Decalcification

Question 1

What is the definition of decalcification?

Answer 1

The controlled removal of calcium from calcified tissues to allow for paraffin infiltration and sectioning.

Question 2

What is the first, crucial step that must be done to tissues before they are treated with decalcifying agents?

Answer 2

Tissues must be properly fixed.

Question 3

Name the four main methods of decalcification.

Answer 3

Acid methods

Ion exchange resins

Electrolytic method

Chelating agents

Question 4

What characteristic of a bone determines whether a strong or weak acid should be used as the decalcifying agent?

Answer 4

Bone size.

Question 5

What are the two main risks associated with incorrect decalcification time?

Answer 5

Over-decalcification: Leads to a loss of nuclear morphology.

Under-decalcification: Makes sectioning difficult.

Question 6

What is the primary danger of using a strongly acidic solution or exposing tissue to acid for an extended period?

Answer 6

It will damage the tissue and render it un-stainable (e.g., no nuclear staining).

Question 7

At what pH do calcium salts become soluble and ionize?

Answer 7

At pH 4.5.

Question 8

What is the typical pH range of decalcifying agents, and what must be done to them before use?

Answer 8

They typically have a pH of 0.5 to 3.0 and must be buffered to pH 4.5 before use.

Question 9

List four acids commonly used for decalcification, including the concentration range.

Answer 9

5%-10% solutions of:

Hydrochloric acid

Nitric acid

Formic acid

Formic acid + formaldehyde (for simultaneous fixation and decalcification)

Question 10

Why is it necessary to change the decalcifying solution frequently and agitate it?

Answer 10

To prevent the solution from becoming saturated with calcium ions. A saturated solution forms a “barrier” that stops further decalcification.

Question 11

Why should heat never be used to speed up the decalcification process?

Answer 11

Heat will result in the swelling and maceration (breaking down) of the tissues.

Question 12

Explain how the Ion Exchange Resin method works and what its main advantage is.

Answer 12

Formic acid is used over a layer of ammonium salt of sulfonated resins. Ammonium ions from the resins are exchanged for calcium ions from the tissue. This keeps the solution free of calcium ions, speeding up decalcification and eliminating the need for frequent solution changes.

Question 13

What are the benefits of the Ion Exchange Resin method on the final tissue quality?

Answer 13

Staining is excellent, and the method is not harmful to the tissues.

Question 14

Briefly describe the setup of the Electrolytic method.

Answer 14

Formic acid and hydrochloric acid are placed in an electroplating device. The bone is attached to the anode (+ pole), and a current is passed through the solution. Calcium ions are attracted to the cathode (- pole).

Question 15

What is a major disadvantage of the Electrolytic method, and how can its effects be mitigated?

Answer 15

The process generates heat, which is detrimental to tissue architecture and can render tissue un-stainable. To mitigate this, the acid solutions must be neutralized with 1% sodium bicarbonate after decalcification.

Question 16

What are the key characteristics of using EDTA for decalcification?

Answer 16

It is very slow-acting but causes little to no tissue damage.

Question 17

What are the three main methods for determining the end point of decalcification?

Answer 17

Physical or mechanical method
Chemical testing
Radiography

Question 18

How is the physical or mechanical method for determining decalcification performed?

Answer 18

By testing the flexibility of the specimen or probing it with a needle or pin.

Question 19

What is a major disadvantage of using the physical or mechanical method to test for decalcification?

Answer 19

It creates histologic artifacts, which can damage the tissue sample and make it difficult to examine under a microscope.

Question 20

Which method is considered the most accurate for determining if demineralization is complete?

Answer 20

Radiography.

Question 21

In radiography, what indicates that decalcification is complete?

Answer 21

Zero opaqueness of the specimen on the X-ray.

Question 22

What important precaution must be taken if using radiography to check decalcification?

Answer 22

Do not use metallic-based fixatives, as they can interfere with the results.

Question 23

What is the first step in the chemical testing method for determining the end point of decalcification?

Answer 23

Take 5 ml of the decalcifying fluid from day 2.
Add ammonium hydroxide to the sample until it is neutralized.
Observe the solution for turbidity (cloudiness) caused by the formation of calcium oxalate.

Question 24

In the chemical test, what does a positive result (+) for turbidity in Step 1 indicate, and what action should be taken?

Answer 24

A positive result (+) means the specimen is still heavily calcified. You should change the decalcifying solution and continue the process.

Question 25

What is the procedure for Step 2 of the chemical test for decalcification?

Answer 25

Add 5 ml of saturated ammonium oxalate to the neutralized sample from Step 1. Mix the solution and let it stand for 30 minutes. Observe for turbidity (cloudiness) caused by the formation of calcium oxalate.

Question 26

In the chemical test, what does a negative result (-) for turbidity in Step 2 indicate?

Answer 26

A negative result (-) means that decalcification is complete. The specimen is ready for further processing.

Question 27

What is the significance of observing turbidity in both steps of the chemical test?

Answer 27

Turbidity indicates the presence of calcium oxalate, which forms when free calcium ions in the decalcifying fluid react with the ammonium hydroxide/oxalate. It signifies that calcium is still being leached from the specimen.

Question 28

What specific problem do micro-calcifications cause during microtomy, and what is the term used to describe the resulting artifact on the tissue?

Answer 28

They cause nicks on the blade. The resulting artifact on the tissue is called "scoring."

Question 29

What is the solution for sectioning a tissue block that contains micro-calcifications?

Answer 29

Perform surface decalcification.

Question 30

What are the three main steps in the surface decalcification process?

Answer 30

Place the exposed surface of the tissue block on decalcification fluid. Leave it for 30-60 minutes. Rinse the block well and section gently.

Question 31

Why is it important to rinse the block well after surface decalcification?

Answer 31

To remove any remaining decalcification fluid, which could interfere with staining or further damage the tissue and blade if left to dry on the block.

Question 32

If a histotechnologist encounters a hard, gritty block that is causing "chattering" or "scoring" of the sections, what is the most appropriate initial troubleshooting step?

Answer 32

Perform surface decalcification by placing the cut surface of the block on decal fluid for 30-60 minutes before attempting to section again.

Question 33

Why is it necessary to section "gently" after surface decalcification?

Answer 33

Even after surface decalcification, the tissue may still be slightly more brittle or delicate than usual. Gentle sectioning helps to prevent the tissue from shattering, crumbling, or folding.