Anda di halaman 1dari 61

Good morning

e
g
u
n
s
i
s
s
i
s
T ce
o
r
P
- By Dr Shruthi

References
Theory and Practice of histological
techniques, 6th Ed - Bancroft.
Cellular pathology technique, 4th EdnCulling
Theory & practice of Histotechnology,
2nd Edn Sheehan
AFIP laboratory methods in
Histotechnology
Histopathology Techniques: Tissue
Processing and Staining, Dr. S.I
Talukder

Presentation outline
Introduction
Specimen handling
Tissue processing
Factors affecting tissue
processing
Stages of tissue processing
Methods of tissue
processing
Conclusion

Introduction
The most commonly used
method of examining tissue
microscopically is by sectioning
Fixation of the tissue stabilizes
the structure and stabilised
tissues must be adequately
supported by infiltrating and
embedding in support medium
before they can be sectioned for
microscopical examination

Specimen handling
Correct labelling &
identification are the first
essentials of processing
Emphasis should be given to
1. A foolproof system of labelling
2. Ensuring the correct label
remains with the specimen
throughout
3. Constant vigilance

Labelling of tissues
(a)Handwritten in soft lead pencil or
waterproof ink
(b)A numbered card label generated by
computer-printer
(c) Automated embossing systems that
permanently etch or emboss tissue
cassettes and slides and produce
serial numbers
(d)Tissue- Tek system where tissue
identity is written on cassette &
retained as permanent record

Tissue Tek System


The cassettes consisted a small
perforated plastic container with
metal or plastic lid
Edges of the cassettes are
roughened enabling them to be
marked in pencil
As the cassette forms a part of
final wax block, the id number
stays with the specimen from the

Tissue processing
Definition
It refers to any treatment of
the tissue necessary to
impregnate them with a solid
medium to facilitate production
of sections for microscopy

Principle of tissue processing


Fick's Law: The rate of solution diffusion
through tissues is proportional to the
concentration gradient as a multiple of
temperature dependant constants for
specific substances.
Tissue processing is concerned with the
diffusion process results from the
thermodynamic tendency of processing
reagents to equalise concentrations
inside and outside blocks of tissue and
thus stabilize it

Factors affecting the rate


of processing

1. Specimen size:
Thicker the specimen, longer will be the
impregnation
Ideally, specimen thickness has to be 34mm
If thicker slices are unavoidable, processing
schedule has to be prolonged

2. Heat:
Increases the rate of penetration & fluid
exchange
Temperature is limited to 45C
Disadvantages:
Hardens tissue, increases brittleness,
shrinkage
Interferes with staining

3. Agitation:
The greater the surface area of the tissue
in contact with processing fluid, more
effective the fluid interchange.
Agitation ensures the effective interchange
between fluid within the tissue & fluid
outside
Commercial tissue processors has
automatic agitation with either vertical or
rotatory movements of specimen container
or by drawing & refilling the containers

4. Viscosity:
Higher the viscosity of processing fluid,
slower the rate of penetration.
Clearing agent like cedarwood oil &
molten wax are of high viscosity and
require longer immersion times to
achive impregnation.
Heating reduces viscosity but this is
avoided when using cederwood oil as
this causes tissue hardening

5. Vaccum:
Vacuum reduces the time necessary
for impregnation by one-half
particularly dense and fatty tissues
Helps in removal of trapped air from
tissues
Assists in the removal of clearing
agents because of increased
volatility
Some Automatic processing
machines incorporated vacuum at
all stages of processing cycle

Stages of processing
Dehydration

Orienting
Permeating
Removal
the tissue
the
tissue
ofsample
dehydrating
within
aasupporting
supporting
solutions
medium
medium
Removal
of water
and
fixative
from the
tissue

Dehydration
First stage of processing concerned with
the removal of unbound water and
aqueous fixatives from the tissue
components
Specimens are processed in a graded
series of reagents of increasing
concentration to prevent its distortion
Excessive dehydration- hard, brittle &
shrunken tissue
Incomplete dehydration- inhibit the
penetration of clearing agents

Duration of dehydration
Duration of dehydration should be kept
to the minimum consistent with the
tissues being processed.
Tissues may be held and stored
indefinitely in 70% ethanol without
harm.
Specimen
For whole organ

Time
24-48 hrs in each

For delicate tissue &


cytological research

2-4 hrs in each according to size

For post mortem tissue of not


> 7mm

70,90,100% (3 changes) for 1-2


hrs in each

Dehydrating fluids
Dehydrating fluids should be
water miscible
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

Concentration of dehydrants
The dehydrant concentration at which
processing is initiated depends largely
upon the fixative employed
70%Specimen

