| Question |
Answer |
| Removal of calcium salts from bone or calcified tissues |
Decalcification |
| failure to decalcify tissue w/large amounts of calcium results in torn/ragged sections and damage to the cutting edge of microtome |
Failure to decalcify tissue |
| 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 disoolve 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 heat 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 |
| interefer with most techniques |
metallic fixatives |
| examined for diagnosis of metallic bone disease |
undecalcified bone |
| neutralizes remaining acid before processing the specimen |
lithium carbonate |
Beef Production
