How does the decalcification process work?

Any tissue containing bone, teeth, or calcifications must be FULLY FIXED prior to decalcification. Decalcification must be performed such that the tissue can be sectioned in the gross room with a blade and/or in histology with a microtome. Scant bony fragments (as in marrow margins) can be frozen fresh (without decal) as long as they are small and can be cut with the cryostat. Non-organic materials such as bone cement, dental fillings, or metals will NOT decalcify and should NOT be submitted in cassettes.

For more extensive tips on grossing complex specimens with bone, see this presentation (pdf).

What are the types of decalcifiers?

  • Strong Acid decalcifiers (HCl, Hydrochloric acid) are standard in pathology labs. They quickly decalcify but destroy nucleic acids.
  • Moderate Organic Acid decalcifiers (Formic acid) are slower but also destroy nucleic acids.
  • Chelating Agents such as ethylenediaminetetraacetic acid (EDTA) are the slowest but also the best at preserving nucleic acids.

When should I use each of these decalcifiers?

For Big / Tumor Specimens: Never decalcify an entire specimen before consulting with an attending first:

  • Check to see if there is a pre-existing recent non-decalcified biopsy.
  • Always try to isolate soft lesional tissue to submit WITHOUT decalcification:
    • Soft tumor next to bone
    • Soft tumor within bone
    • Noncalcified tumor
  • If these above two criteria are NOT met, determine the extent of HCL vs EDTA in discussion with an attending.
    • Examples of these cases might be predominantly ossified tumor (such as osteosarcoma) or tumor diffusely infiltrating bone with no soft component.

For Biopsies: 

  • All non-Heme bone core biopsies should be decalcified in EDTA, as the need for molecular testing cannot always be predicted. Refer to Core Biopsy Grossing Page for additional details.
  • For outreach oral specimens needing decalcification, refer to Oral Biopsy Grossing Page for specific instructions.


Any hard tissue for which molecular testing (NGS, FISH, etc) may be needed should NOT be decalcified in acid (HCl). It should be decalcified with a gentle decalcifier (EDTA). Please ask a PA if you need EDTA.


NO LESION SHOULD BE DECALCIFIED ENTIRELY IN HCl. If multiple sections / cassettes require decalcification, be sure to isolate some for gentle EDTA decalcification, after fixation.


How should I use each of these decalcifiers?

  • For HCl, small tissue: If your tissue to be decalcified is small and already in a cassette, in can be decalcified in the common HCl jar. Be sure to LOG your case and monitor every day for pliability or ability to be cut with a blade. If tissue is not ready after 3 days – please consult with a PA or attending to determine the next best steps.
  • For HCl, large tissue: If tissue is large and intact, it should be placed in its own container labeled with a sticker (patient name, MRN, surg path #) and hand-written HCl DECAL on the container.
  • For ALL HCl: Wash cassettes for 10 minutes under running water BEFORE HCl decalcification (i.e., when transferring from Formalin to HCl) and AFTER HCl decalcification (i.e., when transferring from HCl back to Formalin).
  • For EDTA: There is no common jar currently for EDTA. Please use a separate container labeled with a sticker (patient name, MRN, surg path #) and hand-written EDTA DECAL on the container.
  • When is tissue “done”? Tissue is adequately decalcified when it can be cut with a blade or is pliable.

How is the decalcification process billed?

The billing team verifies the need for the decalcification charge by looking for two items:

  1. The word “decalcified” or phrase “submitted after decalcification” in the gross description / cassette summary. Be sure to state the specific type of solution you used (HCl or EDTA) for each cassette.
  2. A Decalcification charge needs to be ordered on each Container in which tissue was decalcified:
    • The decal charge is automatically linked to certain part-types such as bone biopsy, bone fragments, bone resection, etc. In these cases, if you do not need to decal the tissue, delete the decal charge.
    • If the decal charge is not already linked (incidental calcs in thyroid, leiomyoma, etc.), manually add the decal charge to a cassette. The charge should be added once per container, regardless of the number of cassettes decaled. You CAN add the charge to multiple cassettes that were decalcified, but it is most important that you add it to AT LEAST ONE:
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