DECALCIFICATION

Decalcification

• It removes calcium or lime salts most especially on bones and teeth samples.

• Done after fixation and before impregnation to facilitate the normal cutting of sections. It is only bone in boney specimens.

Decalcification

It prevents obscuring of the microanatomical details of sections caused by bone dust and other cellular debris.

Tissue maceration and Destruction of tissue components which

results in poor staining.

Prolonged decalcification may result to:

Poor cutting of

hard tissues and may cause damage to the knife during

section cutting.

Inadequate decalcification may result to:

Microcalcification

Occasionally encountered in paraffin-embedded or

frozen tissues.

Microcalcification

• May cause resistance and a “grating” sensation when sectioned.

• When staining section with small foci of calcification with

  Hematoxylin and Eosin (H&E) staining, they would appear

  as dark purple granular masses with lighter purple halos.

• To remedy microcalcification (a grating sensation is felt

  during section cutting), remove the block from where it is

  clumped in the chuck of the microtome and place it face

  down on a pad of cotton/gauze saturated with 10% HCl for

  1 hour.

RAPID DECALCIFYING AGENTS

Save time in decalcifying but produces adversely affect

(greater degree of adverse effect) in any subsequent

staining; noticeable in the cell nuclei after staining; in H&E

staining, the nuclear chromatin fails to take up the

hematoxylin and eosin can produce a deep brick-red stain

without differential shading (monotone)

GOOD DECALCIFYING AGENTS

→ Can completely remove calcium salts from tissues without

considerable damage to cells and tissue components.

→ Without adversely affecting staining capacity of the cell,

especially in the nucleus.

Decalcification can be achieved by the use of:

❖ Acids

❖ Chelating agents

❖ Ion exchange resins

❖ Electrical ionization/Electrophoresis

ACID DECALCIFYING AGENTS

→ Most widely used decalcifying agents of large amounts of

boney tissues.

→ Stable, easily available and inexpensive.

→ It removes calcium by forming soluble calcium salts.

→ Examples: Nitric acid, HCl, Formic acid, Trichloroacetic

acid, Sulfurous acid, Chromic acid, Citric acid.

Nitric Acid

→ The fastest and the most commonly used acid decalcifying

reagent because it produces minimal tissue distortion and

is recommended for routine purposes

→ Utilized both as a simple solution or combined with other

reagents.

→ Recommended concentration: 5-10% (when used as an

aqueous solution)

→ Careful: When in high conc., could inhibit nuclear stains

and destroy tissues, especially when concentrated. Highly

concentrated nitric acid could be prevented by combining

nitric acid with formaldehyde and 70% alcohol.

5-10%

Recommended concentration for Nitric Acid?

12-24 hours

Decalcification time for 10% Aqueous Nitric Acid soln.

(1) Rapid

(2) Minimum distortion

(3) Good nuclear staining

(4) Excess acid is removed by 70% alcohol (to stop

decalcification)

(5) Recommended for urgent biopsy, and for needle and

small biopsy because it has decalcification time of 12-24 hrs.

(6) Can be used for large or heavily mineralized cortical

bone specimen ONLY if the decalcification progress is

carefully monitored by an endpoint test

10% AQUEOUS NITRIC ACID SOLN. (PURE CONCENTRATED

NITRIC ACID) ADVANTAGES:

(1) Prolonged used—tissue distortion

(2) Damage tissue stainability (especially in the nucleus)

(3) It imparts a yellow color—impairs staining rxn.

(4) Old stock is damaging (fresh 10% nitric acid soln. should

be used)—more damage than benefits

(5) Strong acid- more damaging to tissue antigen for

immunohistochemical staining—enzymes may be totally

lost

• 0.1% urea can be added to the pure

concentrated nitric acid to make the

discoloration disappear without considerable

effects to the efficiency of the reagent.

10% AQUEOUS NITRIC ACID SOLN. (PURE CONCENTRATED

NITRIC ACID) DISADVANTAGES:

(1) Rapid

(2) Recommended for urgent biopsy because it has a

decalcification time of 1-3 days.

(3) Good nuclear staining

(4) Less tissue destruction compared to 10% Aq. Nitric Acid

Soln. (Formol-nitric acid has additives like formaldehyde)

FORMOL-NITRIC ACID (NITRIC ACID + FORMALDEHYDE) ADVANTAGES:

1-3 days

Decalcification time for Formol-nitric acid

(1) It imparts a yellow color (nitrous acid formation) —

impairs staining rxn → neutralized by 5% sodium sulfate

and washing in running tap water for at least 12hrs to

neutralize yellow discoloration

• Alternative: 0.1% urea added to your conc.

Nitric acid can also remove the discoloration

w/o affecting the efficiency of the acid.

