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CONVENTIONAL TISSUE PROCESSING

Course Information
  • Course Code: HFCT31 LAS
  • Course Title: Histopathologic and Cytologic Techniques
  • Instructor: Turno, Mit OLFU-Valenzuela
  • Lab Course: HPCT311 LAB: HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES.
Learning Objectives
  • Students will:
    • Understand the different tissue processing steps in Histopathology Laboratory.
    • Learn the different techniques and chemicals used in tissue processing.
    • Learn about gross examination and tissue accessioning.

Introduction to Conventional Tissue Processing

  • Main goal: Enable pathologists to diagnose disease through quality tissue sections that allow for microscopic analysis of cellular changes.
  • Solid tissues must be fixed and processed to preserve their structure and be impregnated with a suitable hardening substance to facilitate thin slicing for staining and microscopic evaluation.

Steps in Tissue Processing

  • The essential steps employed in processing solid tissues are:
    1. Fixation
    2. Decalcification
    3. Dehydration
    4. Clearing
    5. Impregnation
    6. Embedding
    7. Trimming
    8. Section-Cutting
    9. Staining
    10. Mounting
    11. Labeling
  • Tissue processing is defined as the steps taken from fixation to a state where tissue is fully infiltrated with histological wax and ready for section cutting on the microtome.

Accessioning

  • Definition: The process of receiving incoming tissue specimens and verifying their patient and tissue identity.
  • Each surgery case is assigned a unique accession number and logged in an accession log, ensuring accurate tracking.
  • Types of Specimens:
    • C: Cytology specimen
    • A: Autopsy specimen
    • S: Surgical specimen
  • Example of Accessioning Number: S-26-0001

Gross Examination

  • Grossing: Examination and dissection of surgical specimens, involving preparation of sections requiring processing.
  • Accurate diagnosis relies on proper identification, handling, and processing of specimens, carried out by personnel such as pathologists, residents, physician assistants, histotechnologists, or biomedical scientists.

Tissue Processing Steps

1. Fixation

  • Importance: Critical first step in histotechnology that preserves tissues from decay and prevents autolysis or putrefaction. Must be performed rapidly post-removal (in surgeries) or post-death (in autopsies).
  • Goals of Fixation:
    • Preserve the morphologic and chemical integrity of cells in a life-like manner.
    • Prevent degeneration of tissues post-removal from the body.
    • Harden and protect the tissue for easier handling during subsequent processing.
    • Proper orientation in cassettes for paraffin embedding and microtomy.
  • Fixatives work by inactivating enzymes, preventing autolysis, and inhibiting bacterial growth.

2. Objectives of Fixation

  1. Stop all cellular activities to preserve the tissue for microscopic examination as if still alive.
  2. Prevent breakdown by inactivating lysosomal enzymes or altering cellular components to make them insoluble.
  3. Stabilize cellular components (proteins, lipids, carbohydrates) to prevent autolysis.

3. Methods of Fixation

Physical Methods:
  • Heating: Rarely used, mostly for smears of microorganisms.
  • Microwaving: Widely used in routine labs.
  • Cryo-Preservation (freeze-drying): Utilized in histochemistry.
Chemical Methods:
  • Fixative Solution: Various chemical agents are employed for this purpose.

4. Chemical Fixatives

Formaldehyde and Formalin
  • Formaldehyde: A gas from the oxidation of methyl alcohol, water-soluble (37-40% w/v).
  • Formalin: A diluted version of formaldehyde (e.g., 10% NBF is a 4% solution). Purchase commercially prepared solutions to ensure safety and effectiveness.
Buffered Formalin
  • Description: 10% neutral buffered formalin, buffered to pH 7 with phosphate buffer. Best for routine histology; excels in preventing autolysis and preserving tissue morphology.
  • Applications: Effective for paraffin embedding, immunohistochemistry, and FISH.
Advantages of Using Formalin:
  1. Cost-effective and readily available.
  2. Compatible with many stains for various techniques.
  3. Avoids over-hardness even with prolonged fixation.
  4. Excellent tissue penetration.
  5. Preserves fat, mucin, and glycogen.
Disadvantages of Using Formalin:
  1. Irritating fumes causing nasal and ocular discomfort.
  2. Skin irritant leading to allergic dermatitis on prolonged contact.
  3. Potential for significant tissue shrinkage.
  4. Soft fixative, inadequately hardening certain cytoplasmic structures for effective embedding.

