Tissue Processing
HISTOPATHOLOGY (LEC)
October 10, 2022
DECALCIFICATION
Calcium may be removed by the following:
• Acid
• Chelating agents
• Ion exchange resins
• Electrical ionization
ACIDS
NITRIC ACID
Most common and the fastest decalcifying agent used so
far. However, it inhibits nuclear stain and destroying
tissues especially in concentrated solutions.
A. Aqueous nitric add solution 10%
Recommended for urgent biopsies and for needle
and small biopsy specimens to permit rapid
diagnosis within 24 hrs. or less.
B. FORMOL-NITRIC ACID
•Rapid acting; recommended for urgent biopsies
•Produce yellow color; remedy: neutralizing
tissue in 5% sodium sulfate and washing in
running tap water for at least 12 hrs. addition of
0.1% urea to pure concentrated nitric acid will
also make discoloration disappear without
affecting the efficiency of the decalcifying agent.
C. PERENYI'S FLUID
• Recommended for routine purposes
• Nuclear and cytoplasmic staining is good.
• Maceration is avoided due to the presence of
chromic acid and alcohol
• Not recommended for urgent work.
• Composed of chromic acid, ethyl alcohol and
nitric acid
D. PHLOROGLUCIN NITRIC ACID
• Most rapid decalcifying agent
HYDROCHLORIC ACID
A. VON EBNER'S FLUID
- Moderately rapid decalcifying agent.
- Does not require washing out before
dehydration
- Recommended for teeth and small pieces of
bone.
FORMIC ACID
•May be used as both fixative and decalcifying agent.
• Recommended for routine decalcification of postmortem
research tissues.
A. FORMIC ACID-SODIUM CITRATE SOLUTION
- Recommended for autopsy materials, bone
marrow, cartilage.
TRICHLORO ACETIC ACID
• Very slow acting; weak decalcifying agent
• Suitable for small spicule of bone.
SULFOROUS ACID
-is a very weak decalcifying solution suitable only for
minute pieces of bone.
CHROMIC ACID (FLEMMING'S FLUID)
• May be used as both fixative and decalcifying agent
• Suggest for minute bone specimens.
• Consider as carcinogenic
CITRIC ACID - CITRATE BUFFER SOL
• pH 4.5
• excellent nuclear and cytoplasmic staining.
• Too slow
• Contains chloroform as preservatives
CHELATING AGENTS
EDTA (VERSENE)
• Most common chelating agent
• Recommended for detailed microscopic studies
• Very slow decalcifying agent
• For small specimens: 1-3 weeks
• 6-8 weeks or longer totally decalcify dense cortical bone.
• Excellent for immunohistochemistry or enzyme staining
and electron microscopy.
• Inactivates ALP; to restored add magnesium chloride.
ION EXCHANGE RESIN
• Not recommended for fluids containing nitric acid or
hydrochloric acid.
• The decalcifying agent is then added, usually 20-30 time
of the volume of the tissue.
• The tissue will stay in solution 1-14 days.
FACTORS INFLUENCE OF DECALCIFICATION
1. Concentration and volume -More concentrated, more
rapidly; more harmful
2. Structure and temperature
ratio: 20:1
higher concentration and greater amount of fluid
will increase the speed of the process.
3. Heat
4. Mechanical Agitation
5. Accelerates the rate of diffusion and speeds up the
decalcification process.
MEASURING EXTENT OF DECALCIFICATION
1. Physical or mechanical test
-Touching or bending the tissue with the fingers.
Inaccurate method.
2. X - ray or radiological method
-Very expensive; most ideal; most sensitive and
most rellable method,
3. Chemical method (calcium oxalate test)
-Simple, rellable and convenient method,
- Cloudiness will signify incomplete
decalcification.
Wong, J.V.
 Tissue Processing
HISTOPATHOLOGY (LEC)
October 10, 2022

