Decalcification

Tissue containing bone requires special treatment to ensure high quality sections for microscopic examination are produced. Inadequate decalcification will result in paraffin sections disrupted by damaged knives. Incorrect application of decalcification solutions will cause loss of cellular staining, immunohistological antigenicity and nucleic acid recovery. 1-5

Decalcification of teeth is rare in routine laboratories but various techniques are published for this purpose.6

Optimising tissue decalcification 1,2
  • Ensure adequate fixation prior to decalcification to preserve cellular structure.
  • Thoroughly wash the specimen after fixation and prior to decalcification; then after decalcification and prior to processing.
  • Agitation will improve uniformity and rate of decalcification. Roller beds or magnetic stirrers are commonly used but vigorous agitation should be avoided to ensure the tissue is not damaged.
  • Decalcification will also be quicker at higher temperatures but due to the possibility of increased damage to cellular structures, room temperature between 18-25°C is recommended.
  • Conversely, refrigeration at 4°C can be used to slow down decalcification if tissues will not be monitored over an extended period.
  • Sufficient volume of solution to tissue is required (20:1) for optimal decalcification.
  • Higher concentrations of solutions will decalcify tissue more rapidly but with greater damage to tissue.
  • Decalcification solution will become depleted over time and should be changed every 24 hours.
  • Mature cortical bone will be slower to decalcify than less developed cortical or trabecular bone.
  • Bone marrow contains fat and specimens will tend to float to the surface of solutions as calcium is removed.
  • End-point of decalcification must be monitored to ensure sufficient calcium is removed without over-decalcification and damage to tissue morphology.
  • Surface decalcification may be required on blocks if focal calcification is present at microtomy.
Decalcification solutions 1
Chelating

Chelating agents decalcify by binding to calcium ions.

Ethylendiaminetetraacetic acid (EDTA)

  • Slow decalcification
  • Not suitable for routine processing
  • Retains morphology
  • Optimal pH  is 7-7.4
  • Will not bind to calcium below pH3
  • Higher pH may damage alkali-sensitive protein linkages

Examples:

  • Formalin-EDTA
  • Aqueous EDTA
Acids

Acids decalcify by forming soluble calcium salts.

Weak organic acids
Formic acid
  • Slower than strong acids
  • Good compromise between chelating and strong acid decalcification 2

Examples:

  • Aqueous formic acid
  • Formic acid-formalin
  • Buffered formic acid
Glacial acetic acid and picric acid
  • Not used as primary decalcifiers but may have an incidental decalcifying effect that is sufficient where minimal decalcification is required when present in fixatives such as Zenker’s, Carnoy’s or Bouin’s.
Strong inorganic acids
Nitric acid
  • Rapid
  • Useful for cortical bone
  • Over-decalcification will cause loss of nuclear staining and poor morphology 

Examples:

  • Nitric acid, 5-10%
  • Aqueous nitric acid
  • Perenyi’s fluid
  • Formalin- nitric acid
Hydrochloric acid
  • Rapid
  • Useful for cortical bone
  • Over-decalcification will cause loss of nuclear staining and poor morphology 

Example:

  • Hydrochloric acid, 5-10%
Bone types and applicable decalcification solutions7
Type of bone
Strong acid
Strong acid + EDTA
Formic acid
Fixative + decalcifier
Cortical
O
NR
NR
S
Cancellous
S
S
O
S
Bone biopsy
NR
O
O
S
Bone marrow trephine
NR
O
S
NR

O = Optimal                S = Satisfactory                NR = Not recommended

Bone Marrow Trephines
  • A range of techniques are available for the decalcification of bone marrow trephines from a rapid methods using an inorganic solution for up to two hours to slower processes using EDTA for 24 hours.2,8,9
  • Opinions vary as the effect of strong acid solutions on the preservation of DNA and must be considered when deciding on a suitable decalcification procedure. 8,9
Evaluating endpoint

Refer to procedures used within your laboratory for specific instructions.

  • Small biopsies may only require 24 hours decalcification and can be physically tested for hardness by gently pressing the specimen.
  • Larger and harder bone specimens must be monitored to detect endpoint of decalcification using:
    • Chemical detection of calcium ions in decalcification solution.
    • Radiographical detection of calcium in specimen.
Surface decalcification
  • Lie block face down on gauze soaked in decalcification solution. (e.g. 10% formic acid or 1% HCl) for 10mins 2 and up to 60 mins if required. 1
  • Decalcification solution will not penetrate very deeply into the block.
  • Repeat after several ribbon of sections are cut if focus of calcification persists or occurs in other areas of the block. 
References
  1. Suvarna KS, Layton C and Bancroft JD. Bancroft's Theory and Practice of Histological Techniques. Churchill Livingstone, 2012.
  2. Freemont AJ, Denton J and Mangham DC. Tissue pathways for bone and soft tissue pathology, The Royal College of Pathologists, London, 2011.
  3. Singh VM, Salunga RC, Huang VJ, Tran Y, Erlander M, Plumlee P and Peterson MR. Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies. Ann Diagn Pathol 2013;17(4):322-326.
  4. Belluoccio D, Rowley L, Little CB and Bateman JF. Maintaining mRNA integrity during decalcification of mineralized tissues. PLoS One 2013;8(3):e58154.
  5. Darvishian F, Singh B, Krauter S, Chiriboga L, Gangi MD and Melamed J. Impact of decalcification on receptor status in breast cancer. Breast J 2011;17(6):689-691.
  6. Silva GA, Moreira A and Alves JB. Histological processing of teeth and periodontal tissues for light microscopy analysis. Methods Mol Biol 2011;689:19-36.
  7. Dimenstein IB. Bone grossing techniques: helpful hints and procedures. Annals of diagnostic pathology. 2008;12(3):191-8.
  8. Brown RS, Edwards J, Bartlett JW, Jones C, Dogan A. Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer. J Histochem Cytochem. 2002;50(1):113-5.
  9. Wickham CL, Sarsfield P, Joyner MV, Jones DB, Ellard S, Wilkins B. Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol : 2000;53(6):336.