350 - Topic 6 (Tissue Processing & Embedding) Flashcards
Four distinct stages in tissue processing:
- fixation: fully fixed before further processing
- dehydration: remove all unbound water and fixative from tissue
- clearing: removes dehydrating agent and acts as solvent for paraffin wax; also raises refractive index of tissue closer to that of glass = high power magnification
- infiltration: support medium; paraffin wax = allows cutting of thin sections
T or F. Dehydrating solutions are hydrophobic
F! hydrophilic! allows them to draw out water form the tissue and into the solution
solutions frequent exchanged with increasingly concentrated solutions which will dilute any remaining water
ethyl alc as dehydrating agent
- routinely used
- infinitely soluble with water
- fast-acting
- non-toxic
- may cause hardening and shrinkage
isopropyl alc as dehydrating agent
- miscible in water but IMMISCIBLE in salt solutions = tissue must be washed following fixation
- isopropyl can be mixed w paraffin so clearing agent not used/required
- not toxic
- does not harden/shrink tissue
methyl alcohol as dehydrating agent
- similar to ethanol (ethyl alc)
- more hazardous/toxic
What happens if a large concentration gradient exists between water and alcohol during dehydration?
cells may be distorted or damaged by flow of solutions
tissue should be subjected to 50-70% alc immediately after fixation, followed by increasingly concentrated solutions = 80%, 95%, then absolute alc
what do clearing agents have in common?
they are soluble in both dehydrants and paraffin
once tissue is dehydrated, tissue is filled with alcohol which does not dissolve in wax, so what does clearing agent do?
acts as a link between these steps = dissolves any remaining alcohol
leaves tissue receptive to non-polar substances = molten wax
T or F. Clearing agents have a high refractive index
T! 1.4-1.51
helps make tissue appear transparent (1.51 to 1.62)
xylene
routinely used
- fast-acting
- miscible with most solvents as well as paraffin
- over-exposure will harden tissue
- flammable, moderately toxic
toulene
- similar to xylene; unlikely to harden tissues
- more volatile than xylene (more vapours)
chloroform
- used for CNS that may become brittle when cleared w xylene; esp. brain and eyes
- non-flammable
- highly toxic
- in presence of oxygen = phosgene = exceptionally hazardous; poisonous gas
Xylene substitute
- short chain aliphatic hydrocarbons; like butane and petroleum jelly
- less toxic and work almost as well as xylene
- intolerant of water so final alcohol must be completely anhydrous
universal solvents
chemicals capable of dehydrating AND clearing tissue
two most commonly used = tetrahydrofuran (THF) and dioxane
neither recommended due to safety concerns and tendency to over-harden tissue
lower melting points =
softer wax
higher melting points =
harder waxes
wax additives and their purpose:
- plasticizers: used to make wax harder and facilitate ribboning
- beeswax: lowers the melting point and makes section ‘sticky’
- rubber: facilitates ribboning by increasing elasticity
- resins: makes wax harder/increase the melting point
factors affecting tissue processing:
agitation
heat
vacuum/pressure
viscosity
how does agitation affect tissue processing?
- ensures solutions surrounding tissue do not become locally saturated
- helps avoid ‘dead zones’ where solutions are not effectively exchanged during processing
- most use magnetic stir bars; some blow air bubbles through solutions; other repeated drain and fill the retort
how does heat affect tissue processing?
- heat will speed up; use w caution
= sustained temps above 40C may alter microscopic appearance of delicate tissue - most recommend no heat at al during F,D,C steps for highest quality and only enough heat to keep paraffin wax molten
how does vacuum/pressure affect tissue processing?
- pressure = force solutions into tissues
- vacuum = open porous structures
- vacuum effective aid during infiltration; lowering pressure in retort = clearing agent more easily replaced by hot wax
how does viscosity affect tissue processing?
- measure of how thick liquid is
- water is standard for low-viscosity
- alcohol and xylene = low-viscosity
- wax = significantly higher
- lower viscosity solvents work more quickly than higher = so timing in each station varied accordingly
how does an enclosed tissue processor work?
