HPCT311 Midterms: Lesson 2 - Decalcification

Process of removing Calcium or Lime Salts from tissues, which is performed on bones, teeth, calcified tissues

Decalcification

If the tissue size is very large, what should you use cutting?

Saw (fine-fret)

Ratio of the volume of decalcfying agent to be used from the specimen

20:1

These accelerates decalcification process, besides suspending a tissue in a decalcifying agent

1. Heat

2. Agitation

This produces effect on nuclear staining, wherein nuclear chromatin fails to take up hematoxylin.

Rapid decalcification

General duration of decalcification

1-20 days

If there is a grating sensation during cutting, what should be done?

place block in 10 % HCl for 1 hour

What are the types of decalcifying agents?

1. Acids

2. Chelating Agents

3. Ion Exchange Resins

4. Electrical Ionization

Most common type of decalcifying agents

ACIDS

MODIFIED TRUE OR FALSE:

Acids are excellent decalcifying agents since it is accessible and stable. However, they are expensive.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

A.

Most common type of acid decalcifying agent and is also considered a rapid decalcifying agent.

Nitric Acid

Nitric acid inhibits nuclear stain. How is it prevented?

by combining with

formaldehyde or alcohol

A type of Nitric acid decalcifying agent which are recommended for both urgent biopsy and needle biopsy.

10% Aqueous Nitric Acid

What is the composition of of 10% Aqueous nitric acid

10mL Conc. Nitric acid + 100 mL dH2O

A type of nitric acid decalcifying agent which is used for urgent biopsies and imparts less tissue destruction than other types of nitric acid decalcifying agent

Formol-Nitric Acid

Composition of Formol-Nitric Acid

Conc. Nitric Acid + 40% Formalin + dH2O

A nitric acid decalcifying agent which is good for nuclear and cytoplasmic staining, which decalcifies and softens calcified tissues

Perenyi’s fluid

Composition of Perenyi’s fluid

10% Nitric acid + 0.5% Chromic acid + absolute ETOH

Composition of Phloroglucin Nitric Acid

Conc. Nitric + Phloroglucin

Most rapid among all nitric acid decalcfyong agent

Phloroglucin Nitric Acid

When dense white fumes are formed in preparing phloroglucin nitric acid decalcifying agent, what should you do next?

add 10% Nitric acid after disappearance of white fumes

When when decalcification is completed and the agent used is phloroglucin-nitric acid, what should be done next?

acid must be removed by 3 changes of 70 to 90% ethanol

A type of acid decalcfying agent which is recommended for surface decalcification.

Hydrochloric Acid

MODIFIED TRUE OR FALSE

Hydrochloric Acid is superior to Nitric Acid in terms of decalcifying calcified tissues. Also, it has good nuclear staining.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.

A type of HCl decalcifying agent which has a good cytologic staining and is recommended for teeth and small pieces of bones.

Von Ebner’s Fluid

MODIFIED TRUE OR FALSE:

Formic Acid decalcifying agent has better nuclear staining but imparts more tissue distortion. However, It is generally safer to handle this type of decalcifying agent han Nitric Acid and HCl decalcifying agents.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.

A type of acid decalcifying agent which is recommended for postmortem research tissues upon the addition of sodium citrate

Formic acid

A type of acid decalcfying agent which is recommended for autopsy materials, BM, cartilage and tissue for research

Formic Acid

A type of formic acid decalcifying agent which permits excellent nuclear & cytoplasmic staining but is slow in action.

10% Formic Acid

A type of formic acid decalcifying agent which acts as both fixative and decalcifying agent.

10% Formic Acid

Composition of 10% Formic Acid

Formic Acid (SG 1.20) + 10% Formol saline

A type of formic acid decalcifying agent which is not recommended for routine purposes but permits better nuclear staining than nitric acid

Formic-Acid Sodium Citrate Solution

Formic-Acid Sodium Citrate Solution requires neutralization in preparing the reagent. What is the agent used to neutralize it?

