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350 > Processing > Flashcards

Processing Flashcards

1
Q

what are the stages to tissue processing

A

fixation
dehydration
clearing
infiltration

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2
Q

what is fixation

A

formalin fixation of biopsies

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3
Q

what QC is in fixation

A

monitor and documentation of fixation time

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4
Q

what is dehydration

A

removing all unbound water and fixative from tissue

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5
Q

what are the 3 functions in clearing

A

removing dehydrating agent

act as solvent for paraffin wax for infiltration

raises refractive index of tissue for high power magnification

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6
Q

what is infiltration

A

impregnated tissue with paaffin wax

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7
Q

T/F dehydrating solutions are hydrophobic

A

F - theyre water loving (hydrophillic) to draw out moisture out of the tissue

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8
Q

what are common dehydrating agents

A

ethyl alcohol

isopropyl alcohol

methyl alcohol

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9
Q

properties of ethyl alcohol

A

routine use - fast acting and non toxic BUT shrinks and hardens tissue

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10
Q

properties of IPA

A

good with water but not salt solutions = tissues NEED to be WASHED

non toxic and does NOT harden or shrinks

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11
Q

properties of methyl alcohol

A

same to ethanol but TOXIC

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12
Q

why is gradual dehydration important

A

cells may be damaged if jumping to high concentrated alcohol

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13
Q

what do all clearing agents have in common

A

they are soluble in both dehydrating and paraffin

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14
Q

what is the refractive index of clearing agents

A

1.40-1.51

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15
Q

3 key steps for processing to ensure proper tissues

A
  1. tissues need to be fully fixed before dehydrating
  2. <5mm thin for good penetration of reagents
  3. processing is separated to size and type of tissue schedules (small biopsy = cores and GI)
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16
Q

what are options for clearing agents

A

xylene
toluene
chloroform
xylene substitute

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17
Q

properties of xylene

A

routine used - fast acting BUT over exposure = harden tissue

flammable and toxic

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18
Q

properties of toluene

A

same as xylene but NO harden

more volatile vapours / flammable than xylene

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19
Q

properties of chloroform

A

used for CNS specimens (brain and eyes)

non flammable but VERY toxic

makes phosgene with oxygen

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20
Q

properties of xylene substitute

A

aliphatic hydrocarbons (butane, petroleum jelly like)

less toxic but needs to be ANHYROUS

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21
Q

what are universal solvents

A

chemicals that can dehtydrate and clear tissue

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22
Q

what are options for universal solvents

A

tetrahydrofuran (THF)
dioxane

cant use either because saftey and over harden

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23
Q

what options for infiltration medias

A

paraffin

DMSO = chemicals enter human skin = DANGER

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24
Q

why is paraffin so good to use

A

non toxic, cheap, easy to use, has different melting points (~56C)

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25
Q

properties of plasticizers

A

make wax harder to help ribboning

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26
Q

properties of beeswax

A

lowers melting point and makes sections sticky

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27
Q

properties of rubber

A

increase elasticity = help ribboning

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28
Q

properties of resin

A

increase melting point for harder wax

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29
Q

what factors affect tissue properties

A

agitation
heat
vacuum/pressure
viscosity

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30
Q

how does agitation affect processing

A

helps solution surround tissue = avoid DEAD ZONES

uses magnetic stir bar

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31
Q

how does heat affect processing

A

speed up but >40C = alter tissues

mostly used in paraffin wax molten infiltration

32
Q

how does vacuum/pressure affect processing

A

force solution into tissues and open pores during infiltration

lower the pressure in retort = clearing agent is replaced by wax

33
Q

how does viscocity affect processing

A

lower viscosity solvents; water, alcohol, xylene work quickly

34
Q

what is a disadvantage of carousel processors

A

exposure to reagent vapours

cant use pressure

35
Q

what processors do we use now

A

enclosed processors = uses retort that fills and empties reagents with vacuum

36
Q

how does a microwave processor work

A

heats tissue from the inside = speeds processing without formalin or xylene

less time in each solution compared to enclosed processor

37
Q

why can small biopsies NOT be processed overnight

A

shrunken and brittle due to prolong exposure to alcohol and hot paraffin

38
Q

during overnight routine where can we find a delay

A

pure alcohol to xylene because clearing needs to be ANHYDROUS

39
Q

aliphatic hydrocarbons clearing pros and cons

A

pro = xylene free

con =
do not clear as fast as xylene (~50% more time needed)

