Decalcification Flashcards
Removal of calcium or lime salts from bones or calcified tissues following fixation (bone, teeth, atherosclerosis, BV, TB lesions); Types of
agents: acids, chelating agents, ion exchange resins, electrophoresis
Decalcification
More concentrated acid solutions decalcify more rapidly but may destroy tissue; ratio____ , room temperature, heat and agitation hasten decalcification, suspension in fluid also hastens decalcification; duration: 1-2 days
20:1
Most common, rapid decalcifying agent; acid can be removed by 70% alcohol; imparts yellow color due to nitrous acid formation; remedy: add 5% Na thiosulfate, urea crystals, concentrated solution
Nitric Acid (5-10%)
Contains formalin, less tissue distortion, rapid for urgent biopsies
Formol Nitric Acid
Decalcifies and softens tissues without maceration; contains chromic acid and alcohol; cannot determine complete decalcification by chemical test
Perenyi’s Fluid
Most rapid decalcifying agent, 12-24°C
Phloroglucin Nitric Acid
Inferior to HNO3, slower, produces more distortion; good for surface decalcification and nuclear staining at 1% concentration
HCl (Hydrochloric Acid)
Contains 36% NaCl, recommended for teeth and small pieces of bone
Von Ebner’s Solution
Moderate acting, slow decalcifying agent, suitable for routine decalcification of postmortem tissues; addition of citrate hastens decalcification; also a fixative; best for nuclear staining
Formic Acid
Better nuclear staining than nitric acid; recommended for autopsy materials, BM, cartilage, and tissues for research
Formic Acid Sodium Citrate Solution
Weak decalcifying agent and fixative, permits good nuclear staining, slow, suitable for small bone spicules
TCA (Trichloroacetic Acid)
Weak decalcifying agent, only for minute pieces of bone
Sulfurous Acid
Both a fixative and decalcifying agent for minute bone spicules; carcinogenic, oxidizing agent
Chromic Acid
Combine with calcium salts to form complexes; excellent for immunohistochemical or enzyme staining and electron microscopy; duration: 1-3 weeks for small specimens, 6-8 weeks for dense bones; pH adjusted to 7-7.4
Chelating Agents (EDTA)
Hastens decalcification when used with formic acid; not recommended with mineral acids like nitric acid or HCl; complete decalcification measured by physical or X-ray method
Ion Exchange Resin (Ammonium form of polystyrene)
Positive calcium ions move towards the negative electrode using an electric charge; shorter calcium removal time; suited for small bone fragments; uses 88% formic acid
Electrophoresis/Electrical Ionization
Faster decalcification technique using a microwave oven
Microwave Oven Decalcification
Bending or touching the tissue with a needle; rubbery texture indicates decalcification; subjective and prone to artifacts; may destroy cellular details
Physical or Mechanical Test (Extent of Decalcification)
Expensive but ideal radiologic method; not suitable for tissues fixed with mercuric chloride due to radio-opacity
X-ray Method (Extent of Decalcification)
Simple, reliable method; detects calcium in acid solutions by precipitation of calcium hydroxide or calcium oxalate; clear solution indicates complete decalcification; cloudiness or precipitation indicates incomplete decalcification
Chemical Method (Extent of Decalcification)
Neutralizes decalcified tissues by immersing in saturated Li2CO3 or 5-10% NaHCO3; rinsing in running tap water; storing in formol saline with 15% sucrose or PBS with 15-20% sucrose at 4°C
Post-Decalcification
Added to hard tissues to facilitate cutting and processing; can be added to dehydrating agents
Tissue Softeners
Decalcifies and softens tissues
Perenyi’s Fluid
Contains 4% phenol; tissues appear swollen and soapy
Lendrum’s Tissue Softener