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Processing
Lab
| Question | Answer |
|---|---|
| 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 |
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CCF
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