(2) Used inside a fume hood (expense of the lab)

FORMOL-NITRIC ACID (NITRIC ACID + FORMALDEHYDE) DIS ADVANTAGES:

(1) Recommended for routine purposes (10% aq. nitric acid

soln. and formol-nitric acid for urgent works)

(2) Decalcifies and softens (double purpose, acid

decalcifying agent and tissue softener)

(3) Good nuclear and cytoplasmic staining

(4) Maceration is avoided (because of the chromic acid and

alcohol additives)

PERENYI’S FLUID (NITRIC ACID + CHROMIC ACID AND

ALCOHOL) ADVANTAGES

(1) Slow decalcifying agent; decalcification time: 2-7 days

(2) Complete decalcification cannot be determined by

chemical tests

• Ppt. formation upon addition of ammonia even

though calcium ion is absent and then ppt can be

dissolved by the addition of glacial acetic acid,

drop by drop, and add 0.5 mL saturated aqueous ammonium oxalate to the soln., resulting to a white

ppt in 30 minutes and that would indicate the

presence of calcium. Therefore, decalcification is

incomplete (false positive)

• Ammonia is used in the chemical test to detect the

extent of decalcification and ppt would indicate

incomplete decalcification

PERENYI’S FLUID (NITRIC ACID + CHROMIC ACID AND

ALCOHOL) DISADVANTAGES:

2-7 days

Decalcification time of PERENYI’S FLUID

(1) The most rapid so far, therefore, recommended for

urgent works; decalcification time: 12-24 hrs.

PHLOROGLUCIN-NITRIC ACID ADVANTAGES:

12-24 hrs

Decalcification time for PHLOROGLUCIN-NITRIC ACID

(1) Poor nuclear staining

(2) Produces extreme tissue distortion (prolonged

decalcification)

• To prevent prolonged decalcification, measure

again the extent of decalcification and when

decalcification is complete, immediately remove

the acid by 3 changes of 70%-90% ethanol to

remove excess acid and wash with running tap

water and prolonged washing will also cause

excessive swelling and deterioration of tissue

samples.

(3) Yellow color (nitrous acid formation) = neutralized with

5% sodium sulfate and washed with running tap water for

at least 24 hours

(4) Complete decalcification cannot be determined by

chemical means (same with Perenyi’s Fluid)

PHLOROGLUCIN-NITRIC ACID DISADVANTAGES:

HYDROCHLORIC ACID

→ Inferior compared to nitric acid because it is much slower

in decalcifying and produces greater distortion to the

sectioned tissue

→ Produces good nuclear staining

→ Recommended for surface decalcification

(microcalcification)

→ An ingredient in a rapid decalcifying agent

→ Can be diluted without affecting the decalcification

process (when too much HCl is placed, efficiency will not

be affected)

• Ex: 1% solution with 70% alcohol used in surface

decalcification for approx. 1 hour

(1) Good cytologic staining

(2) Moderately rapid

(3) Does not require washing out before dehydration (can

be directly placed in a dehydrating agent)

(4) For teeth and small pieces of bone (but not for large,

dense bone spx)

VON EBNER’S FLUID ADVANTAGES:

(1) A chemical test cannot be used when checking for the

completeness of decalcification.

VON EBNER’S FLUID DISADVANTAGE:

FORMIC ACID

→ Moderate acting, not recommended for urgent

examination because it is not rapid.

→ Better nuclear staining w/ less tissue distortion because it

is a weak acid.

→ Safer than the other two (Nitric and Hydrochloric acid).

→ Routine for postmortem research tissues.

→ Used as a primary decalcifying agent (the only weak acid

used as decal. agent).

• Picric acid and acetic acid are both ingredients of a

fixative that also decalcifies a tissue specimen.

→ Addition of citrate may accelerate decalcification

• How? Citrate accelerates by chelating calcium as it is

liberated/released from the bone. As formic acid

works its purpose in the bone, it forms soluble calcium

salts.

(1) A fixative and a decalcifying agent.

(2) Excellent nuclear and cytoplasmic staining.

(3) Recommended for small pieces of bones and teeth

(because it is a weak acid)

(4) Suitable for most routine surgical specimens, especially

when immunohistochemical staining is needed.

FORMIC ACID ADVANTAGES:

(1) Slow-acting (but can be hastened by increasing formic

acid proportion from 10mL to 25mL but the resulting solution

will be opaque which results with staining results like yellow

discoloration)

(2) Requires neutralization w/ 5% sodium sulfate and

washing out.

FORMIC ACID DISADVANTAGES:

(1) Better nuclear staining than nitric acid.

(2) Recommended for autopsy materials, bone marrow,

cartilage, and tissues studied for research purposes.

(3) Recommended for small pieces of bones and teeth

(because it is a weak acid)

(4) Suitable for most routine surgical specimens, especially

when immunohistochemical staining is needed.

FORMIC ACID-SODIUM CITRATE SOLN. (FORMIC ACID +

CITRATE) ADVANTAGES:

(1) Relatively slow; not for routine and dense tissues

(2) Requires neutralization w/ 5% sodium sulphate to

prevent prolonged tissue decalcification.

