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DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY

TISSUE PROCESSING

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CONTENTS:

Introduction
Principles of Tissue Processing
Factors effecting rate of processing
Dehydration
Clearing
Embedding
Vacuum impregnation
Automatic tissue processing
Manual tissue processing
Alternative embedding media
Problems during Tissue Processing
Conclusion
References

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Introduction:

The examination of tissues with a microscope usually


requires a slice of tissue which is thin to transmit light
and preparation of such thin slices is called section cutting
or microtomy. impregnation in a suitable embedding
medium to provide support and a suitable consistency for
microtomy is called tissue processing .

Processing of the tissue involves rendering the tissue firm


so that thin section can be cut and studied under the
microscope. It also helps in preservation of tissue, for
later investigations .

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Transportation: Tissue processing begins with the clinician
surgically removing a tissue specimen from the patient.
The tissue is placed in a container (often containing a
fixative) and transported to the lab. At this stage, the
tissue is called gross tissue.

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Labelling of tissues :

Tissues arriving in the laboratory is done by allocating a unique


number followed an oblique sign(/) and the last two figures of
year which specimen carries until is processed , sectioned ,
reported and filled in thin white card 2x 1 cm with writing in
soft pencil.

The labels should also indicate number of pieces being put into
one cassette,ordinary ink should not be used as this may be
dissolved in the reagents used during processing.

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Tissue and its labell is placed in tissue processing baskets. These
are perforated small metal containers , primarily designed for use
with automatic tissue containers , but may be used in a manual
system.

Small porcelain pots which are perforated have also been used for
this purpose .Printed ,graphite pencilled, typewritten, stencilled
or indian ink written labels are satisfactory .

Tissue to be fixed in a solution containing osmium tetraoxide


should not be labelled by the forgoing methods as the label will be
blackened. In such cases it is advisable to fix a label to the outside
of the jar during fixation .

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More reliable way is use of tissue tek system in which tissue
identification is written over cassette .

Automatic number generation for tissue identification is possible


due to increase use of computers , and numbers can be embossed
by a machine , programmed either manually or by computer
onto cassette .

Better way is use of bar codes,within bar codes are all the
relevant patient details.

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Fixatives :

In the fields of histology, pathology, and cell biology,


fixation is a chemical process by which biological tissues
are preserved from decay, either through autolysis or
putrefaction.

Fixation terminates any ongoing biochemical reactions,


and may also increase the mechanical strength or stability
of the treated tissues.

Chemical fixatives are used to preserve tissue from


degradation, and to maintain the structure of the cell and
of sub-cellular components such as cell organelles (e.g.,
nucleus, endoplasmic reticulum, mitochondria).

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Fixative should be ten times the volume of tissue

Chemical fixatives may be divided into two broad categories—


coagulating and noncoagulating—with respect to their
effects on proteins, which form the framework of virtually
all cells.

Factors Influencing Fixation include temperature, size of the


sample, the volume ratio of tissue to fixative solution, the
duration of fixation, and the pH of the solution.

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Formic Acid Decontamination of Fixed Blocks

For routine diagnostic purposes, thoroughly fixed tissues can be


decontaminated prior to tissue processing.

Fixed and trimmed tissues are placed in processing cassettes and


immersed in 98% formic acid for one hour.

Blocks are then washed in running tap water for 20 minutes,


then returned to fixative prior to tissue processing.

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Principle of tissue processing

Aim – To embed the tissue in a solid medium firm enough to


support the tissue and give it sufficent rigidity to enable thin
sections to be cut.

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Tissue processing is concerned with the diffusion of various
substances into and out of stabilize porous tissues.

The diffusion process results from the thermodynamic tendency of


processing reagents to equalize concentrations inside and outside
blocks of tissue.

Conforming to Fick's Law: the rate of solution diffusion through


tissues is proportional to the concentration gradient (the
difference between the concentrations of the fluids inside and
outside the tissue) as a multiple of temperature dependant
constants for specific substances.

13
Factors influencing the rate of processing :

Agitation :

Using manual processing methods ,it is difficult to achieve and is


time consuming.

Automatic tisuue processing machines use rotation or vertical


oscillation of the tissue basket, and remove & replace the fluid at
frequent intervals.

Rate should not be too slow so that it is ineffective,neither should


be violent as can cause small fragments of tissue to be damaged.

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Heat :
It increases the rate of penetration. Temperatures limited to 45
degrees can be used effectively.
Higher temperatures adversely affect staining and
immunochemistry.

Viscosity :
Most of fluids used during standard dehydration and clearing
have similar properties.

Vacuum :
Reduce the pressure are of use during impregnation by paraffin
wax.
With dense and fatty tissues ,reduces the impregnation time

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Water is present in tissues in free and bound (molecular) forms.
Tissues are processed to the embedding medium by removing
some or all of the free water. During this procedure various
cellular components are dissolved by dehydrating fluids.

