Histopathology.pptx

Histopathology
Dr. Gurjeet Singh
PGT
AMCH

Aims and objectives
Introduction
Procedure
Pathology
Medico-Legal importance
Bibliography
Presentation Title

Introduction
HISTO – Tissue
PATHOS – Disease Suffering
Histopathology is the department of clinical lab
which deals with the study of different types of
tissue.
9/3/20XX Presentation Title 3

Procedure
RECEIPT AND IDENTIFICATION
LABELLING
FIXATION
GROSSING
DEHYDRATION OF THE FIXED TISSUE
CLEARING OF THE DEHYDRANTS
INFILTRATION OF THE TISSUE
EMBEDDING OF THE TISSUE
TISSUE ORIENTATION
MICROTOMY OF THE PARAFFIN BLOCK
DEWAXING OF THE TISSUE
STAINING OF THE TISSUE
DPX MOUNTING OF THE SLIDE

Labelling

Tissue Fixation
The aim of fixation is ‘to
preserve tissue in as life
like a manner as
possible’.
The preservatives must
also allow a variety of
techniques to be
performed upon it without
destroying its integrity.
Usually, whole sample
should be kept in 10%
neutral buffered formalin
for a day to get fixed.

Next day, Size, shape and consistency
of the specimen is noted.
Small sections are taken from whole
specimen (both normal & abnormal
parts)
These sections are again kept in 10%
formalin overnight.
Ideally, tissue sections shouldn’t be
more than 3 - 6 mm thick to permit
rapid penetration and fixation.

Dehydration
• Water in tissues are present in:
(a) Free form
(b) Bound form
• Dehydrants displaces the residual fixatives as well as the cellular water
found in tissues.
• Dehydration is done with acetone (a dehydrant) for minimum of 2 hours.
The acetone is being replaced with fresh acetone every 30 minutes for 4
times.

Some examples of dehydrating fluid-
1. Ethanol
2. Isopropyl alcohol
3. Dioxane
4. ethyl glycerol

Clearing
Clearing of the tissue is done to remove the dehydrants (acetone),from
the tissue.
Clearing agents must be miscible with both the dehydrants, to effectively
remove them and with the ensuing paraffin wax to allow complete
infiltration.
Most widely used clearing agent: xylene

For clearing of the tissues, the tissue
sections are kept in clearing agent,
for 30 minutes. After 30 minutes, if it
is clear, then clearing has been done
correctly. If not, then dehydration is
repeated.
Other clearing agent-
• Toluene
• Chloroform
• Cedarwood oil
• Limonene
• Citrus based clearents- eg -Histo-
Clear , Master- Clear, etc.

Infiltration
• After clearing, tissue sections are infiltrated with paraffin wax to support
the tissue, allowing thin sections to be cut.
• Infiltration must be sufficient to displace the clearants from the tissues,
otherwise, wax will not harden properly and it will interfere with
microtomy.
• Temperature of the embedding wax should be 2-4 degree Celsius above it’s
melting point.
• Embedding is done for 1 & ½ hours to 2 hours.
• An identity code is given for each tissue sample and put along with the
tissue sample while embedding.

Alternative infiltrating media-
• CELLOIDIN- used in dense and/or hard tissues.
• GELATIN - As a routine, frozen sections of fixed or unfixed tissue are
cut without further processing.
• RESIN- it is used for electron microscopy, ultra thin sectioning for high
resolution and undecalcified bone.

