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OBJECTIVES
HISTOLOGY TECHNIQUES
Define dehydration, Clearing, Infiltration/
Impregnation and Embedding process.
Explain the tissue processing functions.
State various dehydration and clearing
solutions.
Elaborating tissue processing at level
dehydration, Clearing, Infiltration/
Impregnation and Embedding including action,
preparation and indication.

SMS2143
LECTURE 3

INTRODUCTION

Cont..

Once fixed or decalcified, the tissue must be

treated to allow the cutting of the thin sections
required for viewing under the microscope.

The procedures designed to prepare the tissue for

embedding by using paraffin wax are collectively
known as Tissue Processing.
Processing.

Then the tissue will be immersed in the liquid

paraffin wax solution before the tissue undergoes
embedding process.

First, the sample is dehydrated by immersion in a

series of aqueous alcohol solutions gradually
moving to pure alcohol.

Finally the tissue is embedded in paraffin wax,

which enables the cutting of sections of between 4 10 microns thickness.

The movement through the series of baths in Tissue

Processing occurs either by hand or by means of an
automated processor.

The tissue is then soaked in an appropriate solvent

to remove the alcohol (clearing).

DEHYDRATION

TISSUE PROCESSING
The

Purpose

: preparation for the tissue for

embedding by using paraffin wax
Divided into 4 steps which are ::

Dehydration
Clearing
Infiltration & Impregnation
Embedding

purpose of the dehydration process is:

To remove water and fixative from tissue

To replace the space with solution that can be mixed
with paraffin
This

is because wet fixed tissue CANNOT be

directly infiltrated with paraffin wax.
This process is done by using ALCOHOL solution
solution..
The technique that is used to perform this process
is SERIAL DILUTION.
DILUTION.
The tissue will be soak in a series dilution of
ascending alcohols such as (70%  95% 
100%)

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List of commonly used DEHYDRANTS

Cont

DEHYDRANT

Tissue

can be brittle if it is left too long in high

conc. of alcohol
Most essential step
Tissue transferred directly from dH2O to high
[alcohol] would cause distortion of tissue elements
because of mixing dH2O + alcohol
More delicate object to be processed, more
numerous steps required
Failure to do this properly will prevent the tissue
from becoming impregnated successfully later with
paraffin wax or celloidin

Other dehydrants agent used in histopath lab

Agents

Advantages

Disadvantages

Ethyl alcohol

Fast action

Shrink tissue

Isopropyl alcohol

Non toxic, good

penetration

Tissue harden

Methyl alcohol

Fast action

Toxic

Butyl alcohol

Less shrinkage & has

hardening effect for
long term

Slow dehydration
capability

Spirit

Fast action

Slow dehydration
capability

2-propanone

Fast action
Less expensive

Shrink tissue

Tetrahydrofuran

Less expensive

Volatile &
flammable

Method

Tissue block are placed
in perforated containers
or cassette

To allow thorough
penetration

Identification number
place in the cassette

Lid securely closed

Tiring fragments of
tissue should be
wrapped in a single layer
of lens paper

Ethanol

Methanol

Acetone

DESCRIPTION
Flammable
Expensive
Mixture of water and organic material
(hydrophilic)
Flammable
Rarely use

Colorless
Flammable
Used for urgent biopsy
Rarely used because it can cause tissue
damage and low in penetration

Other dehydrants can be used, but have major disadvantages.

Dioxane can be used without clearing, but has toxic fumes.

The process can be carried out by:

transferring tissue direct from fixative into methylated spirit

(for 1 6 hrs) or 70%, 80% & 90% alc.
absolute alcohol 1 6 hrs
absolute alcohol 1 12 hrs
Delicate tissue (brain, spinal cord, embryo) better carried
in stages using varying grades of alcohol
Other reagents acetone, cellosolve, butanol and
isopropanol
When dehydrating tissue, a large bulk of fluid must always
be used - 1:10
Dehydrant most common - ethyl alcohol, isopropyl alcohol
(99%) is the best substitute for ethyl alcohol

Cont..
During

dehydration, tissues are passed through a

series of progressively increasing conc. of alcohol

With one change in each conc.

Duration of 1 hr in each

Keep covered to avoid evaporation

Starting [alcohol] is usually 80%

For soft tissue 30%, 50%, 70%

Important that the final bath of alcohol is pure and
change 3X

Free from water

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Cont..

CLEARING
The step following dehydration is called "clearing"
It is also referred as dealcoholization
The purpose of the clearing process ::-

As a result, when the tissue is completely infiltrated with

the clearing agent, it becomes translucent.