POST DECALCIFICATION CELLOSOLVE (EHTYLENE GLYCOL MONOETHYL

• Involves lithium carbonate to wash the tissue after the ETHER)
decalcification is complete. • Cellosolve dehydrates rapidly. The tissue may be
• Also decalcified tissue can rinse in running tap water to transferred from water or normal saline directly to
re moved acids. cellosolve and stored in it for months without producing
hardening or distortion.
TISSUE SOFTENER'S
1. Perenyi's fluid TRIETHYL PHOSPHATE
-May act as both decalcifying and tissue • Used to dehydrate sections and smears following certain
softeners stains and produces minimum shrinkage.
2. 4% aqueous sol
3. Molliflex TETRAHYDROFURAN (THF)
-Tissue immersed in molliflex appear swollen and • Both dehydrates and clears tissue since it is miscible in
soapy. both water and paraffin.

DEHYDRATION
• Starts by placing the fixed specimen in 70% to 95% to
100% ethyl alcohol.
• For delicate tissues, particularly embryonic tissues
starting 30% ethyl alcohol
• The amount of dehydrating agent is should not be less
than 10 times the volume of the tissue to ensure complete
penetration of the tissue by the dehydrating agent.
ALCOHOL
• Anhydrous copper sulfate will accelerate dehydration by
removing water from dehydrating fluid. A blue
discoloration will indicate a complete dehydration.
A. ETHYL ALCOHOL
• Recommend for routine dehydration of tissues
• Best dehydrating agent
B. METHYL ALCOHOL
• Toxic dehydrating agent
• Used for blood and tissue film for smear
preparations
C. BUTYL ALCOHOL
- For plant and animal microtechniques
- Slow dehydrating agent
ACETONE
• Cheap, rapid acting dehydrating agent & for urgent
biopsies which dehydrates 30 mins to 2 hrs
• Limited for only small specimen
• Volatility and inflammable
• Most lipids are removed from tissues with this
dehydrating agent.
DIOXANE (DIETHYL DIOXIDE)
• Excellent dehydrating agent and clearing agent
• Readily miscible with water and paraffin
• Tends tissue ribbon poorly
• Highly toxic in man and expensive
CLEARING
• De-alcoholization; process whereby alcohol or a
dehydrating agent is removed from the tissue and
replaced with a substance that will dissolve the wax with
which the tissue to be impregnated.
• Must be miscible with water, paraffin, and mounting
medium.
COMMON CLEARING AGENTS
XYLENE (XYLOL)
• Colorless clearing agent, commonly used in histology
laboratories.
• Miscible with absolute alcohol and paraffin
• It is cheap; used for celloidin sections
• Highly inflammable
• Not suitable for nervous tissues and lymph nodes
• Xylene turns milky when an incompletely dehydrated
tissue is immersed in it.
TOLUENE
* May be used as a substitute for xylene and benzene. .
More expensive
• Miscible with absolute alcohol and paraffin
BENZENE
• Preferred by some as clearing agent in the embedding
process of tissues because it penetrates and clears tissue
rapidly.
• Carcinogenic, may damage the bone marrow resulting
aplastic anemia
• Miscible with absolute alcohol
CHLOROFORM
• does not make tissue translucent recommended for
tough tissues (skin, fibroid, decalcified tissues)
• toxic to liver
• tissue tend to float in chloroform to avoid this wrapped
the tissues with absorbent cotton gauze to facilitate
sinking of the section in solution.
• Miscible with absolute alcohol

