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
What factors affect fixation (6)?
TEMPERATURE - Routine fixation occurs in ambient temperatures, Cool temperatures slow fixation.
Warmer temperatures speed up fixation.
VOLUME RATIO - 20:1 fixative to tissue ratio
SIZE OF TISSUE - thk less then 3 mm. Smaller tissue would take less fixation time
DENSITY OF TISSUE a) less dense = less time to fix
b) dense tissues = more time to fix (Thick and dense tissues may be more susceptible to autolysis).
Fatty tissues usually require more time to be fully fixed
TIME IN FIXATIVE
- Small biopsies ~ 2 to 6 hours,
- Larger specimens ~ overnight fixation.
- Whole organs sliced for adequate fixation (typ).
Too long in a fixative can also impair staining.
FIXATIVE USED
What factors affect the choice of fixative?
PENETRATION - Varies with each fixative. Temperature can affect penetration.
TISSUE STORAGE - Tissue can be stored in 10% NBF indefinitely. Tissue for Immunohistochemistry should be transferred to 70% alcohol after 10% NBF to stop cross-linking
AGITATION - Speeds up fixation and reduce the length of time required.
USE OF VACUUM - Increases the rate of fixation
pH & BUFFERS - pH ranges outside of 6-8 can cause detrimental changes.
OSMOLALITY - Saline can be used as a holding solution for short periods of time (so tissues don’t dry out) as it is isotonic. It will not shrink or swell cell membranes.
Which fixative/fixative ingredient penetrates faster than others?
Formaldehyde ingredient in fixatives penetrates faster then other ingredients.
The textbook states penetration rates from fastest to slowest: formalin, acetic acid, mercuric chloride, methyl alcohol, osmium tetroxide, and picric acid.
What can form at an acid pH with a tissue in formalin?
Formalin pigment forms at an acid pH.
What are some causes of improper fixation?
- Delay in fixation – must be done as soon as possible
- Inadequate amount of fixative solution
- Improper fixative to tissue ratio. Should be at least 15 to 20:1
- Poor penetration – too large a specimen without making surgical cuts
- Fixative used not suitable for a particular type of tissue
- Prolonged fixation time in a intolerant fixative
How are fixatives classified?
- COMPOSITION: Simple vs. compound.
- LEVEL OF ACTION:
a) MICRO-ANATOMICAL Allows cells to retain former relationship with each other. Ex. Formalin, Bouin’s, Glutaraldehyde
b) CYTOLOGICAL Ex. 95% Alcohol- smears, films. Acetic acid- nucleic acids
c) HISTOCHEMICAL Used to preserve enzymes and other histochemical elements like glycogen and lipids
Ex. Mercuric chloride, Picric acid, Ethanol, Acetone, Zinc - PRODUCT WITH PROTEINS Coagulant vs. Non-coagulant
- TYPE OF RXN WITH PROTEINS Additive vs Non-Additive
What is the difference between a simple fixative to a compound fixative?
A simple fixative has only a single reagent in it.
A compound fixative uses a combination of fixatives that can benefit another, even counter-acting a disadvantage of another
What is secondary fixation? Give example with why 10% NP Formalin may be followed by picric or mercuric chloride.
The fixative ingredients are not combined but rather one follows another to enhance preservation and staining.
Example: Tissues fixed in 10% N.B. Formalin can be further fixed with coagulating fixatives such as picric acid or mercuric chloride to enhance staining
What is a fixation artifact?
Unnatural structure / appearance in tissue
Due to handling, man made
Need to be aware of them and how to prevent.
