Histopathology Techniques - Fixation

Histopathology Techniques - Fixation

• Fixation is crucial for preserving tissue samples for histopathological analysis.
• Cross-linking fixatives like glutaraldehyde effectively preserve ultrastructure.
  • Ideal for ultrastructural studies due to detailed preservation.
  • Rapid fixation, achieving cross-linking quickly, ensures fine cellular detail.
  • High fixation strength preserves subcellular structures (organelles and membranes), optimal for electron microscopy.
• Osmium Tetroxide fixatives preserve lipids and contrast cell membranes.
  • Binds to unsaturated lipids, preserving and contrasting cell membranes.
  • Often used after aldehyde-based fixation for superior ultrastructure preservation.
• Mercuric chloride excels at preserving protein structures and maintaining tissue morphology.
  • Excellent for preserving protein structures and maintaining tissue morphology.
  • Enhances tissue staining for light microscopy by adding density.
  • Often combined with other fixatives like formalin (Zenker's and Helly's solutions).
• Compound fixatives, like formaldehyde-based ones, preserve histomorphometric patterns.
  • Pathologists utilize formaldehyde-based fixatives for preserving histomorphometric patterns.
  • Additional agents are often added to formaldehyde to create specific effects not achievable with formaldehyde alone (e.g., ethanol added to formaldehyde produces alcoholic formalin useful for preserving glycogen).
  • Examples include Carnoy fixative, Susa, and Zenker fixatives.

Disadvantages of Fixatives

• Molecular loss is a common drawback.
• Tissue swelling or shrinkage can occur during fixation.
• Quality of biochemical and immunohistochemical stains may vary.
• Loss of antigens leading to reduced immunorecognition.

Factors Affecting Fixation Quality

• pH: Acidic formalin solutions can cause brown-black insoluble crystalline pigments. Neutral buffer formalin (NBF) is used to avoid this.
• Duration and Specimen Size: Fixation depth is proportional to the square root of the duration (d = k√t).
• Temperature: Increased temperature leads to enhanced molecular diffusion.
• Concentration: Formalin concentrations above 10% can lead to hardening and shrinkage. Ethanol concentrations below 70% don't effectively remove water from tissue.
• Osmolality and Ionic Composition: Hypertonic fixatives cause shrinking; hypotonic fixatives lead to tissue swelling. Slightly hypertonic solutions (400-450 mOsm) often provide the best morphological results.
• Additives: Electrolytes and non-electrolytes added to fixatives can improve fixed tissue morphology.

Fixative Artifacts

• Formalin Pigments: Result from the use of non-buffered formalin and can be removed with saturated alcoholic picric acid.
• Mercuric Pigments: Result from using mercuric chloride and are removed by alcohol.
• Chrome Oxide Pigments: Result from using chrome acid and can be removed with 1% acid alcohol.

This quiz covers fixation techniques used in histopathology, essential for preserving tissue samples for analysis. Key fixatives like glutaraldehyde, osmium tetroxide, and mercuric chloride are discussed, highlighting their specific properties and ideal applications in preserving cellular and subcellular structures. Test your understanding of these crucial techniques and their relevance in ultrastructural studies.