Infiltration and Impregnation in Histology

Infiltration and Impregnation

• Infiltration involves replacing the clearing agent within a tissue with a supporting agent, typically wax or resin. This process facilitates microtomy.
• Impregnation is the process of embedding tissue in a supporting medium, usually wax, to provide structural rigidity and ease of cutting.
• Manual processing is cost-effective but prone to human error, while automatic processing eliminates these errors but is more expensive.
• Vacuum infiltration is the most efficient method for removing air pockets and allowing the infiltrating agent to penetrate.
• Properties of infiltrating media:
  • Miscible with the clearing agent
  • Non-polar
  • Not too dense
  • Cheap and reliable
  • Compatible with reagents used in preceding procedures
  • Supports tissue structure
  • Transparent
• Paraffin is the most commonly used infiltrating medium due to its affordability, reproducibility, and compatibility with routine histopathology.
• Embedding media provide structural support for tissue during sectioning.
• Paraffin is the simplest and most common embedding medium, with a melting point of 60°C. It is recommended for routine histopathology.
• Paraplast is a more elastic and resilient alternative to paraffin, ideal for dense tissues like bones and brains.
• Embeddol is a synthetic wax substitute with a similar consistency to Paraplast.
• Ester wax is harder than paraffin and has a lower melting point. It is soluble in organic solvents.
• Alternative infiltration media are used when:
  • Heat damages cellular components
  • Thin sections are required
  • Existing media cannot support the tissue
• Resins are used for special procedures, providing exceptional support and allowing thinner sections.
• Water-soluble waxes are used when dehydration and clearing are not desired.
• Celloidin is a suitable alternative for paraffin, particularly for large, hollow cavities, dense tissues, and delicate specimens.
• Agar is a last resort for embedding small, friable tissues.
• Important considerations
  • Homogenous agent is crucial to avoid crystallization and proper embedding
  • Temperature monitoring is vital to ensure proper melting and hardening of the embedding medium
  • Use of fresh filtered wax minimizes impurities in paraffin
  • Use of the same material for both infiltration and embedding
• Double embedding involves embedding tissue in two different media, typically paraffin followed by gelatin, for added structural support.

Considerations during Impregnation

• Temperature monitoring
  • Temperature should be checked every 5 minutes.
  • Ensure adequate heating of the infiltrating agent - at least 2-5℃ above its melting point.
• Multiple changes required to ensure complete replacement of the clearing agent.
• Optimal duration of infiltration is minimal for best results.
• Homogenous agent ensures uniform embedding without crystallization.

Paraffin Wax

• Melting point of 56℃ is commonly used in routine histopathology.
• Ambient temperature dictates the appropriate melting point of the wax for optimal results:
  • Room temperature of 20-24°C: Wax with a melting point of 54-58°C
  • Room temperature of 15-18°C: Wax with a melting point of 50-54°C
• Fresh wax must be filtered before use to remove impurities.
• Wiremesh is used for filtering.
• Pure paraffin is used in routine histopathology.

This quiz covers the processes of infiltration and impregnation in histology, particularly focusing on the materials and methods used for embedding tissues. It distinguishes between manual and automatic processing, highlights the benefits of vacuum infiltration, and describes the properties of ideal infiltrating media, especially paraffin. Test your understanding of these vital histological techniques!