Processing Flashcards
What is required (in general) to be able to cut a 3 to 5 um thin section of tissue?
Proper internal (in the tissue) and external support for the tissue.
What are the stages of processing and briefly describe them?
- Dehydration - remove water from tissue so that it can be infiltrated and embedded later, typically done with alcohol.
- Clearing - replace dehydrating fluids with a solution that mixes with paraffin - such as xylene.
- Infiltration - fill tissue with strong internal support to enable cutting - e.g. paraffin.
What happens if the tissues are left too long in the dehydration state?
If left too long - the chemicals will harden the tissue, i.e. denature the proteins, non-additive.
What is the most common dehydrating agent?
Alcohol
What is done to make the dehydrating process more gentle and prevent shrinking of the tissue?
The alcohol (or ethanol) is used on the tissue in a series of steps starting with either 50-60% (or 70%), then 95% and finally 100%.
What % of alcohol (or ethanol) must delicate tissues like brain start out with in the dehydration process?
Delicate tissues like brain must start with 50%.
What do some labs add to the last alcohol step to aid embedding?
Some labs add a bit of eosin dye in the last alcohol step to help find tiny specimens while embedding.
The dye can be washed out with alcohol so that it doesn’t affect the later staining process.
Is there really 100% alcohol? What is the issue for having or maintaining 100% alcohol?
No real 100% alcohol as alcohol absorbs water from the environment. There needs to be a sufficient % of alcohol for the last dehydration step to be effective. In class she suggested if 2-3% water (or 97-98% alcohol that would be sufficient to go to the xylene process. But if more water than that the xylene will not work as it does not like water.
What are the advantages and disadvantages of using ethanol in ascending concentrations?
Advantage: Removes water from tissue gradually.
Disadvantage: Can cause excessive shrinkage and hardening.
What are the hazards associated with alcohol / ethanol?
Moderately toxic, very toxic if methanol is added.
Very flammable.
Small amounts ok down the sink with lots of water (must be <24%) but large amounts need to be collected in a waste container.
What are some substitutes for ethanol?
- Methanol- flammable, poisonous, mostly
used for blood smears - Isopropanol- excellent substitute for ethanol.
Doesn’t harden or shrink tissue as much as
ethanol, flammable, and toxic - Butanol- needs a long time to complete
dehydration. Causes less hardening and
shrinkage than ethanol - Acetone- fast acting, less expensive, causes
excessive shrinking, and extremely
flammable
What happens if there is any water left in the tissue after dehydration?
The clearing agent will not be able to do its job.
What are some signs of incomplete dehydration?
Edges look fine. Center is lighter or whiter and has a soft/mushy consistency. Smells of formalin. Water in cassette or in paraffin bath. When cut, shrinks due to loss of water.
How do you fix incomplete dehydration if not detected until finished processing?
Remove the wax with xylene, then go back into alcohols of decreasing concentration.
What happens if the tissue is excessively dehydrated?
Tissue is very hard and brittle which can cause micro-chatter on tissue.
What gives the stage ‘clearing’ its name?
Called clearing as the tissues become transparent after this stage.
In clearing, the agents used must be _____ with the dehydrating reagents and with the embedding medium.
miscible
Miscible means able to form a solution, i.e. mix well.
What is the most common agent used for clearing?
Xylene
What are the advantages and disadvantages of using xylene for clearing?
Advantages:
- high refractive index, removes
alcohol from tissue, also prepares tissue for
infiltration with wax, relatively inexpensive
Disadvantages:
- hard and brittle if left too long
in solution (soft/mushy if not long enough)
- Toxic - neurotoxin, requires proper ventilation!
- Flammable and volatile
Notes: Melts rubber. Must wear nitrile gloves.
How do you properly dispose of Xylene?
Collected in a waste container. Never pour down the sink. It is heavier than water and does not go down the drain properly - gets caught in the p-trap.
What are some other clearing agents (other than xylene) and their features (i.e. pros/cons)?
- Toluene- doesn’t harden tissue as much
as Xylene, Toluene is flammable and
more volatile than xylene - Benzene- fast reacting, doesn’t over
harden tissue. Carcinogenic. - Chloroform- less brittle penetrates slowly
than xylene, not flammable or
combustible. Extremely toxic. - Essential Oil- expensive and only used
for special projects (would not have on
automatic processor), strong odor - Limonene reagent- tend to harden tissue less than
xylene. Less toxic than Xylene. Strong citrus odor. Some people get headaches from exposure.
Alphatic hydrocarbons – newer substitute, Less toxic
See text for advantages and disadvantages
What typical problems result from incomplete clearing?
Tissue can be: 1. Opaque / cloudy. 2. Soft consistency. 3. Shrinks upon standing. 4. Hard to cut sections. These problems won't be detected until the tissue is finished processing.
How do you correct problems from incomplete clearing?
Remove wax, put into xylene (at least 2 changes - then back into wax (4 changes).
What are universal solvents? Name some examples.
Universal solvents complete dehydration and clearing in one step.
Miscible with water and embedding medium.
