Decalcification Methods Using Acids

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Decalcification Methods Using Acids

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 acids used for decalcification are 5-10% solutions of hydrochloric acid (HCl), nitric acid, and formic acid. These acids can be used alone or in combinations.
The following should be considered before implementing an acid decalcifying protocol in the laboratory.
  • Tissue must be trimmed small and fixed first (unless a fixative combo is used as directed).
  • Decalcifiers with higher concentration of acids act rapidly and affect tissue staining the most.
  • Tissue left in acid too long will lose nuclear staining.
  • Decal solution must be changed frequently because calcium that has leached out will become a barrier to further decalcification.
  • Agitate tissue during decalcification to expose all surfaces to fresh decal agent.
  • Heat should be avoided with strong acid decalcification as swelling of tissue and possible digestion of bone collagen will occur.
  • Tissue must be rinsed in water prior to processing, otherwise acids will continue to decalcify tissue; will also prevent possible chemical reactions with subsequent reagents and contamination of processor reagents.
The following section summarizes several acids used for decalcification and their properties.