Tissue processing Flashcards
what are the steps in tissue processing
dehydration
clearing
infiltration
embedding
- fixation should be complete before processing begins. if not add fixation time to your processing schedule
dehydration
- main use
- how we prepare it
- also used for
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
what is the most commonly used for dehydration
Ethanol ( ethyl alcohol )
what happens if you don’t use graded alcohol ( dehydrate all in one step )
tissue distorted
especially in delicate tissue
closed processors should start with processor with no greater than
approx 60% to avoid precipitated buffered formalin in the lines or even worse tissues
when can dehydration be accomplished in one step
microwave dehydration can be done in one step bc molecules are in motion and therefore rapid diffusion throughout specimen is possible
when may dehydrates be used beside for processing
dehydrates are also used fro tissue section sa part of staining process
Ethyl alcohol advantages
non-toxic(relatively) miscible little shrinkage with graded alcohols may be used for eyes or embryos if graded fast acting reliable
ethyl alcohol disadvantages
expensive long exposure can cause shrinkage and hardening record keeping ( every mL) theft prevention extracts some dyes from tissue
safety - alcohols
- 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
isopropanol advantages
penetrates as well as ethanol
good substitute for ethanol
less shrinkage & hardening than ethanol
less expensive
no government restrictions
isopropanol disadvantages
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
acetone advantages
rapid dehydration
less expensive
doesn’t remove dyes from stained sections
acetone disadvantages
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)
methyl alcohol advantages and disadvantages
works well
may cause blindness or death
denatured alcohol aka methylated spirit
ethanol with 1% methanol added
works well
not subject to alcohol taxations
security measures not needed
no record keeping
which alcohol is the most important
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
added to dehydrates
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
universal solvents
same reagent achieves both dehydration and clearing
advantage is speed
disadvantage is extreme toxicity, cost, and unpleasant aroma
ex. dioxane, tertiary butanol or tetrahydrofuran
what two processing reagents must a universal solvent be miscible with ?
fixative ( usually aqueous)
paraffin
clearing meaning and must be miscible with
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
incomplete dehydration or clearing
prevents paraffin from infiltrating and= mushy blocks
too long in clearing agent
makes tissue too brittle
criteria for choosing clearing agents
speedy removal of dehydrant
ease of removal by molten wax
minimal tissue damage
flammability
toxicity
cost
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
xylene advantages
clears quickly
makes tissue transparent, endpoint obvious
will not dissolve cellodin
does not affect aniline dyes
turns cloudy in presence of water
xylene disadvantages
toxic
prolonged treatment over hardened tissue
hazardous waste
- removed by licensed waste disposal $$
- or may be distilled (recycled)
xylene safety
xylene is a neurotoxin
requires ventilation
can cause CNS damage
flammable
gloves
automatic cover slipping reduces exposure
toluene advantages
doesn’t harden like xylene ( great for CNS aka nerve & brain )
best aromatic hydrocarbon
clears quickly
makes tissue transparent
clear endpoint
more tolerant to water than xylene
toluene disadvantages
TOXICITY
addictive
dermatitis, headaches & dizziness
long term exposure harmful to organs
benzene advantages
fast acting
makes tissue transparent
clear endpoint
hardens less than xylene
quick evaporation from paraffin
less shrinkage than xylene & toluene
Benzene disadvantages
proven carcinogen !
very volatile
chloroform advantages
less hardening than xylene
not flammable
better for uterus, muscles & tendon ( hard tissue )
penetrates well
works for celloidin
chloroform disadvantages
absorbs water from air
slow
volatile
***may damage vacuums pump membranes
disposal $$
doesn’t change refractive index
violent reaction with acetone
acetone advantages
boils off at 58 degrees, less wax contamination and changing
dehydrates and clears
acetone disadvantages
volatile( great ventilation needed)
flammable
***shrinkage ( best graded with xylene)
volume needs 20x that of tissue
interfere with some automatic cover slipping
essential oils advantages
clears quickly
less hardening than other s
little evaporation
essential oils disadvantages
expensive
must be removed with xylene
smells may be nauseating
oil residue makes cutting difficult
which essential oil if the best known
cedar wood oil
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
xylene substitute ( second wave - aliphatic hydrocarbons- alkanes) advantages
low toxicity
combustible
gentle on tissue
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
infiltration
supporting media holds cells & intracellular stricture in proper relationship
paraffin ( best choice )
water soluble wax
celloidin
plastics
epoxy
paraffin is most popular
cheap
easily handled
relatively easy section production
shorter processing time
paraffin additives
beeswax - stickiness
rubber- reduces brittleness
other waxes - smooth texture, smaller crystals
plastic - increases hardness & support
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
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
paraffin under pressure
vacuum speeds up process
removes residual air
*may damage tiny biopsies
pressure shouldn’t exceed 500mm Hg
tissue processors
tissue processors most popular
closed/fluid transfer
- pumps each solution in and out
- allows temp & pressure motion for each station
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
tissue processing by hand
extremely urgent specimens
possible prion CJD infection
special circumstances
new automated instruments may achieve full processing in less than one hour
has proprietary solutions, on board microwave, &thinner sections
sakura expresss
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
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
operators can schedule processors
time, pressure & heat in each station
vacuum is usually applied to at least one station
which processors can be used safely at 37 degrees
all except paraffin
processing station and reagents
- 10% neutral buffered formalin ( stations 1-10, 35 degrees)
- 70% alcohol
- 80% alcohol
- 95% alcohol
- 100% alcohol
- 100% alcohol
- 100 % alcohol
- clearite 3
- clearite 3
- clearite 3
- paraffin ( 60 degrees)
- paraffin (60 degrees)
- paraffin (60 degrees)
- paraffin (60 degrees)
when do you changed reagents
depends on
- age of regents
- # of blocks run
- any obvious contamination
change in rotation not a full change
why does alcohol on tissue processor require frequent changing ?
alcohol absorbs water from the tissues 7 humidity from the air
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 *
other infiltration media
carbowax - water soluble in wax
celliodin - nitrocellulose compound
plastics - glycol methacrylate GMA
epoxy resin
carbowax
advantages
fats can be demonstrated *
some enzymes can be demonstrated
no dehydration or clearing
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
celloidin advantage
no heat used in processing **
- allows very delicate tissues to be processed without distortion
- favoured for brain research **
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
plastics advantages
embedding of calcified bone ***
- cut with a glass knife
great cellular detail
-kidney, bone marrow & lymph h node
plastics disadvantages
difficult microtomy
staining difficult
dangerous chemicals
- use under hood
epoxy resin
used for electron microscopy ***
- diamond knife 60-90nm thick sections
avoid akin contact
agar & gelatin
used for friable tissue
block may be then embedded in paraffin ( double embedding )
30% sucrose
serves as a cryoprotectant
formalin fix, infiltrate with sucrose solution
dry slides well
tissue will sink when infiltrated
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 )
processor precipitation
too high alcohol concentration following NBF
zinc formaalin must have pH below 7.0
over dehydration
evidence - microchatter in tissue **
process biopsy & large specimens separately
decrease dehydration time
poor processing
evidence - poor to absent nuclear staining
water in tissue evident when placed in clearing agent
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
improper processing consequences
makes microtomy difficult
decreases the quality of the sides produced
failure to follow SOP ( not defendable )
forgotten to load a solution
loaded solutions in wrong sequence
solutions not changed often enough
programmed incorrectly
when errors occur
acknowledge
attempt remediation
file incident report
how could this be prevented in the future
open processors
seldom used
have unique problems
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
specimen thickness
4mm or less in cassette
garbage in garbage out - no processing method works if sections are too thick for solution to penetrate
