Decalcification

• Decalcification


  removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut.
• It is a waste of time to put toenails in decalcification solution, because they are composed of insoluble keratin filaments
• three main types of decalcifying agents

  strong mineral acids

  weaker organic acids

  chelating agents.
• ACID DECALCIFYING AGENTS


  most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily available, and relatively inexpensive as compared to other decalcifying agents
• Strong Mineral Acids


  most rapid in action but if used longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues
• Staining by acid dyes will be less affected, although eosin can produce a deep, brick red stain without differential shading. These effects of decalcifying agents on H&E staining can be reduced by post-decalcification and removal, and by appropriately adjusting the staining procedure.
• Nitric acid


  most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents
• Nitric acid


  decalcification agent that inhibits nuclear stains and destroying tissues, especially in concentrated solutions
• Aqueous Nitric Acid Solution 10%


  FORMULA: Concentrated nitric Acid 10 ml. Distilled water added up to 100 ml. DECALCIFICATION TIME: 12-24 hours
• Aqueous Nitric Acid Solution 10%


  It produces good nuclear staining (although less than in slower acting agents).
• Aqueous Nitric Acid Solution 10%

  It is recommended for urgent biopsy, and for needle and small biopsy specimens to permit rapid diagnosis within 24 hours or less.
• Aqueous Nitric Acid Solution 10%


  It imparts a yellow color with nitrous acid, thereby impairing the staining reaction of the tissue.
• Formol-Nitric Acid


  FORMULA:

  Concentrated nitric acid 10 ml.

  Strong formaldehyde, 40% 5 ml.

  Distilled water 85 ml.

  DECALCIFICATION TIME: 1-3 days
• Formol-Nitric Acid


  It produces less tissue destruction than 10% aqueous nitric acid
• Formol-Nitric Acid


  The yellow color imparted by nitrous acid formation will impair staining reaction of the cell. This may be prevented by neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours. Addition of 0.1% urea to pure concentrated nitric acid will also make discoloration disappear without considerably affecting the efficiency of the decalcifying solution.
• Perenyi’s Fluid

  FORMULA:

  Nitric acid 10% 40 ml.

  Chromic acid 0.5% 30 ml.

  Absolute ethyl alcohol 30 ml.

  Mix shortly before use. Chromic acid must be collected for proper disposal.

  DECALCIFICATION TIME: 2 - 7 days
• Perenyi’s Fluid


  . It decalcifies and softens tissues at the same time.
• Perenyi’s Fluid


  Maceration is avoided due to the presence of chromic acid and alcohol
• Phloroglucin-Nitric Acid

  FORMULA:

  Concentrated nitric acid 10 ml.

  Phloroglucin 1 gm.

  Nitric acid 10% 100 ml.

  (To be added after disappearance of dense white fumes formed by combining the first two ingredients.)

  DECALCIFICATION TIME: 12-24 hours
• Phloroglucin-Nitric Acid


  most rapid decalcifying agent so far, recommended for urgent cases
• Phloroglucin-Nitric Acid

  Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.
• When decalcification is complete, the acid must be removed by three changes of 70% to 90% ethanol, since washing in watery solutions will lead to excessive swelling and deterioration of tissue. When the sections are cut, the slides are brought to water and placed in 1% aqueous lithium carbonate for I hour, washed in later for 15 minutes, and then stained.
• Hydrochloric acid


  inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section.
• Hydrochloric acid


  it produces good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended for surface decalcification of the tissue blocks.
• Von Ebner's Fluid

  FORMULA:

  Saturated aqueous solution of NaCl 50 ml.

  36% concentrated hydrochloric acid 8 ml.

  Distilled water 50 ml.
• Von Ebner's Fluid

  recommended for teeth and small pieces of bone

  permits relatively good cytologic staining
• WEAK ACIDS

  better suited to bone marrow, since they are not as harsh. However, they act more slowly on dense cortical 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


  recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations.
• Formic acid


  slower than the strong acid agents, but it is much gentler in action and less likely to interfere with nuclear staining.
• Formic acid

  FORMULA:

  Formic acid (Sp. grav. 1.20) 10 ml.

  Normal saline 10% 90 ml.

  DECALCIFICATION TIME: 2-7 days
• Formic acid


  only weak acid used extensively as a primary decalcifying agent
• Formic acid


  It may be used both as a fixative and decalcifying agent

  permits excellent nuclear and cytoplasmic staining

  recommended for small pieces of bones and teeth

  suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed
• Formic acid is relatively slow; hence, is not suitable for urgent specimens. Decalcification may be hastened by increasing the proportion of formic acid to 25 ml. However, such concentration may make the solution opaque, thereby interfering with the staining results.
• Formic acid requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue
• Formic Acid-Sodium Citrate Solution


  FORMULA:

  Aqueous sodium citrate 20% 50 ml.

  Formic acid 45% 50 ml.

  DECALCIFICATlON TIME: 3 -14 days
• Formic Acid-Sodium Citrate Solution


  recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes
• Formic Acid-Sodium Citrate Solution


  permits better nuclear staining than nitric acid method
• Formic Acid-Sodium Citrate Solution


  requires neutralization with 5% sodium sulfate
• TRICHLOROACETIC ACID

  FORMULA:

  Trichloroacetic acid 5 gm.

  Formal saline 10% 95 ml.

  DECALCIFICATION TIME: 4- 8 days