Histology Week 1 - Intro

Learning Objectives

  • Understand safety protocols and potential hazards in histopathology laboratories.
  • Explore specialized histotechnology techniques and their roles in histopathological analysis.
  • Gain knowledge of the complete histopathology workflow:
    • Sample receival
    • Fixation
    • Grossing
    • Processing
    • Embedding
    • Sectioning
    • Staining

History of Histology and Histopathology

  • 17th Century:
    • Robert Hooke (~1665): Introduced microscopy.
    • Antonie van Leeuwenhoek (~1673): Staining of cells using plant-based dyes.
  • 19th Century:
    • 1867: Introduction of formaldehyde for fixation.
    • Mid 1800s: Development of Toluidine Blue, one of the first synthetic dyes in histology.
  • Early 20th Century:
    • 1922-1924: Establishment of the Biological Stains Commission (BSC) to standardize dye nomenclature.
    • Named staining techniques:
    • Golgi: Silver staining for nervous tissue.
    • Weigert: Stains for elastic tissue and myelin.
    • Grocott: Methenamine silver stain for fungi.
    • Masson: Trichrome staining for connective tissue.

Histopathology Laboratory Workflow

  1. Biopsy/Autopsy: Specimen receival and registration.
  2. Fixation or Freezing: Option for immediate preservation.
  3. Dissection and Selection: Determine blocks for examination.
  4. Processing to Paraffin Wax: Essential for support during slicing.
  5. Embedding in Paraffin Wax: Block preparation.
  6. Cutting Sections: Slicing the embedded specimen.
  7. Staining: Routine and special techniques including immunohistochemistry.
  8. Microscopy: Analyzing stained slides under a microscope.
  9. Reporting: Documenting findings.
  • Labor-intensive processes: Turnaround time for samples typically 24 hours. Most carried out by lab technicians.

Sample Requirements

  • Routine Diagnosis:
    • Tissue in 10% Neutral Buffered Formalin (NBF).
    • Volume: At least ten times the volume of the specimen.
  • Frozen Sections (on request):
    • Fresh tissue without fixative delivered immediately.

Fixation in Histopathology

  • First Critical Step: Maintain specimen integrity by processing tissue immediately.
  • Objectives of Fixation:
    • Preserve clear morphological features and tissue integrity for accurate diagnosis.
    • Prevent further degradation and microbial invasion.

Types of Fixation

  • Chemical Fixatives: e.g., Formaldehyde (most commonly used, 10% NBF):

    • Cross-links proteins but may mask binding sites (requiring antigen retrieval).
    • Changes protein structures, although primary and secondary structures remain intact.

  • Physical Methods:

    • Heating, freeze-drying, and microwave technology.

Factors Affecting Fixation

  1. Buffering: Best around neutral pH (6-8).
  2. Penetration: Varies by fixative; formalin penetrates tissue effectively.
  3. Volume: 10:1 ratio of fixative to tissue is recommended.
  4. Temperature: Increasing temperature enhances fixation speed.
  5. Concentration: Best at the lowest effective concentration to prevent artifacts.
  6. Time Interval: Quick transfer to fixatives critical for quality preservation.

Safety in the Histology Lab

  • Hazards: Consider chemical, physical, and biological risks.

  • Chemical Handling: Proper PPE like gloves and goggles; ensuring ventilation is critical.

  • Biological Risks: Minimize exposure to infectious materials; always use precautions when handling specimens.

  • Occupational Health: Awareness of chemical hazards and the necessity of using safety equipment (e.g., fume hoods).

Infection Risks and Universal Precautions

  • Principles: Proper fixation acts as a bactericide and ensures inactivation of infectious agents.
  • Always handle specimens with gloves and dispose of hazards properly in biohazard bags.

Key Notes

  • Timely fixation is essential for accurate histopathology diagnosis; delays can compromise results.
  • Good practices in specimen handling and fixation lead to optimal preservation.