Processing Flashcards
what are the stages to tissue processing
fixation
dehydration
clearing
infiltration
what is fixation
formalin fixation of biopsies
what QC is in fixation
monitor and documentation of fixation time
what is dehydration
removing all unbound water and fixative from tissue
what are the 3 functions in clearing
removing dehydrating agent
act as solvent for paraffin wax for infiltration
raises refractive index of tissue for high power magnification
what is infiltration
impregnated tissue with paaffin wax
T/F dehydrating solutions are hydrophobic
F - theyre water loving (hydrophillic) to draw out moisture out of the tissue
what are common dehydrating agents
ethyl alcohol
isopropyl alcohol
methyl alcohol
properties of ethyl alcohol
routine use - fast acting and non toxic BUT shrinks and hardens tissue
properties of IPA
good with water but not salt solutions = tissues NEED to be WASHED
non toxic and does NOT harden or shrinks
properties of methyl alcohol
same to ethanol but TOXIC
why is gradual dehydration important
cells may be damaged if jumping to high concentrated alcohol
what do all clearing agents have in common
they are soluble in both dehydrating and paraffin
what is the refractive index of clearing agents
1.40-1.51
3 key steps for processing to ensure proper tissues
- tissues need to be fully fixed before dehydrating
- <5mm thin for good penetration of reagents
- processing is separated to size and type of tissue schedules (small biopsy = cores and GI)
what are options for clearing agents
xylene
toluene
chloroform
xylene substitute
properties of xylene
routine used - fast acting BUT over exposure = harden tissue
flammable and toxic
properties of toluene
same as xylene but NO harden
more volatile vapours / flammable than xylene
properties of chloroform
used for CNS specimens (brain and eyes)
non flammable but VERY toxic
makes phosgene with oxygen
properties of xylene substitute
aliphatic hydrocarbons (butane, petroleum jelly like)
less toxic but needs to be ANHYROUS
what are universal solvents
chemicals that can dehtydrate and clear tissue
what are options for universal solvents
tetrahydrofuran (THF)
dioxane
cant use either because saftey and over harden
what options for infiltration medias
paraffin
DMSO = chemicals enter human skin = DANGER
why is paraffin so good to use
non toxic, cheap, easy to use, has different melting points (~56C)
properties of plasticizers
make wax harder to help ribboning
properties of beeswax
lowers melting point and makes sections sticky
properties of rubber
increase elasticity = help ribboning
properties of resin
increase melting point for harder wax
what factors affect tissue properties
agitation
heat
vacuum/pressure
viscosity
how does agitation affect processing
helps solution surround tissue = avoid DEAD ZONES
uses magnetic stir bar
how does heat affect processing
speed up but >40C = alter tissues
mostly used in paraffin wax molten infiltration
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
how does viscocity affect processing
lower viscosity solvents; water, alcohol, xylene work quickly
what is a disadvantage of carousel processors
exposure to reagent vapours
cant use pressure
what processors do we use now
enclosed processors = uses retort that fills and empties reagents with vacuum
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
why can small biopsies NOT be processed overnight
shrunken and brittle due to prolong exposure to alcohol and hot paraffin
during overnight routine where can we find a delay
pure alcohol to xylene because clearing needs to be ANHYDROUS
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
what are two xylene free clearing methods
aliphatic hydrocarbons
isopropanol and ethanol
how does isopropanol and ethanol processing work
- fixation
- ethanol- water dehydration
- ethanol - isopropanol dehydration
- isopropanol clearing (100%)
- high temp wax infiltration (85C to boil off isopropanol)
what happens when xylene is contaminated with alcohol
miscibility issues with wax
when are reagents changed
based on:
of cassettes used
types of tissues
visual appearance
daily schedule
what instrument measure alcohol quality
hydrometer
what system is used to change repeating reagents (alcohol, xylene, paraffin)
rotation
when does a clean cycle occur in the retort
at the end of infiltration / before embedding
how does a clean/purge cycle work
lines are flushed with xylene followed by alcohol
what happens when dehydration is incomplete
soft and mushy center of tissue during microtomy
water interferes with nuclear staining
what happens when tissues are over processed
brittle tissues
what happens when epithelial tissue when over processed
brittle and MICROCHATTER
purpose of embedding
provides support for microtomy
elasticity and resist the compression during cutting
benefits of harder waxs
more support for harder tissues and better for thin cutting
benefits of softer waxes
easier to ribbon = best for serial sections
how is the size of mold determined for embedding
2-3mm bigger than tissues on all sides
what is the most critical step during embedding
oritentaion
what happens when the oritentation is improper
misdiagnosis, missing area of interest, cannot compare tissue layers
where does orientation of tissue start
grossing bench
how should vas deferens, fallopian tubes, appendix, ducts be embedded
lumen side down
how should skin, intestine, gallbladder be embedded
on edge
how are curettings embedded
clusters but NOT OVERLAPPED
how are elongated tissues embedded
30 degrees offset ti orevebt cinoressuib
how long to hold onto paraffin blocks
30 years prior to discard
what are acrylic resins (methacrylate = MMA/GMA) used for
section undecalcified bone, teeth, or <2um for light microscopy
NOT EM
what resins are useful for EM and why
epoxy resin - can be cut 0.5-1.0 um
why is resin embedding dangerious
organic peroxides that need to harden in exothermic reactions (super hot)
why are alcohols changed and rotated
contaminated with water or formalin = poor clearing and infiltration
white coloured xylene leads to what in slide quality
poor nuclei staining and water droplets
mushy blocks as wax will not infiltrate
how does heat affect processing
speed up processing but >45C = cooks tissue
what happens when the wax infiltrates on too cold or warm temperatures
cold = not penetrate
warm = difficult sections because crumbly
what creates white precepitate in the processor
isopropy alcohol used as dehydrating agent
hazards of ethanol
flammable and irritant
hazards of xylene
flammable, irritant, birth defects, CNS depressant
hazards of benzene
anemia and leukemia
what happens to tissue exposed to ethanol for long time
microchatter and crunchy tissue
what happens to tissue exposed to hot wax for a long time
brittle = sectioning issues
what causes incomplete dehydration
condensation
non absolute alcohol
wrong schedule
