DECALCIFICATION (Lecture)

DECALCIFICATION

Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding tissues of bones

DECALCIFICATION

Not a mandatory step in tissue processing

DECALCIFICATION

It must be done following fixation. Adequate fixation must be first achieved prior to decalcification. Done manually

DECALCIFICATION

Both _________ and processing depend on bone thickness.

DECALCIFICATION

Heat and constant agitation speed up _________

Calcium and lime salts

make tissue hard to cut

1-3 mm

Ideal thickness is

20:1

Ratio of fluid to tissue is

Room Temperature

Optimal temperature:

Autotechnicon

cannot perform decalcification. It can only do the first 4 steps in tissue processing

ELECTROLYTIC METHOD

• Most rapid. Through electricity, calcium is removed.

• Dependent upon the supply of electric current

ELECTROLYTIC METHOD

The positively charged calcium ions are attracted to negative electrodes from the decalcifying solution thus facilitating calcium removal.

ION EXCHANGE RESIN

Calcium is rapidly removed by the decalcifying solution containing formic acid, thereby increasing solubility from tissues

ION EXCHANGE RESIN

The process is longer. Duration: 1-14 days

ammonium form of polystyrene resin

In ION EXCHANGE RESIN, the specimen place in a container. At the bottom of the container, place an _____________. This resin will increase tissue solubility to facilitate removal of calcium.

ION EXCHANGE RESIN and USE OF CHELATING AGENT: EDTA

Not usually carried out in the lab because it is not suited for

urgent biopsy

USE OF CHELATING AGENT: EDTA

It is considered as the best decalcifier in electron microscopy and immunohistochemistry.

magnesium chloride

In USE OF CHELATING AGENT: EDTA, remedy if Inactivate alkaline phosphatase activity.

1-3 weeks

For small specimen

6-8 weeks

Fore dense tissues

USE OF ACID

The most common method of decalcification. Manually done

USE OF ACID

Concentrated solutions are not used. Dilution is needed.

10% Aqueous Nitric acid

• commonly used in routine

• Duration of process: 24 hours (depends on the type of the specimen)

longer time

In 10% Aqueous Nitric acid, Larger tissues:

shorter time

In 10% Aqueous Nitric acid, smaller tissues:

Formol nitric acid

• Nitric acid + formaldehyde

• Requires fume hood

• Duration: 1-3 days

Parenyi’s fluid

• Double purpose: Decalcifying acid

  and tissue softener

• Nitric acid + chromic acid + ethyl alcohol

• Duration: 2-7 days

Phloroglucin Nitric

• Phloroglucin + nitric acid

• Most rapid among other nitric containing acid

HYDROCHLORIC ACID

• Not commonly used. Weak and slow.

• Recommended only for minute pieces of bones

Von Ebner’s

• Hydrochloric acid + sodium chloride

• For teeth and small pieces of bones

• For surface decalcification of blocks

FORMIC ACID, SODIUM CITRATE

For decalcifying cartilage, research specimens, autopsy specimens, bone marrow

TRICHLOROACETIC ACID (TCA), SULFUROUS ACID

Weak and slow decalcifying agents

CHROMIC ACID / FLEMING’S WITH HAC

• Not commonly used

• Considered as an environmental toxin

• HIghly corrosive on the skin

CITRIC ACID, CITRATE BUFFER

Contains chloroform as preservative

PHYSICAL / MECHANICAL METHOD

• Not a reliable method. Easy to do.

• Done by bending the tissue, pricking, probing the tissue with a needle

X-RAY / RADIOLOGIC METHOD

• Most reliable and accurate

• Can detect even the smallest amount of calcium

• Look for opaqueness in the x-ray film

X-RAY / RADIOLOGIC METHOD

Disadvantage: Not suited for mercuric chloride fixed tissues

Opaqueness

In x-ray, decalcification is not yet complete

CHEMICAL METHOD/ CALCIUM OXALATE METHOD

5 mL of discarded fluid in a tube + strong ammonia (to make it alkaline)

ammonium oxalate

In CHEMICAL METHOD/ CALCIUM OXALATE METHOD, If alkaline (check with litmus) add

(+) cloudiness

In chemical method, decalcification is not yet complete, it must be decalcified further

Clear

In chemical method, decalcification is complete

BUBBLE TEST

Carried out by adding calcium carbonate

Presence of bubble

In bubble test, decalcification is not yet complete

DECALCIFIED TISSUES ARE NEUTRALIZED

1. 2. Immersing in saturated lithium carbonate

Immersing in 5-10% sodium bicarbonate.

3. Rinsing in tap water.

4. Storing in formol saline with 15% sucrose of PBS with

15-20% sucrose at 4 degrees celsius

TISSUE SOFTENERS FOR UNDUL Y HARD TISSUES

1. 2% HCl

2. 1% HCl in 70% Alcohol

3. Perenyi’s fluid

4. Molliplex

5. Lendrum’s Method