Fixation PRIOR to decalcification
Fixation of tissue prior to decalcification is the most common and preferred. Complete fixation protects calcified tissue, fibrous elements of bone, and any surrounding soft tissue from the damaging effects of acid decalcification procedures. The integrity of poorly fixed specimens prior to decalcification will be compromised, resulting in poor staining. Tissue is placed in fixative, typically 10% neutral buffered formalin, immediately after collection and inspected for calcifications during the gross examination. Small calcified biopsies are usually processed without decalcification. If the preservation of bone marrow is critical, Zinc formalin, B5, or Davidson's fixative should be used. Large bone specimens (leg bones, ribs) are roughly cut into smaller portions with a saw and placed in fixative. Surrounding soft tissue is typically removed from cortical bone before decalcification. The smaller the tissue, the less time needed for complete fixation. Once fixation is complete, the decalcification process may begin. If hydrochloric acid decalcifiers are used, the tissue fixed in formalin must be washed in water before placement in the decalcifying agent. This will prevent the chemical reaction that forms bis-chloromethyl ether. Tissue treated with other fixatives may also need to be rinsed before decalcification to prevent unwanted chemical reactions and contamination of tissue processor reagents.
Fixation DURING decalcification
Fixation of tissue can also be accomplished during the decalcification process. Long-established fixatives, such as Bouin's and Zenker's, have successfully removed small calcifications from tissue. Zenker fixative was previously used to fix and decalcify bone marrow biopsies simultaneously. The picric acid in Bouin's removes calcium from breast core biopsies. New dual-purpose fixatives/ decalcifiers are introduced to laboratories yearly, many with surprisingly positive results. Most of these dual formulas combine formic acid and formaldehyde to fix and decalcify tissue, which act more slowly than hydrochloric acid. Also, some formulations require initial tissue fixation before decalcification with the fix/ decal combo solution.
Regardless of the fixation route chosen for calcified tissue, ample fixation time relevant to the fixative of choice is still required for optimal results.