Dehydration is effected as follows:

Dilution dehydration, Specimens are transferred through


increasing concentrations of hydrophilic or water miscible fluids
which dilute and eventually replace free water in the tissues.

Chemical dehydration, where the dehydrant, acidified


dimethoxypropane or diethoxypropane, is hydrolyzed by free
water present in tissues to form acetone and methanol in an
endothermic reaction. (50)

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•Dehydration is necessary in all infiltration methods, except where
tissues are simply externally supported by an aqueous embedding
medium.

• Choice of a dehydrant is determined by the nature of the task,


the embedding medium, processing method, and economic
factors.

•Dehydrants differ in their capacity to cause tissue shrinkage. In


the paraffin wax method, following any necessary post fixation
treatment, dehydration from aqueous fixatives is usually initiated
in 60%-70% ethanol, progressing through 90%-95% ethanol,
then two or three changes of absolute ethanol before proceeding
to the clearing stage.

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•While well fixed tissues can be transferred directly to 95%
ethanol, incompletely fixed tissues may exhibit artifacts if placed
directly in higher alcohols.

• The dehydrant concentration at which processing is initiated


depends largely upon the fixative employed.

•Following fixation in anhydrous fixatives such as Carnoy's fluid,


for example dehydration is initiated in 100% ethanol. (50)

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To minimize tissue distortion from diffusion currents, delicate
specimens are dehydrated in a graded ethanol series from water
through 10%-20%-50%-95%-100% ethanol.

Duration of dehydration should be kept to the minimum


consistent with the tissues being processed. 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.
Tissues may be held and stored indefinitely in 70% ethanol
without harm.

Other dehydrants, including universal solvents, are used in a


similar manner to that described for ethanol, though generally in
different concentration increments. (50)

20
Ethyl alcohol has been the dehydrating reagent of choice. The
period of immersion of the tissue specimen in various alcohols
and their various strengths will affect the hardness of the tissue
specimen.

Longer duration in higher percentages (95-100%) ethyl,


isopropyl, and methyl alcohol will cause excessive hardening,
making the tissue specimen difficult or impossible to cut. Heat
and vacuum hasten the process.

Alcoholic dehydrants have a hardening effect.

21
Wet fixed tissues (in aqueous solutions) cannot be directly
infiltrated with paraffin. First, the water from the tissues must
be removed by dehydration. This is usually done with a series of
alcohols, say 70% to 95% to 100%.

22
Other dehydrants can be used, but have major disadvantages.
Acetone is very fast, but a fire hazard, so is safe only for
small, hand-processed sets of tissues. Dioxane can be used
without clearing, but has toxic fumes.

In case of ethanol, tissue is immersed first in 70% ethanol to


100% ethanol. For delicate tissues ,particularly embryonic
and animal tissues,we start with 30% ethanol.

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Dehydrating fluids
ETHANOL INDUSTRIAL METHANOL Propan-2-ol , DIOXANE (DIETHYL
METHYLATED isopropyl DIOXIDE)
SPIRITED ( alcohol
DENATURED CH3CHOHCH3
ALCOHOL)

Clear, physical Clear, colorless, Miscible with Mixes freely with


colorless, properties and flammable liquid, ethanol, water water, alcohol,
flammable use same as highly toxic and most hydrocarbons and
liquid ethanol organic solvents paraffin thus
reducing shrinkage
and hardening

Hydrophilic 1% methanol plus miscible with microwave Toxic and areas of


ethanol ethanol, water processing use should be well
and most organic ventilated
solvents

Expensive More expensive


than alcohol 24
AgtX and ethyl alcohol are miscible with all proportions of
water and clearing reagents (xylene, other cyclic organic
solvents, branched chain aliphatic and D-limonene complexes).

AgtX, like ethyl alcohol, can be used as a dehydrant in tissue


processing with or without heat and vacuum. AgtX has shown
an advantage over ethyl alcohol in not rendering small biopsies
as hard, thus facilitating thin sectioning.

AgtX and ethyl alcohol are both good lipid extractors and are
miscible with all clearing reagents used in tissue processing.

AgtX, as ethyl alcohol, shows little shrinkage of tissue


specimens when used as a graded tissue dehydrant.

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Ethyl alcohol is a controlled substance requiring record keeping
by the end user. AgtX is not a controlled substance and requires
no record keeping.

Isopropyl alcohol is sometimes substituted for ethyl alcohol in


tissue processing. Isopropyl alcohol causes the tissue to be overly
hardened in processing and cannot be used in staining (see
Staining).

AgtX is a more gentle dehydrating agent which causes less


hardening than isopropyl alcohol. Methyl alcohol does not
harden tissue as rapidly as ethyl or isopropyl alcohols, but has
three disadvantages:

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1. Absolute methyl alcohol cannot be uses in most closed
processors.

2. Methyl alcohol is not a universal dehydrant (i.e., can be


used for tissue processing only, not staining. (see Staining).

