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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
properties of plasticizers
make wax harder to help ribboning
26
properties of beeswax
lowers melting point and makes sections sticky
27
properties of rubber
increase elasticity = help ribboning
28
properties of resin
increase melting point for harder wax
29
what factors affect tissue properties
agitation heat vacuum/pressure viscosity
30
how does agitation affect processing
helps solution surround tissue = avoid DEAD ZONES uses magnetic stir bar
31
how does heat affect processing
speed up but >40C = alter tissues mostly used in paraffin wax molten infiltration
32
how does vacuum/pressure affect processing
force solution into tissues and open pores during infiltration lower the pressure in retort = clearing agent is replaced by wax
33
how does viscocity affect processing
lower viscosity solvents; water, alcohol, xylene work quickly
34
what is a disadvantage of carousel processors
exposure to reagent vapours cant use pressure
35
what processors do we use now
enclosed processors = uses retort that fills and empties reagents with vacuum
36
how does a microwave processor work
heats tissue from the inside = speeds processing without formalin or xylene less time in each solution compared to enclosed processor
37
why can small biopsies NOT be processed overnight
shrunken and brittle due to prolong exposure to alcohol and hot paraffin
38
during overnight routine where can we find a delay
pure alcohol to xylene because clearing needs to be ANHYDROUS
39
aliphatic hydrocarbons clearing pros and cons
pro = xylene free con = do not clear as fast as xylene (~50% more time needed) less tolerant to water than xylene
40
what are two xylene free clearing methods
aliphatic hydrocarbons isopropanol and ethanol
41
how does isopropanol and ethanol processing work
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
what happens when xylene is contaminated with alcohol
miscibility issues with wax
43
when are reagents changed
based on: of cassettes used types of tissues visual appearance daily schedule
44
what instrument measure alcohol quality
hydrometer
45
what system is used to change repeating reagents (alcohol, xylene, paraffin)
rotation
46
when does a clean cycle occur in the retort
at the end of infiltration / before embedding
47
how does a clean/purge cycle work
lines are flushed with xylene followed by alcohol
48
what happens when dehydration is incomplete
soft and mushy center of tissue during microtomy water interferes with nuclear staining
49
what happens when tissues are over processed
brittle tissues
50
what happens when epithelial tissue when over processed
brittle and MICROCHATTER
51
purpose of embedding
provides support for microtomy elasticity and resist the compression during cutting
52
benefits of harder waxs
more support for harder tissues and better for thin cutting
53
benefits of softer waxes
easier to ribbon = best for serial sections
54
how is the size of mold determined for embedding
2-3mm bigger than tissues on all sides
55
what is the most critical step during embedding
oritentaion
56
what happens when the oritentation is improper
misdiagnosis, missing area of interest, cannot compare tissue layers
57
where does orientation of tissue start
grossing bench
58
how should vas deferens, fallopian tubes, appendix, ducts be embedded
lumen side down
59
how should skin, intestine, gallbladder be embedded
on edge
60
how are curettings embedded
clusters but NOT OVERLAPPED
61
how are elongated tissues embedded
30 degrees offset ti orevebt cinoressuib
62
how long to hold onto paraffin blocks
30 years prior to discard
63
what are acrylic resins (methacrylate = MMA/GMA) used for
section undecalcified bone, teeth, or <2um for light microscopy NOT EM
64
what resins are useful for EM and why
epoxy resin - can be cut 0.5-1.0 um
65
why is resin embedding dangerious
organic peroxides that need to harden in exothermic reactions (super hot)
66
why are alcohols changed and rotated
contaminated with water or formalin = poor clearing and infiltration
67
white coloured xylene leads to what in slide quality
poor nuclei staining and water droplets mushy blocks as wax will not infiltrate
68
how does heat affect processing
speed up processing but >45C = cooks tissue
69
what happens when the wax infiltrates on too cold or warm temperatures
cold = not penetrate warm = difficult sections because crumbly
70
what creates white precepitate in the processor
isopropy alcohol used as dehydrating agent
71
hazards of ethanol
flammable and irritant
72
hazards of xylene
flammable, irritant, birth defects, CNS depressant
73
hazards of benzene
anemia and leukemia
74
what happens to tissue exposed to ethanol for long time
microchatter and crunchy tissue
75
what happens to tissue exposed to hot wax for a long time
brittle = sectioning issues
76
what causes incomplete dehydration
condensation non absolute alcohol wrong schedule
77

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