Processing Flashcards
What are the steps in tissue processing?
- Fixation
- Decalcification (if required)
- Dehydration
- Clearing
- Infiltration
- Solidification
What factors affect processing?
- Agitation - increased solution mvmnt+ increase reagent flow into specimen
- Heat - increase temp = increase rate of penetration and fluid exchange
- Vacuum - removes reagent in exchange for next reagent (removes trapped air and good for dense/fatty tissue)
- Viscosity of reagents - smaller molecules = less flow resistances into tissue = lower viscosity and reagents permeate quicker
Fixation
- stabilizes and hardens tissue with minimal cellular distortion
-inadequate cross linking/coagulation = macro molecular rearrangement during subsequent steps ~ smudgy nuclei, nuclear bubbling etc
Decalcification
- removes mineralization of calcium in tissue (only performed on specimens with a large amount of calcium)
- 20:1 ratio of volume : tissue
What are the factors that determine the decalcifier?
- urgency
- size
- degree of mineralization - how hard is it?
- scope of investigation - can we put it in molecular studies?
Types of decalcifying techniques
- Acid : strong acids (HCl, nitric), weak acids (formic), ion-exchange resin, electrolytic methods
- Chelation : EDTA, preserves DNA/RNA for subsequent molecular studies
- acetic and picric acid will decal microcalcifications during fixation
Dehydration
- pulls free water from tissues
- most infiltrating media is immiscible with water
- if tissue is not fully dehydrated = clearing agent can’t penetrate = infiltrating media cannot penetrate/solidify tissue properly = under-processed tissue
- don’t want to overdehydrate by removing naturally bound h20 = micro chatter
What reagents are used in dehydration?
Alcohols, acetone and universal solvents
How do you select dehydration reagent?
- Agent selection: must be miscible with the reagent the tissue is in previously and following
- Tissue type: delicate tissue should start in lower levels of alcohol (30-50%) and normal in higher levels (70-80%)
- Fixation: non-aqueous fixative tissues should start in 100% (prevents water entering tissue thru osmosis/hypertonicity)
How do you store dehydrated tissue?
Can safely store in 70% alcohol without morphology changes
- anything lower, tissue will swell
-anything higher, tissue will shrink and become brittle
1: Properties of Ethanol
- clear, colorless and flammable
- not safe to consume as reagent due to addition of isopropanol and methanol
- miscible with water and may contain 1-2%
- used in gradations
- can add eosin/phloxine to 100% to dye tissues pink for better visibility in embedding and microtomy
- may cause autoflorescence
- Properties of Methanol
- aka wood alcohol
- evaporates quick, unpleasant odor, toxic
- rarely used as a reagent
- used in hematology and cytology as fixative - blood smears, cyto preps, Diff-quik staining
Poisonous thru skin; ingestion = blood stream = liver= formaldehyde + formic acid = blindness and death
- Properties of isopropanol
- retains moisture ~ 1% water in it
- doesn’t overharden tissue
- good substitute for ethanol for paraffin infiltration (cannot be used for celloidin technique, not miscible with nitrocellulose)
- cannot be used for some staining solutions = eosin
- mild irritation and toxicity if ingested
- Properties of butanol
- not common in lab
- requires longer dehydration time = acts slower
- has pronounced odor
- good for animal and plant work
Alcohol substitutes for dehydration
- Ethylene glycol - cellosolve
- not easily found
- rapid dehydrant
- not used in graded percentages - Propylene glycol ether (doesn’t fix tissue)
- pro-soft
-not used in graded %
- will not remove bound water = easier for microtomy
- will not fix tissue and cannot be used for staining, not in processing
- Properties of Acetone
- Rapid dehydrant
- but can cause excessive shrinking and hardening
- easily removed by clearing agents - Rapid evaporation ( watch volumes closely)
- Less expensive than alcohols
- Volatile
What are universal solvents?
They are reagents that dehydrate and clear (2 in 1)
- cannot be used with delicate tissue
Common reagents are: (all toxic)
A. Dioxane
B. Tertiary butanol
C. Tetrahydrofuran (THF)
Describe dioxane
- faster than ethanol
- less shrinkage than ethanol; leave in tissues for longer periods than ethanol
- if water is left in tissue = more shrinkage
-very toxic, pronounced odor, carcinogen
Describe tertiary butanol
- pronounced odor
- expensive
- may solidify at room temp
Describe tetrahydrofuran
- miscible with many solvents, water and paraffin
- acts rapidly w/o shrinkage or hardening
-least toxic of group but can cause dermatitis and conjunctivitis
What is a clearing agent?
- called that because it made tissue look translucent
-removes alcohol and sets tissue up for infiltration; must be done with proper dehydrant for adequate infiltration - clearing agents remove lipids
- don’t leave tissue in for too long or else microchatter will occur
How do you pick reagents for clearing?
- Speed at which it removes dehydrate
- Ease of inliftration
- Flammability
- Toxicity
- Cost
Need to be disposed of by hauling away
Refractive index
Tissues with different refractive indices (RI) will have light bend and it will appear hits
Tissues with similar RIs will appear clear/translucent
Name the clearing reagents:
- aromatics hydrocarbons : benzene, xylene, toluene
- chloroform
- essential oils
- limonene
- aliphatic hydrocarbons
- universal solvents (dual role of dehydration and clearing)