Processing CH 2

Question 1

Processing
- Includes steps POST-fixation:

Answer 1

1. Dehydration
2. Clearing
3. Infiltration

Question 2

Dehydration
◦ Alcohols: usually 1-2% water

Answer 2

◦ Ethyl Alcohol
- Drinking Alcohol
- Reliable, fast, possibly the best
- Can precipitate phosphate salts higher than 70%
- 1000ppm PE

Question 3

Dehydration

Answer 3

◦ Removal of FREE water, not bound

◦ Removal of water
-Non aqueous infiltrating media

Question 4

Dehydration
◦ Isopropanol

Answer 4

• Great substitute for ethyl alcohol, eosin is insoluble
• Never absolute
• Does not shrink tissue as much
• 400ppm PEL

Question 5

Dehydration
◦ Methyl Alcohol

Answer 5

• Rarely used
• Fixes blood smears
• Poisonous
• 200ppm PE

Question 6

Dehydration
- Butyl Alcohol

Answer 6

• Mainly used in plant
• SLOW
• Less shrinking than EtOH
• 100ppm PEL

Question 7

Universal Solvents can…

Answer 7

• Can perform both Dehydration and Clearing steps in processing
• Not used on delicate tissue
• Usually very toxic

Question 7

Dehydration
◦ Acetone

Answer 7

• VERY Rapid
• Absorbs atmospheric water
• Flammable
• 1000ppm PEL, per OSHA

Question 8

Universal Solvents
◦ Dioxane

Answer 8

• Can be used long term
• Less shrinkage than EtOH
• Acts fast
• Very toxic, 100ppm PEL

Question 9

Universal Solvents
◦ Tertiary Butanol

Answer 9

• Solidifies at room temp
• 50/50 Tertiary butanol and paraffin required
• 100ppm PEL

Question 10

Universal Solvents
◦ Tetrahydrofuran

Answer 10

• Less toxic than Dioxane
• Fast acting
• Considered the best Universal Solvent
• Useful in reprocessing tissue
• 200ppm PEL

Question 11

Processing
- Clearing Step

Answer 11

• High refractive index
• Renders tissue transparent
• Miscible with alcohols and paraffin
• Incomplete dehydration causes poor, uneven staining due to contamination in Clearing step
• Prolonged exposure creates brittle tissue

Question 12

Clearing Agents
- Xylene

Answer 12

• Most widely used
• Can over harden tissue
• NOT miscible with water
• 100ppm PEL

Question 13

Clearing Agent
◦ Toluene

Answer 13

• Less over hardening affect
• Believed to be the best hydrocarbon
• 50ppm PEL

Question 14

Clearing Agent
◦ Benzene

Answer 14

• Fast acting, less hardening
• Eliminates second paraffin step
• 10ppm PEL
• Carcinogen affecting blood and marrow

Question 15

Clearing Agent
◦ Acetone

Answer 15

• Low boiling point (58C)
• Extreme shrinkage

Question 16

Clearing Agent
◦ Chloroform

Answer 16

• Slow
• Absorbs atmospheric moisture

Question 17

Clearing Agent
◦ Limonene

Answer 17

• Contaminate paraffin
• Biodegradable

Question 18

Clearing Agent
◦ Essential Oils

Answer 18

• Should be removed by aromatic hydrocarbons
• Tends to have strong odor
• Clove, cedarwood, sandalwood

Question 19

Clearing Agent
◦ Aliphatic hydrocarbons (alkanes)

Answer 19

• Propane, butane, petroleum jelly, paraffin
• Incompatible with certain mounting media
• Not miscible with water

Question 20

Processing
- Infiltration Step

Answer 20

• Supporting medium
• Holds cells in structure during microtomy

Question 21

Infiltration
◦ Paraffin

Answer 21

• Most common, readily available and quick
• Considerations include melting point, IHC
• Overheated paraffin causes over hardening of tissue
• Infiltration increased with vacuum

Question 22

Infiltration
◦ Water soluble waxes (Carbowax)

Answer 22

• Infiltrate from aqueous fixatives
• Fat not dissolved
• Blocks must be sealed
• Tissues “float out”

Question 23

Infiltration
◦ Celloidin

Answer 23

• Nitrocellulose compound (Parlodion)
• Graded celloidin infiltration
• Great for CNS tissue
• Rare, very hazardous

Question 24

Infiltration
◦ Plastics
1. Glycol Methacrylate
2. Epoxy Resins

Answer 24

1. Glycol Methacrylate
   - Acrylic resin, miscible with water
   - Undecalcified bone
   - Glass knives, THIN sections
2. Epoxy Resins
   - Propylene oxide “clearing” agent
   - Araldite, Epon, Spurr
   - Cause dermatitis
   - Used for Electron Microscopy

Question 25

Infiltration
◦ Agar and Gelatin

Answer 25

• Used for frozen sections
• 30% Sucrose
• Most common for frozen sections

Question 26

Processing Protocols
- Biopsy specimens
- Fatty specimens

Answer 26

◦ Biopsy specimens
- Smaller tissues
- Shorter cycles

◦ Fatty specimens
- Require longer processing cycles

Question 27

Processing
- Microwave

Answer 27

• Must be well fixed first
• Three reagents
  1. Ethyl Alcohol
  2. Isopropanol Alcohol
  3. Paraffin
• Whats missing? clearing step.

Question 28

Special Techniques
◦ Decalcification
1. Acids
2. Chelating Agents

Answer 28

1. Acids
   - Long periods can diminish nuclear staining
2. Simple Acids dissolve calcium salts
   - Ion-Exchange uses formic acid and ammonium salt***
   - Electrolytic method bone-anode, to cathode (electrolysis)
3. Chelating Agents
   - EDTA binds calcium ions
   - Slow

Question 29

Processing
- Troubleshooting
1.) Precipitation in Tissue processor
2.) Overdehydrating

Answer 29

1.) Precipitation in Tissue processor
- Caused by zinc or phosphate buffered formalin with alcohol over 70%: Fix by starting with 60-65% alcohol
- Zinc formalin pH above 7 precipitates: Fix by
keeping pH below 7
- 5-20% acetic acid can remove the precipitaion

2.) Overdehydrating
- Causes Microchatter! Fix by decreasing time in dehydrants, and having separate protocol for Biopsies

Question 30

Processing
- Troubleshooting
1.) Poor Processing

2.) Sponge Artifact

Answer 30

1.) Poor Processing
- Poor nuclear staining caused by water in tissue: Fix by ensuring no condensation,
absolute alcohol has no water in it.
- Smudgy nuclei can be caused by heat: Fix by only allowing heat on paraffin steps

2.) Sponge Artifact
- Cross hatch artifact when tissue placed between dry sponges: Fix by presoaking the
sponges with fixative