TISSUE PROCESSING

PTH 4311
 OUTLINE
Types of tissue processors.
• Introduction
• Specimen reception and handling
• Fixation
• Principle of tissue processing
• What isTissue processing
-dehydration
-clearing
-embedding and impregnation
• Section cutting
• Staining
• Special techniques/recent advances
• Summary and conclusion
 INTRODUCTION
 Tissue processing deals with the preparation
of tissue for microscopic examination.
 This is accomplished by submitting the total
or a selected part of the tissue presented for
examination to a series of processes.
 The aim of good Histopathological technique
is to produce microscopic preparation of
tissue, usually stained, that represents as
closely as possible their structure in life.

3
 INTRODUCTION
When a tissue sample is removed from a patient, to turn it into final
microscopic slide of good diagnostic quality. It has to pass through a
series of processes. These processes includes –
 Fixation
 Dehydration
 Clearing
 Infiltration/embedding,
Each of these step is very important from the point–
procurement of specimen
selection of the sample
protocol determination
reagents to use
staining and
final diagnosis
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 Tissue processing
• Aims to remove all extractable water from the tissue,
replacing it with a solid medium firm enough to support the
tissue and give it sufficient rigidity to enable sectioning
without damage or distortion.
• Types of processing: Manual or Automated
• Stages of tissue processing:
-dehydration,
-clearing,
-impregnation and embedding
 Specimen reception and
handling
• At the reception d specimen is checked for:
-is d specimen for histological examination
-labelling on d container as well as d
accompanying completed request form
-is d specimen in a fixative, is it sufficient or d
right fluid
• After that, d specimen is registered and given a
laboratory number for identification
• Specimen is collected and stored in d cut-up
room
The quality of the structural preservation of tissue
components is determined by choice of reagents and
exposure time in the reagent during processing.
LABELING OF TISSUE
 Unique code or identification number is assigned to
each tissue sample in the laboratory.
 They may be manually or electronically generated.
 Bar codes and character recognition systems are now
common with most laboratories.
 Automated pre-labeling systems etch or emboss tissue
cassettes or slides permanently.
Chemically resistant:
Pens
Labels
Slides
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Pencils
 Tissue cassettes by
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Tissue cassette writer by thermo
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scientific ltd.
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WHAT IS TISSUE PROCESSING
 Tissue processing refers to treatment of tissue
by passing it gradually through series of reagents
necessary to impregnate it into a solid medium so
that the tissue is rendered sufficiently firm yet
elastic for tissue sections of desirable thickness to
be cut on a microtome.
 The fixed impregnated tissues have an
advantage that they can be easily preserved and
are reproducible.
 This is not the only technique employed for
tissue sections to be produced for microscopy.
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 PRINCIPLE OF TISSUE PROCESSING
 Tissue processing is designed to remove all
extractable water from the tissue.
 The water is then replaced with a solid support
medium.
 The medium provides it with sufficient rigidity to
enable sectioning of the tissue with out distortion
or damage.
 Most tissue fixatives are aqueous fixatives, hence for
proper embedding of tissue in paraffin wax it is necessary
that the water, some lipids and other tissue fluids be
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by 13:15
a variety of compounds.
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STAGES OF TISSUE PROCESSINING

 Complete fixation
 Dehydration
 Clearing
 Infiltration/Embedding

 Complete fixation – post fixing

 Dehydration - removal of water an fixative
 Clearing – removal of dehydrating solutions, making
the tissue receptive to infiltrating solutions.
 Infiltrating- permeating the tissue with a support
medium.
 Embedding – orientating the tissue sample in a support
medium and allowing it to solidify.
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 Dehydration
• This occurs when d water constituent of
tissue is removed and is important because
most embedding media are not miscible
with water thus inhibiting impregnation.
to remove fixative and water from the tissue
and replace them with dehydrating fluid.
There are a variety of compounds many of
which are alcohols. several are hydrophilic
so attract water from tissue.
Types of dehydrating agents:
Ethanol, Methanol, Acetone.
Dehydration
A. Definition: removal of water
B. Rationale: for paraffin
embedding/sectioning
C. Steps
1. wash out fixative
2. graded series of alcohol
a. 70%, 95%, 100%, 100%
3. replace water by diffusion
4. not too long, not too short
 CLASSIFICATION OF DEHYDRANTS
• - Alcohol: ethyl alcohol is d best, non poisonous
-Acetone: good dehydrant, cheap but very
volatile.greater volume required and causes tissue
shrinkage, thus for this not suitable for routine work.
-Dioxane(diethylene dioxide): unique bcos its miscible
with water, alcohol, xylene and paraffin wax with little
tissue shrinkage but has cumulative toxicity.
• Choice of dehydrant depends on Nature of task,
Processing method, Embedding medium and
Economic factors
III. Dehydration
D. Procedure
1. automatic
tissue processor
a. overnight
2. Manual: 50,
70,95,100,100 %
alcohol
 Other dehydrating agents:
Acetone :
• It is clear, colourless volatile inflammable fluid.
• It has a rapid action in dehydrating the tissue but produces shrinkage
and distortion and subsequent brittleness to the tissue.
• Low cost is also an advantage
• Acetone usually dehydrates within 20-30 minutes but four changes of
acetone should be used, it is preferable to use acetone after low
strength of alcohol so that distortion of the tissue is less.

