histopathological_techniques.ppt

Department of Pathology
Faculty of veterinary medicine

Histopathology Techniques
•
Introduced By
•
Dr. Sherein Saied Abdelgayed
•
Assistant Professor
•
Department of Pathology
•
Faculty of veterinary medicine
•
Cairo university

Histopathology Techniques:
•
Tissue Processing and Staining
•
Method of Biopsy Taking:
•
1. Incisional biopsy
•
2. Excisional biopsy
•
3. Punch biopsy
•
4. Core needle biopsy
•
5. Curettage biopsy

Incisional biopsy:
•
* It is performed when removal of
entire lesion is impossible.
•
* Often performed prior to major
surgical procedure.
•
* Is strictly a diagnostic nature.

Excisional Biopsy:
•
* In this technique, the entire lesion is
removed, usually with a rim of normal
tissue.
•
* It is performed when the lesion is
smaller in size.
•
* The procedure serves the diagnostic
and therapeutic function.

Punch Biopsy:
•
* It is done by biopsy
forceps.
•
* It is performed in the lesion
of uterine cervix, oral cavity,
esophagus, stomach, intestine
and bronchus.

Core Needle Biopsy:
•
* It is done with special
type of wide bore biopsy
needle.
•
* It permits a percutaneous
approach to internal
•
structures

Curettage Biopsy:
•
Curetting are
usually done for
diagnosis of
endometrial
disease.

Some General Rules for the biopsy Procedure:
•
1. The larger the lesion, the numerous
the biopsies that should be taken from it
because of the fact that the diagnostic
areas may be present only focally.
•
2. In ulcerated tumour, Biopsies should
be taken from the periphery that
includes normal and diseased tissue.

Some General Rules for the biopsy Procedure:
•
3. Crushing or squeezing of the
tissue with forceps should be
carefully avoided.
•
4. Once the biopsy is obtained,
it should be placed immediately
into container with adequate
volume of fixative.
•

Handling of Specimen
•
* Specimen should be transported in
glass, plastic or metal container or in a
plastic bag in 10%formalin. If formalin
is not available at hand, place the
specimen in refrigerator at 4oC to slow
down autolysis.
•
*The container should have an opening
larger enough so that the tissue can be
•
removed easily after it has
•
hardened by fixation.

General Principle of Gross Examination:
•
1. Proper identification and orientation
of the specimen.
•
2. Unlabelled specimen should never be
processed.
•
3. A properly completed histopathology
requisition form containing patient’s
name, age,sex, relevant clinical data,
surgical findings, nature of operation
and name of tissue submitted.

•
4. Careful search and examination of all the
tissue submitted in order.
•
5. Place the specimen on cutting board in an
anatomic position and record the following
•
information:
•
a. Types of specimen b. Structure included.
•
c. Dimensions d. Weight
•
e. Shape f. Colour
•
g. Consistency
•
h. Surgical margin, whether included
•
or not involved by tumour.

•
6. Measurements are usually given
in centimeter unless the specimen is
very small in which mm can be used.
•
7. Endometrial and prostatic tissue
should be measured by aggregate
pieces in volume.

Sampling for Histopathological Examination:
•
*Tissue submitted for histopathology must not
be more than 3 mm thick and not larger than
the diameter of slides used. Most specimens
from solid tissues are cut in the form of
•
pieces measuring 10 to 15 mm on the slides
and 2 to 3 mm in thickness.
•
*Discrete areas of calcification or ossification
should be taken out and should be decalcified
in nitric acid.
•
*Small fragments of tissue must be wrapped
in thin paper.

Histological Technique:
•
*Histological technique deals with the preparation
•
of tissue for microscopic examination.
•
*The aim of good histological technique to preserve
microscopic anatomy of tissue.
•
*This is achieved by passing through a series of
process.
•
These processes are:
•
1. Fixation 2. Dehydration
•
3. Cleaning 4. Embedding
•
5. Cutting 6. Staining

Fixation
.
•
Difinition:
•
*This is the process by which the
constituents of cells and tissue are fixed
in a physical and a chemical state so that
they will withstand subsequent treatment
with various reagents with minimum loss
of architecture.
•
*This is achieved by exposing the tissue
to chemical compounds,call fixatives

Fixation
•
Mechanism of action of fixatives:
•
*Most fixatives act by precipitating proteins
•
*No fixative will penetrate a piece of tissue thicker than
1 cm.
•
*For dealing with specimen thicker than this, following
methods are recommended:
•
1. Solid organ: Cut slices as necessary as but not
thicker than 5mm.
•
2. Hollow organ: Either open or fill with fixative or pack
lightly with wool soaked in fixative.
•
3. Large specimen, Inject fixative along the vessels or
bronchi as in case of lung so that it reaches
all parts of the organ

