FIXATION

Cards (90)

  • Fixation
    The first and most critical step in the 10 Histotechnological processes. It involves fixing and preserving fresh tissue for examination.
  • Fixation
    • Killing is necessary to stop the metabolic processes that continue to alter the state of tissue to be examined
    • There is not one fixative that solves all fixation processes because there are multiple fixing agents available in the market
  • The container is actually three-fourths full which is the ideal volume for us to meet the fixation process
  • No process of histotechnology is more critical to slide preparation than fixation. If you got fixation wrong, then you would expect that the succeeding processes will not be adequate.
  • Primary aim of fixation
    Cells and its surrounding tissues should be preserved in the morphologic and chemical integrity of the cell in as life-like manner as possible
  • Secondary aim of fixation
    To harden and protect the tissue from the trauma of further handling
  • Degeneration
    The state or process of being or becoming degenerate; decline or deterioration. Deterioration and loss of function in the cells of a tissue or organ.
  • Decomposition
    The process by which organic substances are broken down into a much simpler form of matter
  • Putrefaction
    Decomposition of proteins in a process that results in the eventual breakdown of cohesion between tissues and the liquefaction of most organs. It is one of the seven stages in the decomposition of the body after death.
  • Distortion
    The alteration of the original shape (or other characteristic) of something
  • Additive fixation
    The chemical constituent of the fixative is taken in and becomes part of the tissue
  • Non-additive fixation

    The fixing agent is NOT taken in, but changes the tissue composition and stabilizes the tissue by removing the bound water attached by the hydrogen bonds
  • Coagulant fixatives
    Acts by creating a network that allows solutions to readily penetrate the interior of the tissue
  • Non-coagulant fixatives
    Creates a gel that makes it difficult to penetrate by subsequent solutions. Tissues subjected to non-coagulant fixatives must be cut thinly.
  • Factors involved in fixation
    • Hydrogen ion concentration (satisfactory between pH 6.0 and 8.0)
    • Temperature (commonly room temperature between 20-24°C, electron microscopy 0-4°C)
    • Thickness of section (electron microscopy 1 to 2 mm^2, light microscopy 2 cm^2, no more than 0.4 cm)
    • Osmolality (best using slightly hypertonic solution 400-450 mOsm)
    • Concentration (formaldehyde 10% solution, glutaraldehyde 3% solution)
  • Duration of fixation
    Formalin should have a duration of 2-6 hours during the day the specimen is obtained. Prolonged fixation to formalin will result in shrinkage and hardening of tissue, and inhibit enzyme activity and immunologic reactions.
  • Practical considerations of fixation
    • Speed (tissue must be fixed immediately to prevent autolysis and putrefaction)
    • Penetration (commonly 1 mm per hour, tougher tissues need more penetration)
    • Volume (fixative must be 10-25 times greater than tissue volume, ideal ratio 1:20)
    • Duration (fibrous organs need longer fixation than small/loose tissues, can be reduced by heat, vacuum, agitation, or microwave)
  • Methods of fixation
    • Chemical fixation
    • Vapor fixation
    • Heat fixation
    • Microwave irradiation
    • Ultrasound fixation
  • Brain cells are very sensitive once they have been harvested and deteriorate very quickly so when we receive a section of the brain or the entire brain, it must immediately be fixed in the fixative lasts for about 2-3 weeks.
  • Bone marrow continues to undergo mitosis up to 30 minutes after death when refrigerated.
  • General effects of fixatives
    • Harden soft and friable tissues
    • Make cells resistant to damage and distortion
    • Inhibit bacterial decomposition
    • Increase optical differentiation of cells and tissue components
    • Acts as mordants or accentuators or may inhibit certain dyes
  • Characteristics of a good fixative
    • It must be cheap
    • It must be stable
    • It must be safe to handle
    • It must kill the cells quickly
  • Types of fixatives according to composition
    • Simple fixatives (one component)
    • Compound fixatives (two or more components)
  • Types of fixatives according to action
    • Microanatomical fixatives
    • Cytological fixatives
    • Nuclear fixatives
    • Cytoplasmic fixatives
    • Histochemical fixatives
  • Lipid fixation
    • Mercuric chloride and potassium dichromate
    • Baker's formol-calcium
    • Imidazole osmium tetroxide
    • Digitonin
  • Carbohydrate fixation
    • Alcoholic fixatives
  • Protein fixation
    • Neutral buffered formol saline
    • Formaldehyde vapor
  • Mixture of fixatives
    • Karnovsky's paraformaldehyde-glutaraldehyde solution
    • Acrolein
  • Fixatives that preserve the chemical constituents of cells and tissues
    • Acetone
    • Newcomer's fluid (serves as both nuclear and histochemical fixatives)
  • LIPID FIXATION

    • Mercuric chloride and potassium dichromate
    • Effective preservation of lipids in cryostat sections
  • Baker's formol-calcium
    Preserves phospholipids
  • Imidazole osmium tetroxide
    Post-fixation, improves the ultrastructural demonstration of lipids
  • Digitonin
    • Cholesterol fixation
    • Preserves cholesterol in the tissue
  • CARBOHYDRATE FIXATION
    • Alcoholic fixatives
    • Recommended for glycogen fixation
  • PROTEIN FIXATION
    • Neutral buffered formol saline or formaldehyde vapor
    • Most commonly used fixatives for amino acid histochemistry
  • Mixture of fixatives
    • Karnovsky's paraformaldehyde-glutaraldehyde solution
    • Best known fixative for electron cytochemistry
  • Acrolein
    • Mixture of glutaraldehyde or formaldehyde
    • Rapidly preserves the morphology and enzyme activity at low concentration
    • Useful for immersion fixation of surgical biopsies
  • ALDEHYDE FIXATIVES

    Produce a satisfactory result for routine paraffin sections such as light microscopic studies
  • Formaldehyde
    • Gas produced by oxidation of methyl alcohol (methanol)
    • Most widely used is 10% formalin
    • Pure stock solution (40%)- over hardens the outer layer of tissue and affect staining aversely
    • Usually buffered to pH 7.0 with phosphate buffer
    • Fixation time: 24 hours
    • Recommended for colored tissue photography
    • Tolerant fixative used for mailing specimen because specimen can be left in formalin indefinitely
  • Methanol
    • Preservative added to formaldehyde to prevent its decomposition to formic acid or precipitation to paraformaldehyde
    • Drawback: It denatures protein making formalin unsuitable for electron microscopy