HISTOPATH LAB: DECALCIFICATION

BASED ON STRONG MINERAL ACIDS

BASED ON WEAKER ORGANIC ACIDS

COMPOSED OF CHELATING AGENT

3 MAIN TYPES OF DECALCIFYING AGENTS

ACID DECALCIFYING AGENTS

Most widely used agents for routine decalcifications

STRONG MINERAL ACIDS

Most rapid in action but if used longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues.

NITRIC ACID

Most common and decalcifying agents. Produce minimal distortion. Recommended for routine use. Decalcification Time: 12-24 hours

CONCENTRATED NITRIC ACID 10 ML

DISTILLED WATER UP TO 100 ML

Nitric acid formula:

PERENYI’S FLUID

Decalcifying and softens tissues at the same time. Not recommended for urgent biopsies. Decalcification Time: 2-7 Days

NITRIC ACID 10% 40 ML

CHROMIC ACID 0.5% 30 ML

ABSOLUTE ETHYL ALCOHOL 30 ML

Perenyi’s fluid formula:

12-24 HRS

Nitric acid decalcification time:

2-7 DAYS

Perenyi’s fluid decalcification time:

PHLOROGLUCIN NITRIC ACID

Most rapid decalcifying agent. POOR nuclear staining. Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running water for 24 hours.

DECALCIFICATION TIME: 12-24 hours

CONCENTRATED NITRIC ACID 10 ML

PHLOROGLUCIN 1 GM

NITRIC ACID 10% 100 ML

PHLOROGLUCIN NITRIC ACID FORMULA:

12-24 HRS

PHLOROGLUCIN NITRIC ACID DECALCIFICATION TIME:

HYDROCHLORIC ACID

Slower action than nitric acid. Produce GREATER distortion of tissues. Produce GOOD nuclear staining. Recommended for surface decalcification of the tissue blocks.

VON EBNER’S FLUID

Permits GOOD CYTOLOGIC STAINING. Does not require washing out before dehydration. Recommended for TEETH and small pieces of bone. Decalcification cannot be measure by a chemical test.

SATURATED AQUEOUS SOLUTION OF NACI 50ML

36% CONCENTRATED HYDROCHLORIC ACID 8 ML

DISTILLED WATER 50 ML

VON EBNER’S FLUID FORMULA

FORMIC ACIDS

Moderate acting decalcifying agent. Produce better nuclear staining. Less tissue distortion

FORMIC ACID 10 ML

NORMAL SALINE 10% 90 ML

FORMIC ACIDS FORMULA

FORMIC ACID

Recommended for routine decalcification of POST MORTEM research tissues.

2-7 DAYS

FORMIC ACID DECALCIFICATION TIME

10%

____CONCENTRATION OF FORMIC ACID IS A BEST ALL AROUND DECALCIFIER

FORMIC ACID SODIUM CITRATE SOLUTION

Permits better nuclear staining than nitric acid method. Recommended for autopsy materials, bone marrow, cartilage, and tissues studied for research purposes. DECALCIFICATION TIME: 3-14 DAYS

3-14 DAYS

FORMIC ACID SODIUM CITRATE SOLUTION DECALCIFICATION TIME

AQUEOUS SODIUM CITRATE 20% 50 ML

FORMIC ACID 45% 50 ML

FORMIC ACID SODIUM CITRATE SOLUTION FORMULA

TRICHLOROACETIC ACID

Permits GOOD nuclear staining

Does not require washing out

Weak decalcifying agent, suitable for small spicules of bones. DECALCIFICATION TIME: 4-8 DAYS

4-8 DAYS

TRICHLOROACETIC ACID DECALCIFICATION TIME

TRICHLOROACETIC ACID 5 GM

FORMAL SALINE 10% 95 ML

TRICHLOROACETIC ACID FORMULA

SULFUROUS ACID

Weak decalcifying agent. Suitable for minute pieces of bones.

FLEMMING’S FLUID / CHROMIC ACID

Both FIXATIVE and DECALCIFYING AGENT. Nuclear staining with HEMOTOXYLIN is inhibited. Require frequent changes of solution

Degree of decalcification cannot be measured by routine chemical test

Toxic and corrosive

CHROMIC ACID 15 ML

OSMIUM TETROXIDE 4 ML

2% GLACIAL ACETIC ACID 1 ML

FLEMMING’S FLUID / CHROMIC ACID FORMULA:

CITRIC ACID CITRATE BUFFER SOLUTION

Permits excellent nuclear and cytoplasmic staining. Does not produce cell or tissue distortion. Slow acting. DECALCIFICATION TIME: 6 DAYS

6 DAYS

CITRIC ACID CITRATE BUFFER SOLUTION DECALCIFICATION TIME

4.5 pH

CITRIC ACID CITRATE BUFFER SOLUTION pH

7% CITRIC ACID MONOHYDRATE AQUEOUS SOLUTION 5 ML

7.4% AMMONIUM CITRATE ANHYDROUS AQUEOUS SOLUTION 95 ML

1% ZINC SULFATE AQUEOUS SOLUTION 0.2 ML

CHLOROFORM FEW DROPS

CITRIC ACID CITRATE BUFFER SOLUTION FORMULA

CHELATING AGENTS

Substances which combine with calcium ions and other salts to form weakly dissociated complex and facilitates removal of calcium salts.

VERSENE

Commercial name of EDTA

ETHYLENEDIAMENETETRAACETIC ACID

FULL NAME OF EDTA

EDTA

Preserves nuclear DNA. Excellent bone decalcifier for enzyme or immunohistochemical staining and for electron microscopy.

pH dependent

7-7.6 pH

EDTA: Optimal pH

10.0 pH

EDTA: Increase decalcification time

>8.0 pH

EDTA: Tissue maceration starts due to alkaline sensitive protein bonds

<5.0 pH

EDTA: Works slowly

7.0 pH

EDTA: Generally used

NEUTRAL EDTA

Slow decalcifying agent. Gives excellent results for soft-tissue integrity and best quality of both soft tissue and hard tissue staining. Forms minimal histological artifacts.

1-3 WEEKS

NEUTRAL EDTA DECALCIFICATION TIME FOR SMALL SPECIMENS:

6-8 WEEKS

NEUTRAL EDTA DECALCIFICATION TIME FOR CORTICAL BONES:

ION EXCHANGE RESIN

Hastens decalcification by removing calcium ions from formic acid containing decalcifying solutions which increase solubility from the tissues.

1-14 DAYS

Tissue is allowed to stay in solution of ion exchange resin for _____

ELECTROPHORESIS

Positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution.

ELECTROPHORESIS

Decalcification is shortened due to the heat and electrolytic reaction produced in the process. Used for small bone fragments

MICROWAVE DECALCIFICATION

Faster than routine decalcification. Decalcification time: 3 HOURS

CONCENTRATION

FLUID ACCESS

SIZE AND CONSISTENCY

AGITATION

TEMPERATURE

FACTORS AFFECTING DECALCIFICATIONS

1-3 DAYS

4% AQUEOUS SOLUTION TISSUE SOFTENING

MOLLIFLEX

Appears soapy and swollen. Does not affect tissue staining