Dioxane, Tertiary Butanol and Tetrahydrofuran are examples, highly toxic.
What are the limitations of universal solvents?
Cannot be used on very delicate tissues.
What is infiltration?
Once the tissue has been cleared we must infiltrate
it with a supporting medium. This ensures the components of the tissue stay in place during
microtomy and it allows us to cut thin sections.
What is the most common infiltration/embedding medium?
The most common embedding medium is PARAFFIN
WAX - hydrocarbons produced by cracking of
petroleum (same wax used in embedding).
What are some paraffin additives used to improve microtomy?
- Beeswax – reduces crystal size and
increases stickiness - Rubber – reduces brittleness, increases
stickiness - Other waxes – smaller crystals
- Plastics – increase hardness and support
What are the ranges of melting points of soft versus hard wax?
Melting Point of Paraffin
SOFT WAX: 45ºC - 50ºC
Not the greatest support for hard tissues. Thin sections also become more difficult to obtain but ribboning is easier.
HARD WAX: 50ºC - 60ºC
Paraffin becomes harder and provides great support for hard tissue. Thinner sections can be obtained but ribboning becomes more difficult.
What temperature must the wax be kept at in the processor and embedding stations?
Wax must be kept 2 to 4C above the melting point at the processor and embedding stations.
What is the plastic point?
PLASTIC POINT- lowest temperature at which permanent deformation can occur without fracture. Tends to be a few degrees lower then melting point.
What are the advantages and disadvantages of using paraffin for infiltration?
Advantages:
- non toxic (can dispose in regular garbage after it hardens),
- great for melting and cutting,
- cheap,
- allows ribbons to form,
- can lay out on a water bath to pick up sections on a slide,
- can store block indefinitely,
- leaves no background stain
Disadvantages:
- heat can cause loss of lipids, and if tissues left too long in processor heat can cause hardening and shrinkage of tissue.
- No forgiveness for poor dehydration or clearing (any water will cause poor infiltration).
What are some signs of incomplete infiltration?
“Moist” block - soft
Smells of xylene.
How do you correct incomplete infiltration?
Melt wax block, go through changes of paraffin.
But RRC notes say:
Must remove wax with changes of xylene, then go back through changes of alcohol. Once water is removed go back into xylene followed by paraffin.
Must replace and rotate processor reagents on a set schedule as they become contaminated.
What are some substitutes for paraffin for infiltration?
Water Soluble Waxes – Carbowax
- No dehydration or clearing required
- Fat not dissolved
- Cannot float out ribbons on water bath or put on ice water
Celloidin- Nitrocellulose compound
- Only used in research
- Nitrocellulose is explosive, cannot store blocks, difficult to section
- No clearing required – more details in text
Plastics – Glycol Methacrylate or Epoxy Resins
- Not used for routine specimens
- Very hard block, lots of internal and external support of tissue, can cut extremely thin
Agar & Gelatin
- Used in conjunction with frozen sections – not used often
How is an automatic processor a closed system?
Tissues are never exposed to air.
Reagents not exposed to air.
For fluid exchange tissues remain in retort, solutions are pumped in & out of retort. Many samples fit in the retort at once and vacuum and agitation can be used to help the processing.
What is a typical processor routine?
ROUTINE: 1 HOUR EACH 10% NB FORMALIN – 1 to 2 70% ETHANOL – 1 95% ETHANOL – 2 100%ETHANOL – 3 XYLENE – 2 to 3 PARAFFIN – 3 or 4 Routine run will take 13 to 14 hours.
What is the ‘rapid’ process
RAPID:
reduces time by ½ - good if tissues well fixed and tiny.
What are tasks for general operation that must be done of the processor?
REPLENISHING REAGENTS
DEPENDS UPON USAGE
VIP: every week or routine schedule made dependent on use
“MOVE DOWN” ROTATION
PURGE = CLEANING CYCLE (last 2 reagents on processor)
Run after each run to clean the retort – must remove the wax before you can do another run
XYLENE - removes wax
100% ETHANOL – removes xylene
Replaced frequently as they get contaminated quickly
HOT WATER WASHES
4 hot water bottles replaces first 4 bottles of the processor to perform this task)
ONCE / WEEK or as required
What must be done to maintain quality control on a processor?
Don’t over pack cassettes
Maintain proper reagent schedule, can get contaminated quickly if running often
Monitor alcohol percentage with a hydrometer.
Monitoring heat of wax and topping up
Cleaning retort and processor
What is an hydrometer?
A hydrometer checks the specific gravity of a liquid. There is specific levels for the alcohols you would be using. 100% ethanol has a specific gravity of 0.79.
What are some characteristics of lab model microwaves used for processing?
Lab Model Microwaves:
Tissues well fixed before dehydrating
Heat helps speed up the process
Uses ethyl alcohol, isopropyl and paraffin
Not for large volumes
Quick but can over harden if not done correctly.
Must be vented to proper fume extraction
In a processor on its clean cycle, what is typically used to remove wax? Then what is used to remove what was used to remove the wax?
Xylene is used to remove the wax.
Alcohol is used to remove the xylene.