Tissue Orientation
Correct placement or orientation of
the sample in the tissue block is
required to be able to see the
desired tissue morphology in the
section.
Incorrect placement / orientation may
damage diagnostic tissue elements
during microtomy or obscure them from
microscopic view, preventing a correct
diagnosis

Long tissue sample should be placed diagonally in the block rather than
straight across, because a margin of embedding medium (paraffin wax)
around the tissue also provides additional tissue support

Embedding (Block Making )
Paraffin blocks are made with the help of-
1)paraffin wax
2)spirit lamp
3) “l” bar
4) tray half-filled with water

Paraffin wax is heated over the spirit lamp till it
melts. The “L” bars are arranged and a few drops of
water are put inside it to prevent the block from
getting stuck in the wooden platform. The tissue
section is placed in the bottom with proper
orientation and the melted wax is poured over it till
the rim of the “L” bar. The identity code of the
tissue sample is also placed within the block.
Once the block has gained it’s shape ,place the
block over the tray filled with water ,allowing it to
cool down faster.
9/3/20XX
Presentation Title 21

TYPE OF MOULD-
Leuckhart's L pieces are
simple in construction
and use. By adjusting the
L pieces the shape and
size can be modified
according to the tissue.
Glass Petri Dishes are
convenient embedding
moulds previously
smeared with glycerine.
Metal Petri Dishes are
available commercially,
usually being made from
aluminium. They are very
strong and if treated with
moderate care have a
very long life.

Microtomy
• Microtomy is the means by which tissue is sectioned and attached to the surface
of a glass slide for further microscopic examination.
• Instruments used:
1) microtome
2) water bath with tissue paper
3) fine pointed or curved forceps
4) camel haired brush
5) scalpel
6) clean slides
7) slide labeling instrument

9/3/20XX
Presentation Title
24

Before section cutting, water bath is allowed to be ready for proper temperature (56-58 °C)
The paraffin block is trimmed with a scalpel, such that ,the tissue portion of the block is more exposed than the rest of the tissue
block. It makes microtomy easier.
Before cutting of the block by a microtome, a clean slide is taken and a drop of egg albumin is smeared on it.

When we cut a tissue paraffin
block by a microtome(3–6 μm-
thick sections ) a tissue ribbon is
formed. We put the tissue ribbon
on the pre-heated water-bath
,trailing end first to open it up
properly.
Approximately 30 seconds in the
water is sufficient for the ribbon to
flatten.
Now we take the egg-albumin
smeared glass slide and dip it in
the water-bath so as to receive
the already flattened ribbon on it.

Dewaxing
After the ribbon has been
received on the slide, it is
kept in an incubator for
overnight.
Temperature of the incubator
should be more than the
melting point of the paraffin
wax that has been used.
Incubating the tissue slide
overnight helps in removing
the paraffin wax, that has
been used during
embedding, so that staining
of the slide can be done.

When an overnight
incubation of the tissue slide
has been done, it is dipped
in xylene for 3 minutes to
remove the residual wax.
After 3 minutes, the slide is
transferred to another
container with xylene and
kept there for another 3
minutes.
It is then transferred to a
container with absolute
alcohol for 3 minutes to
remove xylene.

With a regular interval of 3
minutes, the slide is transferred
from absolute alcohol > 90%
alcohol > 70% alcohol > 50%
alcohol > 20% alcohol, so as to
decrease the amount of alcohol
from the tissue in the slide.
After that, the slide is kept under
running tap water to wash off the
remaining alcohol.
Now the slide is kept in a tray for
staining

Frozen Section
It is the rapid tissue section by cooling the tissue with
the help of cryostat to give immediate report of tissue
sample.
Cryostat is the refrigerated cabinet that has
arrangement to freeze the tissue and also to cut the
frozen section for microtomy section

• Principle- When the tissue is frozen, the interstitial water in the
tissue turns to ice and in this state the tissue is firm, the ice
acting as embedding medium.
• The embedding medium is Optimum Cutting Temperature (OCT)
compound. OCT is made of water soluble glycerols and resin.

Temperature
needed for
cryostat
sectioning –
-15 to -23
degree
Celsius for
unfixed tissue
-7 to -12
degree
Celsius for
fixed tissue

Decalcification
In order to obtain satisfactory paraffin section of bone, inorganic calcium
must be removed from the organic collagen matrix, calcified cartilage
and surrounding tissues.
The decalcification process is carried out by chemical agents, either
acids to form soluble calcium salts or chelating agents which binds to
calcium ions.
Acid decalcification agents- strong acids like HCL and nitric acid is used,
while weak acids like formic acid and trichloroacetic acid may also be
used.