Wong, J.V.
 Tissue Processing
HISTOPATHOLOGY (LEC)
October 10, 2022
CEDARWOOD OIL
• Recommended for central nervous system tissues and
cytological studies. (Particularly smooth muscles)
• Requires two changes in clearing solution
• It makes tissue transparent
• Extremely slow clearing agent
ANILINE OIL
• Recommended for clearing embryos, insects and very
delicate specimens.
CLOVE OIL
• Unsuitable for routine clearing process
CARBON TETRACHLORIDE
• Properties are very similar to chloroform
METHYL BENZOATE AND METHYL SALICYLATE
-double embedding technique is needed.
IMPREGNATION
• Process whereby the clearing agent is completely
removed from the tissue and replaced by a medium that
will completely fill the cavities, thereby giving a firm
consistency to the specimen and allowing easier handling
and cutting suitably thin sections without any damage to
the tissue and its cellular components.
I. PARAFFIN WAX IMPREGNATION
•Simplest, most common and best embedding medium,
used for routine processing.
• Very rapid
• Prolong impregnation will cause excessive shrinkage
and hardening making cutting of sections difficult.
• Not recommended for fatty tissues
•For routine work melting point is: 56-58 deg cel
• For the lab temp between 15-18 deg cel melting point is:
50-54 deg cel
• For the lab temp between 20-24 deg cel melting point is
54-58 deg cel
METHODS OF PARAFFIN WAX IMPREGNATION
MANUAL PROCESSING
- Four changes of wax are required at 15 minutes interval.
AUTOMATIC PROCESSING
- Use auto technicon which fixes, dehydrates, clears and
infiltrates tissues. - 2-3 changes of wax are required.
VACCUM EMBEDDING
• Involves the wax impregnation under negative
atmospheric pressure inside an embedding oven to
hasten removal of air bubbles and clearing agent.
• Most rapid
• Recommended in urgent biopsies
SUBSTITUTES FOR PARAFFIN WAX
PARAPLAST
• Mixture of highly purified paraffin and synthetic plastic
polymers
• Melting point: 56-57 degcel
• More elastic and resilient
•For bones and brains
EMBEDDOL
•Synthetic wax substitute similar to paraplast
• Melting point is: 56-58 degcel
TISSUE MAT
-contains rubber
ESTER WAX
•Has a lowering melting point 46-48 degcel
•Harder than paraffin
WATER SOLUBLE WAX (CARBO WAX)
• Melting point: 38-42 degcel or 45-46 degcel
II. CELLOIDIN IMPREGNATION
• Purified form of nitrocellulose soluble in many solvents
• Suitable for specimens with large hollow cavities which
tend to collapse for hard and dense tissues such as bones
teeth and for large tissue sections such as whole embryo.
• Recommended for processing neurological tissue
• Very slow
TWO METHODS FOR CELLOIDIN IMPREGNATION
WET CELLOIDIN METHOD
-Recommended for bones teeth large brain and whole
organs
DRY CELLOIDIN METHOD
-Recommended for whole eye sections -Does not use
alcohol due to the presence of cedarwood oil in the block.
III. GELATIN IMPREGNATION
• Rarely used except for histochem and enzyme studies
• Used for delicate specimens and frozen tissue
• Water soluble
• Tissue should not be more than 2-3mm thick; add 1%
phenol to prevent molds
•Excess gelatin may remove by floating the sections to
paper and trimming them with scissors.
EMBEDDING
•After impregnation, the tissue placed into a mold
containing the embedding medium and this medium allow
solidifying.
• In this process orientation is very important.
•Temperature of melted paraffin is 5-10 deg cel above the
melting point
• Immersed in cold or ref temp to solidify
Wong, J.V.
 Tissue Processing
HISTOPATHOLOGY (LEC)
October 10, 2022

1. Leukhart's embedding mold

-Contains two L- shaped strips of heavy brass.

2. Compound Embedding unit

-Made up of a series of interlocking plates resting on a flat
metal base.

3. Plastic embedding rings and base molds

-Consist of special stainless steel base mold fitted with a
plastic embedding ring, which later serves as the block
holder during cutting

4. Tissue-tek
-Equipped with a warm plate to manage the impregnated
specimen.

5. Disposable embedding molds

- Peel -away Plastic ice trays
- Pacer boats

REMEMBER
• Celloidin or nitrocellulose method - recommended for
embedding hard tissues.
•*double-embedding method-process; in which tissue first
infiltrate by celloidin and embedded in paraffin.

Wong, J.V.