TISSUE PROCESSING

Presented By:
Dr. Dinesh Kumar Yadav
PG Resident,
Dept. of Oral Pathology, KDCH

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 Contents
• Introduction

• Principles of tissue processing

• Factors influencing the rate of processing

• Stages of tissue processing

• Tissue processors

• Tissue Reprocessing

• Conclusion 4
 Introduction
Tissue Processing is a series of physical and chemical procedure
which needs to take place between tissue fixation and sectioning of
paraffin blocks to ensure that the final microscopic slides produced
are of a diagnostic quality.

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 Principles Of Tissue Processing
• To remove all extractable water from the tissue

• To replace it with a support medium that provides sufficient rigidity

• To enable sectioning of the tissue without parenchymal damage or

distortion

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Factors Influencing The Rate Of Processing

An interchange occurs
When tissue is
between the fluid
immersed in
within the tissue and
fluid
the surrounding fluid

Rate dependent upon the exposed surface of the tissue that is in contact with the processing reagents.
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Factors Influencing The Rate Of Processing

1. Specimen size

2. Agitation

3. Heat

4. Viscosity

5. Vacuum

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1. Specimen size
• The thicker the specimen, the longer will impregnation of
processing fluid take place
• No thicker than 3 - 4 mm

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2. Agitation
• Increases the flow of fresh solutions around the tissue.

• Efficient agitation may reduce the overall processing time by up to 30%.

• Mechanisms for agitation in Automated processors:

3.
1. 2. Pressurized removal and
Vertical oscillation Rotary oscillation replacement of fluids at
timed intervals

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3. Heat
• Increases the rate of penetration and fluid exchange.

• Heat must be used sparingly to reduce the possibility of:

1. Shrinkage
2. Hardening
3. Embrittlement

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4. Viscosity : The property of resistance to the flow of a fluid..

smaller the size lower the faster the rate of

of molecules viscosity fluid penetration

larger the size of higher the slower the rate of

molecules viscosity fluid penetration

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5. Vacuum

Using reduced Increase the rate Decreases the time

pressure of infiltration of tissue processing

• Evaporation of clearing agent and impregnation of paraffin wax

• Should not exceed 50.79 kPa to prevent damage and deterioration

to the tissue.
• Removal of trapped air in porous
13 tissue.
 Stages Of Tissue Processing

2.
1. 3. 4. 5.
DEHYDRATIO
FIXATION CLEARING INFILTRATING EMBEDDING
N

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 1. Fixation
• Stabilizes and hardens tissue with minimal distortion of cells.

• First and important step in tissue processing

Stations should be designated

If fixation is not complete
on the processor for this
prior to processing
purpose

If the tissue is The subsequent dehydration

solutions may complete the
inadequately fixed process
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 Post-fixation Treatment
Special fixation techniques require additional steps before processing is initiated:

Picric acid
fixatives • Cassettes placed directly into 70% alcohol for processing
(Bouin’s)

Alcoholic fixatives • Should be placed directly into 100% alcohol.

(Carnoy’s fluid)

To help in the • A few drops of 1% eosin can be added to the specimen

visualization of small container 30 minutes prior to processing.
fragments of tissue • The pink color of the tissue remains during processing, but
during embedding washes out during subsequent staining.
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 2. Dehydration
• Removal of ‘free’ unbound water and aqueous fixatives from
the tissue components.
• Many dehydrating reagents are hydrophilic, possessing strong
polar groups that interact with the water molecules in the
tissue by hydrogen bonding.

Specimens are processed • Should be accomplished slowly.

through a graded series • If the concentration gradient is excessive,
of reagents of increasing diffusion currents across the cell membranes
concentration may increase the possibility of cell distortion.
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 Dehydration

Excessive dehydration
• may cause the tissue to become hard, brittle and shrunken.

Incomplete dehydration
• impair the penetration of the clearing reagents into the
tissue
• leaving the specimen soft and non- receptive to infiltration

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 Dehydration
• Duration of treatment in graded alcohols

• Depend on the size and type of tissue

Giant sections of whole organs:

50% 70% 96% 100% alcohol

• For 24 to 48 hours in each, depending on their thickness

• Three changes of each strength.

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 Dehydration
• For routine biopsy specimens and post-mortem tissue of not more than 7
mm in thickness

70% 90% Absolute alcohol

• Tissues may be stored in 70% alcohol after fixation in intolerant fixatives

such as those containing mercuric chloride.

• 1-2 hours each

Heidenhain's Susa: must be followed directly by 96% alcohol.

