MT112 – Histopathologic and Cytologic Techniques with General There’s a good and bad decalcifying agent and also

Pathology disadvantages and advantages.

DECALCIFICATION

WHAT IS DECALCIFICATION?
DECALCIFYING AGENTS
 It removes calcium or lime salts
 Rapid decalcifying agents
(removal of calcium and lime salts most specially on bones and teeth
samples) – could adversely affect any subsequent staining

 Done after fixation and before impregnation (to facilitate the The adverse effect is noticeable in the cell nuclei after staining
normal cutting of sections) when you read the sample.

 Decalcification is only done on bony specimens
Purpose of Decalcification
 It prevents obscuring (of the microanatomical details of sections
caused by bone dust and other cellular debris)
 Good decalcifying agent:
 Decalcification must be exact (not prolonged or inadequate)
 Prolonged decalcification may result to:
✓ Maceration (of tissues)
✓ Destruction of tissue components
In a H & E staining, the nuclear chromatin fails to take up the
Hematoxylin. The Eosin can produce a brick red stain without
differential staining. So, the tissue sample is just monotone.
✓ Can completely remove calcium salts (from the tissue) without
producing considerable damage (to the cells and tissue
components)
✓ Without adversely affecting staining
capacity of the cell (most importantly on the nucleus)

-this would result to poor staining  Decalcification can be achieved by the use of:
4 Types of Decalcifying Agents
 Inadequate decalcification may result to: ✓ Acids
✓ Poor cutting ✓ Chelating agents
✓ Damage to the knife (during section-cutting) ✓ Ion exchange resins
✓ Electrical ionization/Electrophoresis
 Therefore, it is necessary that the degree of decalcification be
evaluated (this is to ensure complete calcium removal and normal
processing of tissues)
ACID DECALCIFYING AGENTS
 The resulting bone sample should not be too hard for section-
cutting  Most widely used decalcifying agent of large amounts of bony
tissues
MICROCALCIFICATION
 Stable, easily available and inexpensive as compared to others
 Occasionally encountered in paraffin-embedded or frozen
 It works by forming soluble calcium salts
tissues
 Examples:
 May cause resistance and a “grating” sensation when
Nitric acid
sectioned (but the tissue can still be cut or sectioned as it is only a
HCl
tiny block that is barely noticeable)
Formic acid
 Remedy: Trichloroacetic acid
If you feel a ‘grating’ sensation during section-cutting, remove the block Sulfurous acid
from where it is clumped in the microtome or chuck of microtome and Chromic acid
place it face-down on a pad of cotton or gauze that is saturated with Citric acid
10% HCl for approx. 1 hr
I. NITRIC ACID
After, the first few sections of the block after saturation, can be cut
more easily
 The fastest and the most commonly used reagent
Only the surface will be relieved from the microcalcification
- it produces minimal tissue distortion
 dark purple granular masses with lighter purple halos (this - recommended for routine purposes
appears during staining using H & E staining)
 Utilized both as a simple solution or combined with other
H & E = Hematoxylin and Eosin reagents

 10% HCl for 1 hour (remedy)  Recommended concentration:

5-10% (for simple solution)

 You can’t use a concentrated nitric acid because it could inhibit nuclear
stains and it may destroy tissues
 This may be prevented by combining nitric acid with formaldehyde
or 70% alcohol

 Could inhibit nuclear stains and destroy tissues, especially

-the purple granular masses w/ halos is the microcalcification when concentrated.
portrayed in the picture
A. 10% Aqueous Nitric Acid Soln.

To fulfill the removal, we must use decalcifying agents. ADVANTAGES:

There are a lot of decalcifying agents in the market that is 1. Rapid (decalcification of tissues)
suitable to the needs of the laboratory. 2. Minimum distortion (of the tissues)
3. Good nuclear staining (results)
 4. Acid (excess) is removed by 70% alcohol  Ammonia is a reagent used when there’s a chemical test to
5. Recommended for urgent biopsy, and for needle and small detect the extent of decalcification
biopsy
6. Can be used for large or heavily mineralized cortical bone D. Phloroglucin-Nitric Acid
specimen
(only if the decalcification progress is carefully monitored by an ADVANTAGES:
endpoint test) 1. The most rapid so far

