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MLSCI 350 > 3: Fixation, Pigments, and Artifacts > Flashcards

3: Fixation, Pigments, and Artifacts Flashcards

(51 cards)

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1
Q

Functions of fixatives (3)

A
  1. Prevent autolysis
  2. Stabilize tissue morphology (carbs/ lipids)
  3. Enhance staining
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2
Q

2 mechanisms of fixation

A
  1. Denaturation
  2. Formation of cross-links
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3
Q

Denaturation

A

alters secondary and tertiary protein structures (H bonding, hydrophobic interactions, disulfide bonds, and salt linkages)

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4
Q

4 ways fixatives can Denature proteins

A
  1. Heat
  2. Alcohols
  3. Acids
  4. Heavy metals
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5
Q

How does heat denature proteins ?

A

molecules vibrate rapidly = disrupts chemical bonds (hydrogen, hydrophobic interactions)

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6
Q

Why may heat fixation not be preferred over chemical fixation ?

A

It produces random protein structures = reproducibility issues when analyzing tissue

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7
Q

How do alcohols denature proteins ?

A
  • 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
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8
Q

Fixation __ proteins

A

Fixation DENATURES proteins

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9
Q

How do acids denature proteins ?

A
  • hydronium ions (H3O+) react with amino (NH2) and carboxyl (COO-)
  • breaks and form new salt linkages = altered shape
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10
Q

How do heavy metals denature proteins ?

A
  • Breaks and form new DISULFIDE bonds = altered secondary structure
  • React with negatively charged side chains = INSOLUBLE PRECIPITATES
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11
Q

How do Cross-linking fixatives fix tissues ?

A

Stabilizes protein by forming methylene bridges = hardens tissue with minimal shrinkage

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12
Q

How do aldehydes fix tissue ?

A

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

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13
Q

How do oxidizing agents fix tissues ? Give an example

A

OSMIUM forms cross-links with unsaturated carbons (ie. IN LIPIDS)

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14
Q

Additive fixatives

A

Chemically BIND with tissue (components) to alter primary, secondary, and tertiary structure:
- aldehydes
- oxides (osmium)
- heavy metals (mercury, zinc)
- acids

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15
Q

Non-additive fixatives

A

Do not bind to tissue and only alter tertiary structure:
- alcohols (ethanol, methanol, acetone)

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16
Q

List factors that affect fixation (4):

A
  1. Temperature
  2. Tissue thickness/ size
  3. Time
  4. Fixative volume
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17
Q

How does temperature affect fixation ?

A
  • warmer = faster fixation
  • routine light microscopy <45°C; TYPICALLY RT
  • electron microscopy <37°C
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18
Q

How does tissue thickness/ size affect fixation ?

A
  • Fixative will penetrate at different rate
    <4mm thick in formalin
    <1mm thick in glutaraldehyde
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19
Q

How does time affect tissue fixation ?

A
  • transport should be minimized to prevent putrefaction
  • neutral buffered formalin = minimum of 8 hours
  • breast = minimum of 24 hours
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20
Q

How does fixative volume affect fixation ?

A

(At least 20 TIMES ) higher ratios of fixative:specimen volume are better

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21
Q

Neutral buffered formalin contains _% formaldehyde

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A

Neutral buffered formalin contains ~4% formaldehyde

22
Q

Why may manufacturers add 10% methanol to concentrated formaldehyde ?

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A

To prevent formation of PARAFORMALDEHYDE; a white precipitate

23
Q

Formalin: what kind of fixative? Shrinkage ? Hardening ?

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A
  • additive, non-coagulant
  • cross-links with positive amino acids
  • less shrinkage than other fixatives
  • extensively hardens tissues over time
24
Q

Describe fixation by formalin

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A

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)

25
Why is formalin the fixative of choice despite its toxicity ?
Cheap, stable and available for other special stains
26
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
27
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
28
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
29
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
30
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
31
Picric acid: what kind of fixative ? Shrinkage ? Hardening
- additive, coagulant - SEVERE SHRINKAGE - does not harden tissue - HYDROLYZES NUCLEIC ACIDS
32
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
33
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
34
Ethanol: What kind of fixative ? Shrinkage ? Hardening ?
- preserves glycogen and urate crystals - shrinkage - hardens tissue
35
Osmium: What kind of fixative ?
- additive, non-coagulant - fixes LIPIDS for ELECTRON MICROSCOPY - MOST POISONOUS
36
Which two fixatives are used for electron microscopy ?
1. Glutaraldehyde (1mm-thin tissue !!) 2. Osmium
37
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)
38
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
39
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
40
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)
41
Why may zinc sulfate be added to 10%NBF ?
- ?reduced methylene bridges = preserves tissue antigenicity - improved nuclear detail
42
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
43
Endogenous pigment
Melanin
44
What stain is used to demonstrate melanin ?
- Fontana Masson - duplicate slide is bleached using potassium permanganate (oxidizing agent)
45
When does formalin pigment form ?
When Hemoglobin reacts with formaldehyde in ACIDIC conditions - neutral buffered formalin counteracts this
46
Why is formalin pigment undesirable ?
As an Argentaffin substance= binds AND reduces silver solutions= false positive in silver stains
47
How is formalin pigment removed ?
Saturated picric acid+alcohol, followed by a wash
48
How is mercury pigment removed ?
Iodine solution, followed by 5% aqueous sodium thiosulfate (“hypo”) to remove iodine staining
49
How is picric acid discolouration removed ?
Neutralize using 70% ethanol (more effective) or water
50
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
51
Why are aldehyde and oxidizing fixatives unsuitable for immunohistochemistry ?
Non-coagulant fixatives form cross-links that reduce tissue antigenicity

MLSCI 350 (6 decks)

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