3. Methyl alcohol is not miscible with branched chain


aliphatic and D-limonene, two commonly used clearing
reagents.

AgtX is a universal dehydrant that can be used in closed and


open processors, and is miscible with branch-chain aliphatic
and D-limonene clearing reagents

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Additives to dehydrating agents

Phenol:

•Softening agent for hard tissues such as tendon, dense fibrous


tissue & keratin masses.

•4% phenol should be added to 95% ethanol baths.

Anhydrous copper sulfate:

Act as both a dehydrating agent & an indicator of the water


content of the last bath of 100% ethanol.

Layer 1-2 cm of anhydrous copper sulfate in the final


dehydrating bath & cover with a filter paper.
-Water present: anhydrous copper sulfate will turn blue.
-Cannot be used in the pump action enclosed processors.

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Artifacts during Dehydration

The most common problems in processing are caused by


processing both biopsy and large tissue specimens
simultaneously on the same processing program.

This leads to overprocessing and excessive dehydration of the


biopsy tissues and/or underprocessing and incomplete
dehydration of the larger specimens.

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A problem that leads to very poor staining of the nucleus. In
the United States this problem is referred to as smudginess or
blue halo effect, and in the United Kingdom as “nuclear
meltdown.”

This is most often caused by incomplete dehydration prior to


clearing, but using too much heat on the processor will also
cause this same poor staining pattern.

Tissues that are insufficiently dehydrated prior to clearing and


infiltration with paraffin wax will be hard to section on the
microtome, with tearing artifacts and holes in the sections.

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Removal of the dehydrant with a substance that will be miscible
with the embedding medium (paraffin).

The commonest clearing agent is xylene.

When the dehydrating agent has been replaced by most of


solvents the tissue has a translucent appearance; hence the term
‘clearing agent’.

Most clearing agents are flammable liquids.

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Boiling point of the clearing agent gives an indication of its
speed of replacement by molten paraffin wax.Fluids with low
boiling point are more readily replaced .

Viscosity also influences speed of penetration of clearing agent.

Prolonged exposure to most clearing agents causes the tissue to


become brittle and therefore more difficult to section.

Most clearing agents are immiscible with water and require


special arrangements for their disposal.

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Enclosed tissue processors have charcoal filters,thereby
reducing the amount of harmful fumes from clearing
agents entering the environment.

Use of a clearing agent is necessary when the dehydrating


agent,e.g. alcohol,is not miscible with the impregnating
medium e.g. paraffin wax.

As the dehydrant is removed , the tissue clears becoming


translucent as many of these fluids have a similar
refractive index as that of protein.

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‘Agitation’ option on processors is useful as it improves
penetration of reagents and speed of processing. Several
baths of each reagent are required to ensure complete
processing

‘Vacuum’ option on processors is useful for the wax step


as it helps speed up penetration of this slowly permeating
reagent. The length of time in each reagent is dependent
upon the size and structure of the tissue

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Reagents must be changed in accordance with an appropriate
schedule (to account for the number of cycles or number of
cassettes or length of time) as each will become contaminated
with the previous one and contamination can cause incomplete
processing of the block.

This can make sectioning difficult as the block may become


sunken and soft and the resulting stained sections may be of
very poor quality.

Wax must be changed if the odour of the clearing agent can be


detected.

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Clearing is the transition step between dehydration and
infiltration with the embedding medium.

Many dehydrants are immiscible with paraffin wax, and a


solvent (transition solvent, ante medium, or clearant) miscible
with both the dehydrant and the embedding medium is used to
facilitate the transition between dehydration and infiltration
steps.

Shrinkage occurs when tissues are transferred from the


dehydrant to the transition solvent, and from transition solvent
to wax. In the final stage shrinkage may result from the
extraction of fat by the transition solvent.

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The term clearing arises because some solvents have high
refractive indices (approaching that of dehydrated fixed tissue
protein) and, on immersion, anhydrous tissues are rendered
transparent or clear.

This property is used to ascertain the endpoint and duration


of the clearing step. The presence of opaque areas indicates
incomplete dehydration.

Solvents, notably chlorinated hydrocarbons, do not render


tissues transparent and the clearing endpoint (generally when
the specimen sinks in the solvent)is determined empirically.

Transition solvents extract certain tissue substances such as


lipids, but otherwise do not alter tissue reactivity nor behave
as secondary fixatives during processing.

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Criteria for choosing a suitable clearing agent are :

Type of tissues to be processed, type of processing to be


undertaken , processor system to be used .

Intended processing conditions such as temperature, vacuum


and pressure

safety factors

cost and convenience, Speedy removal of dehydrating agent,


Ease of removal by molten paraffin wax.

Minimal tissue damage.

Flammability,Toxicity.

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Technique for clearing

Similar to dehydration

Tissue lightly blotted during transfer from one reagent


to next

Volume 50-100 times that of tissue

Tissue cleared in chloroform and carbon tetrachloride are


left overnight

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Those in xylene, benzene and toulene should be given one change
after 30-60 minutes

Later transferred to wax when seen to be clear.