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 Dehydration
Dehydrating agents
1. Ethanol
2. Industrial methylated spirit (denatured alcohol)
3. Methanol
4. Isopropyl alcohol
5. Butanol
6. Acetone

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 Dehydrating fluids
1. Ethanol (C2H5OH)
• Clear, colorless, flammable liquid

• Hydrophilic, miscible with water and other organic solvents

• Fast acting

• Graded concentrations of ethanol are used for dehydration: 70%, 95%, 100%

• Ethanol ensures total dehydration, making it the reagent of choice for the
processing of electron microscopy specimens.
• For delicate tissue it is recommended that the processing starts in 50% ethanol.
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 Dehydrating fluids
2. Industrial methylated spirit (denatured alcohol)
• Denatured alcohol consists of ethanol, with the addition of
methanol (about 1%), isopropyl alcohol or a combination of alcohols.
• Has the same physical property as ethanol.
• Used in the same manner as ethanol.

3. Methanol (CH3OH)
• Methanol is a clear, colorless and flammable fluid that is miscible
with water, ethanol and most organic solvents.
• It is highly toxic but can be substituted for ethanol in processing
protocols
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 Dehydrating fluids
4. Propan-2-ol, isopropyl alcohol (CH3CHOHCH3)
• Miscible with water, ethanol and most organic solvents

• Used in microwave processing schedules

• Does not cause over-hardening or shrinkage of the tissue.

5. Butyl alcohol (butanol) (C4H9OH)

• Used primarily for plant and animal histology.

• Butyl alcohol is a slow dehydrant causing less shrinkage and hardening of the
tissue
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 Dehydrating fluids
6. Acetone (CH3COCH3)
• Clear, colorless, flammable fluid that is miscible with water,
ethanol and most organic solvents.
• Rapid in action, but has poor penetration and causes brittleness
in tissues if its use is prolonged.
• Acetone removes lipids from tissue during processing.

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 Additives to dehydrating agents
Phenol
• When added to dehydrating agents, it acts as a softening agent for
hard tissues such as tendon, nail, and dense fibrous tissue and keratin
masses.
• Phenol (4%) should be added to each of the 95% ethanol stations .

Glycerol/Alcohol
• Hard tissue can be immersed in a glycerol/alcohol mixture.

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 Universal solvents
• Both dehydrate and clear tissues during tissue processing.

• No longer used for routine processing due to their hazardous

properties

• Not recommended for processing delicate tissues due to their

hardening properties.
Dioxane, Tertiary butanol , Tetrahydrofuran

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 3. Clearing
• Removal of dehydrating solutions, making the tissue components
receptive to the infiltrating medium.
• Since alcohol and wax are not miscible, the alcohol must be replaced
by a wax solvent

• Raise the refractive index of tissue, which makes them appear clear

Why is it called clearing ?

When the dehydrating agent has replaced by the clearing solvents, the tissue has a
translucent appearance.
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• The boiling point of the clearing agent gives an indication of its
speed of replacement by melted paraffin wax
• Fluids with a low boiling point are generally more readily replaced

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Criteria Of Choosing A Clearing Agent
• Rapid penetration of tissues

• Rapid removal of dehydrating agent

• Ease of removal by melted paraffin wax

• Minimal tissue damage

• Low flammability

• Low toxicity

• Low cost 30
Clearing Agents Suitable For Routine Use
1. Xylene

2. Toluene

3. Chloroform

4. Xylene substitutes

5. Citrus fruit oils – limonene reagents

7. Cedar wood oil

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 Clearing agents
Xylene
• Flammable, colorless liquid

• Characteristic petroleum or aromatic odour

• Miscible with most organic solvents and paraffin wax

• Suitable for clearing blocks that are less than 5 mm in thickness and
rapidly replaces alcohol from the tissue.
• Overexposure to xylene during processing can cause hardening of
tissues. 32
 Clearing agents
Toluene
• has similar properties to xylene

• although it is less damaging with prolonged immersion of tissue

• It is more flammable and volatile than xylene.

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 Clearing agents
Chloroform
• Slower in action than xylene but causes less brittleness.

• Thicker tissue blocks can be processed, greater than 1 mm in thickness.

• Tissues placed in chloroform do not become translucent.

• It is non-flammable but highly toxic, and produces highly toxic

phosgene gas when heated.
• It is most commonly used when processing specimens of the central
nervous system. 34
 Clearing agents
Xylene substitutes
• Aliphatic hydrocarbons that exist in long- and short-chained forms.

• Differ in the number of carbon atoms within the carbon chain.

