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350 > fixation, pigments, artifacts > Flashcards

fixation, pigments, artifacts Flashcards

1
Q

what are the functions of fixation

A
  1. prevent autolysis
  2. stabilize tissue morph
  3. enhance staining
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2
Q

how do fixatives work

A

altering tissue proteins by denaturation and forming crosslinks

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3
Q

what is denaturation

A

altering the shape of a protein (secondardy and tertiary structures)

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4
Q

how does heat denature

A

molecules vibrate rapidly = weaken chemical bonds

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5
Q

how does alcohol denature

A

disrupts hydrogen bonds and forms a bond between the alcohol and amino acid chains = tissue hardening

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6
Q

how do acids denature

A

hydronium ions react with amino and carboxy group and arginine, histidine, and lysine = break saltlinkages

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7
Q

how do heavy metals denature

A

affinity for sulfur in disulfide bonds (mercury) = secondary structures altered

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8
Q

what is crosslinking and how does it work

A

chemical reaction with amino acids by forming methylene bridges cross links = harden tissue and reduce shrinkage

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9
Q

what is the most common cross linking fixative

A

aldehydes like formaldehyde and gluteraldehyde

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10
Q

how do aldehydes react with tissue protein

A

First covalently bind to amino groups lysine, cysteine, serine, and threonine to prevent autolysis

then they bind to one another to form methylene bridges (slow process)

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11
Q

examples and fuction of oxidizing agent fixatives

A

osmium tetroxide and potassium dichromate

osmium reacts with lipids for EM
dichromate ion links to carboxyl

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12
Q

what are coagulant fixatives and how to they work

A

distort cytoplasmic content while stabilizing proteins

work fast compared to non coag

tertiary structure affected

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13
Q

what are non- coagulant fixatives and how to they work

A

preserve cytoplasmic content by turning into gel and stabilize by forming cross links

gel stops penetration of reagents = work slower

primary structure affected

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14
Q

additive vs non additive

A

additive = chemically binding proteins

non additive = no chemicals binding = dehydrating agents

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15
Q

examples of additive non caoag

A

formaldehyde, gluteraldehyde, osmium tetroxide, potassium dichromate

aldehydes and oxidizing agents**

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16
Q

examples of additive coag

A

mercuric chloride, chromic acid, picric acid, zinc salt

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17
Q

examples of non additive coag

A

ethanol, methanol, acetone

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18
Q

4 factors that affect quality of fixation

A

temperature
size
time
volume of fixative

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19
Q

what temperature should fixation be

A

<45C for light microscopy but routinely done at RT

<37 for EM

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20
Q

what size should tissue be for dixation

A

<3-4 mm thick

<1mm for gluteraldehyde

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21
Q

how long should tissue be fixed

A

minimum 8 hours using NBF

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22
Q

what volume of fixative to use

A

15-20X the tissue volume

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23
Q

how is formaldehyde prepared in histology

A

37-40% solution in water aka 100% formalin

used as 10% NBF that contains 4% formaldehyde

24
Q

what is paraformaldehyde and how to prevent it

A

white precipitate from concentrated solution

prevented by adding methanol

25
Q

how does formaldehyde work

A

cross linking with amino groups, arginine, histidine, and lysine

26
Q

how does formaldehyde affect tissues

A

less shrinkage but hardens tissue when prolonged

27
Q

fuction and use of gluteraldehyde

A

dialdehyde = crosslink while penetrating **tissue <1mm thin

used for EM

28
Q

function and use of acetic acid

A

used to lyse RBC

cannot fix cytoplasmic contents therefore it is combines with other reagents

29
Q

how does acetic acid affect tissues

A

penetrates tissue fast with no hardening

30
Q

2 reasons why acetic acid is used in compound fixatives

A

preserves nucleoproteins and precipitates DNA

swells tissue

31
Q

function and use of picric acid

A

both a fixative and stain

hydrolyze DNA/RNA

32
Q

how does picric acid affect tissues

A

causes SEVERE shrinkage

decalcify small biopsies

33
Q

storage of picric acid

A

stored wet as it can be explosive when dry

combined with metals = explosive so never pour down the drain

34
Q

how is picric acid neutralized before tissue processing and what happens when it is not neutralized

A

use 70% ethanol or water

staining characteristics lost over time

35
Q

Alcohol fixation affects on tissues

A

harden and shrinks

36
Q

ethanol use for tissue

A

preserve glycogen and urate crystals

37
Q

Use of osmium tetroxide

A

EM for lipids

38
Q

What does bouins fluid contain

A

formaldehyde, acetic acid, picric acid

39
Q

use for bouins fluid

A

trichrome staining

lysing RBC and decal

40
Q

how is swelling from the picric acid countered in bouins fluid

A

using acetic acid

41
Q

what does B plus contain

A

mercuric chloride, sodium acetate, formalin, water

42
Q

uses for B plus

A

best for nuclear detail and rbc in IHC

43
Q

instead of murcury in B plus, what is substitudted

A

zinc chloride

44
Q

what is Clarkes fluide made of

A

3 parts ethanol, 1 part glacial acetic acid thats made fresh

45
Q

uses for clarkes fluid

A

molecular pathology for nucleic acids and lipids

46
Q

what is modified formalin made of

A

alcohol and formalin

47
Q

uses for modified formalin

A

fixes and dehydrates

48
Q

benefits from using modified formalin

A

prevents shrinkage

really good with fatty tissues like breast

49
Q

why may zinc sulfate be added to modified formalin

A

preserve tissue antigenicity during storage

improve nuclear detail

50
Q

what are endogenous pigments and give example

A

naturally occuring in tissue like melanin and lipofuscin

51
Q

what is formalin pigment

A

birefringent dark brown in blood rich tissues

52
Q

how does formalin pigment forms

A

hemoglobin reacts with formaldehgyde in acidic conditions

53
Q

T/F formalin will create false positive in silver stains

A

T - formalin is an argentaffin (reduces silver)

54
Q

what is murcury pigment

A

birefringent brown crystals that is non preventable

55
Q

how is mercury pigment removed after fixation

A

iodine followed by hypo

56
Q

examples of exogenous pigments

A

carbon - smoker lung

tattoo ink - dark and granular in skin

350 (7 decks)

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