Fixation CH 1 Flashcards
1
Q
Fixation
(Definition)
A
- Alters tissue by stabilizing the protein so that it is resistant to further changes.
- Must change the soluble contents of the cell into insoluble substances so that they are not lost during subsequent processing.
- Can be chemical or physical
2
Q
Functions of Fixatives
A
- To prevent putrefaction (bacterial attack during decay or
postmortem activity) - To prevent autolysis (enzyme attack during decay or
postmortem activity) - To help maintain the proper relationship between cells and
extracellular substances - Brings out differences in refractive indexes and to increase contrast between tissues for microscopic evaluation
- Enhance subsequent staining
2
Q
Actions of Fixatives
A
1.) Chemical method – additive or non- additive, coagulant or non-coagulant
2.) Physical method – using heat to fixate tissue
- Microwave Processing
- Overheating alters nuclei staining
- Desiccation
3
Q
Actions of Fixatives
A
- Additive fixation – protein combines chemically with a fixative molecule and protein then becomes insoluble. (Mercuric Chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, osmium tetroxide.)
- Non additive fixation – denaturation causes
protein to become less capable of maintaining an intimate relationship with water, but does NOT combine the protein.
(Acetone, Alcohols)
4
Q
Actions of Fixatives
A
- Coagulant fixative – establishes a network in tissue that allows solutions to readily
penetrate or gain entry into the interior of the tissue
(Alcohol, zinc salts, mercuric chloride & picric acid) - Noncoagulant fixative – act by creating a gel that makes penetration by the subsequent solutions difficult.
(Formaldehyde, glutaraldehyde, osmium tetroxide, potassium dichromate and acetic acid)
4
Q
Factors influencing Fixation
A
- Choice of fixative – staining procedure(s) for tissue must be considered.
- Penetration – fixatives penetrate at different
rates and fixate at different rates. (i.e. formalin penetrates fast but cross-links
slowly) - Tissue storage – certain fixatives do not allow for long term storage. NBF or 70% alcohol are acceptable for long term storage.
5
Q
Factors influencing Fixation
A
1.) Temperature – increase in temperature increases the
rate of fixation (but also increases rate of autolysis &
diffusion of cellular elements).
2.) Size – tissue thickness should be no more than 3mm thick.
3.) Volume ratio – fixative volume should be at least 15
to 20 times greater than the tissue volume.
4.) Time – tissue should be placed in fixative immediately
after surgical removal duration in fixative is crucial; adequate fixation time is needed to complete the fixation process
6
Q
Factors influencing Fixation
A
- pH – applies to aldehyde fixatives (i.e. pH below 5 can cause formation of formalin
pigment) and to immunohistochemisty techniques. - Osmolality – also must be considered for immunohistochemistry techniques.
7
Q
Reactions of the cell with fixatives
A
- Nucleus – preferred fixatives for nucleic acids are acetic alcohol & Carnoy solution
- Proteins – fixation can affect the secondary or tertiary structures of proteins.
- Lipids – several fixatives will preserve but only two (osmium tetroxide & chromic acid) will fix so that they are not lost during subsequent processing
- Carbohydrates – can be lost during fixation
8
Q
- Simple Aqueous Fixatives
1. Acetic acid
2. Formaldehyde
3. Glutaraldehyde
4. Mercuric chloride
5. Osmium tetroxide
6. Picric acid
7. Potassium dichromate
8. Zinc salts
A
- Acetic acid
- Formaldehyde
- Glutaraldehyde
- Mercuric chloride
- Osmium tetroxide
- Picric acid
- Potassium dichromate
- Zinc salts
9
Q
Simple Aqueous Fixatives
- Acetic acid - Noncoagulant
A
- Acetic acid - Noncoagulant
- Concentrated is called glacial acetic acid due to freezing point of 16.6C
- Penetrates rapidly and leaves tissue soft
- Used in preservation of nucleoproteins
- Causes swelling of tissue
- Red blood cells (RBC) are lysed
- Precipitates DNA
- Causes Protein swelling more than any other
- Collagen greatly affected; absorbs water with contact at lower pH’s
10
Q
Simple Aqueous Fixatives
- Formaldehyde
A
- Formaldehyde – Noncoagulant additive
- Gas that comes in 37% to 40% solution with water called formalin
- Commercial stock solutions of 37% to 40% formaldehyde are considered 100% formalin
- In preparing 10% formalin, 1 part commercial stock to 9 parts water (leaving a concentration of 3.7% to 4% formaldehyde)
11
Q
Simple Aqueous Fixatives
- Formaldehyde
A
- Formaldehyde has a strong tendency to polymerize to dimers and trimers.
- Paraformaldehyde is a highly polymeric form of formaldehyle (has uses in EM).
- Commercial formalin contains 10% to 14% methanol to prevent polymerization
12
Q
Simple Aqueous Fixatives
- Formaldehyde
A
- Reacts with the amino group (NH2) on the side chains of amino acids.
- Causes the formation of methylene bridges that cross-link the proteins together.
13
Q
Simple Aqueous Fixatives
- Multiple Fomalin solutions
A
1.) 10% Aqueous Formalin
2.) 10% Formalin saline
3.) Calcium Formalin
- Recommended for phospholipids
4.) Formalin ammonium bromide
- Recommended for CNS
5.) Acetate Formalin
6.) 10% Neutralized & 10% NBF
7.) Modified Millonig Formalin
- Can be used for EM & Light microscopy
8.) Alcoholic Formalin
14
Q
Simple Aqueous Fixatives
- Formalin
A
- Preserves lipids but does not make them insoluble
- Does not fix carbohydrates (but stabilizes proteins in a manner that “traps” glycogen
- Black acid hematin (formalin pigment) tends to form when the pH of the solution drops below 6.0
- Removed with absolute alcohol saturated with picric acid
or 70% alcohol with 3% mL ammonium hydroxide, wash
sections, rinse in 1% acetic acid, wash again
15
Q
Simple Aqueous Fixatives
- Glutaraldehyde
A
- Dialdehyde – extra aldehyde that does not cross- link.
