Bone, teeth, and calcified tissues cannot be sectioned on a microtome without prior removal of calcium salts. Decalcification (decalc) softens the tissue by extracting calcium ions, leaving the organic matrix intact for processing and sectioning. The choice of decalcification method depends on how urgently results are needed and which downstream tests are planned.
Why Decalcify?
Calcium hydroxyapatite crystals (Ca10(PO4)6(OH)2) in bone and pathological calcifications make tissue too hard for microtome sectioning. Even thin bone specimens (e.g., decalcified bone marrow trephine biopsies) will shatter the microtome blade or produce unusable sections if not decalcified. Decalcification is required for all mineralized tissue including bone, teeth, calcified heart valves, atherosclerotic arteries, and tumoral calcifications.
Acid Decalcification
Strong acids (nitric acid, 5-10%, formic acid, 10-20%) decalcify rapidly — small bone fragments in 2-6 hours, cortical bone in 24-48 hours. Nitric acid is the fastest but can hydrolyze nucleic acids and degrade proteins, compromising IHC and molecular testing. Formic acid is gentler and preferred when IHC or PCR is planned.
Acid decalcification mechanism — protons (H+) displace calcium ions from hydroxyapatite, forming soluble calcium salts that diffuse out of the tissue. The reaction rate depends on acid concentration, temperature (increasing temperature accelerates but increases tissue damage), and agitation. End-point is determined by chemical testing of the decalcification fluid for calcium (oxalate or calcium test strip) or by physical assessment (the tissue bends without resistance).
Chelating agents (EDTA, ethylenediaminetetraacetic acid, 10-14%, pH 7.0-7.4) decalcify by binding calcium ions in a stable chelate complex. EDTA is much slower than acids — decalcification takes 2-14 days depending on tissue size and density — but preserves morphology, antigenicity, and nucleic acids better than any acid method. EDTA is the preferred decalcification method when IHC, FISH, or PCR will follow. EDTA decalcification at 4°C is even slower (3-4 weeks) but provides optimal nucleic acid preservation.
Decalcification Protocols
Bone marrow trephine biopsies — fixed in formalin for 2-4 hours, decalcified in EDTA or formic acid for 2-4 hours (small cores) to 24 hours (large cores). Over-decalcification destroys marrow cellular detail and compromises IHC for lymphoid and plasma cell markers.
Femoral heads and large bone specimens — fixed in formalin for 24-48 hours, sectioned on a band saw to 3-5 mm thick slices, decalcified in formic acid-sodium citrate solution or EDTA for 2-7 days. Change decalcification solution daily. Test endpoint with ammonium oxalate — formation of a precipitate indicates calcium is still being released.
Calcified soft tissue tumors — decalcify in EDTA to preserve IHC antigenicity. Acid decalcification may mask markers essential for tumor classification.
End-Point Detection
Chemical testing — add 1 mL of decalcification fluid to 5 mL of ammonium oxalate solution; a white precipitate (calcium oxalate) within 30 seconds indicates decalcification is incomplete. X-ray — specimens can be radiographed to detect residual calcification. Physical — a bent safety pin or scalpel blade should pass through the tissue without resistance; the tissue should feel like firm cheese, not bone.
Artifacts and Problems
Over-decalcification — the most common problem. Excessive acid exposure destroys nuclear basophilia (H&E shows pale, ghost-like nuclei), fragments DNA (PCR failure), and denatures proteins (IHC false negatives). EDTA over-decalcification is less damaging but still should be avoided. Under-decalcification — sections contain hard, gritty areas that tear the section or damage the blade; retreat the block in decalcification solution for additional time. Acid hematin pigment — brown-black birefringent crystals from acid interaction with formalin-fixed hemoglobin; removed by treatment with saturated alcoholic picric acid.
Quality Control
Document decalcification method, time, temperature, and endpoint. Assess nuclear preservation on H&E — well-decalcified tissue shows crisp nuclear detail comparable to non-decalcified tissue. IHC controls must include decalcified tissue to verify antigen preservation. Positive controls for IHC should be decalcified by the same method as the test tissue. Participation in EQA programs for bone marrow and bone pathology includes assessment of decalcification quality.