This change in appearance is often used as an indication of

the effectiveness or completeness of the clearing process.

Important characteristic in choosing clearing agent

alcohol removal from tissue to make it transparent

Clearing agents will replace the alcohol and enable paraffin to
penetrate into tissue

Can be mix with dehydrant

Tissue will seen shining and transparent when soak in

cleaning agent
It consists the replacement of dehydrant with a substance
that will be miscible with the embedding medium (paraffin).
The term "clearing" comes from the fact that the clearing
agents often have the same refractive index as proteins.

Can be mix with paraffin wax

DESCRIPTION

Advantages

Disadvantages

Xylene

Tissue cant be
soak more than
6 hours
 Carcinogenic
 Flammable






Fast action
Non-toxic
Less expensive
Tissue become
transparent





Shrink tissue
Harden tissue
Flammable

Toluene

 More suitable
but dangerous

 Fast action
 Less expensive

Flammable
Slightly toxic

Benzene

 Highly in toxicity  Fast

 Carcinogenic
 Less expensive
 Minimum tissue
shrinkage

Flammable
Toxic
Carcinogenic

Expensive

 Suitable for
CNS tissue
Chloroform  Low in reaction
 Carcinogenic
 Poisonous

 Non flammable
 Minimum tissue
hardening
 Tissue not brittle

Slow clearing
capacity
Toxic

Cont..

The most common clearing agent is Xylene.

Xylene is reasonably cost effective and works well for

short--term clearing of small tissue blocks.
short

Long-term immersion of tissue in Xylene results in tissue

Longdistortions.

INFILTRATION & IMPREGNATION

CLEARING AGENT
Agents

After clearing, the tissue specimen are transferred to a bath of molten

(liquid) paraffin wax for infiltration and impregnation process
During this combined process ::
Xylene is eliminated from tissue by diffusion (infiltration)

wax diffuses into the tissue pores to replace the clearing agent
(impregnation)

Temperature 50 560C

Time 2 3 hrs

Vacuum impregnation oven reduces the time to 15 min
The purpose of the infiltration & impregnation process ::
Empty space (pores) in the tissue after removal of watery solution will
be filled with paraffin wax

Hardens of the tissue which helps in section cutting
Most tissue processing is done using automated machines that carry
out the steps automatically.

AUTOMATED TISSUE PROCESSOR

Paraffin wax is the most common embedding

media

there should be at least 1 change of wax

to completely remove the clearing agent

Types of wax depends :

nature of material to be embedded thickness

temperature of working area

change 3X 1 hrs if needed

Inadequate impregnation leads to drying and shrinking of

tissue
Cracks and crumble develop
High temperature will over harden the tissues
Manual and automated tissue processing
Good technician can produce equally good result with
manual procedure

Problem with automated processing

electricity supply
lack of service support

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Cont..
Automated tissue processing :

arranged in a circle

with a timing device

containers hold reagent and paraffin wax

transfer arm changes position of the casket thru the
reagents

the final dip is in the final paraffin wax

the cassettes are opened and the tissues are embedded
Changing of solution :

More than 100% alcohol, xylene and paraffin wax are kept
in a series

after using more than 2 -3 days the last solution in a series
are replaced

previously used ones are moved forward

while the first one are discarded

EMBEDDING PROCESS
definition of embedding process is to infiltrate,
support & enclose specimens, which in turn
produce tissue block that subsequently cut into
thin sections.
The capability of the embedding medium are
The

substances capable of being converted from liquid to solid

form

has the ability to infiltrate or penetration

The histological cassettes were transfer through the

following solvent ::Solution / Reagents
1.70% alcohol

Duration /
Temperature

Description

1 hr

2.90% alcohol

1 hr

3.100% alcohol

1 hr

4.100% alcohol

1 hr

5.100% alcohol

1 hr

6.100% alcohol

1 hr

7.Histo-Clear (or Xylene)

1 hr

8.Histo-Clear (or Xylene)

1 hr

9.Histo-Clear (or Xylene)

1 hr

10.Molten wax

2 hr, 6065oC

11.Molten wax

2 hr, 6065oC

Steps 1-6 give gentle, but complete

dehydration to remove aqueous fixative
and any tissue water content

Steps 7 - 9 are the 'clearing' fluid which

is totally miscible with the dehydrating
alcohol and wax embedding
agent. Other clearing fluids are Xylene
and chloroform but Histo-Clear is safer
to use routinely.
Steps 10 -11 infiltration & impregnate the
tissue with molten wax for the final
embedding stage which sets specimens
in blocks of paraffin wax from which
sections may be cut.

