1. Factors Affecting Fixation
1.1. Buffers and pH.
1.1.1. Fixation is best carried out close to neutral pH, in the range of 6–8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity.
1.1.2. Common buffers include phosphate and bicarbonate. Commercial formalin is buffered with phosphate at a pH of 7. Unbuffered 4% formaldehyde is slightly acidic.
1.1.3. Fixing in unbuffered 4% formaldehyde results in:
1.1.3.1. Reduced formation of reactive hydroxymethyl groups and cross-linking (can be good for immunohistochemistry).
1.1.3.2. Artifact known as formalin pigment or hematin (brown-black insoluble precipitate produced from hemoglobin by the action of acid formaldehyde). This must be taken in consideration when dealing with patients that have hemoglobin breakdown secondary to hematopoietic diseases.
1.2. Duration of fixation and size of specimens.
1.2.1. Depth (d) reached by a fixative is directly proportional to the square root of duration of fixation (t).
1.2.2. The constant (k) is the coefficient of diffusability. It represents the distance in millimeters that the fixative has diffused into the tissue in one hour.
1.2.2.1. 10% formalin – 0.79
1.2.2.2. 100% ethanol – 1.00
1.2.2.3. 3% potassium dichromate – 1.33
1.3. Temperature of fixation.
1.3.1. Formaldehyde penetrates tissue at a faster rate at higher temperatures. Why? The diffusion of molecules increases with rising temperature due to their more rapid movement and vibration.
1.4. Concentration of fixative.
1.4.1. Optimum fixative concentration is determined by the solubility and effectiveness of each fixative individually.
1.4.1.1. Formalin: concentrations above 10% result in increased hardening, shrinkage and the formation of a white precipitate.
1.4.1.2. Ethanol: concentrations below 70% do not remove free water from tissues efficiently.
1.4.1.3. Glutaraldehyde: lower concentrations (0.25%) can penetrate tissues much better and quicker than strong solutions (4%).
1.5. Osmolality and ionic composition of fixative.
1.5.1. Hypertonic solutions lead to shrinking.
1.5.2. Hypotonic solutions lead to swelling.
1.5.3. Some ions (Na+, K+, Ca+, Mg+) can affect cell shape and structure. Therefore, ionic composition of fluids should be as isotonic as possible to the tissues.
1.6. Additives.
1.6.1. Certain electrolytes/non-electrolytes can improve morphology.
1.6.2. Examples of electrolytes: calcium chloride, potassium thiocyanate, ammonium sulfate, and potassium dihydrogen phosphate.
1.6.3. Examples of non-electrolytes: sucrose, dextran, and detergent.
2. chemical
2.1. **{Coagulate}** Can be organic and non-organic solutions. Coagulate proteins, making them insoluble – when lipoproteins and fibrous proteins (collagen) are coagulated, tissue structure is highly maintained. Not useful for ultrastructural analysis due to: Poor preservation of mitochondria and secretory granules Cytoplasmic flocculation
2.1.1. Dehydrant coagulants (alcohols such as ethanol and methanol, acetone).
2.1.1.1. Remove and replace free water from tissue (destabilize hydrogen bonding).
2.1.1.2. Results in the disruption of the tertiary structure of proteins (denaturation) and subsequent coagulation.
2.1.1.3. Rate of reversal back to a soluble ordered state is impossible even if placed in an aqueous environment again.
2.1.2. Acidic coagulants (picric acid, acetic acid, tricholoracetic acid).
2.1.2.1. Change the charges on the ionizable side chains and therefore, disrupt electrostatic and hydrogen bonding.
2.1.2.2. Acetic acid coagulates nucleic acids but not proteins.
2.1.2.3. Trichloroacetic acid precipitates proteins and extracts nucleic acids.
2.1.2.4. Picric acid forms salts with basic groups of proteins, causing them to coagulate.
2.2. **Compound Fixatives** Compound fixatives are mostly comprised of formaldehyde with other agents. Metallic ions have been used to aid in fixation, most prominently being mercury, lead and zinc. Ethanol in combination with formaldehyde produces alcoholic formalin Better preservation of glycogen and less shrinkage. Good for fatty tissues (esp. breast) since it clears and extracts lipids, thereby allowing for lymph node detection.
2.2.1. Glutaraldehyde Fixation
2.2.1.1. Glutaraldehyde has two aldehyde groups and can form stronger crosslinks better preservation of ultrastructure.
2.2.1.2. Less is known about mode of action.
2.2.1.3. Not as stable as formaldehyde – requires storage at 4°C and a pH of 5
2.2.1.4. Disadvantages:
2.2.1.4.1. Slow penetration of fixative due to strong crosslinks (tissue must be < 0.5mm)
2.2.1.4.2. Negative impact on immunohistochemistry
2.2.2. Osmium Tetroxide Fixation
2.2.2.1. Uses:
2.2.2.1.1. Very important as a secondary fixative for electron microscopy (reacts with lipids and phospholipids in cell membranes)
2.2.2.1.2. Lipid stain in frozen sections.
2.2.2.2. Disadvantages of osmium fixation:
2.2.2.2.1. Toxic.
2.2.2.2.2. Volatile and can readily fix nasal mucosa and the conjunctiva of the eyes.
2.2.2.2.3. Causes clumping of DNA and larges losses in proteins and carbohydrates.
2.2.2.2.4. Limited penetration into tissues.
2.3. **Cross-linking Fixatives** Aldehydes: most commonly used, such as formaldehyde and glutaraldehyde. Metal salts: mercuric chloride, zinc chloride. Other metallic compounds: osmium tetroxide.
2.3.1. Formaldehyde fixation
2.3.1.1. 10% neutral buffered formalin (NBF): Most commonly used fixative in diagnostic pathology.
2.3.1.2. Pure formaldehyde is a vapor. When dissolved in water, it forms a 37-40% solution known as “formalin”.
2.3.1.3. “10% formalin” commonly used in routine fixation is a 10% solution of formalin or 4% formaldehyde.
2.3.1.4. *Formaldehyde Mode of Action*
2.3.1.4.1. Formaldehyde reacts with amino acids and proteins in the cytoplasm, nuclear proteins and nucleic acids in the nucleus, and with unsaturated lipids.
2.3.1.4.2. No interaction with carbohydrates.
2.3.1.4.3. Formaldehyde reacts with different side chains to form reactive hydroxymethyl side groups forms the initial process of covalent addition (primary reaction in short fixation times).
2.3.1.4.4. Formation of these hydroxymethyl groups denatures macromolecules and renders them insoluble.
2.3.2. Mercuric Chloride
2.3.2.1. Used to fix bloody specimens and hematopoietic tissues (bone marrow, lymph nodes, spleen).
2.3.2.2. Diminishing application due to :-)
2.3.2.2.1. Safety and health issues that arise with mercury use.
2.3.2.2.2. Formation of intensely black mercury precipitates.
3. physical
3.1. microwave
3.1.1. Speeds fixation (reduces fixation time from 12 hours down to 20 minutes)
3.1.2. Disadvantage: produces dangerous vapors that can pose safety and health problems
3.2. heating
3.2.1. Simplest form of fixative
3.2.2. Acts by precipitating proteins and rendering them less soluble in water
3.2.3. Rarely used on its own. Used to accelerate other forms of fixation.
3.3. freeze drying
3.3.1. Very useful in research-based labs for the study of soluble materials and small molecules.
3.3.2. Tissue is immersed in liquid nitrogen followed by the removal of water under vacuum at -40°C.