- Left free to react in any method using Schiff reagent (PAS).
- Penetrates slowly and poorly.
- Frequently used for EM as it preserves ultra- structures better than any other of the aldehydes.
- Overhardens tissue with prolong fixation; recommended length no longer than 2 hours.
16
Q
Simple Aqueous Fixatives
- Mercuric chloride
A
- Used in compound fixatives due to it’s ability to enhance staining
- Inhibits freezing of tissue (frozen sections)
- Penetrates poorly and causes tissue shrinkage
- Can harden tissue with prolong exposure
- Can form mercury pigment than can be removed with iodine followed by sodium
thiosulfate - Very toxic compound
17
Q
Simple Aqueous Fixatives
- Osmium Tetroxide
A
- Primary use is for EM
- Used as a post-fixative after aldehyde fixation
- Chemically combines with lipids making them insoluble.
- Does not penetrate tissue deep; sections must be thin
- Extremely hazardous
18
Q
Simple Aqueous Fixatives
- Picric Acid
A
- Only substance used as both a fixative & stain
- Strong coagulant of nucleoproteins but leave them
soluble - Should not be used for subsequent stains for DNA & RNA
- Does not fix lipids or carbohydrates
- Can decalcify small calcium deposits in tissue
- Leaves tissue receptive to eosin
- Causes extreme tissue shrinkage
- Must be washed out of tissue before processing with 50% alcohol with lithium carbonate
- Extremely hazardous explosive compound. Must be kept
in aqueous solution of at minimum 10% moisture.
19
Q
Simple Aqueous Fixatives
- Potassium dichromate
A
- Rarely used alone for fixation
- Can fixate some lipids
- Preserves mitochondria for EM but dissolves DNA
- Increases tissue affinity for eosin
- Chromate based solution; can yield chromate pigments; removed by 1% HCl in 70% alcohol for 30 minutes
- Highly toxic substance
20
Q
Simple Aqueous Fixatives
- Zinc salts
A
- Preserves antigenicity
- Can be used in place of mercury for safely reasons (zinc chloride)
- Can be corrosive on closed tissue processors and cause salt precipitation. Removed with 5%
to 20% acetic acid solution. - Superior nuclear detail and better paraffin infiltration are obtained with zinc formalin
21
Q
Compound or combined fixatives
1. B-5
2. Bouin
3. Gendre
4. Hollande
5. Zenker & Helly
6. Orth
7. Zamboni
8. Zinc Formalin
A
- B-5
- Bouin
- Gendre
- Hollande
- Zenker & Helly
- Orth
- Zamboni
- Zinc Formalin
22
Q
Compound or combined fixatives
- B-5
A
- Mercuric Chloride
- Sodium acetate
- (added to) formaldehyde
- Recommended for hematopoietic & lymphoreticular tissue (nuclear detail)
- Sections must be treated for mercury pigment
- Tissue can not remain in fixative (placed in 70% alcohol).
23
Compound or combined fixatives
- Bouin solution
- Picric acid, formaldehyde, glacial acetic acid
- Lysis of RBC
- Recommended for trichrome stains, endocirne system tissues and antibody staining.
- Yellow coloring from fixative must be washed out with 50% to 70% alcohol saturated with lithium carbonate.
- Remaining tissue placed in 70% to 80% alcohol.
- Maximum fixation time is 24 hour
24
Compound or combined fixatives
- Gendre
- 95% alcohol saturated with picric acid, formaldehyde, glacial acetic acid
- Preservation of glycogen
- Follow Bouin procedures
25
Compound or combined fixatives
- Hollande
- Picric acid, formaldehye, acetic acid, copper acetate
- Cupric acid solution stabilized RBC and the granules of eosinophils
26
Compound or combined fixatives
- Zenker & Helly
- Zenker & Helly stock solution: mercuric chloride, potassium dichromate, DI
- Zenker working solution: Stock with acetic acid
- Lyses RBC, but better nuclear fixative & can decal needle biopsies of bone marrow
- Recommended for Mallory PTAH
- Helly working solution: Stock with formaldehyde
- Preserves RBC
- Neither is recommended for subsequent silver staining
- Maximum fixation time is 24 hours
27
Compound or combined fixatives
- Orth
- Potassium dicrhomate, sodium sulfate, (added to) formaldehyde
- Recommended for chromaffin granules
28
Compound or combined fixatives
- Zamboni PAF
- Paraformaldehyde & picric acid
- Good general purpose fixative
29
Compound of combined fixatives
- Zinc Formalin
- 10% NBF
- Zinc Sulfate
- Distilled Water
- Great for Immunohistochemical methods
- Prevents cross linking
- Antigenicity not lost with long term storage
30
Nonaqueous fixatives
- Acetone
- Non additive coagulant fixative
- Dehydrates tissue and fixes rapidly at room temp
- Mainly used for enzyme preservation
31
Nonaqueous fixatives
- Alcohols
- Used for fixation of water soluble tissue components (glycogen, urate crystals)
- Methyl used for touch smears
- Dissolves fats, and shrinks tissues
32
Nonaqueous Fixatives
- Alcoholic Fixatives
**Carnoy solution**
- Absolute ethyl alcohol
- Chloroform
- Acetic acid
- Good nuclear detail, used in cytology
- Methacarn is same but with methyl alcohol
33
Nonaqueous Fixatives
- Alcoholic Fixatives
**Clarke Fluid**
- Absolute alcohol
- Glacial acetic acid