FORMIC ACID-SODIUM CITRATE SOLN. (FORMIC ACID +

CITRATE) DISADVANTAGES:

3-14 days

Decalcification time for FORMIC ACID-SODIUM CITRATE SOLN.

(1) Good nuclear staining

(2) Does not require washing out (like Von-Ebner’s fluid,

excess acid has to be removed by subjecting to several

alcohol baths of 90% concentration, and by doing this, it does

not only remove excess acid but is also improving the tissue

dehydration)

TRICHLOROACETIC ACID ADVANTAGES:

(1) Weak and is not for dense tissues; suitable only for small

spicules of bone

(2) Very slow-acting; decalcification time: 4-8 days

TRICHLOROACETIC ACID DISADVANTAGES:

4-8 days

Decalcification time for TRICHLOROACETIC ACID

SULFUROUS ACID

Very weak acid therefore, only suitable for minute pieces of bone.

(1) Used both as a fixative and decalcifying agent

(2) May be used for minute bone spicules

CHROMIC ACID (FLEMMING’S FLUID) ADVANTAGES:

(1) Inhibits nuclear staining with hematoxylin

(2) It undergoes reduction and forms ppt at the bottom of

the container (if reagent is not fresh)

(3) It forms insoluble pigment when the tissue is dehydrated

with alcohol (thus tissues must be washed out before

dehydration)

(4) A chemical test cannot be used when checking for the

completeness of decalcification

(5) Chromic acid is an environmental toxin (caution must be

exercised).

• Chromic acid is highly corrosive, carcinogenic,

and suitable PPE is not readily available so it is not

for routine laboratory usage.

• Usage of any solution that has chromium must not

be drained down the sink since it is an

environmental toxin.

CHROMIC ACID (FLEMMING’S FLUID) DISADVANTAGES:

(1) Excellent nuclear and cytoplasmic staining

(2) Does not produce cell or tissue distortion

CITRIC ACID-CITRATE BUFFER SOLN. (PH 4.5) ADVANTAGES:

(1) Too slow for routine purposes (very weak acid);

decalcification time: 6 days

CITRIC ACID-CITRATE BUFFER SOLN. (PH 4.5) DISADVANTAGES:

6 days

Decalcification time for CITRIC ACID-CITRATE BUFFER SOLN. (PH 4.5)

CHELATING AGENTS

→ Combines with calcium ions and other salts (e.g., iron and

magnesium) → forming weakly dissociated complexes →

facilitates removal of calcium salt

→ EDTA- Ethylene Diamine Tetraacetic Acid (commercial

name: Versene)— binds to metallic ions (Ca and Mg);

anticoagulant (hematology studies); water softener; very

slow decalcifying agent

• Takes 1-3 weeks to decalcify a small spx. and 6-

8 weeks or longer to decalcify dense, cortical

bones that has a lot of calcium.

• Using EDTA: must change every 3 days and every

day when near to final stages of decalcification

• Will not bind to Ca ions below pH 3; Faster at pH

7-7.4 (recommended pH); Optimal binding at pH

8 and above, however increasing the pH may

damage alkaline sensitive protein linkages

EDTA & EDTA DISODIUM SALT (10%)

→ Can be a simple aqueous or buffered solution at pH 7 to

7.4 or you can also add it to formalin.

(1) Excellent staining results

(2) Minimal cell and tissue distortion

(3) Minimal histologic artifacts—CO2 bubbles

(4) Extent of decalcification measured by routine chemical

test

(5) Excellent for IHC (immunohistochemical)/enzyme

staining and for electron microscopy)

(6) pH can be adjusted (enzymes requires specific pH

conditions to maintain activity, so good for enzyme

staining)

EDTA & EDTA DISODIUM SALT (10%) ADVANTAGES:

(1) Very slow

(2) Causes slight tissue hardening (decalcification takes too

long)

(3) Inactivates alkaline phosphatase activity (restored by

adding magnesium chloride)

EDTA & EDTA DISODIUM SALT (10%) DISADVANTAGES:

ION EXCHANGE RESIN

→ The ammonium form of polystyrene resin.

→ Hasten's decalcification → removes calcium ions (from

formic acid-containing decalcifying sol’n) → increase

tissue solubility

→ Not for fluids w/ mineral acids (like nitric acid or HCl);

Decalcification time: 1-14 days

→ Extent of decalcification is measured by physical or X-ray

method.

→ Previously used resin can be reused by reactivating it by

immersing in N/10 HCl twice and distilled water three

times.

(1) Well-preserved cellular detail

(2) Decalcification is hastened by increasing tissue

solubility

(3) Daily washing is eliminated

(4) Excellent staining

(5) Minimal cell and tissue distortion

(6) Minimal histologic artifacts—CO2 bubbles

ION EXCHANGE RESIN ADVANTAGES:

(1) Degree of decalcification cannot be measured by

chemical means (only by physical and x-ray methods)

(2) Very slow

(3) Slight tissue hardening

ION EXCHANGE RESIN DISADVANTAGES: