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MLSCI 250 > 350 - Topic 2, 3, 5 (Gross Dissection, Fixation, Decalcification) > Flashcards

350 - Topic 2, 3, 5 (Gross Dissection, Fixation, Decalcification) Flashcards

1
Q

What are the functions of fixatives?

A
  1. Prevent autolysis: alter enzymes so they are no longer effective; also toxic to bacteria and fungi which averts putrefaction
  2. Stabilize tissue morphology: preserve relationship b/w cells and extracell substances; also helps to maintain non-protein elements, esp. carb and lipids
  3. Enhance staining: expose more reactive sites on a protein to increase dye binding = makes stain more intense; some create additional reactive groups or even act as mordants
    NOTE: not al fixatives improve staining (ex: formalin reduces available dye binding sites for an ionic dyes like eosin)
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2
Q

All histological fixatives work by …

A

Altering tissue proteins

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3
Q

Two major mechanisms of fixation:

A

Deanaturation
Formation of cross-links

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4
Q

What is denaturation ?

A

Alteration of the shape of a protein
- always involves 2ry and 3ry protein structures = largely result of hydrogen bonding, hydrophobic interactions, disulfide bonds and salt linkages
- heat, alcohol, acids, heavy metals

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5
Q

Heat (denaturation)

A
  • molecules vibrate rapidly = disrupts weaker chemical bonds
  • will produce random protein structure compared to chemical fixatives = reproducibility issues?
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6
Q

alcohol (denaturation)

A
  • disrupt hydrogen bonding extensively
  • net hydrogen bonds between alcohol and amino acid side chains = stabilizes denatured protein structure and hardening tissue markedly
  • expose hydrophobic domains, unfolding polypeptide
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7
Q

How does alcohol remove water from protein?

A

They are hydrophilic and hygroscopic = will attract and absorb water

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8
Q

Acids (denaturation)

A

Supply hydronium ions in solution which readily react with amino and carboxyl groups in proteins as well as basic AAs (arg, His, Lysine)
- this action = breaks salt linkages, forms new ones = changes shape of protein

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9
Q

Heavy metals

A
  • mercury, lead, zinc
  • behave similarly to acids, though they have a particular affinity for the sulfur found in disulfide bonds (esp. mercury)
  • disrupting disulfide bonds affects the secondary structure of many proteins
  • metal react w/ neg-charged side chains and ionized carboxyl groups = forming insoluble precipitates
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10
Q

What do cross-linking fixatives do?

A

They chemically react with amino acids
- they stabilize morphology by forming methylene bridges bw adjacent reactive areas
- methylene bridge cross-linking harden tissue markedly and tend to reduce shrinkage during tissue processing

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11
Q

The most common cross-linking fixative

A

Aledehydes
- formaldehyde and glutaraldehyde

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12
Q

Reaction of aldehydes with tissue proteins

A
  1. Aldehydes covalent bind to amino groups, as well as lysine, cysteine, serine, and threonine residues; reaction occurs rapidly and prevents autolysis by rendering enzymes ineffective
  2. Adjacent tissue-bound aldehydes link with one another by forming methylene bridges; this cross-linking phase occurs more slowly = days or weeks to complete
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13
Q

Dialdehyde

A

Glutaraldehyde
- it cross-links and stabilizes as it penetrates tissue

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14
Q

Fixation using oxidizing agents

A
  • osmium tetroxide, potassium dichromate
  • poorly understood but known to stabilize tissues by cross-linking reactive groups in close proximity
  • dichromate ion links adjacent carbonyl groups of many proteins; osmium readily reacts with unsaturated carbons (lipids)
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15
Q

Additive

A

Chemically binding with the proteins or other tissue component

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16
Q

Non-additive

A

Disrupting tissue structure without becoming chemically incorporated; ONLY dehydrating agents are non-additive (ethanol, acetone, methanol)

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17
Q

These are all additive fixatives

A

Aldehydes
Acids
Metal salts
Oxidizers

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18
Q

Factors affecting fixation (4)

A
  • temperature: warmer the solution ,faster it will act on tissue
  • size/thickness/ tissue type: different fixatives will penetrate at different rates; formalin (no more than 3-4 mm thick); glutaraldehyde (no more than 1 mm)
  • time: duration of fixation varies considerably; minimize delay (prevent autolysis and putrefaction)
  • volume of fixative: the higher the ratio of fixative to specimen vol, the better ALWAYS; use 15-20 times the tissue volume when aliquoting fixatives
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19
Q

Temp acceptable for light vs electron microscopy

A

Light = up to 45C

Electron = do not exceed 37C

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20
Q

All tissues should be fixed for a minimum of how many hours wen using NBF?