Decalcification time: 12 – 24 hrs - recommended for urgent works

Decalcification time: 12 – 24 hrs

DISADVANTAGES:
DISADVANTAGES:
1.Prolonged used = tissue distortion
2. Damage tissue stainability 1. Poor nuclear staining
(especially on the nucleus) 2. Produces extreme tissue distortion
(due to prolonged decalcification = leads to tissue maceration and
3. It imparts a yellow color = impairs staining reaction
destruction of components)
4. Old stock is damaging
(to the sample)
Can be prevented by:
5. Since nitric acid is a strong acid, it is more damaging to Measure the extent of decalcification and when it is completed,
tissue antigen for Immunohistochemical staining = enzymes immediately remove the acid by three changes/three baths of 70% -
may be totally lost 90% ethanol to remove the excess acid and then wash it with tap
water.
 Immunohistochemical samples are delicate
 This acid is more destructive to the tissues if prolonged usage Be careful or prolonged washing with tap water because it would lead
to another problem like tissue swelling and deterioration of sample
than Formol-Nitric Acid because it is a nitric acid that is just
diluted to 10%
3. Yellow color (nitrous acid formation)
B. Formol-Nitric Acid
Can be removed or neutralized with:
5% sodium sulfate and washed with running tap water for at
ADVANTAGES:
least 24 hours to remove excess acid
1. Rapid
4. Complete decalcification cannot be determined by chemical
2. Recommended for urgent biopsy
means
3. Good nuclear staining
II. HYDROCHLORIC ACID
4. (gives) Less tissue destruction than 10% Aq. Nitric Acid Soln.
- another strong acid
Decalcification time: 1 – 3 days
 Inferior compared to nitric acid
- slower in decalcifying and produces greater distortion to the tissues
 Has additives like Formaldehyde compared to nitric acid
 Produces good nuclear staining
DISADVANTAGES:  Recommended for surface decalcification
 An ingredient in a rapid decalcifying agent
1. It imparts a yellow color (nitrous acid formation) = impairs
 when it is too concentrated, it Can be diluted without affecting the
staining reaction
decalcification process
May be prevented or neutralized by: A. Von Ebner’s Fluid
Addition of 5% sodium sulfate and washing in running tap
water for at least 12hrs ADVANTAGES:
1. Good cytologic staining
 You can also add 10% Urea to the pure concentrated nitric acid and
2. Moderately rapid
this would make the discoloration disappear without considerable
3. Does not require washing out before dehydration
effects to the efficiency of the reagent
4. can be used For teeth and small pieces of bone (not for large
bones)
2. Used inside a fume hood
(Fume hoods equipments are additional expense in the laboratory and
takes up space) DISADVANTAGES:
1. Chemical test cannot be used when checking for the
C. Perenyi’s Fluid completeness of decalcification

ADVANTAGES: Random Fact:

