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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
how does formaldehyde work
cross linking with amino groups, arginine, histidine, and lysine
26
how does formaldehyde affect tissues
less shrinkage but hardens tissue when prolonged
27
fuction and use of gluteraldehyde
dialdehyde = crosslink while penetrating **tissue <1mm thin used for EM
28
function and use of acetic acid
used to lyse RBC cannot fix cytoplasmic contents therefore it is combines with other reagents
29
how does acetic acid affect tissues
penetrates tissue fast with no hardening
30
2 reasons why acetic acid is used in compound fixatives
preserves nucleoproteins and precipitates DNA swells tissue
31
function and use of picric acid
both a fixative and stain hydrolyze DNA/RNA
32
how does picric acid affect tissues
causes SEVERE shrinkage decalcify small biopsies
33
storage of picric acid
stored wet as it can be explosive when dry combined with metals = explosive so never pour down the drain
34
how is picric acid neutralized before tissue processing and what happens when it is not neutralized
use 70% ethanol or water staining characteristics lost over time
35
Alcohol fixation affects on tissues
harden and shrinks
36
ethanol use for tissue
preserve glycogen and urate crystals
37
Use of osmium tetroxide
EM for lipids
38
What does bouins fluid contain
formaldehyde, acetic acid, picric acid
39
use for bouins fluid
trichrome staining lysing RBC and decal
40
how is swelling from the picric acid countered in bouins fluid
using acetic acid
41
what does B plus contain
mercuric chloride, sodium acetate, formalin, water
42
uses for B plus
best for nuclear detail and rbc in IHC
43
instead of murcury in B plus, what is substitudted
zinc chloride
44
what is Clarkes fluide made of
3 parts ethanol, 1 part glacial acetic acid thats made fresh
45
uses for clarkes fluid
molecular pathology for nucleic acids and lipids
46
what is modified formalin made of
alcohol and formalin
47
uses for modified formalin
fixes and dehydrates
48
benefits from using modified formalin
prevents shrinkage really good with fatty tissues like breast
49
why may zinc sulfate be added to modified formalin
preserve tissue antigenicity during storage improve nuclear detail
50
what are endogenous pigments and give example
naturally occuring in tissue like melanin and lipofuscin
51
what is formalin pigment
birefringent dark brown in blood rich tissues
52
how does formalin pigment forms
hemoglobin reacts with formaldehgyde in acidic conditions
53
T/F formalin will create false positive in silver stains
T - formalin is an argentaffin (reduces silver)
54
what is murcury pigment
birefringent brown crystals that is non preventable
55
how is mercury pigment removed after fixation
iodine followed by hypo
56
examples of exogenous pigments
carbon - smoker lung tattoo ink - dark and granular in skin

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