3: Fixation, Pigments, and Artifacts Flashcards
Functions of fixatives (3)
- Prevent autolysis
- Stabilize tissue morphology (carbs/ lipids)
- Enhance staining
2 mechanisms of fixation
- Denaturation
- Formation of cross-links
Denaturation
alters secondary and tertiary protein structures (H bonding, hydrophobic interactions, disulfide bonds, and salt linkages)
4 ways fixatives can Denature proteins
- Heat
- Alcohols
- Acids
- Heavy metals
How does heat denature proteins ?
molecules vibrate rapidly = disrupts chemical bonds (hydrogen, hydrophobic interactions)
Why may heat fixation not be preferred over chemical fixation ?
It produces random protein structures = reproducibility issues when analyzing tissue
How do alcohols denature proteins ?
- disrupts hydrogen bonding = new OH bonds form between alcohol and amino acids = stabilize denatured protein structures and harden tissue
- alcohols are hydrophilic = removes water from protein = exposed hydrophobic domains unfolds the peptide
Fixation __ proteins
Fixation DENATURES proteins
How do acids denature proteins ?
- hydronium ions (H3O+) react with amino (NH2) and carboxyl (COO-)
- breaks and form new salt linkages = altered shape
How do heavy metals denature proteins ?
- Breaks and form new DISULFIDE bonds = altered secondary structure
- React with negatively charged side chains = INSOLUBLE PRECIPITATES
How do Cross-linking fixatives fix tissues ?
Stabilizes protein by forming methylene bridges = hardens tissue with minimal shrinkage
How do aldehydes fix tissue ?
In two steps:
1. RAPID covalent binding to amino acids = prevents autolysis by rendering enzymes ineffective
2. SLOW* linking of adjacent tissue-bound aldehydes = methylene bridges
*NOTE: days to weeks
How do oxidizing agents fix tissues ? Give an example
OSMIUM forms cross-links with unsaturated carbons (ie. IN LIPIDS)
Additive fixatives
Chemically BIND with tissue (components) to alter primary, secondary, and tertiary structure:
- aldehydes
- oxides (osmium)
- heavy metals (mercury, zinc)
- acids
Non-additive fixatives
Do not bind to tissue and only alter tertiary structure:
- alcohols (ethanol, methanol, acetone)
List factors that affect fixation (4):
- Temperature
- Tissue thickness/ size
- Time
- Fixative volume
How does temperature affect fixation ?
- warmer = faster fixation
- routine light microscopy <45°C; TYPICALLY RT
- electron microscopy <37°C
How does tissue thickness/ size affect fixation ?
- Fixative will penetrate at different rate
<4mm thick in formalin
<1mm thick in glutaraldehyde
How does time affect tissue fixation ?
- transport should be minimized to prevent putrefaction
- neutral buffered formalin = minimum of 8 hours
- breast = minimum of 24 hours
How does fixative volume affect fixation ?
(At least 20 TIMES ) higher ratios of fixative:specimen volume are better
Neutral buffered formalin contains _% formaldehyde
Neutral buffered formalin contains ~4% formaldehyde
Why may manufacturers add 10% methanol to concentrated formaldehyde ?
To prevent formation of PARAFORMALDEHYDE; a white precipitate
Formalin: what kind of fixative? Shrinkage ? Hardening ?
- additive, non-coagulant
- cross-links with positive amino acids
- less shrinkage than other fixatives
- extensively hardens tissues over time
Describe fixation by formalin
Since formalin is an aldehyde:
1. RAPID penetration of tissue and binding of positive amino acids
2. SLOW formation of Cross-links = methylene bridges of adjacent proteins = stabilized protein structure (takes days)
Why is formalin the fixative of choice despite its toxicity ?
Cheap, stable and available for other special stains
How does glutaraldehyde differ from formaldehyde (formalin) ?
- has two aldehydes (think shark face) instead of one
- cross-linking occurs simultaneously as penetration BUT
- cross-linking slows down penetration
Glutaraldehyde: What kind of fixative ? Shrinkage ? Hardening ?
- additive, non-coagulant fixative used in electron microscopy
- unstable; must be used fresh and buffered to prevent becoming acidic
- hardens tissue
Since cross-linking slows down further penetration in glutaraldehyde, tissue must be __.
