Decalcification

Question 1

What mineral does bone, dentine, arteries, cartilage and certain other abnormal tissues contain will cause cutting difficulties during microtomy due to its extreme hardness?

Answer 1

Hydroxyapatite, a complex molecule composed of calcium and phosphate.

Question 2

What is the risk in using decalcifying agents? When do you decalcify?

Answer 2

Decalcifying agents are very harmful to the tissue.

Therefore, decalcification is always carried out AFTER appropriate fixation has occurred.

Question 3

Why should decalcification solution be changed frequently?

Answer 3

Calcium ions migrate out of the tissue into the surrounding solution and the solution may become saturated with calcium ions and hence no calcium ions come out of solution.

Question 4

What is the purpose of decalcification?

Answer 4

Purpose – remove calcium salts from tissue (bone, arteries) to allow routine sectioning with a microtome. If they were left in the sample the sections would just shred making it impossible to view under a microscope.

Note: tissues must be thoroughly fixed prior to this process

Question 5

What are the two main methods of decalcification?

Answer 5

There are two main methods of decalcification:

1. Acid Methods
2. Chelating Agents

Question 6

What is the method and/or acid selected based on?

Answer 6

METHOD and ACID SELECTED BASED ON:

1. urgency of case
2. degree of mineralization
3. scope of investigation

Question 7

What does the rate of decalcification depend on?

Answer 7

RATE OF DECAL DEPENDS UPON:

1. size of tissue
2. density of tissue
3. type of decal agent
4. volume of decal agent

Question 8

What should be done after fixation prior to the decalcification procedure?

Answer 8

Wash tissues well with water post fixation and prior to decalcification procedure

Question 9

What affect do fixatives still remaining in the tissue have on decalcification?

Answer 9

• Phosphate buffers in 10%NBF will also buffer the decalcification reagent
• If using HCl, it can combine with formalin to form a toxic bis-chloromethyl-ether
• Zinc in fixatives will form a chelate with EDTA

Question 10

What negative effects will acid have on the tissue when used for decalcification?

Answer 10

• Acids will effect the stainability of tissues
• The stronger the acid = more damage to the tissue and poorer quality stain
• The longer it is an acid = more damage to the tissue and poorer quality stain
• Acids affect the nuclear staining the most

Question 11

How do you mitigate the effect of acids?

Answer 11

1. Fixing the tissue first helps minimize the effects on cells and collagen fibres
2. Must monitor the end point – just leave in as long as required.

Question 12

How do acids act on calcium?

Answer 12

Acids act on the calcium - hydroxyapatite to release a calcium salt

Question 13

How is the pH to be adjusted for the decalcification process?

Answer 13

Calcium salts are soluble at a pH of 4.5
Acids used to remove calcium will have a pH below that (0.5 – 3.0)
If we buffer the acid it will be closer to pH 4.5 not as harmful to the tissue or staining

Question 14

Describe use of NITRIC ACID and HYDROCHLORIC ACID for decalcification.

Answer 14

NITRIC ACID and HYDROCHLORIC ACID
strong acids
5% - 10% used
rapid – urgent, biopsy
1-2 days
may damage tissue

Question 15

Describe use of FORMIC ACID for decalcification.

Answer 15

FORMIC ACID
Weak acid
Slow - routine, non urgent
Up to 10 days
Very little damage & staining maintained

Can be mixed with 10%NBF to fix and decalcify in one step

Question 16

Describe use of commercial acid decalcifiers for decalcification. Name some examples.

Answer 16

COMMERCIAL ACID DECALCIFIERS
CAL-EX, RDO
Contents are a secret
Usually strong acids, buffered to have pH of 4.5
More gentle on tissues and allow better staining
Used frequently in the hospital labs
Are more expensive than other simple acids

Question 17

What are commercial acid decalcifiers really good for?

Answer 17

Great for urgent specimens and bone marrow biopsies

Question 18

Why does the acid solution need to be changed frequently?

Answer 18

As the acid solution allows the Calcium ions to be removed from the tissue they are brought into the solution.
The acid solution can become saturated with the ions and actually prevents further decalcification
The acid solution needs to be changed frequently

Question 19

What aids can be used to speed up decalcification?

Answer 19

Aids to help speed up the decalcification:
Agitation
Vacuum
Suspension in a bag – exposes all surfaces to the action of the decalcification solution, salts go to the bottom of the bag

Question 20

What should one never use to decalcify?

Answer 20

NEVER USE HEAT – causes severe damage to other components of the tissue

Question 21

Describe ion exchange resins use for decalcification (Acid type)?

Answer 21

Ion Exchange Resins
Uses formic acid over a layer of an ammonical salt of a sulfonated resin.
Calcium ions go out of tissue and ammonium ions go in

Question 22

Describe electrolytic method use for decalcification (Acid type)?

Answer 22

Electrolytic Method
Specimen is attached to the anode (positive pole) and when a current is passed through an acid solution, the calcium is drawn toward the cathode (negative pole)
Very quick but may generate heat

Question 23

Describe the chelating agent (EDTA) method of decalcification. Rate, adverse effects, etc.

Answer 23

CHELATING AGENT – EDTA
Uses organic compounds that bind to certain ions such as calcium (also iron, magnesium)
pH more neutral or slightly alkaline
very slow – 6-8 weeks
very  tolerant
Has no adverse effects on staining

Question 24

What is the END POINT DETERMINATION?

Answer 24

Decalcification methods can damage the tissues and negatively affect its staining. We must remove the tissue from the solution as soon as the calcium has been removed. We have to be able to tell when the all of the calcium has been removed.