Cedarwood oil

Poured in a jar with similar quantity of absolute alcohol


superimposed on it.Specimen gently placed in alcohol.

As clearing takes place specimen slowly sinks in cedar wood oil.


Alcohol removed with pipette. Specimen transferred to fresh
cedar wood oil for a few hours. Finally transferred to paraffin
wax

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Artifacts during Clearing

Contamination of clearing agents or coverslipping media


may also produce a bubbled appearance under the
microscope.

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slower than xylene renders tissue less non inflammable but
CHLOROFROM brittle than benzene, highly toxic
toulene and xylene

Similar action to non inflammable but


CARBON chloroform but highly Toxic
TETRACHLORIDE much cheaper

PARAFFIN Cheap Time of immersion


similar to Chloroform

CITRUS FRUIT OILS Non-toxic and Miscible with water so small mineral deposits
strong odour can be discharged in tissue, such as
through ordinary copper
waste pipes.
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XYLENE, BENZENE Fairly rapid in action Tissue becomes
AND TOLUENE clearer as alcohol is
replaced

CEDARWOOD OIL Best reagent for Tissue can be left in Can be used for tissue
research and this reagent for long like skin and dense
treatment of delicate periods without fibrous tissue
tissue as it has least damage
hardening effect.

METHYL BENZOATE slow-acting used when double


AND METHYL embedding
SALICYLATE techniques are
required.

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Ideally an infiltrating and embedding medium should be:
• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C
• translucent or transparent; colorless
• stable
• homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive

In addition the properties of the medium should approach


those of the tissues to be sectioned with regard to density,
elasticity, plasticity, viscosity and adhesion and should be
harmless to the embedded material.

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Infiltration (Interpenetration)

Is the saturation of tissue cavities and cells by a supporting


substance which is generally, but not always, the medium in
which they are finally embedded.

Tissues are infiltrated by immersion in a substance such as a


wax. Alternatively, tissues can be infiltrated with a solution of a
substance dissolved in a solvent, for example nitrocellulose in
alcohol-ether, which solidifies on evaporation of the solvent to
provide a firm mass suitable for sectioning.

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Double embedding is the process by which tissues are first
embedded or fully infiltrated with a supporting medium such as
agar or nitrocellulose, then infiltrated a second time with wax in
which they are also embedded.

Investment generally refers to the practice of embedding wax


infiltrated tissues in another wax, such as piccolyte-paraffin
wax, modified to provide improved tissue support and sectioning
qualities.

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Process Fixation Dehydration Clearing Infiltration

Reagents 10% Neutral Xylene Paraffin


Buffered Formalin 70% Ethanol
95% Ethanol
100% Absolute

Action Stabilize Proteins Removal of water Removal of Interpenetration


alcohol

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IMPREGNATION WITH PARAFFIN WAX

Paraffin wax Is a popular embedding medium for histology.


Is cheap,easily handled,wide range of melting points.

Is a polycrystalline mixture of solid hydrocarbons produced during the


refining of coal and mineral oils. It is about two thirds the density and
slightly more elastic than dried protein.

Wax hardness (viscosity) depends upon the molecular weight of the


components and the ambient temperature. High molecular weight
mixtures melt at higher temperatures than waxes comprised of lower
molecular weight fractions.

Paraffin wax is traditionally marketed by its melting points which


range from 39°C to 68°C.

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Tissue-wax adhesion depends upon crystal morphology of
the embedding medium. Small, uniform sized crystals
provide better physical support for specimens through close
packing.

Crystalline morphology of paraffin wax can be altered by


incorporating additives which result in a less brittle, more
homogeneous wax with good cutting characteristics.

There is consequently less deformation during thin


sectioning. Setting temperature does not appreciably affect
crystal size.

51
Paraffin wax has advantage for the different climatic
regions of the world.

Paraffin wax is a mixture of straight chain hydrocarbons


produced by pressing the residue of vacuum distilled crude oil.

Higher the melting point of paraffin wax the harder it will be


at any given temperature.

The plastic point is generally about 10 degree centigrade


below the melting point & is the temperature at which a
crystalline rearrangement takes place during solidification & at
which the behavior characteristics of the wax change.

52
Low melting point (i.e., softer) paraffin wax is the most suitable
for soft, friable tissue & a higher melting point paraffin wax
(i.e., harder) for tough tissue.

Additives

Various substances have been added to paraffin wax,


primarily to improve the performance during microtomy.

EXAMPLES: ceresin, microcrystalline wax, rubber, bees wax,


& dental wax.

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Increase the hardness of the wax & thus the support for
tougher tissues; to increase the plasticity of the wax & thus
improve the ribboning qualities & presumably change the
sectioning from point to point cleavage to a continuous flow
shearing; & to produce a more uniform crystalline structure
to the wax.

Recently plastic polymers have been added to paraffin wax.


