Histopathology Fixation Techniques

Fixation in Histopathology

• Fixation preserves tissue by hardening it and preventing the loss of tissue molecules.
• Appropriate tissue fixation is crucial for all histology tests.
• Without fixation, tissues degrade, and analysis becomes impossible.
• Fixation maintains clear and consistent morphological features.

Function of Fixatives

• Prevent autolysis (enzyme breakdown of tissue).
• Prevent putrefaction (microorganism breakdown of tissue).
• Preserve cells in a live-like state.
• Protect tissue from damage during processing.
• Aid in staining processes.
• Minimize diffusion of soluble molecules.

Types of Fixatives

• Physical fixatives:

  • Use heat, microwave, or freeze-drying.
  • Not routinely used in practical histopathology.

• Chemical fixatives:

  • Most frequently used in histopathology labs.
  • Involve using liquid fixatives.

Chemical Fixatives: Categories

• Coagulant fixatives:
  • Use organic or non-organic solutions to coagulate proteins, making them insoluble.
  • Cellular architecture mainly maintained by lipoproteins and collagen.
  • Protein coagulation maintains tissue histomorphology at the light microscope level..

Coagulation Fixatives: Subtypes

• Dehydrating coagulant fixatives:

  • Remove water from the tissue by breaking down hydrophobic bonds in the protein structure.
  • Disruption of tertiary protein structure (denaturation) which changes their physical properties and potentially renders them insoluble.
  • Examples: ethanol, methanol.

• Acid coagulant fixatives:

  • Use acids like picric acid and acetic acid to disrupt electrostatic and hydrogen bonds.
  • Can insert lipophilic anions into hydrophilic regions.
  • Disrupt the tertiary structures of proteins.
  • Acetic acid coagulates nucleic acids but not proteins and added to other fixatives to prevent nucleic acid loss.

Cross-Linking Fixatives

• Several chemicals are selected for their potential to form cross-links between proteins and within nucleic acids and proteins.

Formaldehyde

• 10% neutral buffered formalin (NBF): The most common fixative in diagnostic pathology.

• Pure formaldehyde, a vapor, when dissolved in water creates formalin (37-40% formaldehyde solution)

  • The typical 10% formalin used in tissue fixation only has approximately 4% weight-to-volume formaldehyde.

• Formaldehyde is effective in preserving tissue morphology, preventing degradation, and maintaining cellular structure by forming bonds.

• Suitable with stains like H&E, IHC, and ISH.

• Types of Formalin:

  • Acidic formalin: pH below 7 (typically 4-5).
    • Less frequently used.
    • Prolonged fixation can lead to formalin pigment, especially in blood-rich tissue.
    • May impact staining quality.
  • Neutral buffered formalin (NBF): neutral pH (typically 7.0).
    • Minimizes pigment formation and improves tissue preservation.
    • Compatible with a range of histological and molecular techniques.

• Safety Precautions (Formalin)

  • All formalin types are carcinogenic, respiratory irritants, and skin sensitizers.
  • Use proper ventilation, personal protective equipment (PPE), and store in sealed containers.

This quiz explores the essential role of fixation in histopathology, focusing on its function, types, and categories of fixatives. Learn how fixation preserves tissues and prevents degradation, ensuring accurate histological analysis. Test your understanding of both physical and chemical fixatives used in laboratory settings.