A

8 hrs
24 hrs or ore generally preferred
24 hrs for breast

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21
Q

T or F. Formaldehyde readily polymerizes in aqueous solutions

A

T! Concnetrated solutions of formaldehyde may form paraformaldehyde

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22
Q

Paraformaldehyde

A
  • concentrated formaldehyde
  • white powder which precipitates out of solution
  • prevention: add methanol (around 10%) to concentrated solutions
  • dilute formaldehyde does not polymerize
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23
Q

Unlike most other fixatives, acetic acid does not …

A

Fix cytoplasmic proteins
So never used alone; instead, used in compound fixatives

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24
Q

Why is acetic acid used in compound fixatives?

A
  • it preserves nucleoproteins and precipitates DNA (coagulant fixative in this regard)
  • it swells tissue more than any other fixative
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25
Q

This will hydrolyze nuclei acids and must be avoided if DNA/RNA analysis is anticipated

A

Picric acid

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26
Q

Why is it important tat picric acid is completely neutralized prior to tissue processing?

A

If any picric acid remains in the tissue after processing, expected staining characteristics of tissue will be lost over time
- 70% ethanol better than water for this

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27
Q

This is notable for preserving glycogen and urate crystals (gout)

A

Ethanol
- glycogen often detectable in formalin-fixed tissue due to trappings f large molecules within cells, but urates quickly dissolve in aqueous fixatives

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28
Q

Excellent fixative for tissue that will be stained with a trichromatic method

A

Bouin’s
- picric a cts as a mordant for an ionic dyes = exceptionally vivid

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29
Q

B5

A

Mercuric chloride
Sodium acetate
Formalin
Water

30
Q

Excellent fixative for hematopoietic and reticuloendothelial tissues as nuclear detail will be exceptional

A

B5 or B-plus (zinc)

31
Q

Clarke’s fluid

A

3 parts absolute ethanol with 1 part acetic acid immediately before use

32
Q

Molecular pathology fixative

A

Clarke’s

33
Q

Maintains microanatomical structures better than most other fixatives

A

Clarke’s fluid

34
Q

Modified formalin

A

65-70% alcohol used as diluent for concentrated formaldehyde = alcoholic formalin

  • alcohol speeds up tissue fixation and begins dehydration and formalin fixation helps prevent shrinkage associate with alcohol
  • popular with large amounts of adipose (breast, colon)
35
Q

This can be added to 10% NBF in order to preserve tissue antigenicity, esp. with prolonged storage

A

Zinc sulfate or chloride
- effect probably due to a reduction in ‘methylene brige’ formation
- addition of zinc also improves nuclear detail when compared to unmodified 10% NBF

36
Q

an undesirable change in tissue created by lab’s attempts to process it

A

artifact

37
Q

endogenous pigment

A

naturally occurring in tissue
- not artifactual
- melanin (granular dark-brown) and lipofuscin (yellow-brown)

38
Q

how is melanin often demonstrated?

A

Fontana Masson stain
- may be bleached using potassium permanganate or other oxidizing agents

39
Q

acid formaldehyde hematin

A

formalin pigment

40
Q

formalin pigment

A

AKA acid formaldehyde hematin
amorphous, birefringent dark-brown black pigment
- noted in blood-rich tissues
- forms when hemoglobin reacts with formaldehyde in acidic conditions
- using NBF will minimize the amount of formalin pigment in tissue sections

41
Q

T or F. formalin pigment will reduce silver solutions

A

T
it is an argentaffin substance
formalin pigment can potentially produce a false pos when trying to demonstrate spirochetes, fungi, melanin, reticulin

42
Q

how do we remove formalin pigment ?

A

saturated solution of picric acid and alcohol followed by a running water wash
also alcoholic ammonium hydroxide works

43
Q

a crystalline, birefringent brown pigment that may be found in any tissue fixed in mercury-containing fixative

A

mercury pigment

44
Q

T or F. the formation of mercury pigment is non-preventable

A

T! all tissues fixed in mercury is treated with an iodine solution to remove the pigment, followed by 5% aq sodium thiosulfate (hypo) to remove iodine staining

45
Q

stain precipitates

A
  • on top of tissue
  • can confirm by focusing in and out of focus
  • avoided by filtering stains
  • will form if stain is allowed to dry on the slide
46
Q

exogenous pigments

A
  • not naturally occurring
  • must be introduced to patient’s body
  • NOT ARTIFACTS
  • carbon, tattoos
47
Q

T or F. neither carbon or tattoo ink is easily removed from tissues

A

T!
tattoo usually does not obscure pathology

48
Q

why is zinc formalin considered better than formalin for most tissues?