Concentration of
dehydrant

Embryonic tissue in aqueous


fixative

10%-20%-50%-95%-100%

Tissue in carnoys fluid

100%

Most specimens in aqueous


fixative

70%-80%-100%

Use of copper sulphate in final


alcohol:
A layer (1.2 5mm thick) of Anhydrous
copper sulphate added to the final
absolute ethanol on a tissue processor
scavenges any water present
The salt is self-indicating: white when
anhydrous, blue when hydrated
This action not only speeds the
dehydration process of tissue but also
prolongs the life of alcohol

Ethanol
Clear, Hydrophilic, miscible with
water and organic solvents
Rapid, efficient, widely applicable
& most commonly used dehydrant
in histology
Ensures total dehydration, making
it as choice for the processing of
electron microscopic specimens
The volume of alcohol should be
50-100 times that of tissue

Advantages
Nontoxic
Miscible in all
proportions of water
Little shrinkage
Fast acting
Reliable

Disadvantages
Expensive
Longer period may
cause shrinkage
May have prohibitive
taxes
Extracts methylene
blue from sections

Methanol
It is a good ethanol substitute
but rarely used for routine
processing because of its
volatility, flammability and
toxicity
It is miscible with water, ethanol
& organic solvents
In microwave processing it tends
to harden tissues more than

Isopropyl alcohol
Miscible with water, ethanol
and most organic solvents
Often used in microwave
processing schedules
Does not cause overhardening
or shrinkage of the tissue

Acetone
Clear, colourless, flammable
fluid miscible with water,
ethanol and most solvents.
Has characteristic pungent
odour
Rapid in action, with poor
penetration & Causes
brittleness of tissue if use is
prolonged.
Removes lipids from the tissue

Additives
Phenol- (4%) softening agent
for hard tissues like tendon,
nail, keratin masses
Hard tissue can be mixed even
in alcohol-glycerol mixture

Clearing
Clearing is the transition step
between dehydration and
infiltration with the embedding
medium
Dehydrants are immiscible with
paraffin wax, and a solvent
miscible with both the dehydrant
and the embedding medium is
used to facilitate the transition
between dehydration and
infiltration

Why is it named so??


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

Criteria
The type of tissues to be
processed, and the type of
processing to be undertaken
Rapidity of removing
dehydrating agent
Ease of removal by infiltrating
agent
Minimum tissue damage
Toxicity & inflammability

Clearing agents
Xylene & Toulene:
Flammable, colourless, miscible with
most of the organic solvents &
paraffin
Rapid in action, 30-60 min for small
pieces of tissue and about 2-4 hours
for 5mm thick specimens
Toulene has same properties as
xylene but less damaging to tissue
and more volatile

Advantages
Clears quickly
End point is easily
determined
Used for clearing in
staining sequence
Recyclable

Disadvantages
Flammable
Hardens the tissue
Toxic to skin &
various systems of
body
Not miscible with
water

Chloroform:
Commonest agent in routene use by manual
methods because of its tolerance
Often used when processing specimens of
CNS

Advantages
Penetrates well
Makes tissue less
brittle
Thicker tissue
>1mm can be
processed
Non flammable

Disadvantages
Highly toxic due to
phosphogene gas
End point cannot
be determined
Used in well
ventilated room

Cedar wood oil:


Best reagent for research
Tissues can be left for longer period even for
months without damage
Advantages
Rapid clearing
Least hardening &
shrinkage
Less toxic
Excellent for tissues
such as skin, uterus,
muscle & tendon

Disadvantages
Must be removed with
xylene before
impregnation
Expensive
Remanants may cause
difficulty in sectioning

Methyl benzoate and

methyl salicylate:
Slow acting and can be used when
double embedding techniques are
required
Moderate speed of action
Minimal distortion of tissue

Citrous fruit oils:


They are extracted from orange and
lemon
Nontoxic and miscible with water, so
they can be discharged through

Universal solvents
Some fluids are capable of acting as
dehydrating agents and at the same
time they are miscible with molten
paraffin wax, thus avoiding
intermediate clearing stage
Dioxane, 1:4 diethlene dioxide
- highly toxic therefore not used
Ethylene glycol monoethyl ether
(cellosolve)
-less toxic, slow action & requires
further
investigation

Recycling of processing fluids


Distillation equipment is used
Advantages include
Reduced cost
Rapid, efficient
Eliminates the risk of disposal of
toxic chemicals

Impregnation/Infiltration
Infiltration 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

Ideal requisites
Soluble in processing fluids
Suitable for sectioning and
ribboning
Molten between 30C and 60C
Translucent or transparent;
colourless
Stable, non toxic, odorless
Homogeneous
Capable of flattening after
ribboning
Easy to handle

