Decalcification

Principle

• Calcium salts in bone and calcified tissue are removed after fixation so that the tissue can be processed and sectioned.
• Acids dissolve calcium salts to form soluble calcium salts; chelating agents (EDTA) bind calcium ions slowly.

Specimen

• Bone, bone marrow trephines, teeth and calcified soft tissue, well fixed beforehand and cut into thin slices (about 2–5 mm) with a bone saw.

Reagents and equipment

• Strong acids: nitric acid or hydrochloric acid (fast, hours) but damage nuclear staining, antigens and nucleic acids.
• Weak acids: formic acid (commonly about 5–10%, sometimes buffered) — slower, gentler, widely used for routine work.
• Chelating agent: neutral EDTA (about 10–14%, pH about 7–7.4) — slowest (days to weeks) but best preservation for morphology, IHC and molecular testing.

Procedure

• Ensure complete fixation first; acid on poorly fixed tissue causes severe damage.
• Use a large volume of decalcifier (at least 20 times the tissue volume) and change it regularly; agitation helps.
• Check the endpoint regularly.
• After acid decalcification, rinse well (and neutralize if the protocol requires) before processing.

Quality control and pitfalls

• Endpoint testing: radiography (most accurate), chemical test of the used acid for calcium (ammonium oxalate precipitate after neutralizing), or physical probing/bending (least reliable and damaging).
• Over-decalcification: poor, smudgy hematoxylin staining, excessive eosin, and loss of antigens and DNA/RNA.
• Under-decalcification: gritty tissue, torn sections and damaged blades.
• Heat speeds decalcification but increases tissue damage.
• Surface decalcification of the block face can help when small calcified areas are found at microtomy.
• For tissue needing ER/PR/HER2 or molecular testing (e.g. bone metastases), avoid strong acids; EDTA is preferred.

Clinical use

• Diagnosis of bone tumors, metastases, metabolic bone disease and bone marrow disorders (trephine biopsies).