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Tissue processing Flashcards

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1
Q

what are the steps in tissue processing

A

dehydration

clearing

infiltration

embedding

  • fixation should be complete before processing begins. if not add fixation time to your processing schedule
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2
Q

dehydration

  • main use
  • how we prepare it
  • also used for
A

removes water from tissue
-hydrophilic reagents attract water from tissue

gradual increase in concentration used
- known as graded alcohols

dehydrates are also used for tissue sections as part of staining process

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3
Q

what is the most commonly used for dehydration

A

Ethanol ( ethyl alcohol )

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4
Q

what happens if you don’t use graded alcohol ( dehydrate all in one step )

A

tissue distorted

especially in delicate tissue

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5
Q

closed processors should start with processor with no greater than

A

approx 60% to avoid precipitated buffered formalin in the lines or even worse tissues

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6
Q

when can dehydration be accomplished in one step

A

microwave dehydration can be done in one step bc molecules are in motion and therefore rapid diffusion throughout specimen is possible

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7
Q

when may dehydrates be used beside for processing

A

dehydrates are also used fro tissue section sa part of staining process

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8
Q

Ethyl alcohol advantages

A 
non-toxic(relatively) 
miscible 
little shrinkage with graded alcohols 
may be used for eyes or embryos if graded 
fast acting 
reliable
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9
Q

ethyl alcohol disadvantages

A 
expensive 
long exposure can cause shrinkage and hardening 
record keeping ( every mL)
theft prevention 
extracts some dyes from tissue
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10
Q

safety - alcohols

A
  • flammable( usage, storage &disposal )

ethyl is intoxicating
methyl & isopropanol are poison

violet reaction with oxidizing agents including silver nitrate

> 24% needs waste management
24 or less discard does sink

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11
Q

isopropanol advantages

A

penetrates as well as ethanol

good substitute for ethanol

less shrinkage & hardening than ethanol

less expensive

no government restrictions

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12
Q

isopropanol disadvantages

A

not suitable for stain preparations such as eosin (eosin is insoluble in it)

cannot be used for celloidin

nitrocellulose is insoluble in it

mildly irritating

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13
Q

acetone advantages

A

rapid dehydration
less expensive
doesn’t remove dyes from stained sections

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14
Q

acetone disadvantages

A

requires 20X that of tissue

flammable- low flashpoint

excessive shrinkage

best graded with with xylene before paraffin

volatile

melts pastic coverslips ( not recommended for automatic cover slippers)

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15
Q

methyl alcohol advantages and disadvantages

A

works well

may cause blindness or death

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16
Q

denatured alcohol aka methylated spirit

A

ethanol with 1% methanol added

works well

not subject to alcohol taxations

security measures not needed

no record keeping

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17
Q

which alcohol is the most important

A

the last alcohol is the most important
- water content should be <2%

verify alcohol is anhydrous by adding alcohol to xylene
- water turns white in xylene (cloudiness)

specific gravity may be used to determine water content

desiccant may be added (calcium or copper sulfate ) as an indicator of water
- BUT not suitable for pump action processors

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18
Q

added to dehydrates

A

softeners

  • phenol
  • mollifex( alcohol/glycerin mix)
  • dish detergent in DI water

dye

  • eosin ( NOT with isopropanol) or alcian blue
  • may tiny tissue more visible and easier to embed
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19
Q

universal solvents

A

same reagent achieves both dehydration and clearing

advantage is speed

disadvantage is extreme toxicity, cost, and unpleasant aroma

ex. dioxane, tertiary butanol or tetrahydrofuran

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20
Q

what two processing reagents must a universal solvent be miscible with ?