Picric Acid and Acetic Acid are both ingredients of a fixative
1. Recommended for routine purposes that could also decalcify a tissue
2. Decalcifies and softens
3. Good nuclear and cytoplasmic staining III. FORMIC ACID
4. Maceration is avoided
(tissue sample will not be destroyed) - weak acid
 Moderate acting
Why is maceration avoided? - not recommended for urgent decalcification because it is not rapid
> It is because of the chromic acid additive and alcohol in the nitric acid  Better nuclear staining w/ less tissue distortion
 Safer than the other/first two
DISADVANTAGES:  recommended: Routine for postmortem research tissues
 the only weak acid that is Used as a primary decalcifying agent
1. Slow decalcifying agent  Addition of citrate may accelerate decalcification
2. Complete decalcification cannot be determined by chemical
tests  because there’s a ppt formation upon addition of How could citrate accelerate the decalcification?
ammonia even though calcium ion is absent > By chelating the calcium as it is liberated or released from the bone
(or forming calcium salts)
Decalcification time: 2 – 7 days
 Using citrate is only applicable for Formic Aci
 ADVANTAGES:
1. A fixative and a decalcifying agent.
2. Excellent nuclear and cytoplasmic staining
3. Recommended for small pieces bones and teeth (it can’t
decalcify large dense cortical bone samples as it is only a WEAK acid)
4. Suitable for most routine surgical specimens, especially
when Immunohistochemistry staining is needed
Why?
> Because it is a weak acid and samples
Immunohistochemistry are delicate tissues
DISADVANTAGES:
1. Slow
(not recommended for urgent specimens)
 You can hasten the reaction by increasing the formic acid proportion
(e.g., from 10mL to 25mL) but the downside is, the resulting solution
will be opaque and not clear
2. Requires neutralization w/ 5% sodium sulfate and washing
out
A. Formic Acid-Sodium Citrate Soln.
Purpose of the Citrate:
> Hastens the decalcification by chelating calcium being released
ADVANTAGES:
1. Better nuclear staining than nitric acid
2. Recommended for autopsy materials, bone marrow,
cartilage, and tissues studied for research purposes.
DISADVANTAGES:
1. Relatively slow; not for routine and dense tissues
2. Requires neutralization w/ 5% sodium sulfate
IV. TRICHLOROACETIC ACID
ADVANTAGES:
1. Good nuclear staining
2. Does not require washing out
 But always remove the excess acid to avoid prolonged decalcification
How to remove excess acid of Trichloroacetic acid?
> Subject the tissue that is decalcified to several alcohol baths of 90%
concentration
By doing this, you are not only removing the excess acid but is also
improving the tissue dehydration
DISADVANTAGES:
1. Weak and is not for dense tissues
- suitable only for small spicules of bone
2. Very slow-acting
Decalcification time: 4 – 8 days
V. SULFUROUS ACID
 Very weak therefore, only suitable for minute pieces of bone
VI. CHROMIC ACID (FLEMMING’S FLUID)
ADVANTAGES:
1. Used both as a fixative and decalcifying agent
2. May be used for minute bone spicules
DISADVANTAGES:
1. Inhibits nuclear staining with hematoxylin
2. It undergoes reduction and forms precipitate at the bottom of
the container (if the reagent is not fresh)
3. It forms insoluble pigment when the tissue is dehydrated
with alcohol
 It is important to wash out excess chromic acid before dehydration
4. Chemical test cannot be used when checking for the
completeness of decalcification
5. Chromic acid is an environmental toxin
in
 Caution must be exercised not only when using Chromic acid but all
the time when handling chemical reagents
 Chromic acid is highly corrosive, carcinogenic and suitable PPE is not
readily available so it is not practical/routine for laboratory use
 Using this reagent or any solution that contains chromium must not be
drained in the sink because it is an environmental toxin
VII. CITRIC ACID-CITRATE BUFFER SOLN. (pH 4.5)
ADVANTAGES:
1. Excellent nuclear and cytoplasmic staining
2. Does not produce cell or tissue distortion
- so, this is a very weak acid
DISADVANTAGES:
1. Too slow for routine purposes
Decalcification time: 6 days
CHELATING AGENTS
 substances that Combines with calcium ions and other salts
(e.g., iron and magnesium) to form weakly dissociated
complexes and facilitates the removal of calcium salt
 EDTA (Versene)
- Ethylenediamine tetraacetic acid
- Commercial name: Versene
- common chelating agent that is used for decalcification
- binds to metallic ion particularly calcium and magnesium
- it forms an insoluble nonionized complex with the calcium
- also used as an anticoagulant for Hematology studies
- also used as a water softener (hard metals are removed)
- very slow decalcifying agent
✓ 1-3 weeks
(decalcification time for small specimen)
✓ 6-8 weeks
(decalcification time for large specimen or longer time)
✓ the solution must be Changed every 3 days and every day in the
final stage of decalcification (money and time consuming)
✓ EDTA Will not bind to calcium ions below pH3 (EDTA has a
maintaining pH balance)
✓ Faster at pH 7-7.4 (recommended pH)
✓ Optimal binding at >/= pH 8
(Downside: The higher the pH, it may damage the alkaline sensitive
protein linkages of the tissue)
I. EDTA & EDTA disodium salt (10%)
ADVANTAGES
1. Excellent staining results 9after decalcification)
2. Minimal cell and tissue distortion
3. Minimal histologic artifacts = CO2 bubbles (these histologic
artifacts are CO2 bubbles)
4. Extent of decalcification can be measured by routine
chemical test
5. Excellent for Immunohistochemistry/enzyme staining and for
electron microscopy
6. pH can be adjusted (that is why it is good for enzyme staining
because enzymes need specific pH conditions to maintain activity)
DISADVANTAGES:
 1. Very slow  Dependent upon a supply of direct current
2. Causes slight tissue hardening (because the decalcification is  Satisfactory for small bone fragments (one at a time)
too long)  Good cytologic & histologic details
3. Inactivates alkaline phosphatase activity which can be restored (however, they are not always preserved because of the
by adding magnesium chloride
electricity)
Alkaline phosphatase is an enzyme
 The difference between Electrophoresis and Chelating agents is that
Electrophoresis uses electricity (provides heat generation)
ION EXCHANGE RESIN
 The ammonium form of polystyrene resin
 Hastens decalcification by removing calcium ions from the
formic acid containing decalcifying solutions thereby increasing
tissue solubility