Paraffin wax
Most popular due to ease with which
large number of tissue blocks may be
processed in comparatively short
times
Staining presents fewer difficulties
than other media
Cheap, easy handling, inexpensive,
wide range of melting point &
provides quality section
It can be used in different climatic
regions

Properties
It is a mixture of hydrocarbons
produced by sweating and pressing the
residue to vacuum distilled crude oil.
Melting point range is between 40-70c,
normally for routine use 54-58c is
satisfactory
Higher the melting point of wax, harder
the wax at any given temperature
Plastic point governs the behaviour of
the wax. Plastic point [at which
crystalline rearrangement occurs] is

Paraffin wax additives


Plasticizers or other resin
additives to provide desired
hardness
Substances added to paraffin are
bees wax, rubber, ceresin, plastic
polymers
Additives with higher melting
point than paraffin can make the
tissue brittle

Time of impregnation
Duration & number of changes
depends on:
Size & type of the tissue
The clearing agent employed
Use of vaccum-embedding
oven

Size and type of


tissue:
Size:
Thicker the tissue, longer will be the
time required for wax to penetrate
Thick tissue will carry more of the
clearing agents, & hence requires
more change of wax to remove it.

Type:
For dense tissue impregnation time is
twice that of soft tissues (liver and
kidney)

The clearing agents


employed:
Some clearing agents are replaced
faster than others.
Xylene, chloroform, toluene and
food oils require two changes of
wax. While cedarwood oil requires
several changes

Vacuum impregnation:
Normal paraffin wax impregnation with
two changes of wax takes over a
period of 4 hours. By use of vacuum
impregnation this time may be halved.
This procedure not only speeds up
impregnation but also rapidly remove
air bubbles and clearing agents from
the tissue
The degree of vacuum should not
exceed 500 mm of mercury
Tissues benefited from this are lungs,
muscles, spleen, decalcified bone, skin
and central nervous system tissue.

Methods of processing
Manual tissue processing

Manual tissue processing


Manual tissue processing has now
largely been superseded by automatic
tissue processing but there are occasion
when it may be necessary to adopt this
procedure.
These circumstances are

power failure or breakdown of a tissue processor


requirement for a non-standard processing
schedule like:
rapid processing of an urgent specimen
delicate material
very large or thick tissue blocks
hard, dense tissues (nitrocellulose

Advantages
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
Use of fluids on grounds of
inflammability, volatility or cost would
be unacceptable in automated

Manual processing schedules

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 controlled
environment
Automated machines are most
widely used to overcome the
possibilities of human error and
forgetfulness leading to

Types
Tissue-transfer processors

Tissue-transfer Processors
These processors are characterised
by the transfer of tissues, contained
within a basket, through a series of
stationary reagents arranged in-line
or in a circular carousel plan
The rotatory or carousel is the most
common model of automatic tissue
processor
Fluid agitation is achieved by
vertical oscillation or rotary motion
of the tissue basket

Fluid-transfer Processors
In fluid-transfer units processing
fluids are pumped to and from a
retort in which the tissues
remain stationary
Depending upon the model
these machines can process
100-300 cassettes at any one
time
Agitation is achieved by tidal
action

Processing Schedules

Microwave processors
Rapid manual microwave-stimulated paraffin
wax processing gives excellent results
compared to tissues processed by longer
automated non-microwave methods
Processing is undertaken in a dedicated
microwave oven which is fitted with precise
temperature control and timer, and an
interlocked fume extraction system to
preclude accidental solvent vapour ignition.
Toxic and flammable solvent vapours
generated during processing cannot always
be adequately vented from these ovens and
present an ignition hazard if the electrical
system is unprotected

Rapid Automated Processing


The enclosed system machine have
the facility to use vacuum and
heating at all stage which can greatly
increase the speed of processing.
Spleen, muscle, skin, decalcified
tissue and tissues containing blood
clot to become unduly hardened if
high temperature are used
This can be minimised by using
limited temperature during fixation,
dehydration and clearing

Techniques for increasing


speed of processing:
The use of warm (40-50C) 45 C for
30 min, fixative to ensure that
fixation is complete before
commencing dehydration.
Dehydration commencing at 95%
alcohol stage
The use of a fast acting clearing
agents
The use of a vacuum infiltration at
all wax stage.
Agitation at all stage even during
fixation.

Conclusion
Producing quality slides for
diagnosis is not an accident
rather it essentially requires
skills that are developed by
continued practice &
experience

u
o
Y
k
n
Tha

To be conscious that you are


ignorant is a great step to
- Sir Benzamin Disreli
knowledge