Dioxane :
• It dehydrates and clears at the same time.
• It is miscible with paraffin and with water and alcohol, tissue from
Dioxane can be transferred straight to paraffin.
• There is less shrinkage of tissues
• Tissues can be left in Dioxane without danger of hardening for longer
period of time.

Disadvantage: It is more expensive than alcohol and It is toxic.

 Use of solid dehydrants:
• Anhydrous copper sulphate is used in higher grade of dehydrating
alcohols.

• A layer 1-2.5 cm thick is placed at the bottom of a dehydrating vessel

or beaker and is covered with 2 or 3 filter papers to prevent
contamination of the tissues.

• Anhydrous copper sulphate is white, it removes water from alcohol

which in turn has been diluted upon absorption of water from the
tissues.

• The change of colour of copper sulphate from white to blue indicates

that both alcohol and water should be changed.

• Use of copper sulphate enhances the process of dehydration and also

prolongs the life of alcohol.
FACTORS AFFECTING DEHYDRATION

 Agitation
 Heat
 Copper sulphate
 Excessive dehydration – shrink, hard and brittle
 Incomplete dehydration – prohibits infiltration of
clearing agent and wax
 Duration of dehydration- Tissue blocks 1 mm thick
should receive up to 30 minutes in each alcohol,
blocks 5 mm thick require up to 90 minutes or longer
in each change.
 Clearing
• Is a process of removal of alcohol (de
alcoholisation) Replacing the dehydrating fluid with
a fluid that is totally miscible with both the
dehydrating fluid and the embedding medium
• Clearing means appearance of tissue after it has
been treated by the fluid chosen to remove the
dehydrating agent.
• clearing agent also serve as an ante-media for wax
impregnation
• Ante media have the property of making tissue
transparent.
 Clearing
• The clearing agent is a reagent that is miscible with
paraffin wax and acts as a link between the dehydrating
agent (non-miscible) and the paraffin wax itself.
• The most commonly used clearing agents for routine
tissue processing are hydrocarbons such as xylene,
toluene, chloroform and petroleum solvents.
• are flammable, cause skin degreasing and have varying
degrees of toxicity.
• Toluene is a possible carcinogen, chloroform has a
narcotic effect and xylene, may cause headaches as a
side-effect to inhalation of the vapour where adequate
extraction facilities are not provided (usually in
coverslipping and manual staining areas).
 cont
CLEARING AGENT
1.Xylene (Xylol) very good clearing agent, cheap
and cleared tissue within 20 – 30mins. When
the hydration is not complete it turns milky.
2.Toluene (toluol) slower than Xylene and toxic
3.Benzene – known to be carcinogenic.
4.Chloroform – ideal clearing agent for CNS, LN &
Embryo.
 cont
5. Cedar wood oil – slow in action, expensive
causes little damage to tissue, is ideal for
research. Disadvantage is diff to eliminate oil
from wax.
6. Others include carbon tetrachloride, carbon
disulphate, paraffin wax, petrol clove oil, methyl
benzoate.
IV. Clearing
A. Paraffin
solvent
B. Xylene,
“clearing
agent”
C. Makes tissue
appear “clear”
 IMPREGNATION &
EMBEDDING
Impregnation is the process whereby cavities,
spaces, and interstices of the tissue are filled with
a medium which solidify to form a sufficient firm
consistency that can give considerable support
during the cutting of section without distortion to
or alteration of the tissue and cellular
constituents. Impregnation: It is the complete
removal of clearing reagents by substitution of
paraffin or any such similar media.
 Impregnation / Infiltration
Is the saturation of tissue cavities and cells by a supporting
substance. Eg molten paraffin wax