5% Na sulfate

Composition of Formic-Acid Sodium Citrate Solution

45% Formic Acid + 20% Na Citrate

A weak decalcifying agent which is suitable only for small bone spicules but permits good nuclear staining.

Trichloroacetic acid

Composition of TRICHLOROACETIC ACID (TCA)

TCA + 10% Formol Saline

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

Sulfurous Acid

A fixative and decalcifying agent, which is not recommended for nuclear staining and is also carcinogenic and corrosive to skin

Chromic Acid (Flemming’s Fluid)

Composition of Flemming’s Fluid

Chromic acid + Osmium tetroxide + glacial HAc

pH of Citric Acid-Citrate Buffer

4.5

Composition of Citric Acid-Citrate Buffer

7% Citric Acid + 7.4% Ammonium Citrate + 1% Zinc sulfate + Chloroform (preservative)

Substances which combine with Calcium ions and other salts (Fe, Mg)

Chelating Agents

Most common chelating agent

EDTA/Versene/Sequestrene

EDTA primary mode of action is to chelate calcium. However, in what condition it does not bind to Calcium?

if Ca is at pH below 3.0

EDTA inacivates what type of enzyme? What is the solution?

Alkaline phosphatase; add magnesium and chloride

A type of decalcifying agent which is excellent for immunohistochemical or enzyme staining for microscopy

Chelating Agents

MODIFIED TRUE OR FALSE

EDTA permits excellent staining of tissues. However, it is a very slow decalcifying agents.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

C.

Hastens decalcification by removing Ca ions from Formic

acid-containing decalcifying solns

Ion Echange Resins

Ion Exchange Resins are also known as

Ammonia form of Polystyrene Resin

What causes the artifact production of ion exchange resins?

CO2 bubbles

process whereby positively charged Ca ions are attracted to a negative electrode and subsequently removed from the decalcifying agent

Electrophoresis (Electrical Ionization)

A type of decalcifying agent which is satisfactory for small bone fragments and uses electricity to decalcify tissues.

Electrophoresis

What are the solutions used in decalcifying tissues using electrophoresis?

Formic Acid 88%, Conc. Hydrochloric Acid, distilled water

Types of tests for completeness of decalcification

1. Physical/Mechanical

2. X-ray/Radiological

3. Chemical

A test used for completeness of decalcification which is done by bending or touching the tissue using a needle.

Physical/mechanical

A test for completeness of decalcification which is very expensive and is not ideal

X-ray/Radiological

TRUE OR FALSE

Radiological test for completeness of decalcification can be used for tissues fixed with mecuric chloride

FALSE

Reagent used for chemical test for completeness of decalcification?

Calcium Oxalate

List the process of testing the completeness of decalcifying agent

1. BLUE litmus paper + 5mL discarded decalcifying agent

2. Litmus paper becomes RED due to acidity

3. Add NH3 drop by drop to neutralize (litmus change to BLUE )

4. If CLOUDY → still with calcium;

5. if CLEAR → add ammonium oxalate,

6. 30 mins → CLOUDY if incomplete

Give at least 3 reagenst used as tissue softeners

1. Perenyi’s Fluid– 12-24 hours

2. 4% Aqueous phenol – 1-3 days

3. Molliflex (swollen & soapy appearance)

4. 2%HCl

5. 1%HCl in 70%alcohol

In post decalcification how do you remove the acid?

Saturated lithium carbonate solution or 5-10% aqueous NaHCO3 for several hours

If EDTA is used in decalcifying, what agent should be used in removing the acid

70% alcohol

Ideal time for rate decalcification

24-48 hours

MODIFIED TRUE OR FALSE

The more the concentrated the acid solution is, the more rapid it decalcifies tissues. Also, it is more it is harmful to the tissue.

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

C.

MODIFIED TRUE OR FALSE

Heat slows down decalcification. Also, it increases the damaging effect of acids to tissues

A. Statement 1 is true

B. Statement 2 is true

C. Both statements are true

D. Both statements are false

B.