Addition of Dimethyl Sulfoxide (DMSO) to Paraplast is
particularly beneficial & that Histoplast special & Ralwax I
compounded specially as final embedding media, are helpful in
cutting tough tissues or producing thin sections (i.e., 2µm).

It is recommended that paraffin wax (melting point 58


degree centigrade) be available for routine embedding , &
histoplast special for tough or decalcified sections.

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Many of additives have a higher melting points than paraffin
wax and consequently made the tissue more brittle .

Microcrystalline waxes obtained from petroleum distillation


have a much finer crystalline structure than paraffin wax but
a higher melting point. Adding upto 5% to paraffin wax
produces a hard medium,but greater % causes undue
hardness.

To decrease melting point: add spermaceti or phenanthrene.

To improve adhesion between specimen and wax (alter


crystalline morphology): add 0.5% ceresin, 0.1-5% beeswax,
rubber, asphalt, bayberry wax, or phenanthrene.

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Technique for impregnation

1. After clearing blotting is done with filter paper and


transferred to molten paraffin wax.

2 If vacuum embedding oven not being used


then wax in open glass containers or copper pots is
kept in the embedding oven whose temperature is
regulated at 56ºC for wax with melting point of 54ºC
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3 volume of wax – 25 to 30 times of tissue

4 must be changed atleast once during impregnation

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TIME FOR IMPREGNATION

Length of time and number of changes depend on-

1 . Size and type of tissue


The thicker the tissue more time for wax to penetrate to
centre

Thick tissue will carry more clearing tissue so more changes


Tissue containing more proportion of blood, muscle and
fibrous strands has tendency to over-harden and become
brittle so time to be kept minimum.

Even small amount of clearing agent contaminating the wax


will cause crystallization & crumbling of the sections during
cutting. Dense tissue such as bone, skin & CNS will require
nearly twice as long as soft tissue such as liver & kidney.

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2 Clearing agent employed

• xylene,toulene and benzene – 1 change

• chloroform and carbon tetrachloride- 2 changes

• Xylene, toluene, chloroform require two changes of wax


as a safe routine.

• Cedar wood oil will require several changes dependent


on the size of the tissue.

• Small pieces should be given at least three changes &


large pieces correspondingly more.

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3 Use of vacuum embedding oven

Half the time used in normal paraffin oven

To remove any residual air bubbles tissue can be


transferred after clearing to a heat bath of paraffin wax from
which air can be evacuated

The degree of vacuum not to exceed 500mmHg .

Using the normal paraffin over tissue as a routine given two


changes of paraffin wax over a period of 4 hours.

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Vacuum impregnation :

To speed up impregnation and remove any residual air bubble


,particularly in organs such as lung,tissue can be trans ferred
after clearing to a heated bath of paraffin wax from which air
can be evacuated.

The degree of vacuum should not exceed 500mmhg.This


treatment can reduce impregnation time by upto one half.

If there is a continous stream of bubbles coming from the tissues


at this stage then there has been inadequate dehydration or
clearing.

61
Tissues which benefit from vacuum impregnation are lung,
muscle,spleen, decalcified bone,skin and tissue from the
central nervous system.

Although other media are available paraffin wax remains


popular due to the ease with which large number of tissue
blocks may be processed in comparatively short times
.Sectioning and later staining presents fewer difficulties than
other media.
.

62
The apparatus consists of an air tight embedding oven
attached to an exhaust pump, the degree of vacuum achieved
being controlled by an attached mercury manometer or
vacuum gauge.

It consist s of an outer bath, normally controlled at 56 degree


centigrade (or 2 degree centigrade above the melting point of
the wax being used) into which vacuum chamber fits.

The vacuum chamber is a circular flat bottomed brass vessel,


which has a thick plate glass lid resting on a thick rubber ring.
There are two valves, one being connected to the exhaust
pump, the other being used to admit air to restore
atmospheric pressure at the end of impregnation.

63
To avoid contamination of the wax in the bath by water or oil used
in the vacuum pump, the pump is connected to a thick walled
vessel (flask or bottle) which act as a trap; this in turn is connected
to the vacuum chamber and to a mercury manometer

64
Inadequate infiltration with paraffin

If a tissue is inadequately infiltrated with paraffin


during processing it will spread out rapidly when
floated on the water bath

This causes the tissues to become pulled apart on the


glass microscope slide.

It may also be the result of inadequate fixation,


dehydration, clearing or insufficient time in molten
wax

Inadequate infiltration with paraffin can also result


in wrinkles that run in all directions.

65
66
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.

Alternatives to paraffin embedding include various plastics


that allow thinner sections. Such plastics include methyl
methacrylate, glycol methacrylate, araldite, and epon.

Methyl methacrylate is very hard and therefore good for


embedding undecalcified bone. Glycol methacrylate has the
most widespread use since it is the easiest to work with.

67
•Araldite is about the same as methacrylate, but requires a
more complex embedding process. Epon is routinely used for
electron microscopy where very thin sections are required.