• Short-chained aliphatics have the same evaporation properties as

xylene, and have no affinity for water.
• Long-chained aliphatics do not evaporate rapidly and may cause
contamination of the paraffin wax on tissue processors.
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 Clearing agents
Citrus fruit oils – limonene reagents
• Limonene reagents are extracts from orange and lemon rinds; they
are non-toxic and miscible with water.
• Disposal is dependent upon the water treatment centers and
local/national standards.
• The main disadvantages are that they can cause sensitization and
have a strong pungent odor that may cause headaches.
• Extremely oily and cannot be recycled.
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 Clearing agents
Cedar wood oil
• Best reagent for research and treatment of delicate tissues, since it
has the least hardening effect.
• Used for hard tissues such as skin and dense fibrous tissue since
sections are easier to cut after such treatment.

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 4. Infiltration
• Permeating the tissue with a support medium

• Paraffin wax: the most popular.

• Why infiltration?
• Tissue slices need to be approximately 4-6 μm in thickness
• For highly cellular tissues, such as lymph node, 2-3 μm may be more
appropriate
• When examining larger cells and processes, such as those of the
nervous system, 10-20 μm.

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 5. Embedding
• Embedding involves the enclosing of properly processed,
correctly oriented specimens in a support medium that
provides external support during microtomy.

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Infiltrating And Embedding Reagents
Paraffin wax
• A mixture of long- chained hydrocarbons produced in the cracking of
mineral oil
• It has a wide range of melting points, which is important for use in
the different climatic regions of the world
• Its properties are varied depending on the melting point used,
ranging from 47 to 64°C
• Permeates the tissue in liquid form and solidifies rapidly when cooled
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Infiltrating And Embedding Reagents
Paraffin wax
• To promote desirable ribboning during microtomy, paraffin wax of
suitable hardness at room temperature should be chosen.
• It is more difficult to obtain thinner sections but ribboning is easier.

Higher melting point paraffin wax Lower melting point paraffin wax
• Provides better support for harder tissues • Softer
(bone) • Provides less support for harder tissues
• Can allow production of thinner sections
• May cause difficulty with ribboning

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 Requirements For Successful
Paraffin Use
• Rapidly converted from solid to liquid form upon heating

• Permeates tissue in a liquid state

• Solidifies rather quickly on cooling

• Provides support for tissue cellular structure

• Ability to cut thin or thicker sections

• Cost effective

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 Paraffin..… What is it?
• Paraffin is a blend
• simple hydrocarbon chains will penetrate first
• long polymer chain needs time to fully infiltrate

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 Paraffin..… What is it?
• Resulting in A wide range of crystals

• Causes paraffin to harden unevenly with large & small crystals

• Vast melting point range

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• Dependent on the size or molecular weight of each component
• Paraffin with no additives will penetrate fast due to simple carbon
chains
• Adding branched carbon chain waxes or polymers increases the time
of infiltration proportional to its size

• Larger the molecule the higher the viscosity and thus slower rate of
penetration

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 Paraffin Selection
• Most labs have adopted a paraffin that is in the mid-range, not
too hard and not too soft

Mid-Range
55˚-58˚

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Complete Infiltration is a necessity

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Paraffin infiltration- What happens in the tissue
processor
• Air (arrows) being pulled from the tissue
interstices by the action of vacuum
• Long chain paraffin wax hydrocarbons (small
dashes) entering and filling voids (interstices)
left by the effects of vacuum
• Compacted paraffin wax hydrocarbon molecule
(dark areas) due to vacuum followed by pressure
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 What is the Issue?
• Problem: Fatty Tissue, mushy

• Causes : Processing has failed to remove lipid from

areas of the specimen and these areas have not been
properly infiltrated with wax and are unsupported for
cutting.

• Solution: Reprocessing, using a schedule of sufficient

length, should produce blocks that can be sectioned

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 Infiltrating And Embedding
Paraffin
Infiltrating Paraffin Embedding Paraffin
• Used in Tissue Processors • Used for Embedding

• Pro: Infiltrates quickly • Pro: May be able to cut thinner

• Con: Too soft for cutting thin • Con: May not be recommended for
sections use in tissue processors

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Paraffin wax additives
1. Beeswax
2. Rubber
3. Ceresin
4. Plastic polymers
5. Diethylene glycol distearate

• The amount of additive will impact the rate of infiltration.

• Many of these additives have a higher melting point than paraffin

wax, consequently making the tissue more brittle
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Alternative embedding media
• Resin : For electron microscopy, ultra-thin sectioning for high
resolution and also for undecalcified bone
• Agar: As a cohesive agent for small friable pieces of tissue after
fixation, a process known as double embedding.
• Gelatin: In frozen sectioning.

