Tissue processing

Embedding
Decalcification
Frozen section

Reference: Chapter 2

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 Objectives
• Tissue processing:
– 1 – 3, 6 – 7, 11 – 12, 14, 16,18
• Embedding:
– 19 – 22
• Decalcification
– 23 – 26
• Frozen section
– 30

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 Grossing Embedding

Tissue processing
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 Tissue processing

What is the aim of tissue processing?

Firm
Rigid
Soft

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 Rotary tissue processor

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 Fluid Exchange
Processor

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 Definitions
Take-home activity

• Dehydration
• Clearing
• Infiltration
• Miscible
• Universal solvent
• Ion exchange
• Chelating agent

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 Tissue processing
Dehydration Clearing Infiltration
F 60 95 95 100 100 P P P P
% % % % %
I X X A A A A
A A A A A
X Y Y R R R R
L L L L L
A L L A A A A
C C C C C
T E E F F F F
O O O O O
I N N F F F F
H H H H H
V E E I I I I
O O O O O
E N N N N
L L L L L
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 Dehydration
• Controlled removal of FREE water (not molecularly-
bound water) from tissues fixed in aqueous reagents
• To prepare tissue for embedding in non-aqueous
medium
• Excessive dehydration:
– Removal of molecularly bound water
– Result: hard and brittle tissue
• Incomplete dehydration:
– Free water remains in the tissue
– Result soft, mushy tissues

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 How does the dehydrant remove water?

1. Hydrophilic dehydrants will attract water

from the tissue
2. Diluting dehydrants will dilute aqueous tissue
fluids.

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 Dehydration reagents
• Alcohols
– Ethanol
– Methanol
– Isopropanol
– Butanol
• Acetone
• Universal solvents (dioxane, tertiary butanol,
tetrahydrofuran)

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 Ethanol
• Clear, colorless, flammable – don’t dump down the drain
• Controlled by the government (record keeping is a must)
• (Ideally) Must be used denatured.
• Reliable and fast-acting
• Hydrophilic
• Must be used in ascending grade to reduce tissue shrinkage.
• Dehydration must start @ 60% after fixation with buffered formalin
- PO4 salts will precipitate in the tissue if ethanol is > 70% and this
will cause microtomy problems.
• Long exposure - will cause excessive shrinkage and hardening of
the tissue.

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 Methanol
• Flammable, clear, colorless, unpleasant odor.
Rarely used alone.
• Primary use - fixation of blood smears
• Poisonous – when ingested, it breaks down as
formaldehyde

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 Isopropanol
• Excellent substitute for ethanol
• Certain dyes are insoluble in isopropanol
(eosin) - DO NOT use as a dye solvent
• Nitrocellulose is insoluble in isopropanol - DO
NOT use in celloidin technique.
• Lesser tissue hardening and shrinkage than
ethanol
• Mildly irritating but toxic by ingestion.

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 Butanol
• For dehydrating plant and animal tissues
• Very low dehydrating power - excellent for
slow processing procedures
• Lesser tissue hardening and shrinkage than
ethanol.

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 Acetone
• Very fast acting, less expensive, very
flammable (flashpoint -17°C)
• Causes excessive shrinkage
• Very volatile - maintain volume
• Absorbs atmospheric moisture - maintain
concentration

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 Universal solvents
• Reagents that perform both the dehydration
and clearing steps.
• Unsuitable for delicate tissues - will cause
tissue distortion.
• Examples:
– Dioxane
– Tertiary butanol
– Tetrahydrofuran

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 Dioxane
• Less shrinkage than ethanol
• Very toxic
• Does not show undesirable changes in the tissue
• Faster dehydrant than ethanol but must be used in
large volumes
• Contains water (if left in the tissue after dehydration
will cause 50% shrinkage)
• Expensive; must reclaim or recycle - treat with
anhydrous CaCl2 or CaO for 18-22 hours.

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 Tertiary butanol
• Odorous, expensive
• Solidifies @ RT
• After tertiary butanol, the first step during
infiltration is tertiary butanol-paraffin mixture
• Can be used as dehydrating agent in the
staining procedures - miscible with water,
ethanol, xylene.