- tissue remains stationary in retort
- reagents pumped in and out according to a programmed processing schedule
- reduces tech exposure to reagents and allows use of vacuum/pressure to speed processing/infiltration
microwave processors
- speeds up processing and improve TAT
- heat tissues from the inside = speeds up diffusion of solutions
- processes in minutes
-formalin and xyelene free = safer - EXPENSIVE
xylene-free processing
xylene substitutes
- safer
- aliphatic hydrocarbons dont clear alcohols as quickly so duration of clearing steps increased (up to 50%)
- less tolerant of water contamination than xylene; anhydrous ethanol must be at least 97% pure to be effectively cleared
some labs = mixture of isopropanol and ethanol; no clearing agnet at all
- fixation
- ethanol dehydration (50%)
- continued dehydration/clearing (80% ethanol/20% isopropanol)
- isopropanol clearing (100% isopropanol)
- high temp paraffin infiltration = first wax bath should be held at 85C to effectively eliminate isopropanol
this is used to monitor alcohol quality
hydrometer
clean or purge cycle
before another processing cycle can begin, paraffin must be removed from the retort and lines of instrument
flush lines w xylene (to dissolve wax) and then alcohol to dissolve xylene
alcohol remaining in lines is no issue; can mix w fixative at beginning of next run
xylene and alcohol used for cleaning are not used during processing and have non-rotating or dedicated positions
most common cause of inadequate processing
incomplete dehydration
- microtomy = soft, mushy area at centre of tissue (wax failed to infiltrate) = hole on the finished slide ; staining = interfere with nuclear staining
overprocessed = esp small biopsies; brittle, and easily fragments when sectioning; microchatter is a frequent microscopic finding
embedding purpose
- support medium for microtomy; thin sections
- provides elasticity to resist compression caused by microtomy
hard wax
- higher MPs
- great support for harder tissues
- better suited for cutting thin (2-3 um) sections
soft waxes
- lower MP
- easier to ribbon = ideal for serial sectioning
appropriate mold size
2-3 mm bigger than tissue on all sides to ensure good support
the most critical step in the embedding process
orientation
tissue with lumens
cut and embedded to show a cross-section of lumen
vas deferens
fallopian tube
appendix
arteries
ducts
T or F. cut side down is what will be cut first
T
tissues with layers
should be embedded on edge = all layers demonstrated on each slide
skin
intestine
gallbladder
embedding tissue with soft layers
embed so that tissue is oriented in a way that softest layer will contact blade first = reducing compression
embedding elongated tissue
best to offset tissue slightly (up to 30 degree angle from horizontal)
- important for tougher/harder tissues like bone and uterus to prevent compression-type artifacts
T or F. Paraffin blocks are stored indefintely
T! wet specimens (in formalin) discarded after 8 weeks
T or F. Paraffin blocks are stored indefinitely
T! wet specimens (in formalin) discarded after 8 weeks
blockss retained for at least 30 yrs
T or F. Paraffin blocks are stored indefinitely
T! wet specimens (in formalin) discarded after 8 weeks
blocks retained for at least 30 yrs
these are used to section undecalcified bone, teeth, or to cut thin sections (<2 um) for light microscopy
acrylic resins such as methyl methacrylate (MMA) or glycol methacrylate (GMA)
methacrylates more convenient as the formulation can be varied to make a harder or softer block
T or F. acrylic resins not useful for electron microscopy
T! they are unstable in the electron beam
epoxy resins
used for electron microscopy
also ideal for cutting semi-thin (0.5-1.0 um) ad ultra thin (~800 nm) sections
3 rules to achieve high quality processing results
- must b completely fixed prior to dehydration
- tissues must be thin <5mm to allow adequate penetration
- processing schedules must be tailored to sizes and types of tissue processed
high volume labs = reagent schedule
processor always full
reagents changed on a set schedule
lower volume labs = reagent schedule
reagents should be changed according to how much tissue has been processed
potential causes of incomplete dehydration
- condensation (heat used during processing)
- absolute alcohol is not absolute
- wrong processing schedule
- processor fault (mechanical failure)
potential causes of over-processing
- excessive dehydration
- processing biopsy samples using overnight/routine schedule
- extended contact w paraffin wax
crunchy tissue
over-processing
variation in staining, periphery of specimen lighter than centre
zonal fixation
formaldehyde binds to end terminal AAs as does eosin
formalin fixation reduces eosin binding sites
BUT if incomplete fixation = alcohol finishes fixation so more eosin binding sites = darker eosin in centre
xylene on tissue processor is xanthochromic and turbid
water contamination
turbid is the big thing! clear and yellow = ok!
replace absolute alcohol as well
