Decalcification

• Most rapid decalcifying agent is Pholoroglucin-Nitric Acid
• serves as Decalcifying agent and tissue softener is Perenyi's Fluid
• recommended for surface decalcification of tissue block

  HCl (Von Ebner's Fluid)
• The recommended ratio of fluid-tissue volume is

  20:1
• Optimum temperature for decalcification


  18-30C
• Most common and fastest decalcifying agent


  Nitric acid
• 10% Aqueous nitric acid solutions' decalcification time

  12-24hrs
• Impart a yellow color with nitrous acid

  10% Aqueous nitric acid solution
• Decalcification: 1-3 days

  Produce less tissue destruction than 10% aqueous nitric acid

  The solution should be used inside a fume hood

  Formol-nitric acid
• Decalcification: 2-7 days

  Nuclear and cytoplasmic staining is good

  Complete decalcification cannot be determined by chemical test

  Complete decalcification cannot be determined by chemical test


  Pereyni’s fluid
• Decalcification: 12-24 hrs

  Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours


  Phloroglucin-nitric acid
• Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours
• addition of 0.1% urea to pure Nitric Acid will make the discoloration disappeared
• Strong acids are at concentrations of 5-10%

  Hydrochloric acid
• Decalcification: 2-7 days

  Used as both fixative and decalcifying agent

  Safer to handle than Nitric Acid or HCl 


  10% Formic acid
• weak organic Acid:

  • Decalcification: 3-14 days
  • Recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes
  • Permit better nuclear staining than nitric acid method

  Formic acid-sodium citrate solution
• Weak organic Acid:

  • Weak decalcifying agent
  • Decalcification: 4-8 days


  Trichloroacetic acid
• Weak Organic Acids:

  • May be used both as fixative and decalcifying agent
  • May be used for decalcifying minute bone spicules
  • Nuclear staining with hematoxylin is inhibited


  Chromic acid (Flemmings fluid)
• Very Weak decalcifying agent


  Sulfurous Acid
• pH of Citric acid-citrate buffer acid solution is at pH 4.5
• Decalcification: 6 days

  • contains chloroform that serves as a preservatives


  Citric acid-citrate buffer acid solution
• When decalcification is complete, the acid must be removed by 3 changes of 70-90% ethanol
• • pH dependent
  • Generally used at pH 7.0: excellent result
  • Works more rapidly at pH 10.0 but tissue elements are damaged


  Ethylene diamine tetra acetic acid (EDTA)
• 1-3 weeks 


  (small specimens)
• 6-8 weeks 


  (dense cortical bone)
• Require manipulation, bending probing or trimming of the specimen to feel for remaining calcified areas


  Physical Test
• Also known as calcium oxalate test


  Chemical Test
• 5mL of discarded decalcified fluid is neutralized with 0.5 N sodium hydroxide, 1 mL of 5g/dL ammonium oxalate is added, allowed to stand for 30 minutes

  Chemical Test
• Turbidity or Cloudiness: presence of calcium
• Clear: complete decalcification
• Faxitron machine with exposure setting of 10-110 kv 3ma tube current and kodak xomat xray film is used
• washing time for small specimen

  30 mins
• washing time for large specimen

  1-4 hours
• Immersion of the decalcified bone in either saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate solution for several hours
• Removal of calcium ions from formic acid-containing decalcifying fluid, thereby, ensuring rapid rate of solubility of the calcium from the tissue

  Ion Exchange Resin
• Positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution


  Electrophoresis (Electrical Ionization)