Cont

Embedding, the final stage of the tissue processing

The embedding process is carried out on a Embedding centre

(machine) using stainless steel cassette moulds of several routine sizes
(suitable for large numbers of small and moderate dimensions).

The plastic labeled cassettes are incorporated into the finished block

The tissue is finally transformed into a tissue block of wax

The tissue block gives the tissue support and shape for the next step,
which is sectioning (cutting).

The

common embedding media that are used

currently are
paraffin
carbowax
celloidin
plastic

The tissues, trimmed prior to processing to fit inside

cassettes (25 x 20 x 4 mm maximum), may have
shrunk considerably after the process and are
orientated in the wax mould to present required
features/planes on the final cutting surface.

1. Paraffin embedding

solid at normal room temperature

heat renders paraffin fluid permeate the tissue
hardness of paraffin matched the hardness of the tissue
soft parafins advantages: finer grained & less brittle.
(melting point: 45 C to 50 C only used in cool temp. 59 F
Hard parafins advantages; thinner section & better
support.
(melting point: 56 C to 62 C suitable for lab temp 72 F
Routine work paraffin with high melting point 5656-58 C

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Paraffin embedding techniques

Cont

Cakes of paraffin placed in clean metal melted down in

paraffin oven
After tissue completely dehydrated & cleared, they are
immersed in melted paraffin for 22-4 hours
Usually changes of paraffin needed to eliminate traces of
solvent (clearing agent) which prevent the paraffin from
hardening properly
Rapid chilling of the melted paraffin is recommended
because slow cooling of a liquid form crystallized block
(large crystals)
Rapid cooling fine crystalline structure capable of fitting,
closely to the individual cells give adequate support
The surface of the section to be cut should be placed
parallel to the bottom of the mould

After dehydration, the tissues is embedded in paraffin

(melting temperature 5656- 58 C)
The blocks are transferred to pure paraffin in the oven for 1
hour & second pot of melted paraffin for 22-3hours block is
completely infiltrated with melted paraffin
Always use bluntblunt-nose warm forceps for making the transfer
of tissues to the mould

Then pour the melted paraffin over the tissue

Warm the forceps again & orient the tissue

Pathologist may notch the opposite side to be cut

Make sure that there is no air bubble

2. Wax embedding

Cont..

Identification label must accompany the specimen
through all types of tissue processing
Label

Place the label against the side of the mould containing

the molten wax

Adjacent to the tissue

Label must not be in the way of the knife blade

The encased blocks are removed by tapping on the
bench

The hard wax is ready to be cut

Involves

the use of alcohols, inflammable

solvents and waxes on automatic tissue
processors
Bunsens: represents a fire risk
Electrical plugs and sockets should be made
sparkles to lessen the chances of vapor igniting
Wax baths of tissue processors should have
safety cutcut-outs to prevent over
over--heating

3. Plastic embedding
Resin plastic embedding mainly used for electron
microscope and the resins must be handled with care as
many are toxic can cause dermatitis and must worn a
gloves
Eg. butyl or methyl methacrylate produce harmful fumes
and best handled in a fume cupboard. Methyl
methacrylate is very hard & good for embedding
undercalcified bone.
The soft plastics sectioned with a standard steel
microtome blade do not require glass or diamond
knives

COMPARISON OF TISSUE PROCESSING METHOD

BETWEEN
LIGHT MICROSCOPE & ELECTRON MICROSCOPE
Description
Sample Size
Fixative

Light

Electron

1 cm3

1 mm3

Formaldehyde

Glutaradehyde

Post-Fixation

None

Osmium Tetroxide

Dehydration

Graded Alcohol

Alcohol or Acetone

Clearing Agent

Xylene / Toluene

Propylene Oxide

Embedding Material
Microtome Knife
Section Thickness
Stains

Paraffin Wax

Various Plastics

Standard Steel Blade

Glass or Diamond

5 - 10m

60 - 90 nm

Colored dyes

Heavy Metals

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BRING SECTION TO WATER

Bring section to water is the term used to rehydrates

the tissue specimen again.
These procedures are in reverse order of tissue processing
method or against the Histotechnique procedure in
histopathology laboratory.
Normally in Histotechnique the tissues specimen are
processed in order to removes the water substances from
the tissue
However the Bring section to water method are done
to introduce back the water substance into the tissue again
This methods are done normally for staining or grossing
procedure

Cont
Bring section to water procedure are
done in the following steps

The

Xylene
3 minutes
Xylene
3 minutes
Xylene
3 minutes
Alcohol
3 minutes
Alcohol
3 minutes
90% alcohol 3 minutes
70% alcohol 3 minutes
Tap water / Distill Water
Staining / Grossing