A

fixative ( usually aqueous)

paraffin

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21
Q

clearing meaning and must be miscible with

A

removing alcohol from tissue and replacing it with clearing agent
- dealcoholization

must be miscible: dehydrating alcohols, infiltrating media such as paraffin, mounting media such as permount (slides)

many change the refractive index in tissue

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22
Q

incomplete dehydration or clearing

A

prevents paraffin from infiltrating and= mushy blocks

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23
Q

too long in clearing agent

A

makes tissue too brittle

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24
Q

criteria for choosing clearing agents

A

speedy removal of dehydrant

ease of removal by molten wax

minimal tissue damage

flammability

toxicity

cost

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25
Xylene & substitution protocol
one of the best clearing agents & is used widely changing to a new agent would require splitting specimens , processing with both clearing agents and comparing the results with a large number of antibodies
26
xylene advantages
clears quickly makes tissue transparent, endpoint obvious will not dissolve cellodin does not affect aniline dyes turns cloudy in presence of water
27
xylene disadvantages
toxic prolonged treatment over hardened tissue hazardous waste - removed by licensed waste disposal $$ - or may be distilled (recycled)
28
xylene safety
xylene is a neurotoxin requires ventilation can cause CNS damage flammable gloves automatic cover slipping reduces exposure
29
toluene advantages
doesn't harden like xylene ( great for CNS aka nerve & brain ) best aromatic hydrocarbon clears quickly makes tissue transparent clear endpoint more tolerant to water than xylene
30
toluene disadvantages
TOXICITY addictive dermatitis, headaches & dizziness long term exposure harmful to organs
31
benzene advantages
fast acting makes tissue transparent clear endpoint hardens less than xylene quick evaporation from paraffin less shrinkage than xylene & toluene
32
Benzene disadvantages
proven carcinogen ! very volatile
33
chloroform advantages
less hardening than xylene not flammable better for uterus, muscles & tendon ( hard tissue ) penetrates well works for celloidin
34
chloroform disadvantages
absorbs water from air slow volatile ***may damage vacuums pump membranes disposal $$ doesn't change refractive index violent reaction with acetone
35
acetone advantages
boils off at 58 degrees, less wax contamination and changing dehydrates and clears
36
acetone disadvantages
volatile( great ventilation needed) flammable ***shrinkage ( best graded with xylene) volume needs 20x that of tissue interfere with some automatic cover slipping
37
essential oils advantages
clears quickly less hardening than other s little evaporation
38
essential oils disadvantages
expensive must be removed with xylene smells may be nauseating oil residue makes cutting difficult
39
which essential oil if the best known
cedar wood oil
40
xylene substitutes ( first wave Limonene)
"goo gone" hardens less than xylene citrus odor more changing of paraffin$$$ greasy waste disposal may cause problems with mounting slides
41
xylene substitute ( second wave - aliphatic hydrocarbons- alkanes) advantages
low toxicity combustible gentle on tissue
42
xylene substitutes( second wave ) disadvantages
intolerant to wattle ( humidity ) not compatible with all mounting media three station on processor ( xylene need just 2) deparaffinizing section needs 3 sections instead of 2
43
infiltration
supporting media holds cells & intracellular stricture in proper relationship paraffin ( best choice ) water soluble wax celloidin plastics epoxy
44
paraffin is most popular
cheap easily handled relatively easy section production shorter processing time
45
paraffin additives
beeswax - stickiness rubber- reduces brittleness other waxes - smooth texture, smaller crystals plastic - increases hardness & support
46
commercially supplied paraffin
increased melting temp = hard wax - thinner sections ( ribboning is difficult ) decreased temp= softer wax - better ribboning - harder to get thin section & less tissue support * most labs use lower melting point ( 55-58) in an effort to preserve antigens for immunochemistry temps must be monitored & adjusted when necessary
47
paraffin on the tissue processor
tissue should be in paraffin for a short time to avoid shrinkage & hardening ( can cause more damage than overheating the embedder, oven or floatation bath ) wax should be kept at 2-4 degrees above melting point change frequently to avoid clearing agent contamination - rotation of 4 baths - odor is a sign of contamination
48
paraffin under pressure
vacuum speeds up process removes residual air *may damage tiny biopsies pressure shouldn't exceed 500mm Hg
49
tissue processors
50
tissue processors most popular
closed/fluid transfer - pumps each solution in and out - allows temp & pressure motion for each station
51
microwave processors
