DECALCIFICATION

• Decalcification


  A procedure whereby calcium or lime salts are removed from tissues (most especially bones and teeth) following fixation
• Decalcification


  1. Use of chemical agents

  2. Either acids to form soluble calcium salts

  3. Or chelating agents that bind to calcium ions
• Decalcification should be done after fixation and before impregnation
• Inadequate decalcification may result in poor cutting of hard tissues and damage to the knife edge during sectioning
• Bones and calcified tissues


  • Usually cut into small pieces with a fine fretsaw and trimmed with a hand razor to permit complete penetration of the Decalcifying solution with minimal surface damage and tissue distortion
• Microcalcification


  • Can be seen as dark purple granular Masses with lighter purple halos after hematoxylin staining
• Calcium may be removed by

  • Acids
  • Chelating agents
  • Ion exchange resins
  • Electrical ionization (electrophoresis)
• Acid decalcifying agents


  • Nitric acid
  • Hydrochloric acid
  • Formic acid
  • Trichloroacetic acid
  • Sulfurous acid
  • Chromic acid
  • Citric acid
• Nitric acid


  Most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents
• Aqueous nitric acid solution 10%


  • Rapid in action
  • Produces minimum distortion of tissues
  • Produces good nuclear staining
  • Easily removed by 70% alcohol
  • Recommended for urgent biopsy, and for needle & small biopsy specimens to permit rapid diagnosis within 24 hrs or less
  • Can be used for large, heavily mineralized cortical bone specimen if decalcification progress is carefully monitored by a decalcification endpoint test
• Formal-nitric acid


  • Rapid-acting, hence recommended for urgent biopsies
  • Produces less tissue destruction than 10% aqueous nitric acid
• Perenyi's fluid


  • Recommended for routine purposes
  • Decalsifies and softens tissues at the same time
  • Nuclear and cytoplasmic staining is good
  • Maceration is avoided due to the presence of chromic acid and alcohol
• Phloroglucin-nitric acid


  • The most rapid decalcifying agent so far, recommended for urgent works
  • Nuclear staining is poor
  • Prolonged decalcification produces extreme tissue distortion
• When decalcification is complete, the acid must be removed by 3 changes of 70% to 90% ethanol, since washing in watery solutions will lead to excessive swelling and deterioration of tissue
• Hydrochloric acid


  Inferior compared to nitric acid, slower action and greater distortion of tissue produced, but will produce good nuclear staining
• Von Ebner's fluid


  • Permits relatively good cytologic staining
  • Moderately rapid decalcifying agent
  • Does not require washing out before dehydration
  • Recommended for teeth and small pieces of bone
• Formic acid


  Moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or hydrochloric acid
• Formic acid


  • May be used both as a fixative and decalcifying agent
  • Permits excellent nuclear and cytoplasmic staining
  • Recommended for small bones and teeth
  • Suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed
• Formic acid-sodium citrate solution

  • Permits better nuclear staining than nitric acid method
  • Recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes
• Trichloroacetic acid


  • Permits good nuclear staining
  • Does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration
• Sulfurous acid is a very weak decalcifying solution suitable only for minute pieces of bone
• Chromic acid (Flemming's fluid)

  • May be used both as a fixative and decalcifying agent
  • May be used for decalcifying minute bone spicules
  • Nuclear staining with hematoxylin is inhibited
  • Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol; hence, tissues must be washed prior to dehydration
• Chromic acid is highly corrosive to skin and mucous membranes, it is carcinogenic, and drain disposal is not a legitimate option for any solution containing chromium
• Citric acid-citrate buffer solution (pH 4.5)


  • Permits excellent nuclear and staining
  • Does not produce cell or tissue distortion
• Chelating agents


  Substances which combine with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salt
• EDTA


  The most common chelating agent, combines with calcium forming an insoluble non-ionized complex
• EDTA


  • Very slow decalcifying agent, takes 1-3 weeks for small specimens, 6-8 weeks or longer for dense cortical bone
  • Solution should be changed every 3 days, and in the final stage, everyday, to facilitate decalcification
  • Faster at pH 7-7.4, but pH 8 and above will give optimal binding
• EDTA and EDTA disodium salt (10%) can be simple aqueous or buffered solutions at neutral pH of 7-7.4, or added to formalin
• EDTA sodium salt


  • Permits excellent staining results
  • Produces minimal cell and tissue distortion
  • Causes slight tissue hardening
• Decalcification time


  1. 3 weeks for small specimens, 6-8 weeks or longer for dense cortical bone
• Decalcification process


  1. Change solution every 3 days, change daily in final stage

  2. EDTA will not bind to calcium below pH 3, faster at pH 7-7.4

  3. pH 8 and above gives optimal binding but may damage alkaline sensitive protein linkages
• EDTA solutions


  • Simple aqueous or buffered solutions at neutral pH 7-7.4, or added to formalin
  • EDTA tetrasodium solution is alkaline, pH should be adjusted to 7.4 using acetic acid
• EDTA decalcification solution


  • EDTA sodium salt 5.5 gm
  • Distilled water 90 mL
  • Formaldehyde (37-40%) stock 10 mL
• EDTA decalcification


  • Permits excellent staining results
  • Produces minimal cell and tissue distortion
  • Forms minimal histological artifacts
  • Inactivates alkaline phosphatase activity, can be restored by adding magnesium chloride
  • Extent of decalcification can be measured by routine chemical test
  • Excellent for immunohistochemical, enzyme staining, and electron microscopy
• EDTA decalcification disadvantages


  • Very slow, not recommended for urgent or routine purposes
  • Causes slight tissue hardening
• Ion exchange resin


  Hastens decalcification by removing calcium ions, not recommended for fluids containing mineral acids
• Ion exchange resin decalcification

  1. Layer of resin spread on bottom of container, specimen placed on top

  2. Decalcifying agent added, usually 20-30 times volume of tissue

  3. Tissue may stay in solution 1-14 days, degree of decalcification measured by physical or X-ray method

  4. Used resin can be reactivated by immersing in HCl and washing with water
• Ion exchange resin decalcification

  • Cellular detail well-preserved
  • Decalcification hastened
  • Permits excellent staining results
  • Produces minimal cell and tissue distortion
  • Forms minimal histological artifacts
  • Causes slight tissue hardening
  • Extent of decalcification cannot be measured by chemical means
• Electrophoresis (electrical ionization)


  Process where positively charged calcium ions are attracted to a negative electrode and removed, shortening decalcification time
• Electrophoresis solution


  • Formic acid 88% 100 mL
  • Concentrated hydrochloric acid 80 mL
  • Distilled water 1000 mL