Properties of an Ideal Fixative:
•
1. Prevents autolysis and bacterial decomposition.
•
2. Preserves tissue in their natural state and fix all
components.
•
3. Make the cellular components insoluble to reagent
used in tissue processing.
•
4. Preserves tissue volume.
•
5. Avoid excessive hardness of tissue.
•
6. Allows enhanced staining of tissue.
•
7. Should be non-toxic and non-allergic for user.
•
8. Should not be very expensive.
•

Amount of fixative fluid:
•
*This should be
approximately 10-20 times
the volume of the
specimen.
•
*Fixative should surround
•
the specimen on all sides.
•

Classification of Fixatives
•
A. Tissue fixatives
•
a. Buffered formalin b. Buffered gluteraldehyde
•
c. Zenker’s formal saline d. Bowen’s fluid
•
B. Cytological fixatives
•
a. Ethanol b. Methanol c. Ether
•
C. Histochemical fixatives
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a. Formal saline b. Cold acetone
•
c. Absolute alcohol

Tissue Processing:
•
*Tissue processing is a long procedure and
required 24 hours. Tissue processing can be
done by manually or mechanically.
•
*It is done in stages. It can be subdivided
into; dehydration, clearing, impregnating and
embedding.
•
*It is important that all specimens are
•
properly labeled before processing is started.
•
*For labeling, pen containing ordinary ink
should not be used. Printed, or graphite pencil
written, are satisfactory.
•

Sequence of manual tissue processing:
•
A. Dehydration:
•
*Tissues are dehydrated by using
increasing strength of alcohol; e.g.
50%, 70%, 90% and 100%.
•
*The duration for which tissues are kept
in each strength of alcohol depends upon
the size of tissue,fixative used and type
of tissue.
•
*The volume of alcohol should be 50-
•
100 times that of tissue.
•

B. Clearing:
*The next step alcohol should be replaced by paraffin
wax.
•
*As paraffin wax is not alcohol soluble, we replace
alcohol with a substance in which wax is soluble.
•
This step is call clearing.
•
*Clearing of tissue is achieved by any of the following
reagents:
•
• Xylene Chloroform Benzene
•
• Carbon tetrachloride Toluene
•
*Xylene is commonly used.
•
*Small piece of tissue are cleaned in 0.5 – 1 hour;
•
*whereas larger (5cm or more thick) are
•
cleaned in 2-4 hours.
•

C.Impregnation with Wax:
•
*This is allowed to occur at melting point temperature
of paraffin wax, which is 54-60oC.
•
* Volume of wax should be about 25-30 times the
volume of tissues.
•
*The duration of impregnation depends on size and
types of tissues and the clearing agents employed.
•
*Total duration of 4 hours is sufficient for routine
•
impregnation.
•
*Types of Wax employed for Impregnation:
•
1. Paraffin wax 2. Water soluble wax
•
*Paraffin wax is used routinely. It has hard
consistency, so section of 3-4 micron thickness can
be cut.

D. Blocking:
•
*Impregnated tissues are placed in a mould
with their labels and then fresh melted wax is
poured in it and allowed to settle and solidify.
Once the block has cooled sufficiently to form
a surface skin it should be immersed in cold
water to cool it rapidly.
•
*After the block has completely cooled it is
cut into individual blocks and each is
trimmed.
•
•
*Labels are made to adhere on
•
the surface of the block.

Staining:
*Staining is a process by which we give
colour to a section.
*There are hundreds of stains available.
•
*Classification of Stains:
•
Generally the stains are classified as:
•
A. Acid stains B. Basic stains
•
C. Neutral stains

Classification of Stains:
•
Acid Dyes:
•
*In an acid dye the basic component is coloured and the acid
component is colourless.
•
*Acid dyes stain basic components
•
*e.g. eosin stains cytoplasm red.
•
Basic Dyes:
•
*In a basic dye the acid component is coloured and the basic
component is colourless.
•
*Basic dyes stain acidic components
•
*e.g. basic fuchsin stains nucleus blue.
•
Neutral Dyes:
•
*When an acid dye is combined with a basic dye a neutral dye is
formed.
•
*As it contains both coloured radicals, it gives
•
different colours to cytoplasm and nucleus
•
simultaneously. This is the basis of Leishman
stain.

Procedure of staining
•
*Every stain is to be used according
to a specified method.
•
*Staining can be done either manually
or in an automatic stainer.
•
*Haematoxylin and Eosin staining:
•
It is the most common used routine
stain in histopathology laboratory

Special Stains:
1.PAS (Periodic Acid Schiff) stain: This stain demonstrates
glycogen
•
2.Stains for micro-organism:
•
a. Gram-stain:
•
b. Ziehl_Neelsen stain: This stain detect acid fast bacilli.
•
c. PAS stain: It is used for fungi, amoeba and Tricomonas.
•
d. Modified Giemsa (2% Giemsa in water):For Helicobacter pylori.
•
3.Congo-red: It is used for identification of amyloid.
•
4.Sudan-Black: It is used for fat staining.
•
5.Masson’s Trichrome:It is used for differentiation of
•
connective tissue
•
•

histopathological_techniques.ppt

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