Decalcification methods that employ acids are most widely used in pathology laboratories. Since calcium is soluble at a pH of 4.5, acids quickly and easily dissolve the calcium salts. There are two types of acids used in decalcification procedures:
- Strong mineral acids
- Weak organic acids
The most common decalcified acids are 5–10% solutions of hydrochloric acid (HCl), nitric acid, and formic acid. These acids can be used alone or in combinations.
Before implementing an acid decalcifying protocol in the laboratory, the following should be considered.
- Tissue must be trimmed small and fixed first (unless a fixative combo is used as directed).
- Decalcifiers with higher concentrations of acids act rapidly and affect tissue staining the most.
- Tissue left in acid for too long will lose nuclear staining.
- Decal solution must be changed frequently because calcium leaching out will become a barrier to further decalcification.
- Agitate tissue during decalcification to expose all surfaces to fresh decal agent.
- Heat should be avoided during strong acid decalcification, as tissue swelling and possible digestion of bone collagen will occur.
- Tissue must be rinsed in water before processing; otherwise, acids will continue to decalcify tissue. This will also prevent possible chemical reactions with subsequent reagents and contaminating processor reagents.
The following section summarizes several acids used for decalcification and their properties.
