Decalcification

Question 1

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

Answer 1

Decalcification

Question 2

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

Answer 2

20:1

Question 3

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

Answer 3

Nitric Acid (5-10%)

Question 4

Contains formalin, less tissue distortion, rapid for urgent biopsies

Answer 4

Formol Nitric Acid

Question 5

Decalcifies and softens tissues without maceration; contains chromic acid and alcohol; cannot determine complete decalcification by chemical test

Answer 5

Perenyi’s Fluid

Question 6

Most rapid decalcifying agent, 12-24°C

Answer 6

Phloroglucin Nitric Acid

Question 7

Inferior to HNO3, slower, produces more distortion; good for surface decalcification and nuclear staining at 1% concentration

Answer 7

HCl (Hydrochloric Acid)

Question 8

Contains 36% NaCl, recommended for teeth and small pieces of bone

Answer 8

Von Ebner’s Solution

Question 9

Moderate acting, slow decalcifying agent, suitable for routine decalcification of postmortem tissues; addition of citrate hastens decalcification; also a fixative; best for nuclear staining

Answer 9

Formic Acid

Question 10

Better nuclear staining than nitric acid; recommended for autopsy materials, BM, cartilage, and tissues for research

Answer 10

Formic Acid Sodium Citrate Solution

Question 11

Weak decalcifying agent and fixative, permits good nuclear staining, slow, suitable for small bone spicules

Answer 11

TCA (Trichloroacetic Acid)

Question 12

Weak decalcifying agent, only for minute pieces of bone

Answer 12

Sulfurous Acid

Question 13

Both a fixative and decalcifying agent for minute bone spicules; carcinogenic, oxidizing agent

Answer 13

Chromic Acid

Question 14

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

Answer 14

Chelating Agents (EDTA)

Question 15

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

Answer 15

Ion Exchange Resin (Ammonium form of polystyrene)

Question 16

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

Answer 16

Electrophoresis/Electrical Ionization

Question 17

Faster decalcification technique using a microwave oven

Answer 17

Microwave Oven Decalcification

Question 18

Bending or touching the tissue with a needle; rubbery texture indicates decalcification; subjective and prone to artifacts; may destroy cellular details

Answer 18

Physical or Mechanical Test (Extent of Decalcification)

Question 19

Expensive but ideal radiologic method; not suitable for tissues fixed with mercuric chloride due to radio-opacity

Answer 19

X-ray Method (Extent of Decalcification)

Question 20

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

Answer 20

Chemical Method (Extent of Decalcification)

Question 21

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

Answer 21

Post-Decalcification

Question 22

Added to hard tissues to facilitate cutting and processing; can be added to dehydrating agents

Answer 22

Tissue Softeners

Question 23

Decalcifies and softens tissues

Answer 23

Perenyi’s Fluid

Question 24

Contains 4% phenol; tissues appear swollen and soapy

Answer 24

Lendrum’s Tissue Softener