3. DECALCIFICATION

decalcification

the process of removing calcium salts from the organic ECM, calcified collagen and surrounding tissues of the bone

20:1

ideal fluid to tissue volume ratio for decalcification

24 - 48 hrs

ideal time require for decalcifying tissue

14 days

how long does it take to decalcify dense bones?

2-3 mm

ideal thickness for decalcification?

1. concentration of decalcification agents

2. ratio of decalcifying agent to tissue

3. mechanical agitation

4. temperature

5. ideal time required

ENUMERATE

factors affecting the rate of decalcification

18-30 °C

ideal temperature for decalcification?

1. Ion exchange resin

2. Acid decalcifying agent

3. Chelating agent

4. Electrophoresis

ENUMERATE

methods of decalcification

False

it is done manually

T or F

acid decalcifying agent can also be done manually

acid decalcifying agent

are the most widely used agents of large amounts of bony tissues because they are stable, easily available, and relatively inexpensive

• nitric acid

• hydrochloric acid

• formic acid sodium citrate,

• TCA

• sulfurous acid

• chronic acid/fleming’s with hac

• citric acid citrate buffer

ENUMERATE

ex of acid decalcifying agent

gauze bag

As soon as fixation is complete, the selected pieces of tissues are usually placed in a?

nitric acid

most common and the fastest decalcifying agent used so far, utilized both as a simple solution or combined with other reagents.

Formol Nitric Acid

Nitric acid with formaldehyde

5-10%

conc of nitric acid?

24 hrs

decal time of Aqueous Nitric Acid Solution 10%

5 - 10 % solution

Aqueous Nitric Acid Solution 10% is routinely diluted to __________________

1-3 days

decal time of Formol-Nitric Acid

Perenyi’s Fluid

decalcifying agent + soften tissue at the same time

2 - 7 days

decal time of Perenyi’s Fluid

• nitric acid

• chromic acid

• absolute ethyl alcohol

ENUMERATE

composition of Perenyi’s fluid

yellow

nitric acid can impart what color?

0.1% urea to pure concentrated nitric acid

what agent can remove the color imparted by nitric acid?

fume hood

the Formol-Nitric Acid should be used inside a?

Phloroglucin-Nitric Acid

It is the most rapid decalcifying agent so far, recommended for urgent cases.

False

Nuclear staining is poor.

T or F

Phloroglucin-Nitric Acid nuclear staining capabilities is excellent

HYDROCHLORIC ACID

is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section.

HYDROCHLORIC ACID

reagent recommended for surface decalcification

Von Ebner's Fluid

the only decalcifying agent that contains HCl (36%)

Von Ebner's Fluid

it is a weak sol’n and it is not routinely used

• NaCl

• HCl

• Distilled H2O

ENUMERATE

composition of Von Ebner's Fluid

formic acid sodium citrate

used for cartilage, bone marrow, research / autopsy spx.

• trichloracetic acid (TCA)

• sulfurous acid

decal agent that is weak and slow

chromic acid / fleming’s with HAC

decal agent that contains HCl; not common ; toxic and corrosive

CITRIC ACID CITRATE BUFFER

Contains chloroform as a preservative.

chloroform

_______________ acts as a preservative and a decalcifying agent

• formic acid

• chromic acid

• sulfurous acid

• trichloroacetic acid (TCA)

ENUMERATE

chemicals used both as fixative and decalcifying agent

5% formic acid

what is the best general decalcifying agent

chromic acid

a decal and fixative agent that is carcinogenic, corrosive to skin and mucus membrane

trichloroacetic acid

a “weak” decal agent

sulfurous acid

a “super weak” decal agent

chelating agents

substances that combine with calcium ions and other salts to form weak dissociated complexes and facilitate removal of calcium salts.

• electron microscopy

• enzyme

• immunohistochemical staining

ENUMERATE

chelating agents is ideal for:

Versene

commercial name of EDTA

EDTA (versene)

most common chelating agent

detailed microscopic studies

EDTA us only recommended for?

• small specimens: 1-3 weeks

• cortical bone: 6-8 weeks (or longer)

Identify decal time (EDTA)

• small specimens:___________

• cortical bone: ____________

3 days

replace EDTA every ___ days

7 - 7.4 pH

what should be the pH of EDTA when using it

Ion Exchange Resin Method

Formic acid dissolves calcium, resin increases tissue solubility

CAL-EX

HCl acid + EDTA

mineral acids

ion exchange resin is not suitable for?

20-30x

in ion exchange resin the decalcifying agent is usually ________ the volume of the tissue

electrophoresis

process whereby Ca- ions which are positively charged are attracted to e negatively charged electrode and removed decalcifying solution satisfactory for small bone fragments

• 88% formic acid

• concentrated HCl

• distilled h2O

• NaCl

ENUMERATE

solutions used for electrolytic decalcification

• perenyi’s fluid

• lendrums

• mollifax

• 2% HCl

• 1% HCl in 70% alcohol

ENUMERATE

tissue softeners

Physical Method/Mechanical

Bedding the tissue; pricking the tissue; probing the tissue with a needle. Easy to do but is not reliable

X-ray/Radiologic Method

Most reliable and accurate method. Can detect even the smallest amount of calcium. In this method we look for the opaqueness of the film

not suited for mercuric chloride fixative

disadvantage of X-ray/Radiologic Method

Bubble Test

Carried out by adding calcium carbonate as a reagen

not yet complete

The presence of bubbles (meaning the decalcification process is ____________)

ammonium hydroxide

used to screen the presence of Ca- salts