HISTOPATHOLOGICAL TECHNIQUES

Concerned with the demonstration of minute tissue

structures in disease. Before such structures can be
demonstrated, the tissue must be prepared in such a
manner that it is sufficiently thin (one or two cells thick)
to be examined microscopically and the many and
complex structures which go to make up tissue may be
differentiated.

This involves collection of morbid tissues from biopsy or
necropsy, fixation, washing, dehydration, clearing,
embedding, section cutting, staining and microscopical
examination.
Fixation
Rapid killing of tissue elements and their hardening so that
they are preserved in as near as possible the same relations they had
in living body.
The foundation of all good histopathological preparations
is adequate and complete fixation.
Fixation is required to:-
Prevent post-mortem changes such as putrefaction and
autolysis.
Preserve various cell constituents in as life-like manner as
possible.
Protect by hardening of naturally soft tissues thereby
allowing easy manipulations during subsequent processing.
Convert the normal semi-fluid consistency of cell to an
irreversible semisolid consistency.
Aid in visual differentiation (alteration in refractive indices)
of structures by application of biological dyes and chemicals.
To accomplish the objectives of the fixation of
tissues:-
Should be placed in the fixative immediately upon removal
from the body or as soon as possible after death.
Tissue blocks should be cut thin enough (3-5 mm thick) so
that the fixative readily penetrates throughout in a
reasonably short time.
The volume of fixing fluid should be 15 to 20 times that of
the tissue to be fixed.
The tissues which tend to float (lungs and bone marrow)
should be covered with a piece of gauze or absorbent cotton.

Several reagents are used for fixing tissues.
The choice of a fixative will be governed by the type of
investigation required, both immediately and in future.
The most common fixative used is the formaldehyde
(formalin). It is commonly used as 10 per cent solution i.e. 1
volume 40 per cent formaldehyde solution (formalin) with 9
volumes of water (simple 10% formalin) or with normal saline
(formol saline).
It is desirable to render the formalin solution neutral by
adding 4.0 gm sodium phosphate monobasic and 65 gm.
anhydrous disodium phosphate per litre.
This constitutes the buffered neutral formalin which is
considered the best overall fixative and is therefore strongly
recommended for routine use.
Tissues up to 5 mm thickness are adequately fixed in
24-48 hours depending upon the temperature.
Factors affecting the fixation:
- pH.
- Temperature.
- Penetration of fixative.
- Volume of tissue.
According to previous factors we can determine the
concentration of fixative and fixation time.
Types of fixative:
Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde,
Methanol and Picric acid.
Dehydration
Dehydration is removal of all extractable water
by a dehydrant diffusing through the tissues and in
the process diluting itself 2-4%.

Some of the dehydrants used are ethyl alcohol
(ethanol), acetone, dioxane, tetrahydro furan,
isopropyl alcohol, rectified spirit.

Most common are ethyl alcohol or acetone.
Ethyl alcohol :
It is the most commonly used dehydrant and ascending
grades of alcohol are used for this purpose.
The concentration of alcohol in the first bath depends on
the fixation, size and type of tissue to be dehydrated.
After fixation in aqueous solutions, delicate tissues need to
be dehydrated slowly starting in 50% ethyl alcohol whereas
most tissue specimens may be poured into 70% alcohol.
Tissues immersed in too great a concentration of alcohol
after an aqueous fixative will usually show a high degree of
shrinkage due to the too rapid removal of the water.
Tissues from alcoholic fixatives such as carnoys fluid may
be placed in higher grade alcohols or even in absolute alcohol
but it should be remembered in these instances that several
changes are needed to remove acids.
The minimum duration of treatment in
graded alcohols will depend on the size and
type of tissue.
For routine purposes, tissues are dehydrated
in 70%, 80%, 90% and absolute alcohol for 2-4
hours in each dilution.
The volume of reagent (alcohol) should be 50
times the bulk of specimen.
To speed the process of dehydration, the
tissue may be agitated either mechanically in
an automatic tissue processor or by shaking the
container periodically.
Clearing
Since the dehydrating agents are not miscible with
commonly employed embedding materials
(paraffin), these must be removed by clearing
agents.
Removal of the dehydrant makes the tissue some
what translucent (clear) except in chloroform.
The most common clearing agents in use are
chloroform, carbon tetra chloride, xylene or xylol,
benzene, toluene, cedar wood oil.
Most commonly used clearing agent is benzene or
cedar wood oil.
Clearing
Replacing the dehydrating fluid with a fluid that is totally
miscible with both the dehydrating fluid and the embedding
medium.
Choice of a clearing agent depends upon the
following:
- The type of tissues to be processed, and the type of
processing to be undertaken
- The 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

Benzene:-
Benzene is fairly rapid in action and small
pieces of tissues are cleared in to 1 hour.
Possesses the advantage that the tissue
becomes clearer as the alcohol is replaced.
possible to determine the end point with
some accuracy.
Over exposure of tissues to it may result
in hardening of tissues.
Cedar wood oil:-
Best reagent for research and treatment of delicate tissues since
it has the least hardening effect.
Tissues may be left in this clearing agent for long periods, even
for months without damage.
In some institutions it is used as a clearing agent for hard
tissues such as skin and dense fibrous tissue. Even small pieces
of tissue need to be left overnight in cedar wood oil.
Following clearing in this reagent, several extra changes of
paraffin wax will be required to remove the oil.
In order to avoid extra changes of paraffin wax, tissues after
clearing in cedar wood oil may be placed for few minutes in
xylene. This treatment will remove excess of cedar wood oil from
the tissues.
Impregnation with paraffin or
Embedding
After clearing, the tissues are impregnated with
paraffin wax in an electric oven heated to 54-62
o
C,
the temperature depending on the melting point of
the wax in use.
The temperature of the oven should be slightly (1-
2
o
C) above the melting point of paraffin wax. The
most popular melting point for routine work under
Indian conditions is 60-62
o
C which gives support for
hard tissues and yet allows easy ribboning of
sections.
After blotting lightly with filter paper, tissue is
transferred from the clearing agent to molten paraffin
wax in open glass containers or copper pots kept in
embedding oven.
The volume of wax should be about 25-50 times the
volume of the tissue.

