly regulated; labs must keep records for tax exemption.

      • Denatured alcohol is used to avoid consumption restrictions.

      • PEL (Permissible Exposure Limit): 1000 ppm; denatured alcohol: more toxic.

      • Disposal: best practice is to recycle unless contaminated with clearing agents.

    1.b Methanol (Methyl Alcohol)

    • Properties:

      • Flammable, clears smears, rarely used alone.

      • Very toxic (PEL: 200 ppm); can lead to blindness or death.

      • Disposal: recycle.

    1.c Isopropanol (Isopropyl Alcohol)

    • Preferred substitute for ethanol for paraffin embedding:

      • Less tissue shrinkage.

      • Not totally 'absolute'; about 1% water remains.

      • Serious considerations for its use with certain staining solutions (e.g., eosin).

      • PEL: 400 ppm.

    1.d Butanol (Butyl Alcohol)

    • Suitable for plant and animal material dehydration:

      • Slow process, less shrinkage; strong odor.

      • PEL: 100 ppm.

    Acetone

    • Advantages: Rapid and cost-effective dehydration.

    • Disadvantages: Can cause excessive shrinkage; difficult to maintain volume in open processors.

    • PEL ranges from 250 to 1000 ppm.

    Tissue Effects of Improper Dehydration
    Insufficient Dehydration:

    • Results in excess moisture, leading to inadequate clearing and infiltration, with soft or mushy tissue impacting microtomy.

    Excessive Dehydration:

    • Leads to overhardening and brittleness, complicating microtomy.

    Step 2 - Clearing

    • Agents used must be miscible with both dehydrating agents and the embedding medium (usually paraffin).

    Clearing Agents

    • Common agents include:

      • Xylene

      • Toluene

      • Benzene

      • Chloroform

      • Acetone

    • Xylene:

      • Rapid and effective; but prolonged exposure can over-harden tissues.

      • Cloudiness indicates residual water.

      • PEL: 100 ppm.

    • Toluene:

      • Greater tolerance to water compared to xylene; less hardening effect.

      • PEL: 50 ppm.

    • Benzene:

      • Toxic and volatile, risk outweighing benefits. PEL varies greatly.

    • Chloroform:

      • Not as efficient at making tissues transparent; disposal methods complicated. PEL: 50 ppm.

    Step 3 - Infiltration

    • Importance of a solid support medium for facilitating microtomy.

    • Infiltration agents must fill the intracellular spaces left by clearing agents.

    Infiltration Media

    1. Paraffin: Most commonly used; easy and efficient.

    2. Water Soluble Waxes: Reserved for special projects.

    3. Glycol Methacrylate (GMA): Good support for hard tissues.

    4. Epoxy Resins: Utilized for ultrastructure examination.

    5. Agar and Gelatin: Ideal for friable tissues, ensuring orientations during embedding.

    Properties of Paraffin

    • Different formulations tailored for specific melting points:

      • Regular melting point for routine use (55-58 C) provides sufficient support and good cutting ability.

    • Processing Considerations:

      • Heating and vacuum speeds up infiltration, but caution advised for small specimens.

    Cleaning Cycle in Closed Processors

    • Steps for cleaning and preparing for a new processing cycle:

      1. Clean with xylene or substitutes to remove paraffin.

      2. Use 100% ethanol to eliminate clearing agents and residues.

    Troubleshooting Tissue Processing
    Issues and Remedies:

    • Overdehydration: Dried, brittle samples causing microchatter.

    • Underdehydration: Leads to Concave paraffin blocks and stained nuclei issues.

    • Reprocessing Approaches: Reverse processing or returning to begin processing can address incomplete dehydration.

    Quality in Tissue Processing

    • Importance of proper maintenance and documentation for reagent quality. Procedures include:

      • Filtration and pH adjustments, tracking rotations

      • Ensuring reagents are within proper levels and contamination checks.

    References

    • Carson, F.L., Cappellano, C. (2020). Histotechnology, A Self-Instructional Text (5th ed.). ASCP Press.

    • MLTD1069/2019 Histotechnology Lab Manual, NSCC, Fall 2022.