DECALCIFYING AGENTS

ACID NITRIC ACID o o o Most common and the fastest decalcifying agent As a simple solution- 5-10% Has the disadvantage of inhibiting nuclear stains and destroying tissues, especially in concentrated solutions ADVANTAGES - rapid (12-24 hrs) -produces minimum distortion of tissues -can be used for large or heavily mineralized cortical bone specimen if decalcification progress is carefully monitored by a decalcification endpoint -rapid (1-3 days) -relatively good nuclear staining -less tissue destruction than 10% aqueous nitric acid -decalcifies and softens tissues -good nuclear and cytoplasmic staining -no need for maceration due to the presence of chromic acid and alcohol -most rapid decalcifying agent (12-24 hrs) DISADVANTAGES NOTES -imparts a yellow color with nitrous Addition of buffer acid, impairing the staining reaction of the tissue -old solutions are particularly damaging

10% Aqueous Nitric Acid Solution

Formol-Nitric acid

-imparts yellow color -must be used under a fume hood

Perenyis fluid

-complete decalcification cannot be determined because precipitation occurs upon the addition of ammonia even in the absence of Calcium ions

- 2-7 days (slowest) -add glacial acetic acid drop by drop. Add 0.5 mL of satd ammonium oxalate. Reappearance of a white precipitate within 30 mins confirms incomplete decalcification

Phloroglucin-Nitric Acid

-poor nuclear staining -complete decalcification cannot be determined by chemical means -imparts yellow color

HYDROCHLORIC ACID o Compared to nitric acid: o Slower action o Greater distortion of tissue produced on the section decalcified Good nuclear staining if used in 1% solution with 70% alcohol Von Ebners solution -relatively good cytologic staining -moderately rapid -does not require washing out before dehydration -recommended for teeth and small pieces of bone -extent of decalcification cannot be measured by a chemical test

ADVANTAGES

DISADVANTAGES NOTES

FORMIC ACID o o o o o Better nuclear staining with less tissue distortion Safer to handle than nitric acid and hydrochloric acid Recommended for routine decalcification of postmortem research tissues, although not for suitable for urgent examinations The only weak acid used extensively as a primary decalcifying agent Addition of citrate accelerates decalcification Formic Acid -fixative and decalcifying agent -excellent nuclear and cytoplasmic staining -recommended for small pieces of bones and teeth -immunohistochemical staining -slow -requires neutralization with 5% sodium sulfate 2-7 days Formic acid-sodium citrate -better nuclear staining than nitric acid method -for autopsy materials, bone marrow, cartilage and tissues (research purposes)

ADVANTAGES

DISADVANTAGES NOTES

-slow -5% sodium sulfate 3-14 days

TRICHLOROACETIC ACID Decalcification time ADVANTAGES DISADVANTAGES NOTES 4-8 days -good nuclear staining -does not require washing out -weak decalcifying agent, suitable only for small spicules of bone -very slow acting

SULFUROUS ACID o A very weak decalcifying solution suitable only for minute pieces of bone

CHROMIC ACID (FLEMMINGS FLUID) DECALCIFICATION TIME ADVANTAGES DISADVANTAGES

NOTES

-fixative and decalcifying agent -minute bone spicules -nuclear staining with hematoxylin is inhibited -frequent changes of solution -insoluble pigments are formed when decalcified tissue is dehydrated with alcohol, hence, tissues must be washed out prior to dehydration -degree of decalcification cannot be measured by routine chemical test -highly corrosive, carcinogenic

CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5) DECALCIFICATION TIME ADVANTAGES DISADVANTAGES NOTES 6 days -excellent nuclear and cytoplasmic staining -does not produce cell or tissue distortion -too slow

CHELATING AGENTS o o Combine with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salt. EDTA (Versene) o Advantages: Minimal cell and tissue distortion Extent of decalcification measured by routine chemical test Excellent bone decalcifier for immunohistochemical or enzyme staining, and for electron microscopy o Disadvantages; Very slow

ION EXCHANGE RESIN o o o Ammonium form of polystyrene resin Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions ADVANTAGES: o Cellular detail is well-preserved o Daily washing of solutions is eliminated o Minimal cell and tissue distortion o Extent of decalcification measured by routine chemical tes

ELECTROPHORESIS o Satisfactory for small bone fragments

Factors influencing rate of decalcification o o Concentration and volume of decalcifying agent Temperature at which the reaction takes place

Measuring extent of decalcification o o o Physical or mechanical test (poke, tease, probe) Radiological method (radiopacity) Chemical method (Calcium oxalate test)

DEHYDRATION
Characteristics of an ideal Dehydrating Solution: o o o o o o o Dehydrate rapidly without producing considerable shrinkage or distortion of tissues Should not evaporate very fast Should be able to dehydrate even fatty tissues Should not harden tissues excessively Should not remove stains Nontoxic Should not be a fire hazard