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 Tetrahydrofuran
• Miscible with lower grade alcohols, water, ether,
chloroform, acetone, benzene, toluene, xylene
melted paraffin.
• Not a cumulative toxin
• Acts rapidly without causing excessive shrinkage and
hardening
• For reprocessing inadequately dehydrated and
cleared tissues.
• It will not dissolve dyes, but will dissolve some
mounting media.
• Very volatile (flashpoint -14.5°C)

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 Clearing
• Controlled removal of dehydrating agents from the
tissues (de-alcoholisation)
• Clearing agents have high index of refraction and will
render tissue transparent.
• Clearing agents are miscible with both the
dehydration agent and the infiltration medium
• Inadequate dehydration inadequate clearing
inadequate infiltration
– Result: soft and mushy tissues
• Prolonged exposure:
– Result: hard brittle tissues

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 Xylene
• Most widely used; tissues that are adequately
dehydrated and then cleared with xylene
become transparent
• Prolonged exposure:
– Result: over-hardened tissues, especially fibrous,
muscular, CNS, and cartilaginous tissues

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 Action of xylene
• Displaces alcohol rapidly; miscible with
paraffin
• Intolerant of water  turns cloudy if
contaminated; change reagent ASAP
• Flammable and may not be poured down the
sink
• Neurotoxin and a de-fatting agent.

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 Toluene
• More tolerant reagent than xylene  will not cause
over-hardening upon over processing
• Can tolerate atmospheric moisture better than
xylene
• Incomplete clearing with toluene
– Result: subsequent uneven H&E staining and poor nuclear
chromatin patterns.
• Used especially for open processing system.
• Flammable and more volatile than xylene.

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 Benzene
• Very fast-acting
• Does not over-harden the tissues, in general,
except muscle, tendon, uterus
• Highly volatile; evaporates from paraffin baths
rapidly  less changing of paraffin bath but
difficult to maintain the volume.
• Very toxic

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 Chloroform
• Over-clearing will not leave tissues brittle
• It absorbs atmospheric moisture readily
• It can dessicate connective tissues but excellent for uterus,
muscle and tendon
• Does not make the tissue transparent
• Very volatile
• Not flammable or combustible, therefore, cannot be
incinerated.
• Do not heat - will produce a toxic gas, PHOSGENE.
• Carcinogenic.

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 Acetone
• Very low boiling point (58°C), it will boil off
and be replaced by paraffin (MP > 58°C).
• Does not contaminate paraffin baths
• Tissues cleared in acetone will show more
shrinkage

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 Essential oils
• Clove, origanum, sandalwood, cedarwood oil -
volatile, very expensive
• Odorous (plant-like)
• Not readily replaced by paraffin; oil will remain in the
tissues
• Must be removed by xylene before infiltration
• Cedarwood oil will clear tissues after dehydration
with 95% alcohol; will not damage tissues even after
prolonged exposure.

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 Limonene reagents
• Overpowering citrus odor
• Less hardening of the tissues
• Can cause more contamination of the paraffin
• Irritant and sensitizer
• Biodegradable but not water-soluble

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 Aliphatic hydrocarbons
• Alkanes - low reactivity and low toxicity
• Lightweight short chain aliphatics -
– Will penetrate tissues faster
– Remove fat more effectively
– When used in staining technique, will allow coverslip to dry
in the usual fashion.
• Intolerant for water
• Incompatible with some mounting media
• Do not use in automated coverslipping instruments
• Non-irritating, non-sensitizing

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 Infiltration
• Controlled removal of clearing agents and
replace the reagent with infiltrating
(supporting) medium.
• Hold the cells and intercellular structures in
their proper relationships while thin sections
are cut.
• Incomplete infiltration are manifested on the
H&E-stained section.

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 Paraffin
• Most popular medium made up of inert
hydrocarbons (from cracking of petroleum) +
additives:
– Beeswax - reduces crystal size and increase stickiness and
adhesion)
– Rubber - (reduces brittleness, increases stickiness and
allows for good ribboning)
– Other waxes - produce smooth texture and smaller crystal
size
– Plastic - increase hardness and support

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 Paraffin
• Variable melting point
– Higher MP, the paraffin becomes harder - for hard tissue 
thinner sections can be obtained but ribboning is difficult
– Lower MP, the paraffin becomes soft, provides less support
for hard tissues  thinner sections are difficult to obtain but
ribboning becomes easier
• Plastic point –
– Lowest temp at which permanent deformation can occur
without fracture
• IHC staining - uses low MP to preserve antigens
• MP of 55°C to 58 °C - commonly used.