Since cross-linking slows down further penetration in glutaraldehyde, tissue must be 1mm thin ! (Faster penetration)
NOTE: used in ELECTRON MICROSCOPY
Acetic acid: What kind of fixative ? Shrinkage ? Hardening ?
- coagulant (precipitates DNA + preserves nucleoproteins)
- CANNOT fix cytoplasmic proteins; MIXED IN A COMPOUND FIXATIVE
- swells tissue the most (can counteract shrinkage)
- does not harden tissue
Picric acid storage/ handling requirements
- must be stored WET; wipe up small spills before they dry = dry picric acid is explosive
- NEVER POUR DOWN DRAIN = can combine with metal pipes = picrate salts are explosive even when wet ! womp womp
Picric acid: what kind of fixative ? Shrinkage ? Hardening
- additive, coagulant
- SEVERE SHRINKAGE
- does not harden tissue
- HYDROLYZES NUCLEIC ACIDS
Why is picric acid used in trichrome stains ?
- picric acid enhances all anionic dyes by providing additional reactive groups
- routinely used as a fixative/ pre-mordant for trichrome stains
Why must picric acid be completely neutralized before processing ?
If any picric acid remains, expected staining characteristics of tissue will be lost over time
- use 70% ethanol (more effective) or water
Ethanol: What kind of fixative ? Shrinkage ? Hardening ?
- preserves glycogen and urate crystals
- shrinkage
- hardens tissue
Osmium: What kind of fixative ?
- additive, non-coagulant
- fixes LIPIDS for ELECTRON MICROSCOPY
- MOST POISONOUS
Which two fixatives are used for electron microscopy ?
- Glutaraldehyde (1mm-thin tissue !!)
- Osmium
What is Bouin’s Fluid ?
A compound fixative:
1. formaldehyde (cheap, stable, available)
2. acetic acid (counteracts shrinkage)
3. aqueos picric acid (SEVERE SHRINKAGE but soft tissue facilitates cutting and staining with anionic dyes = CRISP)
B5 vs B-plus
- compound fixatives
B5: MERCURIC chloride, sodium acetate, formalin, water
- NUCLEAR DETAIL in HEMATOPOIETIC (bone marrow) and lymphoreticular tissues
B-plus: replaces mercury with ZINC for comparable results without safety issues of use/ disposal surrounding mercury
What is Clarke’s fluid ?
- 3 parts ethanol and 1 part acetic acid are mixed before use
- PRESERVES NUCLEIC ACIDS, extracts lipids and maintains micro-anatomical structures best = FOR MOLECULAR PATHOLOGY
What is alcoholic formalin ?
- 70% alcohol is used as a diluent
- speeds up fixation and begins dehydration in one step
- popular FOR HIGH ADIPOSE TISSUE (breast, colon)
Why may zinc sulfate be added to 10%NBF ?
- ?reduced methylene bridges = preserves tissue antigenicity
- improved nuclear detail
What are Accustain and FineFIX ? Why are they not more commonly used ?
Formalin-free fixatives:
- safer
- better penetatrate fatty tissues (breast)
- improve tissue antigenicity
- enhance recovery of nucleic acids
BUT routine methods are based on formalin-fixed tissue
Endogenous pigment
Melanin
What stain is used to demonstrate melanin ?
- Fontana Masson
- duplicate slide is bleached using potassium permanganate (oxidizing agent)
When does formalin pigment form ?
When Hemoglobin reacts with formaldehyde in ACIDIC conditions
- neutral buffered formalin counteracts this
Why is formalin pigment undesirable ?
As an Argentaffin substance= binds AND reduces silver solutions= false positive in silver stains
How is formalin pigment removed ?
Saturated picric acid+alcohol, followed by a wash
How is mercury pigment removed ?
Iodine solution, followed by 5% aqueous sodium thiosulfate (“hypo”) to remove iodine staining
How is picric acid discolouration removed ?
Neutralize using 70% ethanol (more effective) or water
Differentiate coagulant vs non-coagulant fixatives
Coagulants: destroy organelles but stabilize connective tissue
ie. ALCOHOLS
Non-coagulants: forms cross-links/ bridges between adjacent reactive groups
ie. ALDEHYDES and OSMIUM
Why are aldehyde and oxidizing fixatives unsuitable for immunohistochemistry ?
Non-coagulant fixatives form cross-links that reduce tissue antigenicity
