DECALCIFICATION

Cards (49)

  • 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