1 Fixation

Cards (90)

  • 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