Condition:
> If you use Ion Exchange Resin, you have to use formic acid
containing decalcifying solutions

 Not recommended for fluids w/ mineral acids like nitric acid or HCl
 The extent of decalcification can be measured by:
> Physical or X-ray method FACTORS INFLUENCING RATE OF
DECALCIFICATION
 Can’t use chemical method to measure the extent of decalcification
 You can reuse the previously used resin (can be recycled) by reactive
plating  CONCENTRATION
✓ The higher the concentration the faster it decalcifies
Can be reactivated by immersing the resin in:
 N/10 HCl 2x (twice) & washing with distilled water 3x (thrice) Downside of high concentration:
> Produces greater tissue distortion
Decalcification time: 1 - 14 days
 VOLUME
How do you perform Ion Exchange Resin? ✓ The higher the volume the faster it decalcifies
✓ 20 to 1
- Recommended ratio for fluid to
tissue
20 parts decalcifying agent
1 part tissue volume

 TEMPERATURE
✓ (proven fact) Heat hastens decalcification
✓ Produces heat examples:
Microwave
Sonication
This picture is an example of Ion Exchange Resin Method Electrolytic methods
> At the bottom: ½ inch thick layer of the Ion Exchange Resin ✓ 37°C
(the black portion) - there will be impaired nuclear staining of Van Gieson’s stain
> On top of the Resin: A gauze wrapped tissue specimen for collagen fibers
> The formic acid solution will be the decalcifier ✓ 55°C
- the tissue will undergo complete digestion in 24 – 48 hrs
Volume of the decalcifying agent: ✓ 18°C - 30°C
> 20 – 30 times the volume of the tissue - optimum room temperature range

ADVANTAGES:  MECHANICAL AGITATION

✓ Fluid exchange  faster rate of diffusion  faster
1. Well-preserved cellular detail
decalcification
2. Decalcification is hastened (by increasing the tissue solubility)
✓ Gentle agitation
3. Daily washing is eliminated
4. Excellent staining – low-speed rotation, rocking, stirring, or bubbling air
5. Minimal cell and tissue distortion ✓ Sonication
6. Minimal histologic artifacts = CO2 bubbles – a vigorous agitation that may cause disruption of tissue that
would result to formation of cellular debris at the floor of the
DISADVANTAGES: container

Suspend the tissue to avoid touching of the cellular debris on the floor
1. Degree of decalcification cannot be measured by chemical
of the container
means Cellular debris will affect the staining result
2. Very slow
3. Slight tissue hardening
 SUSPEND THE TISSUE IN DECALCIFYING SOLUTION
ELECTROPHORESIS ✓ For hastening decalcification
✓ For greater fluid access
How do Electrophoresis works?
 attracts positively charged Calcium ions & negative electrode
How to suspend:
(cathode) and subsequently removed from the calcium
✓ Place the tissue in a gauze bag suspended it in liberal
 Shortened decalcification time (due to the heat and electrolytic amount of decalcifying solution (20 parts) by paraffin coated
reaction) thread
 Principle is same with chelating agents (removes calcium ions)
 Purpose of suspension of the tissue: ➢ Add 0.5 mL saturated aq. soln. of ammonium oxalate and let
> To protect it from any precipitate that settles at the bottom it stand for 30 mins
(clear = to confirm complete decalcification)
The coating of the thread by the melted paraffin wax is due to the
➢ Cloudiness would signify incomplete decalcification
corrosive action of the acid.
(subject the tissue to another 24 hrs or so
decalcification)
 SIZE & CONSISTENCY
✓ The smaller the size sample the faster it decalcifies Clear = complete decalcification
✓ 24 - 48 hours (ideal time for decalcifying tissues)
✓ 14 days or longer (required for dense bone tissues) POST DECALCIFICATION
 removal of decalcifying fluid from the tissue (to avoid prolonged
Decalcifying agent must be changed daily to ensure better penetration. decalcification)

