1 Fixation

• Fixation is the process by which the constituents of cells and tissues are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture.
• Fixation is achieved by exposing the tissue to chemical compounds called Fixatives

  • the quality of the section in the slide is as good as the quality of the fixed tissue specimen
• To preserve or maintain tissue with anatomical and structural characterization, the most important reaction is the stabilization of protein
• • The primary aim of fixation is to preserve the morphological and chemical integrity of the cell
  • The secondary aim is to harden and protect the tissue, thus easier to cut
• Fixation is the first and most critical step in histopathologic techniques/steps
• Fixation preserves the tissue by stopping all cellular activities so that the cells can be viewed under the microscope as if they are still in their original living state

  • Hypotonic Solution → cells cause to swell
  • Hypertonic Solution → cells cause to shrink
• Additive Fixation is a mechanism where the chemical constituent of the fixative is taken in and becomes part of the tissue.

  example:

  • Formalin → form cross-linked/molecular complexes and gives stability to the protein
• Non-Additive Fixation is a mechanism where the fixing agent is not incorporated in the tissue but alters the tissue composition and stabilizes tissue.

  example:

  • Alcohol fixatives → removes bond water that is attach on a certain grp within a molecule
• Hydrogen Ion Concentration pH in fixation should be 6.8 and 8.0
• Routine temperature in fixation is room temperature = 20-25°C; 0-4°C temperature for Electron Microscopy & Histochemistry
• Thickness of Section in fixation for:

  • EM → 1-2mm
  • Light Microscopy → 2cm
• For best result, slightly Isotonic solutions (400-450 mOsm) is used
• Concentration for fixation depends on the size and number of specimen:

  • Formaldehyde → 10% soln
  • Glutaraldehyde → 3%
  • Immunoelectron Mic → 0.25% (ideal concentration)
• Prolonged fixation may cause shrinkage and hardening of the tissue, duration should be:

  • 2-6 hrs for small tissues
  • 24 hrs for big tissues
  • 3 hrs for EM
• Specimen should be placed in fixative immediately to prevent autolysis and putrefaction

  • Standard: 4-6 hrs
• Penetration of tissue should be 1 mm/hour

  • The recommended/standard of Tissue: 2x2 cm, not more than 4 mm thick
• Volume should be 20x the tissue volume

  • routinely → 1:20
  • traditionally → 10-25x vol of tissue
• Fixatives hardens soft and friable tissues, which makes handling and cutting easier; acceleration of alcohol during dehydration
• Fixatives makes cells resistant to damage and distortion to achieve right osmolality
• Fixatives acts as mordant or accentuator thereby facilitating staining process
• Fixation reduces the risk of infection during handling and actual processing of tissue because all tissue specimen is hazardous
• Simple fixatives are composed of one component substance
• Simple fixative: Aldehyde

  • Formaldehyde
  • Glutaraldehyde
• Simple fixative: Metallic Fixatives

  • Mercuric Chloride
  • Chromate Fixatives
  • Lead Fixative
  • Heat
• Compound fixatives are made up of two or more fixatives.
• Compound fixatives: According to Action

  • Microanatomical fixatives permits general microscopy study
  • Cytological fixatives preserve particular microscopic elements of the cell
• Cytoplasmic fixatives never contains Glacial Acetic Acid because it will destroy Mitochondria and Golgi Apparatus; has a pH of >4.6
• Nuclear fixatives always contain Glacial Acetic Acid which is a primary component due to its affinity for nuclear chromatin; has a pH <4.6
• Histochemical fixative preserves chemical constituents of cells and tissues
• 20-22°C native DNA and RNA does not react with formaldehyde
• If reaction mixture are heated: 45°C in RNA, 65°C in DNA reaction will begin.
• Ethanol and methanol, including Carnoy’s solution are commonly used fixatives for nucleic acids.
• Ethanol is the most usable in DNA fragment for PCR
• Formaldehyde limits the size of fragments
• Aldehyde fixatives are satisfactory for routine paraffin sections; for electron microscopy, Histochemical and enzyme studies
• Formaldehyde or Formalin is the most common, widely used; it is a gas produced by oxidation of methanol

  • 10% recommended, buffered to 7.0
  • Commercially available → 35-40% gas by weight
  • Pure stock solutions → >40%
  • Fixation time: 24 hrs at 35°C, 48 hrs at 20-25°C
• 10% formol saline is made up of formaldehyde diluted to 10% NaCl; recommended for CNS tissues and post-mortem tissues for histochemical exam
• 10% neutral buffered formalin or phosphate-buffered formalin is used for preservation and storage of surgical, postmortem and research specimens and dx pathology

  • Fixation Time: 4-24 hrs
  • Recommended and routinely used
• Formol is corrosive, recommended for routine postmortem tissues

  • Fixation Time: 3-24 hrs
• Alcohol formalin fixative coagulates mucus and can be used to fix sputum, which is why it is recommended for sputum specimens