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 Factors affecting paraffin infiltration
• Time - prolonged exposure causes shrinkage and
hardening
• Temp - 2ºC to 4ºC above MP
– Overheated paraffin causes over-hardening of tissues
– Refrain from processing biopsy with large tissues OR fatty
tissues on the same processing cycle.
– Over-processed biopsy tissues will result in hard, friable
tissues that are difficult to section; would present
microtomy artifacts and staining artifacts.

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 Examples of processing programs
• Page 38
• Protocols 1 and 2

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 Protocol 1: Routine overnight
SOLUTION TIME AND CONDITIONS
10% Formalin 2 h – no heat, no vacuum
Alcoholic formalin 1 h – no heat, no vacuum
Alcoholic formalin 1 h – no heat, no vacuum
95% Alcohol 1 h – vacuum only
95% Alcohol 45 m – no heat, no vacuum
100 % Alcohol 45 m – vacuum only
100 % Alcohol 1 h – no heat, no vacuum
Xylene 1 h – no heat, no vacuum
Xylene 1 h – no heat, no vacuum
Paraffin 30 m – no vacuum
Paraffin 1 h – no vacuum
Paraffin 1.5 h – vacuum only

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 Protocol 2: Rapid for Bx
SOLUTION TIME
65% Alcohol 15 m
95% Alcohol 15 m
95% Alcohol 15 m
100% Alcohol 15 m
100% Alcohol 15 m
Xylene 15 m
Xylene 15 m
Paraffin 15 m
Paraffin 15 m
Paraffin 15 m

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 Quality control of paraffin processing
• Adequate volume
• Rotate or change reagents regularly following a
routine schedule of maintenance
• Check alcohols for contamination using hydrometers
• Record daily temp, and adjust if necessary
• Use three changes of paraffin.
• Last paraffin must be the cleanest paraffin bath

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 Other embedding media
Take-home activity - make a chart

• Water-soluble waxes
• Celloidin
• Plastics
– Glycol methacrylate
– Epoxy resins
• Agar and gelatin
• 30% sucrose

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 Microwave oven processing
• Reagents used: ethanol, isopropanol, paraffin
• Duration: ~ 45 min
• Fix tissues for 30 min
• Rinse tissue cassettes in water before using
the microwave to prevent salt formation.
• See procedure on page 39

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 Microwave oven processing
• 100% ethanol – to rinse off water
• 100% ethanol – 67ºC for 5 min
• Isopropanol – 74ºC for 3 min
• Paraffin (molten at 60ºC) – agitate the rack to
remove isopropanol. Drain. Pour used paraffin in
a pot that is at 84ºC to allow isopropanol to boil
off)
• Paraffin (molten at 60ºC) - 65ºC for 2 min then
84ºC for 5 min.

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 Decalcification
• Controlled removal of calcium from calcified tissues
for paraffin infiltration (use plastic infiltration for un-
decalcified bones)
• Properly fixed tissues are treated with decal agents
• Uses the following methods
– Acid methods,
– Ion exchange resins,
– Electrolyte method,
– Chelating agents

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 Acid methods
• Strong acidity of the solution and extended
exposure to these acids will cause damage to
tissue and render them un-stainable
– No nuclear staining
– Calcium salts are dissolved (soluble @ pH 4.5) and
then ionized. Decal agents have pH 0.5 to 3.0.
They must be buffered to pH 4.5 before use.

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 Acid methods
• Concentration: 5%-10% solutions
– Hydrochloric acid
– Nitric acid
– Formic acid
– Formaldehyde + formic acid (for simultaneous fixation and
decalcification)
• Calcium ions migrate out of tissues into surrounding
solution until this solution becomes saturated with
calcium ions (forms a “barrier”)
• Change the solution frequently and agitate.
• Do not use heat - will result in swelling and maceration of
tissues

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 Ion exchange resins
• Use of formic acid over a layer of an ammoniacal salt
of sulfonated resins
• Ammonium ions from resins are exchanged for
calium ions
• Solutions are free of calcium ions, speeds up
decalcification and don’t have to change solutions
frequently
• Staining is excellent, and not harmful to the tissues.

