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Processing

Decal & Frozens

QuestionAnswer
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
Created by: CCF