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CSMLS > HIS Fixation, Pigments, and Artifacts > Flashcards

HIS 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

5 ways fixatives can Denature proteins

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

How do alcohols denature proteins ?

A
  • alcohols are hydrophilic = removes water from protein = exposed hydrophobic domains unfolds the peptide
  • disrupts hydrogen bonding = new OH bonds form between alcohol and amino acids = stabilize denatured protein structures and harden tissue
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6
Q

Fixation __ proteins

A

Fixation DENATURES proteins

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7
Q

How do Cross-linking fixatives fix tissues ?

A

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

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8
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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9
Q

Non-additive fixatives

A

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

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10
Q

List factors that affect fixation (4):

A
  1. Temperature
  2. Tissue thickness/ size
  3. Time
  4. Fixative volume
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11
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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12
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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13
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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14
Q

How does fixative volume affect fixation ?

A

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

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15
Q

Neutral buffered formalin contains _% formaldehyde

A

Neutral buffered formalin contains ~4% formaldehyde

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16
Q

Why may manufacturers add 10% methanol to concentrated formaldehyde ?

A

To prevent formation of PARAFORMALDEHYDE; a white precipitate

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17
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
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18
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)

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19
Q

Why is formalin the fixative of choice despite its toxicity ?

A

Cheap, stable and available for other special stains

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20
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
21
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
  • minimal shrinkage; hardens tissue
22
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

23
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
24
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
25
Q

Picric acid: what kind of fixative ? Shrinkage ? Hardening

A
  • additive, coagulant
  • SEVERE SHRINKAGE
  • does not harden tissue
  • HYDROLYZES NUCLEIC ACIDS
26
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
27
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

28
Q

Ethanol: What kind of fixative ? Shrinkage ? Hardening ?

A
  • preserves glycogen and urate crystals
  • shrinkage
  • hardens tissue
29
Q

Osmium: What kind of fixative ?

A
  • additive, non-coagulant
  • fixes LIPIDS for ELECTRON MICROSCOPY
  • MOST POISONOUS
29
Q

Which two fixatives are used for electron microscopy ?

A
  1. Glutaraldehyde (1mm-thin tissue !!)
  2. Osmium
29
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

30
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)

31
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
32
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)
33
Q

Why may zinc sulfate be added to 10%NBF ?

A
  • ?reduced methylene bridges = preserves tissue antigenicity
  • improved nuclear detail
34
Q

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

A

Formalin-free fixatives:
- safer
- better penetrate fatty tissues (breast)
- improve tissue antigenicity
- enhance recovery of nucleic acids

BUT routine methods are based on formalin-fixed tissue

35
Q

Melanin is an __ pigment

A

Melanin is an ENDOGENOUS pigment

36
Q

What stain is used to demonstrate melanin ?

A
  • Fontana Masson
  • duplicate slide is bleached using potassium permanganate (oxidizing agent)
36
Q

When does formalin pigment form ?

A

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

37
Q

Why is formalin pigment undesirable ?

A

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

38
Q

How is formalin pigment removed ?

A

Saturated picric acid+alcohol, followed by a wash

39
Q

How is mercury pigment removed ?

A

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

40
Q

How is picric acid discoloration removed ?

A

Neutralize using 70% ethanol (more effective) or water

41
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

42
Q

Why are aldehyde and oxidizing fixatives unsuitable for immunohistochemistry ?

A

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

43
Q

How to differentiate between fixation pigment and stain precipitate ?

A
  • fixation pigments occur within tissue; will be in the same field as tissue
  • staining precipitates are found on top of tissue; will not be in the same field when focused on the tissue
44
Q

Why must tissue be fully fixed prior to processing ?

A

Incomplete fixation:
1. Tissue degradation
2. Alcohol used for dehydration will fix the tissue instead = undesirable bc tissue becomes too brittle and difficult to section

45
Q

Why must tissue be fully fixed prior to processing ?

A

Incomplete fixation:
1. Tissue degradation
2. Alcohol used for dehydration will fix the tissue instead = undesirable bc tissue becomes too brittle and difficult to section

CSMLS (46 decks)

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