•Plastics require special reagents for dehydration and


clearing that are expensive. For this reason, and because few
tissues are plastic embedded, the processing is usually done
by hand.

•A special microtome is required for sectioning these blocks.


Small blocks must be made, so the technique lends itself to
small biopsies, such as bone marrow or liver.

68
Embedding tissue to produce wax block

•Embed tissues as soon as processing is finished. Excess time in


molten wax will harden tissue and lead to poor sectioning.
Tissue is embedded by transferring from final wax bath to a
mould filled with molten wax

•Open one cassette at a time to prevent misidentification of


tissues.

69
•Select an embedding base mould (rubber, metal or plastic, or
use L pieces) large enough to give a clear rim of wax all around
the tissue.

70
•Place the surface to be sectioned face down in the embedding
mould. Gently press flat into solidifying wax onto a cool surface
to ensure a flat and even orientation for sectioning.

71
•With Tissue tek system ,paraffin wax is dispensed
automatically from a nozzle into a suitably sized mold which is
then placed on a small cool area to allow the wax at the base
of the mold to semi-congeal.

•This allows easy orientation of block.

72
•When this has been done the base of cassette is placed on top
and together they are placed on the cold plate so the paraffin
wax can cool quickly, thus ensuring a small crystalline
structure.

•After the paraffin wax has solidifies the mold is removed and
the block is then ready for sectioning and afterwards can be
immediately filed .

73
Different moulds that can be used -
Leuckhart’s L Pieces

•Two L pieces of metal usually brass laid on metal or glass


plate to form an oblong.

•Adjusting them the size and shape can be modified . Various


sizes are available.

•Minimum cleaning required.

Paper boats
Cheap and convenient. Tissue can be stored still in their paper
boats . One or more tissue can be blocked in one boat.

Ice trays: Ice trays of the plastic type used to produce


domestic ice cubes are useful. Each tissue is blocked into its
own individual compartments, thus saving on the labour
involved in separating blocks. 74
Embedding cassettes: A plastic cassette which may be
provided with either a metal snap on lid or an integral
plastic one, holds the tissue during processing.

A roughened surface is provided on which the reference


number may be written. Embedding is performed in specially
made base mould & the cassette inverted over the tissue &
filled with wax.

After solidification of the wax, it is removed, complete with


cassette. The cassette fits into a purpose made microtome
clamp which has the advantage of a simple one-handed
spring action.

After cutting, the block is stored with the cassette still in


place, the identification number therefore only having to be
written once & accompanying the tissue through all stages.
Moulds are available in a variety of sizes & cassettes in a
variety of depths & colours.
75
Glass Petri dishes

Several pieces of tissue can be embedded at one time.


Should be previously smeared with glycine.

Metal Petri dishes

Made from aluminum, very strong

Long life

Size depends on number of specimen received daily


Usually 6 inches diameter and 2.5 inches deep

76
Watch glasses
For small pieces of tissue
Glycerin should be used

Test tubes
For small fragments which have been processed in
them throughout

77
Artefacts resulting from the embedding process

•Entrapped air around specimens and multiple embedding of


tissues of varied consistencies occurs. Air may be trapped
around the specimen within the paraffin block.

This allows the tissue to fall out or vibrate during the cutting
procedure. Vibration of the tissue specimen results in an
artefact that resembles a venetian blind, with compressed
zones of tissue separated by open spaces .

•The causes of wrinkles include a dull knife or inadequate


infiltration of the tissue with embedding medium

78
These wrinkles usually extend the length of the section
and stain more intensely since the stain has access to
both surfaces of the wrinkle.

Wrinkles may also result if the embedding medium is


harder than the infiltrated tissue specimen, in which
case cracks in the section, parallel to the cutting edge of
the knife, may be encountered.

In addition, wrinkles occur if the temperature of the


water bath is too hot or too cold: tissue will not spread
out adequately (cold) or will spread out excessively (hot).

79
•An unclean water bath may also contain remnants of previous
tissues, which may become incorporated into the present tissue
section

The surface of kidney tissue demonstrates the presence of fungi,


which may have been present in the water bath or may have
been on the glass slide before mounting

80
liver tissue present in the lung

81
Orientation of tissues :

specimen orientation during embedding is important for


demonstration of proper morphology. Improper orientation
results in diagnostic tissue being damaged during microscopy.

Products are available to ensure proper orientation : marking


systems , tattoo dyes, biopsy bags , sponges and paper.

Orientation of tissue should provide least resistence of the


tissue against knife during sectioning. Most tissues are
embedded : margin of embedding media are the tissue,
ensure support of the tissue.

82
Tubular structures ,eg arteries and vas deferens are cut in
cross section.

Skin and other epithelial biopsies are cut in a plane at


right angles to the surface and oriented so the surface is
cut first.

Muscle biopsies are sectioned in both transverse and


longitudinal planes.A particular tissue feature may be
present on one aspect only.