• Celloidin: Use limited to neuropathology

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 Paraffin Wax Embedding

Most laboratories use modular embedding

centers, consisting of :
1. a paraffin dispenser
2. a cold plate
3. a heated storage area for moulds
and tissue cassettes.

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 Paraffin Wax Embedding
Types of Moulds:

1. Leuckhart’s Moulds: L shaped brass pieces with adjustable

opposite vertices

2. Ice trays

3. Paper ‘boats’

4. Embedding cassettes

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Paraffin Wax Embedding

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 Paraffin Wax Embedding
Paraffin wax is dispensed automatically from a
nozzle into a suitably sized mould.

The tissue is oriented in the mould

A cassette is attached, producing a flat block face

with parallel sides.

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 Paraffin Wax Embedding
The mould is placed on a small cooling area to allow the paraffin
wax to solidify.

The quick cooling of the wax ensures a small crystalline

structure, producing fewer artifacts when sectioning the tissue.

Once solidification is complete, care must be taken that it is

complete block may be removed from the mould.

Mould release spray should ensure that this happens easily

enough , but if it does stick, a sharp tap should release it.
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 Paraffin Wax Embedding
Orientation of tissues
• For the demonstration of proper morphology.

Products are available that help

ensure proper orientation:
• Marking systems
• Tattoo dyes
• Biopsy bags
• Sponges
• Papers
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Orientation of tissues
• Must be cut at right angles to the surface, so that the full
thickness of the epithelium is visualized.
• Wedge shaped biopsies may present special problems

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Orientation of tissues
• In general, the longest axis of the specimen should be parallel to the
blade.
• However, it is sometimes advantageous when cutting, particularly
hard tissue, such as bone or teeth, to orientate the specimen so that
the blade meets a corner of the specimen first.

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Orientation of tissues
• It is often advantageous to embed skin so that the epithelial
surface, particularly where it has a tough surface such as that
found on palms and the soles of feet, meets the blade after the
softer, underlying connective tissue has been cut.

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Orientation of tissues
• Tubes should be sectioned at right angles so that the lumen is
clearly visible

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Orientation of tissues
• Multiple tissue pieces are aligned across the long axis of the
mould, and not placed at random.

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 Double Embedding
• More than two embedding medium for same tissue

• Used when the tissues require external support

• Agar-paraffin embedding or celloidin(nitrocellulose)-paraffin

embedding
• Tissue is first impregnated with celloidin, and subsequently blocked in
paraffin wax
• Organs benefited: hard tissues such as bone, brain, muscle, large
pieces of dense fibrous tissue
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 Automated Tissue Processing
• The basic principle for tissue processing requires the exchange of fluids using a series of
solutions for a predetermined length of time in a controlled environment.

The carousel-type
processor
(tissue transfer)

Types of tissue
processors

The self-contained fluid

exchange systems

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 The Carousel-type Processor
• Transports tissue blocks contained in baskets through a series of
reagents housed in stationary containers.
• The length of time the specimens were submerged in each
reagent container was electronically controlled.
• Rotation or Vertical oscillation of the tissue into the reagent
containers provided the agitation needed for the processing of
the tissue.

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 Vacuum Tissue Processor
• The enclosed, self-contained vacuum tissue processor

• A microprocessor was used to program this instrument.

Tissues were loaded into a retort chamber where they remained throughout the process.

Reagents and melted paraffin wax were moved sequentially into and out of the retort chamber
using vacuum and pressure.

The advantages of this system are:

Vacuum and heat can be used at any stage
Customized schedules for tissue processing are possible
Here is fluid spillage containment
67 and elimination of fumes.
 Microwave Processors
• Shortens the processing time from hours to minutes

• Stimulates the diffusion of the solutions into the tissue by increasing

the internal heat of the specimen, thus accelerating the reaction.
• Tissues are manually transferred from container to container of
reagent.
• Reagents: ethanol and paraffin.

• Temperatures must be maintained between 70 and 85°C

• The size of tissue sample is critical

68 (2 mm)
 Microwave Processors
• Xylene and formalin are not used in this process, which eliminates toxic
fumes and carcinogens.
• Precise temperature controls, timers, and fume extraction systems.

• Graded concentration of solutions is not required.