less tolerant to water than xylene

40
Q

what are two xylene free clearing methods

A

aliphatic hydrocarbons

isopropanol and ethanol

41
Q

how does isopropanol and ethanol processing work

A
  1. fixation
  2. ethanol- water dehydration
  3. ethanol - isopropanol dehydration
  4. isopropanol clearing (100%)
  5. high temp wax infiltration (85C to boil off isopropanol)
42
Q

what happens when xylene is contaminated with alcohol

A

miscibility issues with wax

43
Q

when are reagents changed

A

based on:

of cassettes used
types of tissues
visual appearance
daily schedule

44
Q

what instrument measure alcohol quality

A

hydrometer

45
Q

what system is used to change repeating reagents (alcohol, xylene, paraffin)

A

rotation

46
Q

when does a clean cycle occur in the retort

A

at the end of infiltration / before embedding

47
Q

how does a clean/purge cycle work

A

lines are flushed with xylene followed by alcohol

48
Q

what happens when dehydration is incomplete

A

soft and mushy center of tissue during microtomy

water interferes with nuclear staining

49
Q

what happens when tissues are over processed

A

brittle tissues

50
Q

what happens when epithelial tissue when over processed

A

brittle and MICROCHATTER

51
Q

purpose of embedding

A

provides support for microtomy

elasticity and resist the compression during cutting

52
Q

benefits of harder waxs

A

more support for harder tissues and better for thin cutting

53
Q

benefits of softer waxes

A

easier to ribbon = best for serial sections

54
Q

how is the size of mold determined for embedding

A

2-3mm bigger than tissues on all sides

55
Q

what is the most critical step during embedding

A

oritentaion

56
Q

what happens when the oritentation is improper

A

misdiagnosis, missing area of interest, cannot compare tissue layers

57
Q

where does orientation of tissue start

A

grossing bench

58
Q

how should vas deferens, fallopian tubes, appendix, ducts be embedded

A

lumen side down

59
Q

how should skin, intestine, gallbladder be embedded

A

on edge

60
Q

how are curettings embedded

A

clusters but NOT OVERLAPPED

61
Q

how are elongated tissues embedded

A

30 degrees offset ti orevebt cinoressuib

62
Q

how long to hold onto paraffin blocks

A

30 years prior to discard

63
Q

what are acrylic resins (methacrylate = MMA/GMA) used for

A

section undecalcified bone, teeth, or <2um for light microscopy

NOT EM

64
Q

what resins are useful for EM and why

A

epoxy resin - can be cut 0.5-1.0 um

65
Q

why is resin embedding dangerious

A

organic peroxides that need to harden in exothermic reactions (super hot)

66
Q

why are alcohols changed and rotated

A

contaminated with water or formalin = poor clearing and infiltration

67
Q

white coloured xylene leads to what in slide quality

A

poor nuclei staining and water droplets

mushy blocks as wax will not infiltrate

68
Q

how does heat affect processing

A

speed up processing but >45C = cooks tissue

69
Q

what happens when the wax infiltrates on too cold or warm temperatures

A

cold = not penetrate

warm = difficult sections because crumbly

70
Q

what creates white precepitate in the processor

A

isopropy alcohol used as dehydrating agent

71
Q

hazards of ethanol

A

flammable and irritant

72
Q

hazards of xylene

A

flammable, irritant, birth defects, CNS depressant

73
Q

hazards of benzene

A

anemia and leukemia

74
Q

what happens to tissue exposed to ethanol for long time

A

microchatter and crunchy tissue

75
Q

what happens to tissue exposed to hot wax for a long time

A

brittle = sectioning issues

76
Q

what causes incomplete dehydration

A

condensation
non absolute alcohol
wrong schedule

77
Q
A

350 (7 decks)

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