Purpose
 To remove clearing agent from tissues for them to be completely
permeated by paraffin wax which is subsequently allowed to harden to
produce a block from which sections may be cut.
To provide an internal support to tissue
IMPREGNATING MEDIA
-Paraffin wax mp – 54 – 60
-Paraplast mp 56 – 57
-Embeddol mp 56 -58
-Celloidin and low viscocity nitrocellulose
 Paraffin Wax Impregnation
• Simplest, most common, best, 56 C
• Ease in cutting
• Permanent paraffin blocks
• Good staining results
• Not recommended for fatty tissues
• Overheated paraffin  brittle specimen
• Inadequate impregnation  clearing agent retainedImpregnation
with wax: Impregnation with paraffin wax takes place in an oven
heated to 56-60°C depending upon the melting point of the wax in
use.
• Frequent check of the temperature of paraffin baths is required
since temperature 5°C above the melting point of the paraffin will
cause tissue shrinkage and hardening
Impregnation
A. Replace xylene with
paraffin
B. Immerse in melted
paraffin
1. ~55o C MP
C. Remove all bubbles,
xylene
D. Procedure
1. Two baths of melted
paraffin
 Factors influencing the rate of
impregnation
 A tissue immersed in fluid interchange occurs between tissue fluid and
surrounding fluid.

 The process continues through all stages of processing from fixation to

final impregnation.

• Agitation - Tissue placed in liquid is agitated so that the fluid

immediately in contact with the surface of tissue which is mixed by
tissue fluid is replaced by the fresh immersing liquid.

• It can be achieved by a pumping system which removes and replaces

fluid at selected intervals or by rotation and vertical oscillation
method.

• Efficient agitation reduces the processing time by 25-30% with

improved impregnation of the tissue.
 Heat - Heat increases the rate of penetration.

 Viscosity - Larger the molecule the higher is the viscosity

slower is the rate of penetration.

 Ultrasonic : Use of ultrasonic increases the penetration rate.

 Vacuum : Use of reduced pressure is well known in the

impregnation of tissue by molten paraffin wax.
• It hastens the process.
• Use of vacuum during dehydration and clearing has little
advantage except removal of air bubble trapped within the
tissue.
 Embedding
• is the process by which tissues
are surrounded by a medium
such as agar, gelatin, or wax
which when solidified will
provide sufficient external
support during sectioning.
Embedding:
• It is the orientation of tissue in melted
paraffin wax which when solidified
provides a firm medium for keeping intact
all parts of the tissue when sections are cut.
• Examples of embedding mould are:
Leuckhart's L pieces
Plastic ice cube tray
Petri dish
 VI. Embedding
EMBEDDING: is the process of
casting the tissue in a paraffin
wax and allowing them to set
to facilitate a cutting section.

A. Orient tissue
1. cross section
2. longitudinal section
B. Dissection orientation
C. Avoid bubbles
 Embedding media:
• Like there is no perfect fixatives for tissue , so there is no perfect embedding
medium ,

The Ideal qualities of embedding medium

• Easily available, cheap
• Uniformity from one batch to another
• Solubility in dehydrating agents
• Low viscosity as monomer for penetration
• Uniform polymerization
• Little volume change during polymerization
• Good preservation of fine structure
• Good sectioning quality that includes homogeneity, hardness, plasticity and
elasticity
• Resistance to heat generated by sectioning
• Adequate specimen stainability
• Examples are;
Paraffin wax:
• It is the most commonly used embedding medium in both normal and
pathological histology
• Cutting qualities are good, the blocks are durable and their storage present
no special problems
• Polymerize uniformly and rapidly
• Thin sections (1-3mu) can be cut and it is cheap and therefore use routinely

Ester wax
• It has a lower melting point than paraffin wax, it is hard when solidified.
• It is suitable for cutting thin (2-3u) section with minimal shrinkage

Araldite:
• Polymerize uniformly with little change in volume (as low as 2%)
• Relatively stable in electron beam
• Relatively high viscosity
 Water soluble wax (polyethylene glycol):
• Use to embed tissue directly from aqueous fixatives, thus avoiding
the need for dehydration and clearing
• Shrinkage of tissue is reduced but the cutting and manipulation of
section is more difficult then with paraffin wax
• The embed with this medium must be kept in a dry atmosphere

Cellulose nitrate (celloidin and low viscosity nitrocellulose):

• This involved dehydration and impregnation of tissue with solution of
cellulose nitrate in a suitable solvent (mixture of alcohol and ether)

• The solvent is allow to evaporate to produce a block of suitable or

required consistency
• Double embedding:
• Is a combination of paraffin and cellulose nitrate methods design to use the virtue
of each.
 Synthetic resin:
• Are use particularly for the embedding of
specimens of undecalcified bone, for the
preparation of section for EM and LM (1-2u)

Freeze-drying and freeze substitution:

• This are used mainly for Histochemical
investigation

Gelatin and other aqueous media:

• Are mainly use to support tissue to be cut on the
freezing microtome and in the Gough and
Wentworth method
• Others like plastic embedding blocks (tissue Tek base mold system)
VI. Embedding
D.Procedure
1. Place tissue cassette
in melted paraffin
2. Fill mold with paraffin
3. Place tissue in mold
4. Allow to cool
VII. Sectioning –
Trimming the Block
Untrimmed tissue block

Trimmed block with

excess paraffin
removed and block
face in a trapezoid
shape
•Schematic representation of the conventional
overnight tissue processing
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42
 FACTORS INFLUENCING THE RATE OF
PROCESSING
When tissue is immersed in fluid, interchange occurs between the fluid within the
tissue and the surrounding fluid.

