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Processing

Dehydration, Clearing, & Processors

QuestionAnswer
What is Drierite? Anhydrous calcium sulfate, added to tissue to indicate completeness of dehydration
Hard, brittle tissue in blocks caused by? Excessive dehydration (bound water removed) Excessive clearing time
Soft, mushy tissue in blocks caused by? Incomplete dehydration Inadequate clearing - causes inadequate infiltration
2 types of dehydration reagents? 1) hydrophilic reagents- attract water from tissue 2) reagents that dehydrate by repeated dilution of aqueous fluids
Test if absolute alcohol's water content >2% Add few mL alcohol to few mL toluene or xylene, persistent turbidity means >2% water.
Chemicals used to color tissue to aid in ID at embedding? Eosin and Drierite
Ethanol Best dehydrant, fast, toxic, regulated, PEL 1000 ppm
Denatured alcohol Ethanol w/ methanol or isopropanol, no regulation
Methanol Rarely used alone for dehydration, poisonous, PEL 200 ppm
Isopropanol Less shrinkage & hardening, flammable, used in microwave processing, PEL 400 ppm
What is a good substitute for ethanol in processing? Isopropanol
Butanol Slow, less shrinkage & hardening, also good for plants, PEL 100 ppm
Acetone Fast, shrinks, cheap, absorbs atmospheric water, volatile; evaporates from paraffin
Universal solvents Both dehydrate & clear; dioxane, tertiary butanol, tetrahydrofuran
Dioxane Fast, less shrinkage, v toxic, PEL 100 ppm
Tertiary butanol Expensive, solid @ room temp; After use : 50-50 tertiary butanol/paraffin for first infiltration, PEL 100 ppm
Tetrahydrofuran Best universal solvent, fast, less shrinking/hardening, even for delicate tissue, PEL 200-250 ppm
Good universal solvent for reprocessing tissue? Tetrahydrofuran
Clearing agent must be miscible in? Both dehydrant & infiltration medium
Clearing agent Dealcoholization agents, remove dehydrant so tissue receptive to infiltration medium
Most widely used clearing agent? Xylene
Aromatic hydrocarbon clearing reagents cause tissue to? Become transparent if adequate dehydration & clearing
Xylene Hardens over time, CNS damage, defatting agent, flammable, cloudy if water
Toluene Less hardening, flammable, volatile, best aromatic hydrocarbon clearing agent
Aromatic hydrocarbon clearing agents? Xylene, benzene, toluene
Benzene Fast, less hardening (except mm. tendon, uterus), evaporates from paraffin, volatile, toxic, carcinogen
Chloroform Slow, less brittle tissue, good for mm. tendon, uterus, absorbs atmospheric moisture, volatile, carcinogen, not combustible/flammable
Clearing agent that does not make tissue transparent? Chloroform
Tissue-coloring chemical that can be used in closed processors? Eosin
Tissue-coloring chemical that can't be used in closed processors? Drierite
Alcohol that eosin & Drierite can't be used with? Isopropanol
Cedarwood oil Clears tissue dehydrated in 95%, less damaging, store tissue indefinitely, must remove w/ xylene before infiltration, clears celloidin
Limonene reagents Less hardening, paraffin contamination, less toxic than xylene, not as good, greasy - staining section loss, sensitizer
Aliphatic hydrocarbons Alkanes; less reactive/toxic, NO water, comparable to xylene, remove fat, can't use w/ xylene/toluence mounting media, most are proprietary
What problem may occur when phosphate-buffered formaldehyde solutions are used for fixation? Phosphate salts may precipitate in tissue if concentration initial processing alcohol >70%
Isopropanol is not a good substitute for ethanol in? Staining & celloidin technique b/c many stains & nitrocellulose insoluble in
What is a good substitute for ethanol in staining? Tertiary butanol
Essential oils Clearing agents, must remove w/ aromatic hydrocarbon before infiltration, expensive, volatile; clove, cedarwood, sandalwood
What chemical can be used to indicate presence of water in alcohols? Copper sulfate
Dehydration of very delicate tissues may begin in? 30% alcohol
Biopsy processing protocol example 70% 95% x2 100% x2 Xylene x2 paraffin x3 15 minutes each
Overnight open processor protocol example Alcoholic formalin - 3 hr Alcoholic formalin x2 - 1 hr ea 95% x2 - 1 hr ea 100% x2 - 1.5 hr ea Xylene x2 - 1 hr ea Paraffin - 30 min Paraffin - 1.5 hr Paraffin - 1 hr
Overnight closed processor protocol example 10% formalin - 2 hr Alcoholic formalin - 1.5 hr Alcoholic formalin - 1 hr 95% - 1 hr 95% - 45 min 100% - 45 min 100% - 1 hr Xylene x2 - 1 hr ea Paraffin - 30 min Paraffin - 1 hr Paraffin - 1.5 hr
Microwave processing best for what type of tissue? Biopsies b/c small, tissue must be well-fixed before microwave processing
RMS Reagent management system, plan for changing processing reagents
Open tissue processor Cassette basket moved from one solution to next, few advantages, disadvantages include: evaporation & carryover of reagents, odor
Closed tissue processor Reagents pumped in & out of chamber, advantages: less evaporation, carryover & odor, programmable, add agitation, heat vacuum to any step, disadvantages: req. backup power, can't use some reagents (acetone, chloroform)
Processing problem: hard tissue In xylene too long
Processing problem: dry, over-dehydrated tissue In alcohols too long
Processing problem: soft, mushy tissue Tissue under-processed
Processing problem: poor staining Tissue under-processed
Processing problem: desiccated, dry tissue Tissue dried out between reagents
Created by: CCF