83
If the tissue is fairly homogeneous (like a piece of liver) and
roughly equal in vertical and horizontal dimensions, it doesn't
really matter how you orient it in the block.

If the shape of the tissue is much longer in one dimension than


the other, it is better to embed it parallel to the microtome
knife edge, or diagonal in the block, rather than perpendicular
to the knife edge.

If the tissue has layers of different consistency, like skin, the


knife should pass through the softer layers first.

84
For example, in a cross section of skin, the knife should cut
through the subdermal fat first, then the collagenous dermis,
then the epidermis and finally the keratinous layer.

In sectioning the end of a long bone like a femur, the knife


should pass through the articular cartilage first, before passing
into the actual bone.

Tough tissues like bone and cartilage should not be oriented


parallel to the knife edge, but diagonally, so that the knife does
not contact the entire length of the specimen simultaneously.

When multiple small specimens are embedded in one block,


they should be spread out horizontally as much as possible, so
that they are not all cut by the exact same area of the knife
edge

85
Automated Tissue Processing :
these have reduced the time
taken to process routine tissue
by manual methods at least
24 hrs.

are of 2 types-

Traditional ‘carousel’ type and


the enclosed pump fluid type.
Both have the facility for 12
separate stages in processing.
advantage of not using the
clearing agent .

86
On the older carousel type machine , minimum time at any
stage was 30 minutes. The newer carousel machines can be
programmed electronically and , like the enclosed processor
type, minimum of 15 minutes per station can be achieved.

There are now processing machines that utilize microwave


technology to speed the time taken to produce a paraffin
wax impregnated block of tissue.

: Usually have a twelve stage cycle with the last two usually
reserved for paraffin wax infiltration. Static beakers are
provided to hold the processing fluids & tissues held in
individual containers & cassettes in a suspended basket are
mechanically moved from one to another. Agitation is
provided either by vertical or rotating movements of the
basket.

87
ENCLOSED PUMP FLUID TYPE TISSUE PROCESSOR

The major design difference is


that the tissue cassettes are held
in a static Reaction chamber &
the processing fluids pumped in
& out. It reduces the risk of fire
and spillage. By using single
reaction chamber, it has been
possible to provide facilities to
process tissues with the
application of heat & vacuum at
all stages. If there is any fault
with the electronics / mechanics
than the machine stops & sounds
an alarm.

88
Processing machine maintainance :

changing of fluid depends upon the number and sizes of tissues


processed. Any odour of clearing agent in the final paraffin
wax bath indicates that the paraffin wax must be changed.

Discarding of 1st 100% ethanol bath is done ,and moving down


the others , so that the final bath has fresh 100% ethanol
agent . same method is used for clearing agent and dilute
ethanols.

89
Important points:
Fluid and wax containers must be filled to appropriate level
and correctly located in machine.

Any spillage should be wiped away.

Accumulation of wax on any surface must be removed .


Wax bath thermostats are set atleast 3 degrees above the
melting point of wax.

Care should be taken while attaching basket on carousel type


machine.

Timing should be checked when loading machine ,particularly


with those which have a delay machine.

Paraffin wax baths must be checked to ensure that the


electric plugs are in contact and that the wax is molten.
90
Automated processing schedule : in most laboratories an
overnight schedule is used.with schedules of approximately
16-18 hrs duration.

1. Overnight schedule :There is no need to apply vacuum or heat


with this schedule. If additional fixation is required, the schedule
can be modified to include the first bath with 10% formalin for
a time of 3 hours – can be done by subtracting 1 ½ hour from
the total 100% alcohol time & 1 ½ hour from the second wax
bath. Can be performed on either Carousel or Enclosed tissue
processor.

91
container fluid Time(hrs)
1. 10% FORMALIN 0
2. 70% ALCOHOL ½
3. 95% ALCOHOL ½
4. 100% ALCOHOL ½
5. 100 % ALCOHOL 1
6. 100% ALCOHOL 1
7. 100% ALCOHOL 1
8. 100% ALCOHOL / XYLENE ½
9. XYLENE 1
10. XYLENE 2
11. WAX 2
12. WAX 4

92
2. Short processing schedules

For small biopsies or for urgent work. Recently excised


endoscopic biopsies & needle biopsies can be adequately
processed in 2-5 hours using heat (35 or 45 degree
centigrade) & vacuum. Can be performed on enclosed
processor.

93
CONTAINER FLUID VACUUM HEAT TIME, MINUTES
1. 10% FORMALIN Y 45 20

2 95% ALCOHOL Y 45 5

3 95% ALCOHOL Y 45 5

4 100% ALCOHOL Y 45 5

5 100% ALCOHOL Y 45 5

6 100% ALCOHOL Y 45 5

7 100% ALCOHOL Y 45 5

8 ABSOLUTE ALCOHOL Y 45 5

9 XYLENE Y 45 5

10 XYLENE Y 45 5

11 WAX Y 45 5

12 WAX Y 45 5 94
Manual Tissue Processing :

It has largely been superseded by automatic tissue processing . it


is used -
When the electrical supply has failed or there is a breakdown of
the processing machine .