And test the extent of decalcification to avoid prolonged decalcification. A. Neutralized chemically
✓ Saturated lithium carbonate soln.
MEASURING EXTENT OF
✓ 5-10% aq. sodium bicarbonate soln.
DECALCIFICATION
I. PHYSICAL OR MECHANICAL TEST B. Running tap water
✓ 30 minutes (for small specimens)
A. By touching or bending tissue
✓ 1-4 hours (for large specimens)
- determining the consistency of the tissue
 Quick rinse (for small needle biopsies)
 Decalcified tissue is softer to touch and have diminished consistency
(bendable)
Acid decalcified tissues for frozen sections:
 However, it is subjective and inaccurate
 must be thoroughly washed with water
B. Pricking with a fine needle or probe ✓ store it in Formol saline containing 15% sucrose or
- it is bound to leave traces such as needle tracks which could
potentially destroy important cellular details ✓ Phosphate-buffered saline (PBS) w/ 15- 20% sucrose (this
is alkaline/neutral) at 40C before freezing
DISADVANTAGE:
o Disrupt soft tumor This is done to stop or neutralize the acid decalcifying agent.
o False positive microfractures of fine trabeculae (leads to potential
Tissues decalcified with EDTA solutions should not be placed directly
misdiagnosis)
in 70% alcohol because the residual EDTA in the tissue sample would
o Small calcified foci may not be detected precipitate in the alcohol and within the tissue.

II. X-RAY OR RADIOLOGICAL METHOD To remove the residual EDTA:

> rinse it with water or
 Most ideal > store it overnight in Formol-Saline or Phosphate-Buffered Slaine
 Most sensitive
This is to prevent the crystalline precipitate formation
 Most reliable
 Very expensive
SOFTENERS
- could detect the smallest focus of calcium which appears as an
opaque in an X-ray plate  Additional step aside from decalcification to unduly hard
- could detect microcalcification tissues
- similar to specimen radiography
I. PERENYI’S FLUID
Uses: ✓ Decalcifier and tissue softener
 FAXITRON and Kodak X-OMAT X-ray film ✓ 12-24 hours
- Submerged the selected part of the tissue where there is an
Downside:
unduly hard portion
 Not recommended for mercuric chloride fixed tissues (because
✓ 1-2 hours
this fixative has a characteristic of radio opacity which interferes the
correct interpretation) - cut surface of the block

II. 4% AQUEOUS PHENOL SOLUTION

III. CHEMICAL METHOD ✓ 1-3 days
✓ produces w/o marked deleterious effect and tissue distortion
• Calcium Oxalate Test
✓ Simple, reliable, and convenient Wash it out and immerse the tissue to the 4% aqueous phenol solution
✓ For routine purposes for 1 – 3 days for considerable tissue softening and easier cutting of
✓ Precipitates insoluble calcium hydroxide or calcium oxalate blocks

Precipitate is the indication that there is an incomplete decalcification III. MOLLIFLEX

- when used, tissues appear swollen and soaky/soppy
✓ Performed on a discarded decalcifying fluid - it does not affect the normal processing of the staining
✓ Cloudiness is an indication for incomplete decalcification
IV. 2% HYDROCHLORIC ACID

How to perform Calcium Oxalate Test: V. 1% HYDROCHLORIC ACID IN 70% ALCOHOL

➢ Perform litmus paper test (blue to red) on the discarded
decalcifying fluid
➢ Add strong ammonia drop by drop
(to neutralize the acidity)
➢ Precipitation indicates incomplete decalcification
(subject the tissue to another 24 hrs or so
decalcification)