becoming less popular - complete fixation before processing - reagents manually moved * - isopropanol used as "intermediary"(can dehydrate & clear ) - wax is non-polar, & invisible to microwave radiation - allows temp & pressure option for each station
52
tissue processing by hand
extremely urgent specimens possible prion CJD infection special circumstances
53
new automated instruments may achieve full processing in less than one hour
has proprietary solutions, on board microwave, &thinner sections sakura expresss
54
processing protocol
fluid transfer -set allows embedding first thing in the morning microwave -possibly at intervals during the day ( 2-3) runs continuous throughput instruments - new batch every 40 mins
55
what is the weakness of having all cassettes in fluid waiting to be embedded
tissues are all processed the same regardless if size or type tiny biopsies may require shorter time in reagent
56
operators can schedule processors
time, pressure & heat in each station vacuum is usually applied to at least one station
57
which processors can be used safely at 37 degrees
all except paraffin
58
processing station and reagents
1. 10% neutral buffered formalin ( stations 1-10, 35 degrees) 2. 70% alcohol 3. 80% alcohol 4. 95% alcohol 5. 100% alcohol 6. 100% alcohol 7. 100 % alcohol 8. clearite 3 9. clearite 3 10. clearite 3 11. paraffin ( 60 degrees) 12. paraffin (60 degrees) 13. paraffin (60 degrees) 14. paraffin (60 degrees)
59
when do you changed reagents
depends on - age of regents - # of blocks run - any obvious contamination change in rotation not a full change
60
why does alcohol on tissue processor require frequent changing ?
alcohol absorbs water from the tissues 7 humidity from the air
61
sources of contamination ( carryover)
alcohol - water from tissues in aqueous vocative - water from air clearing agent - alcohol - water if the last alcohol contains more than 2% * paraffin - clearing agent - alcohol if clearing agent was not changed *
62
other infiltration media
carbowax - water soluble in wax celliodin - nitrocellulose compound plastics - glycol methacrylate GMA epoxy resin
63
carbowax | advantages
fats can be demonstrated * some enzymes can be demonstrated no dehydration or clearing
64
carbowax disadvantages
cannot be floated on water ; instead use *** - potassium dichromate & gelatin in water - diethylene glycol, gelatin, formaldehyde, carbowax must be store in desiccated plastic bags ** will nit infiltrate if tissue is extremely fatty
65
celloidin advantage
no heat used in processing ** - allows very delicate tissues to be processed without distortion - favoured for brain research **
66
celloidin disadvantages
process ,may take weeks to months*** - gradual increases in concentration 2-14% blocks must be stored in 80% alcohol *** will tolerate no water
67
plastics advantages
embedding of calcified bone *** - cut with a glass knife great cellular detail -kidney, bone marrow & lymph h node
68
plastics disadvantages
difficult microtomy staining difficult dangerous chemicals - use under hood
69
epoxy resin
used for electron microscopy *** - diamond knife 60-90nm thick sections avoid akin contact
70
agar & gelatin
used for friable tissue block may be then embedded in paraffin ( double embedding )
71
30% sucrose
serves as a cryoprotectant formalin fix, infiltrate with sucrose solution dry slides well tissue will sink when infiltrated
72
OTC
water based solutions used to prepare tissue for frozen sectioning leaves no discolouration of slide or tissue allows cutting of small bone fragments - this can be an advantage for diagnosis of possible bone tumor( decal& other infiltrates may damage making diagnosis more difficult )
73
processor precipitation
too high alcohol concentration following NBF zinc formaalin must have pH below 7.0
74
over dehydration
evidence - microchatter in tissue **** process biopsy & large specimens separately decrease dehydration time
75
poor processing
evidence - poor to absent nuclear staining water in tissue evident when placed in clearing agent
76
poor processing solutions
change reagents more frequently change fume hood control as recommended may be condensation in processor may be mechanical problems use heat for paraffin only
77
improper processing consequences
makes microtomy difficult decreases the quality of the sides produced
78
failure to follow SOP ( not defendable )
forgotten to load a solution loaded solutions in wrong sequence solutions not changed often enough programmed incorrectly
79
when errors occur
acknowledge attempt remediation file incident report how could this be prevented in the future
80
open processors
seldom used have unique problems
81
sponge artifact
sponges or lens paper may be used to secure tiny specimens in cassette throughout processing sponges should be presoaked in fixative use biopsy paper or lens paper
82
specimen thickness
4mm or less in cassette garbage in garbage out - no processing method works if sections are too thick for solution to penetrate

Histology (15 decks)

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