Fixation Flashcards
What happens when the blood supply to a tissue has been cut off?
Once the blood supply has been cut off to tissues, changes start to occur almost immediately to cells and their contents.
What is fixation?
FIXATION is a chemical process that preserves cells in as LIFE-LIKE MANNER AS POSSIBLE.
Fixation is very critical as the damage is irreversible!
How do fixatives preserve tissues?
- Kills the tissue by denaturing proteins to prevent decay or autolysis and putrefaction. Fixatives alter the tissues by stabilizing proteins – resist changes.
- Prevent desiccation (drying out)
- Prevents osmotic swelling and shrinkage
- Solidification of colloid material - changes tissue to semi-solid
- Hardening of tissue - handling
- Optical differentiation - refraction indices
- Stainability - improved in most
What happens to tissue outside body if not fixed?
Autolysis and Putrefaction:
- Autolysis occurs when enzymes in tissue continue metabolic process even after being removed from the body. Organs such as pancreas, liver and brain more susceptible to autolysis.
- Putrefaction is the decomposition by bacteria and molds
What does solidification of colloid material mean?
SOLIDIFICATION OF COLLOID MATERIAL
Converts the normal semi-fluid consistency of cells (solution) into an irreversible and semi-solid consistency (gel). Prevents distortion.
How does hardening of tissue in fixation help?
HARDENING OF TISSUE
Provide easy manipulation of soft tissue
Helps with protection of tissue from harmful effects of tissue processing reagents
What optical properties does fixation change?
OPTICAL DIFFERENTIATION
Alters refractive indices which allow the tissue components to be more easily seen.
How is stainability affected by fixation?
STAINABILITY
Most staining is enhanced by fixation. Tissues that have not been fixed will stain poorly. Fixation allows different stains to attach to tissue more easily. There are a few exceptions (IHC)
What special step is required for immunohistochemistry?
IHC - Immunohistochemistry requires an antigen retrieval step following fixation.
What affects does autolysis have on staining on the nuclei?
NUCLEI
`1. PYKNOTIC (condensation)
2. KARYORRHEXIC (fragmentation)
3. KARYOLYTIC (eventual disappearance)
What affects does autolysis have on staining on the cytoplasm?
CYTOPLASM
- SWOLLEN
- GRANULAR
- HOMOGENEOUS
- LOSE USUAL STAINING REACTION
How does putrification appear on sections under the microscope in comparison to autolysis?
Similar appearance as autolysis
Gas formation = Bubbles
What are different physical and chemical methods of fixation (list 5)?
PHYSICAL:
1. DRY HEAT - Not used for pieces of tissue,
Used for fixing blood smears or bacterial smears
2. MICROWAVE / MOIST HEAT (NONIONIZING RADIATION)
3. DESSICATION - Rarely used, Fixes proteins.
Used when air drying touch preps for Wrights stain
CHEMICAL:
4. VAPOUR FIXATION
5. LIQUID FIXATION
The way the fixative affects tissue varies with the fixative being used
Describe how microwaves are used to fix tissues.
MICROWAVE / MOIST HEAT (NONIONIZING RADIATION)
- Water molecules oscillate very quickly producing heat
- Be careful temperature does not exceed 68 °C
- Takes less time than chemical solution
- More even penetration
Describe uses and limitations of vapour fixation.
VAPOUR FIXATION
Fumes from volatile fixatives in concentrated form
Fix fresh tissue, can be used with cryostat
Tissue does not touch the fixative solution
Describe uses and limitations of liquid fixation.
LIQUID FIXATION - Most common
- Uses one or more chemical reagents
- Immersion - penetration by diffusion
- Rate of penetration varies with the type of fluid and type of tissue
- Limited by the size of the tissue
- Perfusion – inject with liquid fixative into veins or arteries
What are the two types of reactions a fixative can have with a tissue?
- Additive
2. Non-additive
Describe what an additive fixative is.
Additive fixative
1. Certain fixatives can combine chemically by either ionic or covalent bonds with the proteins,
2. Can change the charge at the site of attachment and can alter the shape
E.g. Formalin, Glutaraldehyde, picric acid, mercuric chloride, osmium tetroxide, zinc chloride, etc.
Describe what a non-additive fixative is.
Non-additive fixative
These fixatives do not enter into any direct combination with protein but will alter the shape of proteins. Protein crosslinks occur without forming bridges. No additional chemical groups are added to the tissue
Can cause shrinkage and hardening
E.g. Methyl Alcohol, Ethyl Alcohol, Acetone (organic compounds)
What are the two categories of fixative based on the type of product formed by the reaction between the fixative and the protein in the tissue?
- Non-coagulant fixatives (Non-precipitating, Tolerant)
2. Coagulant fixatives (Precipitating, Intolerant)
What are non-coagulant fixatives? Name some examples.
Non-coagulant fixatives (Non-precipitating, Tolerant)
1. Fixative combines with proteins and transforms the cytoplasm from a solution to a gel consistency.
2. Will not cause excessive hardening or shrinkage of the tissue
3. Might cause slight swelling
E.g. Formalin, Glutaraldehyde, osmium tetroxide
What are coagulant fixatives? Name some examples.
Coagulant fixatives (Precipitating, Intolerant)
1. Fixative combines with proteins to produce a coagulum (a mass that resembles a clot)
2. Causes excessive shrinkage and hardening
E.g. Mercuric chloride, Picric acid/Bouin’s, Acetone, Ethanol/Alcohol, Zinc.
How can you remember what fixatives are additive and which ones are non-additive?
Additive fixatives- don’t start with “a” ex. Formalin, Mercury, Osmium, Glutaraldehyde
Non-additive fixatives- start with “a” ex. Alcohol, Acetic acid, Acetone
How can you remember what fixatives are coagulants and which ones are non-coagulants?
Memorize the coagulants and the reset are non-coagulants!
Coagulants- ZAPAM (Zinc, Alcohol, Picric acid, Acetone, Mercury)
Non-coagulants- rest of fixatives