For speed of processing when an enclosed processor is not


available .

For very large slices of tissue when the allocations of time on an


automatic processing machine is limited.

95
Alternative embedding media :

1. WATER SOLUBLE WAXES-


e.g.. Polyethylene glycol
eliminating the necessity for dehydration and clearing, thus
avoiding deleterious effects of these fluids, especially shrinkage.
Tissue may be transferred directly from aqueous fixatives to
molten wax, but there are difficulties in handling the sections, as
they may only be floated out of water with great difficulty. The
blocks require carefully controlled storage in a dry atmosphere.

2. CELLOIDIN (Low Viscosity Nitrocellulose; LVN)


It has a rubbery consistency allowing a continuous shearing
type of cutting. Infiltration and embedding of tissues is achieved
simply by dehydration followed by impregnation with celloidin in
solution. The solvent is allowed to evaporate ,producing a block
of embedded tissue, avoiding completely damaging effects of
heat.

96
4.RESINS-
-Used for the production of ultra thin sections of electron
microscopy.
-Tissue is dehydrated and infiltrated with resin in monomeric
form which is subsequently polymerized, either chemically or
physically to give a hard, glass clear block.
-Extra hardness provided by resins permits sections of upto
0.5 -2.0µm.

Both methacrylates and epoxy resins have been used.


-Resin embedding is also used for embedding very hard
tissues such as undecalcified bone and teeth.

5. GELATIN- Mainly used for the production of sections of whole


organs in the Gough-Wentworth technique and in frozen
sections.

6. AGAR-
-It mainly act as a cohesive agent for small friable pieces of
tissue after fixation. These fragments are embedded in molten
Agar and when solidified and trimmed , processed to paraffin
wax 97
Restoration of tissue dried in processing

Sometimes due to overfilling of the cassette basket or under


filling the reagent bath results in tissue drying before wax
impregnation and such tissues cannot be regarded as normal.
This tissue can be treated with –

70% Ethanol- 70ml


Glycerol - 30ml In a sealed vessel
Dithionite - 1g

For several hours, followed by processing commenced at the


dehydrating stage in the usual manner which will allow some
interpenetration of the stained sections.

98
ARTIFACTS

"Floaters" are small pieces of tissue that appear on a slide


that do not belong there--they have floated in during
processing.

Floaters may arise from sloppy procedure on the cutting


bench. Dirty towels, instruments, or gloves can have tissue
that is carried over to the next case.

99
(A) Bone marrow in the brain appears to be a focus of
inflammation

100
(B) A piece of small
intestine adjacent to the
brain.

This occurred either during


the trimming of the gross
tissue specimen for
processing and embedding
or within the tissue
processor

101
During macro-sectioning of the fixed gross specimens
prior to processing, care should be taken to keep the
surface of the cutting block free of tissue debris.

If an automatic processor is improperly adjusted, or a


power failure occurs, the basket of cassettes may remain
elevated, and the tissue specimens may become
dehydrated by exposure to air.

Inadequately filled solution containers would produce a


similar artefact which will result in the tissue gaining a
dry homogenous appearance.

102
CONCLUSION:

After removal of tissue sample from patient, tissues are


exposed to series of reagents that fix,dehydrate,clear and
infiltrate with final embedding in medium that provides
support for the tissue.

Each step in tissue processing is important which requires


skills that are developed through continued practice and
experiences.

As new technology and instrumentation develops,the role of


histology laboratory in patient care will continue to evolve.

103
REFERENCES

•Theory and Practice of Histological Techniques.John D .


Bancroft, Marilyn Gamble.5th edition.85-107.

•A Handbook of Medical Laboratory Technology. V.H.Talib.2nd


edition.155-175.

•Implementation of a new rapid tissue processing method


Advantages and challenges. Julie Munkholm , Maj
LisTalman,ThomasHasselager. Pathology – Research and Practice
204 (2008) 899–904

•Fixation, tissue processing, histology and Immunohistochemistry


procedures For diagnosis of animal tse (Bse, scrapie, atypical
scrapie) Histopathology, pathology department, VLA weybridge.
104
Comparison of three methods of tissue processing.Pritam panja,
Sriram J, Saraswathi TR. JOMFP. 2007 .Vol11, Issue1.

•Morphological artifacts induced in intracellularly stained


neurons by dehydration: Circumvention using rapid dimethyl
sulfoxide clearing. Neuroscience Volume 16, Issue 2, October
1985, Pages 461-475

•http://www.nationaldiagnostics.com/article_info.php/articles_id/
104

•Http://in.Answers.Yahoo.Com/question/index?Qid=201104030
41752aaamfzq

•Http://www.Alneurosciencecenter.Uab.Edu/word%20docs/core%
20c/histology-problems%20with%20staining.Pdf
105
THANK YOU

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