A
  • prevents cross-linking; good for preserving antigenicity for IHC
  • better nuclear detail
49
Q

vapour fixative

A

ALDEHYDES
can continue fixing even as a gas
- useful for disinfection

50
Q

how to differentiate between formalin pigment vs melanin

A

formalin = birefringent; can dissolve by alcoholic picric acid

melanin = only within cells; can remove by any oxidizers

51
Q

decalcification

A

process of removing calcium from tissues
- some inherently calcified (bone)
- others = calcification is a pathological process
- removal of calcium required prior to sectioning paraffin blocks; if not = will not section = tears, holes, major disruptions

52
Q

what do most labs use to decalcify tissue?

A

acids
- calcium salts soluble at pH 4.5 or less, so almost any weak or strong acid may be used
- formic acid combined with formalin is used routinely, and HCl is used when more rapid decalcification is required

53
Q

strong acid for decalcification

A

hydrochloric, nitric
- 5-10%
- fast <48 hrs
higher danger

54
Q

weak acid for decalcification

A

formic
5-10%
slow (3-10 days)
lower danger

55
Q

T or F. tissue must be fixed prior to decalcification

A

T! if not, decalcifying agent will destroy delicate tissue components and alter tissue basophilia
- using formic aicd/formalin solution will allow simultaneous fixation and decalcification = ADVANTAGE

56
Q

bischloromethyl ether

A

extremely hazardous carcinogen
- wash thoroughly after formalin fixation bc residual formalin may react with HCl to produce this

57
Q

decalcification methods

A
  • simple acid
  • ion-exchange resins
  • EDTA
58
Q

simple acid to decalcify tissue

A
  • submerge tissue in an appropriate decalcifying solution
  • tissue should be suspended to allow to work on all surfaces
  • agitation to prevent solution surrounding tissue from becoming saturated with calcium ions
  • solution is discarded after saturation and replaced with fresh fluid
59
Q

ion exchange resins to decalcify tissue

A
  • improvement over simple acids
  • resin will exchange ammonium inos for calcium ions preventing solution from becoming saturated = quickening decalcification and reducing need for solution replacement
  • typically formic acid decalcifiers
60
Q

EDTA to decalcify tissue

A
  • selectively binds metal ions from the tissue without affecting any other tissue c components
  • EDTA (neutralized with sodium hydroxide) is popular esp when acids might destroy labile tissue elements we hope to demonstrate = enzymes, molecular targets
  • disadvantage = TAT; may trake weeks
61
Q

under-decalcified

A

will not section well

62
Q

over-decalcified

A

will demonstrate poor staining and morphology

63
Q

three methods to detecting endpoint

A

chemical
radiography
physical

64
Q

CHEMICAL ENDPOINT DETECTION

A

aliquot of used decal solution neutralized with ammonium hydroxide - reacted with ammonium oxalatae

any calcium ions present => insoluble calcium oxalate (white precipitate)
if calcium detected = assumed that calcium persists in tissue and is placed in fresh decal solution

if no precipitate forms = decalcification is complete

fats and accurate method

65
Q

radiography endpoint detection

A

calcium is radio-opaque = solid white on x-rays

  • x-raying specimens = easily see endpoint of decal

cost of equipment may be prohibitive for labs that do not regularly perform this

66
Q

physical endpoint detection

A

endpt determined by bending, probing, or attempting to cut sample
- method is fast and relatively easy to perform, it is unreliable and small deposits of calcium may be missed

  • this may also create artifacts in tissue; NOT RECOMMENDED
67
Q

what happens after decalcification?

A

tissue may be further trimmed/sectioned

carefully washed in running tap water

submitted for routine processing

neutralization of residual acid is an important step to ensure good processing

some labs use lithium carbonate to ensure neutralization

68
Q

the usual rot cause for over/under-decal

A

poor endpt detection methods

69
Q

when do we recognize under decalcified tissue

A

during microtomy
- during sectioning = obvious scores, tears will e noted
- resistance with handwheel may be felt if large amounts of calcium present
- to correct = melt block to remove tissue and ‘reverse process’ = expose to xylene, absolute alcohol, graduated alcohols and running water

  • place tissue in decal solution until complete
70
Q

an easy and efficient way to remove small calcifications

A

surface decalcification
- if only a section or two is desired
- once block has been faced, place cut-side down in small amount of strong acid for 15-30 mins
- wash well in running water, chill, attempt to section once again
- acid will only act on exposed surface of tissue so try to get one of the first couple sections

71
Q

how to recognize overdecal tissue

A

marked reduction in nuclear staining
- nuclei will appear pale red and contrast poorly with cytoplasm
- loss of basophilia
- difficult to reverse
- some basophilia may be restored by placing affected slides ina 5% solution sodium bicarb overnight
next morning = wash with water and re-stain

MLSCI 250 (35 decks)

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