Decalcifying Procedures

Decalcification is required for preparing histologic sections of bone, dentine, cementum, calcified vessels, and calcification in lesions, such as granulomas and tumors. Decalcification solutions are commercially available but we found formic acid decalcification optimal for most purposes. The solution is easy to prepare, inexpensive, and causes little tissue damage. The composition of the solution is as follows:

FORMIC ACID DECALCIFICATION FLUID Formic acid decalcification fluid: 80 mL Neutral buffered formalin (see page 130), 20 mL Formic acid.

For very soft bone specimens and autopsy samples that do not need to be processed urgently, ethylenediamine tetraacetic acid (EDTA) is recommended:

EDTA DECALCIFICATION FLUID EDTA Decalcification Fluid: 4 g Disodium ethylene diamine tetra acetic acid (EDTA); 40 mL Neutral buffered formalin (see page 130).

PROCESSING OF SPECIMENS The samples should not be thicker than 3 mm. For each piece, 100 mL of decalcification fluid should be used. Change and agitate solution daily or more often. Exact end-point determination is essential because staining properties will be lost if fluid is not washed out immediately after decalcification is completed. Formic acid decalcification should not last longer than 2 d; EDTA decalcification may last 2-5 d. The formic acid must be removed by washing the specimen for 30 min in running tap water; EDTA preparations should be processed without washing in tap water.

Fixation with an aqueous solution of calcium acetate, glut-araldehyde, and formalin, followed by decalcification in neutral 10% EDTA reportedly prevents the loss of antigens and the fading of ferritin iron and enzymes (6).

Decalcification time depends on numerous factors such as the size and texture of the specimen, the type and temperature of the solution, and the use of agitation and electrolysis. A small specimen in an acid bath that is exposed to the heat and agitation of the Autotechnicon will be decalcified in little more than 2 h; a protective acid-resistant insert must be used. The speed of decalcification can also be increased by electrolysis. The specimen is placed in acid decalcifying fluid with platinum electrodes; the acid serves as the electrolyte. Use of ultrasound is another method to increase the speed of decalcification. With this method, the fixation process can be combined with the use of acid or chelating decalcifiers (7).

To achieve histologic slides of the best quality, the decalcification process should not be unnecessarily prolonged. Piercing the sample with a needle or blade or bending the specimen usually permits one to judge roughly when decalcification is complete. Another indicator is the decrease or disappearance of CO2 bubbles from the specimen. Among the many methods for end-point determination of decalcification, serial roentgenograms permit the most precise control. For small specimens, dental films can be used.

For additional decalcification methods as well as information on fixation, staining, and other procedures, the reader should consult one of the current textbooks and manuals listed in the beginning of Part II.

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