Processing

Question 1

What is required (in general) to be able to cut a 3 to 5 um thin section of tissue?

Answer 1

Proper internal (in the tissue) and external support for the tissue.

Question 2

What are the stages of processing and briefly describe them?

Answer 2

1. Dehydration - remove water from tissue so that it can be infiltrated and embedded later, typically done with alcohol.
2. Clearing - replace dehydrating fluids with a solution that mixes with paraffin - such as xylene.
3. Infiltration - fill tissue with strong internal support to enable cutting - e.g. paraffin.

Question 3

What happens if the tissues are left too long in the dehydration state?

Answer 3

If left too long - the chemicals will harden the tissue, i.e. denature the proteins, non-additive.

Question 4

What is the most common dehydrating agent?

Answer 4

Alcohol

Question 5

What is done to make the dehydrating process more gentle and prevent shrinking of the tissue?

Answer 5

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%.

Question 6

What % of alcohol (or ethanol) must delicate tissues like brain start out with in the dehydration process?

Answer 6

Delicate tissues like brain must start with 50%.

Question 7

What do some labs add to the last alcohol step to aid embedding?

Answer 7

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.

Question 8

Is there really 100% alcohol? What is the issue for having or maintaining 100% alcohol?

Answer 8

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.

Question 9

What are the advantages and disadvantages of using ethanol in ascending concentrations?

Answer 9

Advantage: Removes water from tissue gradually.
Disadvantage: Can cause excessive shrinkage and hardening.

Question 10

What are the hazards associated with alcohol / ethanol?

Answer 10

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.

Question 11

What are some substitutes for ethanol?

Answer 11

• 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

Question 12

What happens if there is any water left in the tissue after dehydration?

Answer 12

The clearing agent will not be able to do its job.

Question 13

What are some signs of incomplete dehydration?

Answer 13

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.

Question 14

How do you fix incomplete dehydration if not detected until finished processing?

Answer 14

Remove the wax with xylene, then go back into alcohols of decreasing concentration.

Question 15

What happens if the tissue is excessively dehydrated?

Answer 15

Tissue is very hard and brittle which can cause micro-chatter on tissue.

Question 16

What gives the stage ‘clearing’ its name?

Answer 16

Called clearing as the tissues become transparent after this stage.

Question 17

In clearing, the agents used must be _____ with the dehydrating reagents and with the embedding medium.

Answer 17

miscible

Miscible means able to form a solution, i.e. mix well.

Question 18

What is the most common agent used for clearing?

Answer 18

Xylene

Question 19

What are the advantages and disadvantages of using xylene for clearing?

Answer 19

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.

Question 20

How do you properly dispose of Xylene?

Answer 20

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.

Question 21

What are some other clearing agents (other than xylene) and their features (i.e. pros/cons)?

Answer 21

• 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

Question 22

What typical problems result from incomplete clearing?

Answer 22

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.

Question 23

How do you correct problems from incomplete clearing?

Answer 23

Remove wax, put into xylene (at least 2 changes - then back into wax (4 changes).

Question 24

What are universal solvents? Name some examples.

Answer 24

Universal solvents complete dehydration and clearing in one step.
Miscible with water and embedding medium.
Dioxane, Tertiary Butanol and Tetrahydrofuran are examples, highly toxic.

Question 25

What are the limitations of universal solvents?

Answer 25

Cannot be used on very delicate tissues.

Question 26

What is infiltration?

Answer 26

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.

Question 27

What is the most common infiltration/embedding medium?

Answer 27

The most common embedding medium is PARAFFIN WAX - hydrocarbons produced by cracking of petroleum (same wax used in embedding).

Question 28

What are some paraffin additives used to improve microtomy?

Answer 28

- Beeswax – reduces crystal size and increases stickiness - Rubber – reduces brittleness, increases stickiness - Other waxes – smaller crystals - Plastics – increase hardness and support

Question 29

What are the ranges of melting points of soft versus hard wax?

Answer 29

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.

Question 30

What temperature must the wax be kept at in the processor and embedding stations?

Answer 30

Wax must be kept 2 to 4C above the melting point at the processor and embedding stations.

Question 31

What is the plastic point?

Answer 31

PLASTIC POINT- lowest temperature at which permanent deformation can occur without fracture. Tends to be a few degrees lower then melting point.

Question 32

What are the advantages and disadvantages of using paraffin for infiltration?

Answer 32

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).

Question 33

What are some signs of incomplete infiltration?

Answer 33

"Moist" block - soft | Smells of xylene.

Question 34

How do you correct incomplete infiltration?

Answer 34

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.

Question 35

What are some substitutes for paraffin for infiltration?

Answer 35

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

Question 36

How is an automatic processor a closed system?

Answer 36

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.

Question 37

What is a typical processor routine?

Answer 37

``` 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. ```

Question 38

What is the 'rapid' process

Answer 38

RAPID: | reduces time by ½ - good if tissues well fixed and tiny.

Question 39

What are tasks for general operation that must be done of the processor?

Answer 39

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

Question 40

What must be done to maintain quality control on a processor?

Answer 40

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

Question 41

What is an hydrometer?

Answer 41

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.

Question 42

What are some characteristics of lab model microwaves used for processing?

Answer 42

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

Question 43

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?

Answer 43

Xylene is used to remove the wax. | Alcohol is used to remove the xylene.