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 Electrolytic methods
• Use formic acid and hydrochloric acid placed in an
electroplating device
• Bone is attached to the anode (+ pole) and pass a current
through the solution
• Calcium ions are attracted to the cathode (- pole)
• Decalcify bones within 2-6 hours
• Heat is generated which is detrimental to the integrity of
tissue architecture. Will render tissue un-stainable.
• Neutralize acid solutions with 1% sodium bicarbonate

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 Chelating agents
• Organic compounds that can bind to certain
metals
• EDTA can bind calcium
• Very slow
• Can bind only to ionized calcium

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 End point of decalcification
• Determination by:
– Physical or mechanical method - testing the flexibility of
the specimen; probing the specimen with needle or pin;
creates histologic artifacts
– Chemical testing - two step method (on next slide)
– Radiography - most accurate in determining complete
demineralization (zero opaqueness = complete
decalcification) … Do not use metallic-based fixatives!

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 Chemical testing
• Step 1:
– Check 5 ml decal fluid
from day 2
– Add ammonium
hydroxide until neutral
– Observe turbidity (+
means still heavily
calcified, therefore
change solution); (-
means proceed to step
2)
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• Step 2:
– Add 5 ml saturated
ammonium oxalate
– Mix and let stand for 30
min
– Observe turbidity (+
means still heavily
calcified, therefore
change solution); (-
means decalcification is
complete)
50
 Surface decalcification
• Micro-calcification would pose microtomy
problem - “scoring” due to nicks on the blade.
• Place exposed surface of tissue block on decal
fluid for 30 -60 min. Rinse the block well and
section gently.

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 Frozen sections
• Method of processing
– When rapid diagnosis is desired
– When staining technique does not work on routinely
processed tissue
• Used for demonstration of fat
• Used for enzyme and immunohistochemical
techniques
• Immunofluorescence and lymphocyte surface marker
studies

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 Cryostat

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 Frozen sections
• Use cryostat (microtome in a cabinet kept @ -20°C).
Heat extractor (to alleviate slow freezing artifacts)
• Tissues are embedded in cryotechnique medium and
snapped frozen in isopentane (-150 °C)
• Disadvantages:
– Flammable liquid
– Susceptibility to extreme cold temp
– Cutting unfixed tissues

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 Cryostat or frozen sectioning
• http://www.pathologyinnovations.com/frozen
_section_technique.htm

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 Chuck, embedding medium and
cryostat

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 Embedding
• “casting or “blocking”
– Enclose tissue in embedding medium
– Allow to solidify
• Embedding medium
– Melting point (high MP good support for hard tissues; low
MP not enough support for hard tissues)

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 Embedding
• Makes tissue block suitable for microtomy
• After wax infiltration, tissues are oriented in
hot liquid wax, then allowed to solidify into a
paraffin block.
• Proper orientation of tissue is important
• Uses embedding centre, cassettes, molds

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 Retort: cassette storage

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 Embedding steps SOP #3
• Select correct size mould
• Pick up tissue specimen from
the opened cassette with
heated forceps and look for
the flat or marked side
• Fill the well of the mould with
molten paraffin
• Place tissue against the base
of the mould flat or marked
side down.

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 Embedding steps
• Orient the tissue diagonally
then introduce the “initial
chilling”
• Apply gentle pressure on the
tissue so that it will be
embedded flat.
• Place cassette on top and add
more wax through the
perforations.

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 Embedding steps
• Transfer the mould-
cassette “system” on to the
cooling plate (temp: -4° C
to -6° C)
• BE PATIENT! Remove
blocks from molds
• File and sort in ascending
order

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 Final product

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 Points to remember
• Wax temp must be 2 - 4° C above MP
• Select correct mould size for the tissue
specimen
• Embed flat side of tissue down
• Embed notched side up and inked side down
• Apply light pressure when orienting tissue
against the base of the mould

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 Points to remember
• All tissues - Embed tissues diagonally (p.43)
and centrally. Must have at least 2 mm margin
around the tissue(s).
• Tissues with wall - embed on edge to show
layers
• Tubular structures - embed in cross section
• Layered tissues - embed to show all layers

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 Points to remember
• More than one piece of large tissues - embed in the
same orientation in the mould.
• Open and embed one cassette at a time
• Wipe embedding forceps between blocks to avoid
“forceps metastasis” (p. 43)
• Do not allow bottom wax to solidify before adding
more wax
• Cool blocks rapidly to avoid large paraffin crystals.

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 Points to remember
• Record the number of tissue pieces +
orientation instructions (during grossing)
• Number of blocks or pieces for each case must
be checked against the log at the end of
embedding process
• Check total number of pieces or slides at the
end of staining process.
• Match the block against the slide

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 Filing the blocks after microtomy

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 Paraffin block and stained slide

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