Decalcification and End Point Detection of Calcified Tissues

chemical endpoint detection procedure

1. Tissue for decalcification should be first well fixed in formalin, and washed well before placing in decalcification solution
2. Indicate in the Log book that the tissue has been send for decalcification by entering “decal” into the comment section by the accession number.
3. Place tissue for decalcification solution in individual covered containers of filtered decalcifying fluid (in the fume hood).  Containers must be labelled with the patient’s full name and the accession number.  Alternatively, small pieces of tissue such as bone marrow biopsies may be placed into the decal solution in a labelled cassette.
4. Small pieces are checked after 3-4 hours; larger pieces after 6-8 hours. If at any time the decalcification solution appears cloudy, it should be changed.
5. Tissues are checked by a chemical endpoint test. This test relies on the detection of calcium in the decalcifying solution. Decalcification solutions should be changed frequently, and the chemical test performed on the fluid to be discarded.
6. To perform chemical end point detection
   • In a clean test tube, place 2 ml of ‘used’ decalcifying fluid (Cal Ex) which should have been in contact with the tissue 3-12 hours
   • Add a piece of litmus paper to the test tube or use a pH meter with magnetic stirrer. Add strong ammonium hydroxide just until the litmus paper turns blue or solution is neutral (pH 7).
   • If the solution is cloudy immediately after adding the ammonium hydroxide, there is no need to continue further (formation of calcium hydroxide, decal not yet complete). Change decalcifying solution and resume decalcification.
   • If after step 2 the solution is still clear, add 2 ml of saturated aqueous ammonium oxalate. Mix and let stand 30 minutes.
   • If precipitation occurs, calcium oxalate has been formed and decalcification is not complete. Change decalcifying solution and resume decalcification.
   • If the solution is still clear after the 30 minutes, it is safe to assume decalcification is complete.
7. Once the tissue is decalcified, fill in grossing log and submit in appropriately labelled cassettes with ’Decal’ written on the side.
8. NOTE: HCl (used in many decalcifying solutions) and formalin, when mixed together, form a carcinogen (bis-chloromethyl ether). After decalcification has been completed using the decalcifier, the tissue should be washed well in running water or neutralized by immersing in lithium carbonate or aqueous sodium bicarbonate solution before returning to formalin.
9. Process tissue, embed, and take to microtomy. Cut sections should be placed on charged slides to aid in tissue adhesion.