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

3: Fixation, Pigments, and Artifacts Flashcards

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

A

Neutral buffered formalin contains ~4% formaldehyde

22
Q

Why may manufacturers add 10% methanol to concentrated formaldehyde ?

A

To prevent formation of PARAFORMALDEHYDE; a white precipitate

23
Q

Formalin: what kind of fixative? Shrinkage ? Hardening ?

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

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
Q

Why is formalin the fixative of choice despite its toxicity ?

A

Cheap, stable and available for other special stains

26
Q

How does glutaraldehyde differ from formaldehyde (formalin) ?

A
  • has two aldehydes (think shark face) instead of one
  • cross-linking occurs simultaneously as penetration BUT
  • cross-linking slows down penetration
27
Q

Glutaraldehyde: What kind of fixative ? Shrinkage ? Hardening ?

A
  • additive, non-coagulant fixative used in electron microscopy
  • unstable; must be used fresh and buffered to prevent becoming acidic
  • hardens tissue
28
Q

Since cross-linking slows down further penetration in glutaraldehyde, tissue must be __.

A

Since cross-linking slows down further penetration in glutaraldehyde, tissue must be 1mm thin ! (Faster penetration)

NOTE: used in ELECTRON MICROSCOPY

29
Q

Acetic acid: What kind of fixative ? Shrinkage ? Hardening ?

A
  • 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
Q

Picric acid storage/ handling requirements

A
  • 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
Q

Picric acid: what kind of fixative ? Shrinkage ? Hardening

A
  • additive, coagulant
  • SEVERE SHRINKAGE
  • does not harden tissue
  • HYDROLYZES NUCLEIC ACIDS
32
Q

Why is picric acid used in trichrome stains ?

A
  • picric acid enhances all anionic dyes by providing additional reactive groups
  • routinely used as a fixative/ pre-mordant for trichrome stains
33
Q

Why must picric acid be completely neutralized before processing ?

A

If any picric acid remains, expected staining characteristics of tissue will be lost over time
- use 70% ethanol (more effective) or water

34
Q

Ethanol: What kind of fixative ? Shrinkage ? Hardening ?

A
  • preserves glycogen and urate crystals
  • shrinkage
  • hardens tissue
35
Q

Osmium: What kind of fixative ?

A
  • additive, non-coagulant
  • fixes LIPIDS for ELECTRON MICROSCOPY
  • MOST POISONOUS
36
Q

Which two fixatives are used for electron microscopy ?

A
  1. Glutaraldehyde (1mm-thin tissue !!)
  2. Osmium
37
Q

What is Bouin’s Fluid ?

A

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
Q

B5 vs B-plus

A
  • 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
Q

What is Clarke’s fluid ?

A
  • 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
Q

What is alcoholic formalin ?

A
  • 70% alcohol is used as a diluent
  • speeds up fixation and begins dehydration in one step
  • popular FOR HIGH ADIPOSE TISSUE (breast, colon)
41
Q

Why may zinc sulfate be added to 10%NBF ?

A
  • ?reduced methylene bridges = preserves tissue antigenicity
  • improved nuclear detail
42
Q

What are Accustain and FineFIX ? Why are they not more commonly used ?

A

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
Q

Endogenous pigment

A

Melanin

44
Q

What stain is used to demonstrate melanin ?

A
  • Fontana Masson
  • duplicate slide is bleached using potassium permanganate (oxidizing agent)
45
Q

When does formalin pigment form ?

A

When Hemoglobin reacts with formaldehyde in ACIDIC conditions
- neutral buffered formalin counteracts this

46
Q

Why is formalin pigment undesirable ?

A

As an Argentaffin substance= binds AND reduces silver solutions= false positive in silver stains

47
Q

How is formalin pigment removed ?

A

Saturated picric acid+alcohol, followed by a wash

48
Q

How is mercury pigment removed ?

A

Iodine solution, followed by 5% aqueous sodium thiosulfate (“hypo”) to remove iodine staining

49
Q

How is picric acid discolouration removed ?

A

Neutralize using 70% ethanol (more effective) or water

50
Q

Differentiate coagulant vs non-coagulant fixatives

A

Coagulants: destroy organelles but stabilize connective tissue
ie. ALCOHOLS

Non-coagulants: forms cross-links/ bridges between adjacent reactive groups
ie. ALDEHYDES and OSMIUM

51
Q

Why are aldehyde and oxidizing fixatives unsuitable for immunohistochemistry ?

A

Non-coagulant fixatives form cross-links that reduce tissue antigenicity

MLSCI 350 (6 decks)

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