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Stack #171156

Decalcification

QuestionAnswer
Removal of calcium salts from bone or calcified tissues Decalcification
_____ tissue w/large amounts of calcium results in torn/ragged sections and damage to the cutting edge of microtome Failure to decalcify
for routine dx purposes use formalin, use it unbuffered since calcuim phosphate present in the bone serves as an adequate buffer to keep the pH above 6.0, Fixation
Nucleic acids are suscpetible to ribonuclease digestion or digestion by mineral acids,if formalin fixation is prolonged more than two days Nucleic acids
acid methods (acid, ion exchange, electrolytic method) chelating method Two Routine decalcification methods
The stronger the acidity of solution,the longer the specimen remains in it, the more subsequent staining will demonstrate injurious effects of the decalcification. most pronounce effect-nuclear bsophilia Acid method
may result in a total lack of nuclear staining over decalcification
calcium salts dissolve and then ionize. principle of acid method
soluble at a pH of 4.5 calcium salts
pH between 0.5-3.0 decal solutions
used in concentration of 5%-10% simple acids
decalcify fairly rapidly hydrochloric and nitric acids
can cause serious deterioration of tissue beyond 48 hours Nitric acid
slower acting, can remain in solution for two weeks Formic acid
great for simultaneous fixation and decal formic acid and formaldehyde
should be suspended in an embedding bag to expose all the surfaces of the specimen specimen
at the initial stage aids in infiltrating the specimen w/decal solution and will draw off carbon dioxide bubbles that form on the specimen surface Vacuum
migrate out of the tissue into the surrounding solution. solutions around the tissue may become saturated, so the solution should be changed frequently calcium ions
change frequently solution
never use _____to speed up decal process. heat
it increases the effects of decalcifying fluids on other tissue components, swelling and maceration will most likely occur heat
involves use of formic acid over a layer of an ammoniated salt of a sulfonated resin Ion exchange resins
exchanged for calcium ions, this keeps solution free of calcium ions and speeds up the reaction. solution doesnt need to be changed frequently ammonium ions
the best decal method ion exchange resins
utilizes a mixture of formic and hydrochloric acid placed in an apparatus based on a simple ectroplating device electrolytic method
The bone is attached to the anode (+) and a current is passed through the solution. The calcium ions (+ charge) are attracted to the cathode (-). electrolytic method
decal process takes 2-6hours, one sample per day can be processed. electrolytic method
heat generated by this method has a potential for tissue destruction, a total loss of cellular detail and stainability electrolytic method
organic compounds that have the property of binding certain metals chelating agents
ethylenediaminetetraacedic acid EDTA
solution should be between 5.0-7.2 Chelating agents
Binds calcium ions EDTA
Very slow method but many enzyme methods can be used chelating agents
sectioning is difficult underdecalcification of tissue
stain is very poor overdecalcified tissue
three basic method - mechanical/physical, chemical, radiographic end of decalcification
testing flexibility of specimen, probing the specimen with needle or pin, Mechanical method
least desirable method, it is inaccurate and can create artifacts mechanical method
depends on the precipitation of calcium oxalate Chemical method
mixing a sample of the used decal solution w/a solution of ammonium hydroxide and ammonium oxalate. if solution remains turbid it indicates the presence of calcium Chemical method
keep retesting decal solution until free of calcium Chemical method
Yields a visual evidence that demineralization is complete. most accurate method. Radiography
Do not use on metallic fixed tissue such as Zenker or B-5 solution. metal will render the specimen radiopaque Radiography
wash tissue w/running wather or lithium carbonate to neutralize any remaining acid, then routinely process the specimen after decalcification
Glycol methacrylate is the most frequently used embedding media. undecalcified bone
section of bone may be ground with waterproof sandpaper to a thick of 75-100 microns. these ground sections may be stained and mounted on glass slides undecalcified bone
alcohol, buffered formalin or calcium formalin fixatives of choice
interefere with most techniques metallic fixatives
examined for diagnosis of metallic bone disease undecalcified bone
neutralizes remaining acid before processing the specimen lithium carbonate
Created by: nperez