• Clearing agents are not necessary because the temperature of the final
paraffin step facilitates evaporation of the alcohols from the tissue.
• Properly controlled processing provides uncompromised morphology
and antigenicity of the specimens.
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 Alternative Rapid Processors
• Advances in technology have led to the development of a ‘continuous
input rapid tissue processor’.
• A robotic arm moves the tissue cassettes through four stations which
contain acetone, isopropanol, polyethylene glycol, mineral oil and
paraffin.
• Microwaves and agitation are used to accelerate the diffusion of solvents
in tissue.
• A patented microwave technology is utilized, which operates at a
continuous low power instead of pulsing high levels of microwave energy
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Overnight Processing Schedules

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 Rapid Processing Schedules For
Small Biopsies
• Most small specimens will fix
prior to processing.
• If fixation is not complete,
processing should begin in a
station containing 10% NBF.

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 Manual Tissue Processing
Manual tissue processing is usually done for the following reasons:
• Power failure or breakdown of a tissue processor.
• A requirement for a non-standard processing schedule as for rapid
processing of an urgent specimen.
• Delicate material.
• Very large or thick tissue blocks.
• Hard, dense tissues (nitrocellulose methods).
• Special diagnostic, teaching or research applications.
• Small scale processing requirements.
• Resin embedding.
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 Manual Tissue Processing
• The main advantage of manual processing over automated
methods lies in the flexibility of reagent selection, conditions
and schedule design to provide optimum processing for small
batches of tissues.
• Exposure of tissues to the deleterious effects of some reagents
can be carefully monitored and regulated through observation
and precise timing.

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 Manual Tissue Processing
• Manual processing is accelerated using microwave ovens or
ultrasonics.
• By using a microwave oven, heat is generated from within the
tissues which warms the tissue block uniformly in a short time.
• This results in faster penetration of tissue processing chemicals
inside tissues resulting in rapid processing for making paraffin
blocks.

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 Manual Tissue Processing
• Permanent series of solutions in wash bottles simplifies
processing small single specimens
• Tissues are processed in tubes and agitated on a rotor

• Reagents are pipetted

• Multiple specimens or large blocks are economically processed

in large lidded jars of processing fluids
• The specimen to reagent volume ratio should be at least 1:50

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 Rapid Manual Tissue Processing Schedule
Fix the specimen in Carnoy's fluid for 45 min.

Dehydrate in absolute ethyl alcohol for 15 min.

Clear in xylene 1 for 10 min.

(Drury and Weilngton. 1980)
Clear in xylene 2 for 15 min. (or until clear).

Impregnate in paraffin wax 1 for 20 min.

Impregnate in paraffin wax 2 for 45 min

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 Tissue Reprocessing
• In spite of great care to prepare quality specimens, some are not
properly fixed or processed. To fix this problem the tissue can be
reprocessed.
The tissue is removed from the paraffin by immersing the sample in multiple changes of xylene .

The paraffin-cleared tissue is then hydrated through a descending alcohol series and
refixed in formalin.

The tissue is reprocessed and embedded.

• The Ventana Renaissance Tissue Processor that automates this

manual method.
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 Restoration Of Tissue Dried In
Processing
• The following treatment may help provide slides of adequate
diagnostic quality.

Tissue restoration solution:

70% ethanol 70 ml
Glycerol 30 ml
Dithionite 1g

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 Quality Control
• Temperature of all paraffin wax dispensers, flotation water
baths and automated processors are carefully monitored,
maintained and documented.
• The histology laboratory should have a policy and procedure
manual that addresses quality issues and corrective actions
taken.

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• The aim of the present study was to compare the cytoplasmic and
nuclear details as well as staining characteristics of tissue sections
processed by conventional, rapid manual, and microwave techniques.
• Result : The effects of the three methods of histoprocessing on
cytoplasmic and nuclear details of epithelial, fibrous, and glandular
tissue showed no statistically significant variation. The microwave
technique was comparable or slightly better than the manual methods.
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 Conclusion
• Tissue processing is critical step.

• They should not be under processed or over processed, as it may

hamper the tissue details.

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 References
• Bancroft’s theory and practice of histological techniques 7th edition-
S.Kim Suvarna, Christopher Layton, John D.Bancroft
• Cellular Pathology Techniques 4th edition-C.F.A Culling, R.T.Allison, W.T
Barr
• Carson FL. Histotechnology. 2nd ed. Chicago: ASCP Press, 2007

• Drury R A B and Weilngton E A. Carleton Histological Techniques, fifth

edition,Oxoford University press, London 1980; 3:
• https://www.slideshare.net/ahmededrissi1/routine-histological-techniqu
es?from_action=save 83
Thank You !

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