5. Mechanisms of Fixation

  • Additive Fixation: Chemical constituents become part of the tissue by forming cross-links, stabilizing proteins (e.g., formalin, mercury).
  • Non-Additive Fixation: The agent modifies tissue composition but is not incorporated (e.g., alcoholic fixatives).

6. Practical Considerations for Optimal Fixation

  • Quickly transfer specimens to fixative (<1 hour).
  • Maintain an adequate volume of fixative, ideally 20:1 or at least 10:1 ratio to tissue.
  • Remove anatomical barriers to enhance penetration (e.g., incising fascia).
  • Use sufficient time for fixation based on tissue type to achieve the best results.

7. Main Factors in Fixation

  1. Volume: Typically 10-20x the tissue volume.
  2. pH: Best conducted at neutral pH of 6-8.
  3. Temperature: Commonly performed at 40°C.
  4. Thickness of Sections: 1-2 mm for electron microscopy; 2 cm for light microscopy.
  5. Osmolality: Use isotonically buffered solutions to prevent cellular damage.
  6. Concentration: Use lowest concentration effective for fixation (e.g., 10% formaldehyde).
  7. Duration: Fixation time varies by tissue type and structure.
  8. Time Interval: Immediate fixation to prevent autolysis and putrefaction.

8. Effects of Fixatives

  • Reduce infection risks.
  • Harden soft tissues for easier handling.
  • Enhance optical differentiation of cells for examination.
  • Modify staining processes and impact dye effectiveness.

9. Characteristics of a Good Fixative

  • Inexpensive, stable, safe to handle, rapid action with minimal distortion.
  • Should inhibit autolysis and bacterial growth, allow even penetration, and facilitate subsequent staining procedures.

10. Types of Fixatives

  1. Aldehydes: Formaldehyde, glutaraldehyde.
  2. Oxidizing Agents: Osmium tetroxide, potassium permanganate.
  3. Alcohol-Based: Methyl alcohol, ethyl alcohol, acetic acid.
  4. Metallic Fixatives: Mercuric chloride, picric acid, chromate.

11. Secondary Fixation

  • Involves placing already fixed tissue into a second fixative:
    • Facilitates demonstration of specific substances.
    • Enables special staining techniques using the secondary fixative as a mordant.
    • Ensures thorough hardening and preservation.

General Precautions in Handling and Fixation of Specimens

  1. Proper labeling and identification are essential.
  2. Fix surgical specimens post-removal or refrigerate to prevent drying.
  3. Avoid freezing unfixed tissues to prevent ice crystal artifacts.
  4. Treat all fresh tissues as potentially infectious.
  5. Prevent drying of specimens to avoid distortion of cellular detail.
  6. Ensure adequate fixative volume (10-20 times the tissue volume).
  7. Prolonged fixation requires more than 50 times the tissue volume.
  8. Wash fixed tissues to remove contaminants before staining.
  9. Adhere to necessary fixation times to avoid excessive brittleness.

Difficulties Caused by Improper Fixation

  1. Early autolysis due to delays or insufficient fixative.
  2. Removable substances leading to diagnostic issues.
  3. Artefact pigments from incomplete washing of fixatives.
  4. Incomplete fixation resulting in soft tissue consistency.
  5. Loss or inactivation of enzymes needed for further studies.
  6. Over-fixation causing shrinkage or swelling.
  7. Brittle and hard tissue blocks from prolonged fixation.
References
  • McKay, C. (2024). Laboratory Manual for Mohs Micrographic Surgery.
  • Roopao, R. (2010). Grossing in Oral Pathology: General Principles and Guidelines.
  • Bruce-Gregorios, Jocelyn H. (2017). Histopathologic Techniques.