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Decal & Frozens
Lab
| Question | Answer |
|---|---|
| Decal bone when embedding w/? | Paraffin; can't cut bone |
| Two methods of decal? | Acid & chelating agents |
| Acid decalcifiers work by? | Causing Ca salts to dissolve & ionize |
| Overdecalcification effect on staining? | Lack of nuclear staining, IHC may not be possible b/c antigenic sites damaged, tissue structure may be damanged, |
| pH range of acid decalcifiers? | 0.5 to 3.0 pH |
| Common simple acid decalcifiers? | HCl, nitric, formic acids; Ca2+ ions may build up in solution w/ this method |
| Nitric acid | Fast, damages stainability after >48 hr |
| Formic acid | Slow, rarely affects tissue stainability even after 2 weeks |
| Hydrochloric acid | Fast, rinse after fixation, formaldehyde may react w/ HCl & form carcinogen |
| If solution saturated with Ca2+? | Decal may stop; to avoid change frequently, agitate solution |
| Never try to speed acid decal w/? | Heat; will affect other tissue components, cause swelling, breaking down |
| Ion-exchange resins decal | Formic acid w/ ammoniated salt of sulfonated resin; NH4+ exchanged for Ca2+; solution stays free of Ca2+, staining excellent, time non-critical |
| Best form of decal? | Ion exchange resins |
| Electrolytic decal method | Formic acid & HCl, electric current, bone on anode, Ca2+ attracted to cathode; v fast, generates heat, damages tissue, loss of stainability |
| Chelating agents | Organic compounds w/ property of binding certain metals |
| EDTA | Ethylenediaminetetraacetic acid, chelating agent, binds only ionized Ca |
| Best pH for EDTA decal? | 5.0 to 7.2, slightly acidic preferred |
| Decal by chelation | V slow (up to weeks), good for enzyme methods |
| Correct decal end point important b/c? | Underdecalcified - difficult to section Overdecalcified - stains poorly |
| 3 methods to determine decal endpoint | Mechanical/physical, chemical, radiographic |
| Mechanical method | Test flexibility of specimen, probe, scrape; most inaccurate method, cause artifacts |
| Chemical method to determine decal endpoint | Mix used decal w/ NH4OH & ammonium oxalate, if calcium oxalate forms solution turbid |
| Radiographic method | Most accurate, x-ray gives visual evidence demineralization complete |
| Can't use radiographic method on? | Metal-fixed tissues (Zenker's, B-5) b/c metal renders tissue radiopaque |
| Remove decal acid by? | Rinsing w/ water Alkaline solution (lithium carbonate) to neutralize acid |
| Frozen sections good for? | Rapid diagnosis, fat demo, enzyme & IHC techniques |
| Ways to freeze tissue? | Cryostat liquid nitrogen Liquid Freon dry ice dry ice-acetone slurry |
| Cryostat freezing__ | Slow, allows ice crystals to form |
| Liquid nitrogen freezing__ | Better, gas bubbles may form & impede freezing |
| Prevent bubbles during liquid nitrogen freezing? | Container of tissue in isopentane into nitrogen; Dust tissue w/ talc submerge directly into nitrogen |
| Bone decal not required when embedding w/ | Plastic media (GMA) |
| Ca2+ soluble at? | pH 4.5 |
| 3 types acid decal? | Simple acids Ion exchange resins Electrolytic methods |
| Solution for simultaneous fixation & decalcification? | Formic acid & formaldehyde |
| Solution for simultaneous fixation and decalcification of small biopsy specimens? | Zenker's |
| Ideal thickness of specimen for decal? | 3 to 4 mm |
| Areas of dark bluish-purple staining in bone sections may be__ | Calcium remaining in the tissue, insufficient decal |
| Ways to help acid decal? | Agitation, vacuum to help solution get in tissue, suspending sample in middle of container, changing solution as needed |
| Acid safety precautions: | Add acid to water, use acid under hood, carry acid bottles in acid carrier, PPE, only 1% acid down the drain |
| It is important to thoroughly rinse all acid from bone after decal because? | Remaining acid will compromise block, damage staining over time, block itself will look wrong |
| Frozen thick and thins are probably caused by? | A dull blay |
| Incomplete frozen sections, or portions of the block not sectioning are probably caused by? | Incorrect cryostat temperature, fatty tissues req. colder temps. |
| Unfixed cryostat sections are picked up by? | A clean, warm (room temp.) slide |
| Cryostats in daily use should be decontaminated? | Once a week, per CAP |
| When are frozen sections required? | When immediate microscopic evaluation is needed |
| Cryostat preventative maintenance includes? | Keeping refrigerant coils free of dust |
| Cryosectioning fixed tissue may be improved by: | Infiltrating w/ 30% sucrose before freezing |
| Cryostat sections catching on the anti-roll plate is likely caused by: | Damage in the edge of the roll plate |
Created by:
CCF
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