Fixation

Fixation is a process in which tissue & cells are preserved in a state as close to life as possible. No, micro & macro structures should be lost.

OR

It is the process by which constituent of cells and tissues are fixed in a physical and partly in a chemical state so that they will withstand subsequent treatment with various reagents, with minimum loss, distortion and decomposition.

Fixatives

Chemical or Physical Agents which are used to prevent and preserve the tissue are called fixatives.

Ideal Fixative

• It should preserve the tissue volume so that tissue should not change its shape and size.
• To prevent hardness of tissue and should maintains proper tissue consistency.
• It should be non-toxic and non allergic.
•  It should penetrate the tissue quickly.
• Tissue should be as near to the living state as possible especially in Research work.
• Should prevent short and long term destruction of tissue by stopping the activity of catabolic enzymes and autolysis and bacterial/fungal attack.
• Nonflammable.
• Should be cost effective.
• Should support good staining with H&E both initially and after storage of the paraffin blocks for at least a decade.
• Should be used for variety of tissues e.g. fatty, lymphoid, and neural tissues.
• It should preserve small and large specimens and support histochemical, immunohistochemical and other specialized procedures.
• They have 1 year of lifespan and also are Stable.
• Compatible with modern automated tissue processors.
• The fixative should be easy to dispose off.
• Support long-term tissue storage giving excellent microtomy of paraffin block.

Factors Involved In Fixation

1. Buffers and hydrogen ion concentration
2. Temperature
3. Penetration
4. Osmolality
5. Substances added to vehicles
6. Concentration of fixative
7. Duration of fixation

1. Buffers and hydrogen ion concentration:

• Normal range for fixative PH 6-8
• Adjusted to the physiological range

e.g.  Gastric Mucosa = Acidic 5.5

Esophagus         = Neutral 7.0

• For EM glutaraldehyde with increasing pH
• Hypoxia of tissues lowers the pH
• For the prevention of uncontrolled or excessive acidity. There should be buffering capacity into a fixative.
• Some common buffers are bi-carbonate, phosphate, vernol and cacodylate.
• The formalin which is available commercially have pH neutral 7.0 with phosphate buffer.

2. Temperature:

• Surgical specimens are fixed at room temperature.
• For electron microscopy, tissue should be fixed at 0-40’C because at this temperature, the rate of autolysis slow down.
• To fix the blood film and bacteria there should be heat fixation is needed.
• For rapid fixation, formalin is heated at 600’C but the risk of tissue distortion increases.

3. Penetration of fixative:

• Penetration of tissues depends upon the diffusibility of each individual fixative which is a constant.
• It is very important phenomenon of fixation.
• As it is slow process tissue block should be of small size otherwise various zones are formed in the tissue.
• Formalin and alcohol penetration is best.
• Glutraldehyde is worst.
• d=k √ t
• Depth penetrated to the square root of time.

4. Osmolality of the fixative solution:

• Isotonic is normally physiological saline
• Hypertonic solutions cause shrinkage of cells.
• Hypotonic solution cause swelling of cells.
• Slightly hypertonic is ideal.
• For electron microscopy, hypertonic solution is preferable.

5. Concentration of fixative:

• This depends on following factors:
  1. Cost        2. Availability        3. Effectiveness
• Formalin is best at 10%.
• For EM glutar-aldehyde is generally made up at 0.25% to 4%.

6. Duration of fixation:

• This depends on the type of fixative e.g. in formaldehyde, duration period is very short otherwise, shrinkage and hardness of tissue will take place.
• In a glutar-aldehyde fixation is prolong.

7. Substances added to vehicles:

• Different substances are added in a fixative to enhance their function e.g. sodium sulphate and NaCl included in a fixative having mercuric chloride.
• NaCl increases the binding of mercuric chloride with protein amino acids.

Secondary fixation

• Secondary fixation of tissue section is done with iodine soln. to remove black crystals.
• Tissue blocks fixed in gluteraldehyde for EM are commonly fixed osmium tetraoxide.
• Post fixation with dichromate has been recommended for mitochondria.

Fixation Artefacts

• Formalin pigment is a brown/black pigment formed in tissues fixed with acidic formalin.
• Eliminated by treatment with phenolic formalin.