The length of time and the number of changes
required for thorough impregnation of tissue
with wax largely depends on the size and type
of tissue, the clearing agent employed and
the use of a vacuum embedding oven.

As a routine, 3-4 changes of 1 hour each are
sufficient. By the use of vacuum embedding
oven this time may be halved.
Blocking
Two L-shaped moulds are
arranged in the form of a
rectangle over a glass plate.
Melted paraffin is poured into
the mould and the tissue from
the final wax bath is so oriented
that the surface to be cut rests on
the base of mould.
Block is then allowed to cool.
When the wax has set, the L
pieces are easily removed and
are ready for next block.
Section Cutting
The cutting of good sections depends on
thorough knowledge of the equipment used and
the practical experience.
Trim the block with a sharp knife until 1/8
inch wax remains on all sides of tissue and the
edges of block are parallel.
Fix the block to metal block holder by
heating the latter and pressing it firmly against
the back of paraffin block.
The block holder together with block is
dipped in cold water.
Fix the block holder with
block on the microtome in
such a position that it will be
clear of the knife when this is
in position.

Turn backs the feed
mechanism on the
microtome almost as far as it
will go.

Insert the appropriate
knife in the knife holder and
screw it tightly in position.
Check that the tilt of the knife is set
at the correct angle.

Move the block holder forward or
upward or adjust the feed mechanism
until the wax block is almost touching
the knife.

Ensure that leading edge of block is
parallel to the edge of the knife.
To trim the surface of block, set the section
thickness gauge to about 15 microns and
operate the microtome until complete sections
of the tissue are being cut.
Set the thickness gauge to 4-5 u and operate
the microtome gently until complete sections
are cut.
With practice an easy rhythm is achieved
with the right hand operating the microtome
and the left hand holding the sections away
from the knife either with a needle or forceps.
Fixing the sections to slides
During cutting, paraffin wax embedded sections
become slightly compressed and creased.
Before being attached to slides these creases must
be removed and the sections flattened.
This is achieved by floating the ribbon on warm
water in thermostatically controlled baths known as
tissue flotation water bath.
These baths are commercially available with the
inside coloured black and are maintained at a
temperature 5-6
o
C below the melting point of
paraffin wax.
When the sections are flattened, these are lifted
on to the glass slide.
For this, a clean or albuminized slide (slides
smeared with Mayers egg albumin which contain
equal amount of egg white and glycerin) is half
submerged in the water and brought into contact
with the section.
The slide is then withdrawn bringing the
flattened sections with it.
The water is drained from beneath the sections
and these are then dried in an oven at 50-60oC.
The slides are now ready for staining.
Staining of Paraffin Sections

The most common method of staining of paraffin cut
sections for routine histopathological studies in Haematoxylin
and Eosin (H & E).
The usual procedure is:
Deparaffinize the sections in 2-3 changes of xylene for 5-10
minutes each.

Place in absolute alcohol 90% alcohol, 80% alcohol and 70%
alcohol for 1-23 minutes each.

Wash in tap water.

Stain with haematoxylin for 5-10 minutes.

Wash in tap water

Differentiate in acid alcohol, 2-5 quick dips depending upon the
intensity of stain.

Check the differentiation with a microscope.
Nuclei should be distinct and the background very light or
colourless.

Wash in tap water for 2-5 minutes.

Dip in ammonia water or lithium carbonate water until sections
are bright blue.

Wash in running tap water for 15-20 minutes. This step is
important because in case the washing is inadequate, eosin will
not stain evenly.

Stain with eosin for 15 seconds to 2 minutes depending on the
concentration of eosin and depth of the counterstain desired.

Dehydrate in ascending grades of alcohol 70%,
80%, 90% and absolute, 1-2 dips each.

Clear in xylene, 2-3 changes of 2 minutes each.

Mount in D.P.X. and examine under the microscope.

Results: Nuclei blue
Cytoplasm various shades of pink
Mounting of sections

Histopathological sections which need to be
examined for any length of time or to be stored must be
mounted under a cover slip. The media which are commonly
employed for mounting are:
i) Canada Balsam
ii) D.P.X.
Canada Balsam is solid resin from the Canadian fir
tree (Abies balsamea) and is usually dissolved in xylene; a 55
per cent solution is suitable for routine use.
It is not commonly used these days because

i) it turns yellow with time
ii) sections stained with aniline dyes tend to fade and Prussian
blue is slowly bleached
iii) it dries slowly and
iv) excess mountant is not easily removed.

In most of the histopathological laboratories, Canada balsam
has been replaced by D.P.X., a synthetic resin. It preserved
most routine stains, dries quickly and the slides can be
cleaned of excess mountant simply by stripping it off after
cutting around the edge of the cover slip.

Composition of D.P.X.
Distrene 80 - 10 gm
Dibutylphthalate - 5 ml
Xylene - 35 ml