 AGITATION:- Increases the flow of fresh solutions around the

tissue.
- The rate of fluid exchange is dependent upon the
exposed Surface of tissue in contact with fluid.
- Rotary or vertical oscillation or pressurized
removal/replacement of fluid at timed intervals is the mechanism for agitations.
- Agitation can reduce overall processing time by 30% if efficient.

 HEAT : - Increases the rate of penetration and fluid exchange.

- Most be used carefully to prevent and reduce shrinkage, hardening and
brittleness of the tissue.

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 - Temperature limited to 450C can be used effectively, higher
temps may have effect on IHC staining.

 VISCOSITY: - Is the property of fluid that resists flowing: the

property of a fluid or semi fluid that causes it to resist
flowing.
- The rate of fluid penetration is faster if the size of the
molecules in the fluid is smaller.
- If the molecular size is larger, the rate of
exchange is slower.
- Clearing and dehydrating agents have same viscosity,
while paraffin has lower viscosity.

 VACUUM: - Using reduced pressure to increase the rate of

infiltration decreases the time necessary to c omplete other steps in
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tissue processing.
 TISSUE PROCESSORS
Tissue processors are instruments used to prepare
tissue samples for analysis by fixing, dehydrating,
staining, decalcifying. they help to achieve high-
quality histology and efficient laboratory workflow.

The carousel-type processors were the first type of

automatic tissue processors.
 Then came the enclosed self-contained vacuum
infiltration tissue processor.
 Recent advances has also led to the production of
specialty microwave oven processors and
continuous in-put rapid tissue processors.
 Recent processors employ microprocessors used to
programme the instrument.
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 They also employ alarm system and diagnostic
programmes for trouble-shooting malfunction of
the instrumentation.
 The advantages of recent systems are that:
 Vacuum
 Heat can be applied at any stage
 No spillage of fluids (contained)
 No fume
 They save time (Microwave oven) from hrs to
min.

 The continuous in-put rapid tissue processors, uses

microwave technology, vacuum infiltration and
reagents that are molecular-friendly.
 It uses isopropanol, acetone, Polyethylene glycol,
mineral oil and paraffin.

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 Microwave and agitation are used to accelerate rate of
diffusion of solvent in tissue.

 Advantages of the continuous in-put rapid tissue processors.

 Acceptance of tissue samples into the system at timed

intervals
 Improved turn around time
 Environmentally safe and friendly reagents
 Eliminating toxic vapours
 Good morphology and quality of the specimen consistent
with that of traditional tissue processing.

 Disadvantages

 Cost of the tissue processor.

 Grossing of tissue samples.

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 Automatic Tissue Processor

PARTS OF A TISSUE PROCESSOR

Reagent baths, Power sources, Alarm mechanism, Timer
Automatic transfer mechanism, Agitation mechanism
Wax bath, Delay mechanism
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TPC 15: Closed Linear Tissue Processing Canter
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Vaccum Tissue
Processor
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KS: Rapid Multifunctional Microwave Processor : processing: hours, not days! Bone marrow ; Rapid bone
decalcification; transplants biopsies; Special stains .

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The Tissue-Tek® brand Xpress® x50 processor and Tissue-Tek® brand Xpress®
x120 rapid tissue processor.

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extww02a.cardinal.com
 References

1. Bancroft, J.D. and Gamble. M., (2008) Theory and Practice of histological
techniques ed. 6th, Churchill Livingstone inc. . Edinburgh. London,
Melbourne and New York.

. Avwioro,
2Bancroft, O.G.
J.D. and , Histochemistry
Stevens, and tissue
A.: theory and practice pathology principle and
of histological
techniques. , Ed.
techniques ed.3, 1 Claverianum
Churchill Centre.
Livingstone inc. P.O. BoxLondon,
1990. Edinburgh. 4017. U.P.O., Ibadan
Melbourne and New York.

3. Horobin, R.W. (2002) Theory of staining and its practical implications. In

Theory and Practice of Histological Techniques, 5th edn (eds J.D.
Bancroft and M. Gamble). Edinburgh: Churchill Livingstone.

4. Kiernan, J.A. (2001) Histological and Histochemical Methods, 3rd edn.

Oxford: Hodder Arnold.

www.leicamicrosystems.com
www.google.com.ng/images
www.medscape.com

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