Untitled Quiz

Fixation Techniques

• Fixation preserves tissue by hardening it and preventing the loss of tissue molecules.
• Appropriate tissue fixation is crucial for all histology tests. Without proper fixation, tissue degrades, 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 living-like state.
• Protect the tissue from the harmful effects of processing.
• Aid in the staining process.
• Minimize the diffusion of soluble molecules.

Types of Fixatives

• Physical Fixatives: Use heat, microwave, and freeze-drying; not commonly used in routine practice.
• Chemical Fixatives: Most commonly used in histopathology labs; use liquid fixatives.

Categories of Chemical Fixatives

• Coagulant fixatives: Organic and non-organic solutions that coagulate proteins, making them insoluble. Cellular architecture is maintained primarily by lipoproteins and fibrous proteins (like collagen). Coagulation of these proteins preserves tissue histomorphology at the light microscope level.

  • Dehydrating Coagulant Fixatives: Remove water from tissue by breaking down hydrophobic bonds responsible for protein structure. Protein tertiary structure disruption (denaturation) change physical properties, potentially causing insolubility. Examples include ethanol and 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. Disrupts tertiary protein structure. Acetic acid coagulates nucleic acids but doesn't fix or precipitate proteins; it's added to other fixatives to prevent nucleic acid loss.

• Cross-linking fixatives: Several chemical fixatives form cross-links between proteins, individual proteins, within nucleic acids, and between nucleic acids and proteins.

Formaldehyde

• 10% neutral buffered formalin (NBF): The most common fixative in diagnostic pathology. Pure formaldehyde is a vapor; when dissolved in water, it forms a solution containing 37-40% formaldehyde—formalin. 10% formalin usually contains approximately 4% formaldehyde by weight-to-volume.
• Types of Formalin:
  • Acidic formalin: pH below 7 (usually around 4-5). Less common for general fixation, it may lead to formalin pigments (especially in blood-rich tissues). It can also affect staining quality in some protocols.
  • Neutral buffered formalin (NBF): pH is neutral (typically at 7.0). Most commonly used, it minimizes pigment formation and offers better tissue preservation while being compatible with many histological and molecular techniques.
• Safety Notes: All formalin types are carcinogenic, respiratory irritants, and skin sensitizers. Use ventilation, and appropriate personal protective equipment (PPE) like gloves, goggles, and lab coats. Store in sealed containers.