Decalcification

Question 1

What is decalcification?

Answer 1

The removal of calcium or lime salts from bones or calcified tissues following fixation.

Question 2

What is the ideal ratio for decalcification solutions?

Answer 2

20:1 (solution to tissue).

Question 3

What factors hasten decalcification?

Answer 3

Heat, agitation, and suspension of tissue.

Question 4

What is the typical duration of decalcification?

Answer 4

1 to 2 days.

Question 5

At what temperature is decalcification typically performed?

Answer 5

Room temperature.

Question 6

What effect do more concentrated acid solutions have on decalcification?

Answer 6

They decalcify more rapidly but may destroy tissue.

Question 7

What are the four main categories of decalcifying agents?

Answer 7

Acids, Chelating Agents, Ion Exchange Resins, and Electrophoresis.

Question 8

What is the most widely used agent for routine decalcification?

Answer 8

Nitric Acid (5-10%).

Question 9

Why is Nitric Acid considered the most common decalcifying agent?

Answer 9

It’s rapid and effective for routine decalcification.

Question 10

What causes the yellow color during nitric acid decalcification?

Answer 10

The formation of nitrous acid.

Question 11

How can the yellow color from nitric acid be remedied?

Answer 11

By adding Sodium thiosulfate or Urea Crystals.

Question 12

How can nitric acid be removed from tissues?

Answer 12

By 70% alcohol.

Question 13

What is Perenyi’s Fluid?

Answer 13

A nitric acid decalcifying agent that decalcifies and softens tissues at the same time without maceration due to chronic acid and alcohol.

Question 14

Which nitric acid agent is considered the most rapid decalcifying agent?

Answer 14

Phloroglucinol Nitric Acid.

Question 15

What is Formol Nitric Acid used for?

Answer 15

For urgent biopsies, providing rapid decalcification.

Question 16

What is Hydrochloric Acid (HCl) best used for in decalcification?

Answer 16

Good for surface decalcification of tissue blocks.

Question 17

What is Von Ebner’s solution?

Answer 17

A solution containing 36% NaCl recommended for teeth and small pieces of bones.

Question 18

What is Formic-Nitric Acid?

Answer 18

A slow to moderate acting decalcifying agent suitable for routine decalcification and post mortem research tissues.

Question 19

How can formic-nitric acid decalcification be hastened?

Answer 19

By the addition of citrate.

Question 20

What is Formic Acid Sodium Citrate solution particularly good for?

Answer 20

It has better nuclear staining compared to nitric acid and is suitable for autopsy materials, bone marrow, cartilage and tissues for research.

Question 21

What is the primary characteristic of Trichloroacetic Acid (TCA)?

Answer 21

A weak decalcifying agent that is fixative and permits good nuclear staining, though slow.

Question 22

What is Sulfurous Acid primarily used for?

Answer 22

A weak decalcifying agent used only for minute pieces of bone.

Question 23

What is another name for Chromic Acid?

Answer 23

Flemming’s Fluid.

Question 24

What are the characteristics of Chromic Acid as a decalcifying agent?

Answer 24

Both fixative and a decalcifying agent, used for minute bone spicules.

Question 25

What concentration of nitric acid produces very good nuclear staining?

Answer 25

1% concentration.

Question 26

Why might hydrochloric acid be preferred over nitric acid in some cases?

Answer 26

HCl is inferior compared to nitric acid (HNO₃) because it is slower and produces poorer nuclear staining.

Question 27

How do chelating agents work in decalcification?

Answer 27

They combine with calcium salts and other salts to form complexes and facilitate removal of calcium.

Question 28

What is the most commonly used chelating agent?

Answer 28

EDTA (versene or disodium EDTA).

Question 29

What is the duration for decalcification using EDTA?

Answer 29

1-3 weeks for small specimens, 6-8 weeks for larger and denser bones.

Question 30

What pH should be maintained when using EDTA?

Answer 30

pH should be adjusted to 7 to 7.4.

Question 31

For what types of procedures is EDTA particularly excellent?

Answer 31

For immunohistochemical or enzyme staining and for electron microscopy.

Question 32

How do ion exchange resins aid decalcification?

Answer 32

They hasten decalcification by using formic acid containing decalcifying solutions in ammonium form of polystyrene.

Question 33

What types of solutions are not recommended for use with ion exchange resins?

Answer 33

Fluids containing mineral acids such as nitric acid and hydrochloric acids.

Question 34

What is the recommended ratio when using formic acid decalcifying agent with ion exchange resin?

Answer 34

Formic acid decalcifying agent added is 20-30 times the volume of the tissue.

Question 35

What is the principle behind electrophoretic decalcification?

Answer 35

Positively charged calcium ions move toward the negative electrode using an electric charge.

Question 36

What are the advantages of electrophoresis for decalcification?

Answer 36

Shorter time of calcium removal.

Question 37

What sized specimens is electrophoresis suited for?

Answer 37

Small bone spicules.

Question 38

What concentration of formic acid is used in electrophoresis?

Answer 38

8% formic acid.

Question 39

What are the three main methods to measure the extent of decalcification?

Answer 39

Physical or Mechanical Test, X-ray Method, and Chemical Method.

Question 40

What does the Physical or Mechanical Test involve?

Answer 40

Bending or touching the tissue or using a needle, though this is prone to produce artifacts and may destroy cellular detail.

Question 41

Why is the X-ray Method considered ideal but limiting?

Answer 41

It's very expensive but most ideal; not suitable for mercuric chloride fixed tissues because of radio-opacity.

Question 42

What is the Calcium Oxalate Method also known as?

Answer 42

The Chemical Method.

Question 43

How does the Calcium Oxalate Method work?

Answer 43

Simple, reliable, and convenient detection of calcium in acid solutions by precipitation of calcium hydroxide or calcium oxalate; uses concentrated NH4OH and saturated (NH4)2C2O4.

Question 44

What indicates complete decalcification in the Calcium Oxalate Method?

Answer 44

Clear solution means complete decalcification while cloudiness or precipitation suggests incomplete decalcification.

Question 45

What color change occurs in litmus paper during the Calcium Oxalate test?

Answer 45

Litmus paper turns blue to red.

Question 46

What neutralization methods are used after decalcification?

Answer 46

Immersing in saturated Li2CO3 or 5-10% NaHCO3, rinsing in running tap water, or storing in formal saline containing 15% sucrose or PBS with 15-20% sucrose at 4 deg C.

Question 47

What may be added to hard tissues to facilitate cutting and processing?

Answer 47

Tissue softeners.

Question 48

What are some examples of tissue softeners used after decalcification?

Answer 48

Landrum's 4% phenol and 2% HCl or 1% HCl in 70% alcohol.