2 Decalcification

• Decalcification is the process of removing Calcium or Lime Salts from tissues

  • It prevents poor cutting of hard tissues/knife damage
• A fine-fret saw is used if the tissue size is very large
• Decalcifying agent should be changed regularly
• “Grating” sensation during cutting is a poor processing = place block in 10% HCl for 1 hour to fix
• Rapid decalcification produces effect on nuclear staining
• Acid decalcifying agents:

  • HNO3
  • HCl
  • Formic
  • TCA
  • Sulfurous
  • Chromic
  • Citric
• Chelating decalcifying agent:

  • EDTA (Versene)
• Ion exchange resin decalcifying agent:

  • Ammonium form of Polystrene
• Electrical ionization decalcifying agent:

  • Electrophoresis
• Acid Decalcifying Agent is most widely used; it is stable, easily available, and cheap
• Nitric Acid is a most common acid decalcifying agent; it is most rapid

  • 5 – 10% concentrations is recommended
  • Decalcification time is 12-24 hours
• 10% Aqeous Nitric Acid is rapid, causes minimal tissue distortion; recommended for urgent biopsy, such as needle biopsy

  • 10 mL Conc. Nitric Acid + 100mL dH2O
  • decalcification time: 12-24 hours
• 10% aqueous nitric acid disadvantages:

  A) Tissue distortion

  B) yellow

  
• Formol–Nitric Acid is rapid acting, has good nuclear staining, and less tissue destruction than 10% Aqueous nitric acid

  • Nitric Acid + 40% Formalin + dH2O
  • decalcification time: 1-3 days
• Formol-nitric acid major disadvantages:

  A) fume hood

  B) 5% sodium sulphate

  C) 0.1% urea

  
• Perenyi’s Fluid decalcifies and softens tissues at the same time; it is a good nuclear and cytoplasmic staining

  • 10% Nitric acid + 0.5% Chromic acid + absolute ETOH
  • Maceration avoided by chromic/ethyl
  • decalcification time: 2-7 days
• Phloroglucin–Nitric Acid is the most rapid nitric acid and is recommended for urgent work

  • Nitric + Phloroglucin → dense white fumes are formed, add 10% Nitric acid after disappearance
  • decalcification time: 12–24 hours
• In Phloroglucin-nitric acid, when decalcification is complete, acid must be removed by 3 changes of 70-90% ethanol, because watery solution can cause tissue distortion
• Hydrochloric Acid is inferior compared to Nitric Acid as a decalcifying agent; it has slower action, and greater tissue distortion

  • Good nuclear staining (use 1% solution + 70% alcohol)
  • Recommended only for surface decalcification (e.g., Scalp)
• Von Ebner’s Fluid is a good cytologic staining and moderately rapid decalcifying agent; it does not require washing out before dehydration

  • NaCl + HCl + H20
  • Recommended for teeth and small pieces of bones
• Formic acid has a better nuclear staining with less tissue distortion; it is safer to handle than Nitric and HCl

  • Recommended for postmortem research tissues
• Addition of citrate in formic acid probably accelerates decalcification by chelating the calcium as it is liberated from the bones
• 10% Formic Acid acts as both fixatives and decalcifying agent; it permits excellent nuclear & cytoplasmic staining

  • Formic Acid (SG 1.80) + 10% Formol saline
  • Relatively slow
  • decalcification time: 2-7 days
• Formic Acid-Sodium Citrate Solution permits better nuclear staining than Nitric Acid; it is recommended for autopsy materials, bone marrow and cartilage

  • 45% Formic Acid + 20% Na Citrate (++ for acceleration in penetration to bones)
  • decalcification time: 3-14 days
• Formic acid-sodium citrate solution major disadvantage:

  A) 5% Na sulphate

  
• Trichloroacetic acid permits good nuclear staining

  • TCA + 10% Formol Saline
  • decalcification time: 4-8 Days
• Sulfurous acid is a very weak decalcifying agent; it is suitable only for minute pieces of bone
• Chromic acid or Flemming’s Fluid is both a fixative and a decalcifying agent

  • Chromic acid + Osmium tetroxide + glacial HAc
• Chromic acid/Flemming's fluid major disadvantage:

  A) hematoxylin

  
• Chromic Acid is an environmental toxin highly corrosive to skin and mucous membrane carcinogenic
• Citric acid-citrate buffer solution permits good nuclear and cytoplasmic staining and it does not produce cell or cell distortion

  • 7% Citric Acid + 7.4% Ammonium Citrate + 1% Zinc sulfate + Chloroform
  • pH of 4.5
  • decalcification time: 6 days
• Chelating agents are substances which combines with calcium ions and other salts
• EDTA (Versene, Sequetrene) is the most common chelating agent

  • EDTA disodium salt + H2O + HCHO
  • Does not bind to calcium below pH 3.0
  • Faster at pH 7.0-7.4
  • Permits excellent staining; but very slow
  • decalcification time for small specimen: 1-3 weeks
  • decalcification time for dense cortical bones: 6-8 weeks
• EDTA inactivates alkaline phosphatase, magnesium chloride is added to activate
• Ion Exchange Resins is an ammonia form of Polystrene Resin; it hastens decalcification by removing Ca ions from Formic acid-containing decalcifying solutions

  • decalcification time: 1-14 days
  • degree of calcification cant be measured by chemical means
  • artifacts produced, usually caused by CO2 bubbles
  • very slow
• Electrophoresis or electrical ionization is the process whereby positively charged Ca ions are attracted to a negative electrode and subsequently removed from the decalcifying agent, satisfactory for small bone fragments
• Solutions used in electrophoresis:

  • 88% Formic Acid
  • Concentrated HCl
  • Distilled water
• Ratio of decalcification should be 20:1 = Fluid to tissue
• Heat hastens decalcification, but increases damaging effect of acids to tissues
• Optimum temperature in decalcification is 18-30°C