Staining
After the slide has
been placed on a
rack, it is flooded
with hematoxylin and
kept for 5 minutes.
We now wash the
slide under running
water for 5-10
minutes.
We now pour 1%
acid alcohol over the
slide and allowed to
stay for about 1
minute.
Wash in running
water for 5-10
minutes.

Now pour 1% eosin and
allow to stay for 30-60
seconds.
Wash under running
water for 5-10 minutes.
Now the slide is
dehydrated either by air
drying , incubation or
placing it in varying
concentrations of alcohol.

Mounting
When the slide has dried, it is mounted with DPX so as to
preserve the stain from the slide.
The slide is placed on a plain surface. Few drops of DPX are put
over the slide. Now place the coverslip over the DPX and allow it
to spread and dry.
The slide is now ready for the microscopic examinations

Pathological slides

Tuberculosis

Lobar pneumonia

Normal liver

Cirrhosis. There is cirrhosis of the liver with steatosis. The hepatocytes are shrunken and demonstrate decreased
eosinophilia. The nuclei are glassy-appearing and pyknotic. The cells here are “fading away” as opposed to dying in
hepatic necrosis or apoptosis, where there is coincident inflammation. The fibrous bands can be appreciated with a
trichrome stain. Also note that lymphocyte nuclei can fragment and become lobular in decomposition. Please take care
not to confuse these with neutrophils

Heart

Under light microscopy, within 0.5 to 4 hours, waviness of fibers at
the periphery of the tissue is seen. Glycogen is depleted. At 4 to 12
hours, the myocardium undergoes coagulation necrosis and edema.
At 12 to 24 hours, the gross specimen becomes dark and mottled

At 1 to 3 days, there is a loss of nuclei, and at 3 to 7 days,
macrophages appear to remove apoptosis cells. At 7 to10 days,
granulation tissue appears.

Kidney

Diffuse thickening of the basement membranes of nonatrophic renal
tubules in diabetic kidney disease.

A glomerulus shows prominent thickening of Bowman capsule in diabetic
kidney disease.

Soot inhalation in the airway. There is soot (arrow) deposited in the main bronchus.
There is no coagulative necrosis of the epithelium, suggesting that the individual was
alive at the time the fire started and died of inhalation rather then thermal injuries. You
can see evidence of smoke inhalation and thermal injury of the bronchus together. In
such circumstances both likely play a role in death. This finding should be correlated
with the carbon monoxide level.

Trachea. There is coagulative necrosis of the tracheal
epithelium (asterisk) with nuclear streaming (arrow). No soot
is seen in this section

Acute contusion (4–12 hours). Acute hemorrhage with
marked neutrophilic infiltration.

Remote contusion (> 24 hours). Section of subdermal
adipose tissue with erythrocyte “laking” (arrows), or loss of
erythrocyte borders during close association.

Medico-Legal Importance
To identify the cause of death.
To know the exact pathology of the deceased.

Bibilography
• Mohan H, Damjanov I. Textbook of pathology. 8ED,New Delhi: Jaypee
Brothers Medical Publishers; 2019.
• Suvarna KS, Layton C, Bancroft JD. Bancroft’s theory and practice of
histological techniques. Elsevier; 2019.
• Young B, Woodford P, O’dowd G, Wheater PR. Wheater’s functional
histology : a text and colour atlas. 6th ed. Edinburgh: Churchill Livingstone;
2014.
• 4. Fineschi V, Karch SB. Color Atlas of Forensic Histopathology.
Boca Raton, FL: CRC Press; 2021.

Thank you .

Histopathology.pptx

Recommended

Recommended

More Related Content

Similar to Histopathology.pptx

Similar to Histopathology.pptx (20)

More from Dr. Gurjeet Singh

More from Dr. Gurjeet Singh (8)

Recently uploaded

Recently uploaded (20)

Histopathology.pptx