Histopathologic

Techniques
Katrina Joyce Tolentino-Flores, DMD,
MaEd
UPHS – Binan, Laguna
College of Dentistry
 WHAT IS
HISTOPATHOLOGY?

HISTOLOGY + PATHOLOGY

Histopathology is a branch of pathology concerned with

demonstrating minute structural alteratinos in tissues as a result of disease
Sources for tissue study in histology

ANIMAL TISSUE CADAVER

Autopsy
Biopsy
• Needle Biopsy
• Punch Biopsy
• Bone Marrow Biopsy
• Endoscopic Biopsy
• Surgical Biopsy
Areas where histopathological diagnosis is
helpful:

• Useful in establishing pathogenesis and pathology of any disease caused by

bacteria, virus, chlamydia, rickettsia, mycoplasma, parasite, toxin, poision,
etc
• Diseases wherein examination of tissues is the only alternative to diagnose
the disease, e.g. Bovine spongiform encephalopathy
• Cases wherein tissues from dead animals are the only available method for
laboratory diagnosis
Areas where histopathological diagnosis is
helpful:

• Histopathologic procedures produce permanent slides which are considered

as the most reliable technique
• Histopathologic techniques are useful in carrying out the retrospective studies
• The presence of causative agents can be demonstrated in tissue sections using
routine histopathologic techniques or special staining
• Gram staining procedure – demonstrates bacteria
• Hematoxylin and eosin – demonstrates viral inclusions
• Seller’s stain – demonstrates Nigri bodies for diagnosis of rabies
• Detection of chemicals in tissues like enzymes, lipids etc is included in
histochemical examination
 Collection of sample:
1. Small piece of tissue is removed as early as possible
Sample 2. The piece of tissue must be removed with a sharp
knife
collection and 3. At the time of collection, keep in mind that the
representative tissue must include a part of the
preservation lesion and a part of normal tissue
4. Collected tissue must be collected directly in a
fixative and not in any other pot or water
5. Samples taken from hollow organs must be cue
transversely
6. Samples from encapsulated organs must include the
capsule of covering
 Labelling
• Sample tissue must be
accompanied by a tag or label
bearing lab number it is assigned
Sample with.
collection and • A thin white card with writing in
pencil may be used.
preservation
Specimen
Accessioning
• Tissue specimen received in the
laboratory has a request form that
bears the patient information,
history and description of the site of
origin
• The specimen is accessioned by
assigning a number that will identify
each specimen for each patient.
 Information must include:
• Patient identification
• Identification of individual requesting examination
• Procedure date
• Adequate clinical history and physical examination
Specimen • Organs resected or biopsied
Accessioning • Orientation if required
• Intraoperative findings
• Prior surgery or pathologic diagnosis
• Prior treatment
• Mention if rapid diagnosis is required
Gross Examination

It involves:
• Accurate naked eye description of intact specimen
• Correct method of sectioning
• Gross examination of cut surface
• Selection of proper tissue blocks for microscopy
• Instructions for embedding and block making

Important not only to establish tissue diagnosis but is also crucial in

clinical management and provide important prognostic data
 • A process by which constituents of

Fixation cells and tissue are fixed in a

physical and chemical state so that
they will withstand subsequent
treatment with various reagents
with minimum loss of architecture
• Achieved by exposing tissues to
chemical compounds called
fixatives
• Purpose: to preserve tissues
permanently in life-like state as
possible
 • Most fixatives act by denaturing or
Mechanism of precipitating proteins which then form a
sponge or meshwork, tending to hold the
action of other constituents
• In order to achieve a good fixation which is
fixatives important in the production of satisfactory
results in histopathology, the following
factors are important:
• Fresh tissue
• Proper penetration of tissue by fixatives
• Correct choice of fixatives
 • No fixative will penetrate a piece of
tissue thicker than 1cm (10mm)
• When dealing with thick specimens, the
following methods are recommended

Specifications 1. Solid organ: Cut slices as necessary but

not thicker than 5mm
in Fixation 2. Hollow organ: Either open and fill with
fixative or pack lightly with wool
soaked in fixative.
3. Lage specimen: requires dissection,
inject fixative along the vessels or
bronchi as in case of lungs so that it
reaches all parts of the organ
 Prevent autolysis and bacterial decomposition

Preserves tissue in their natural state and fix all components

Make cellular components insoluble in liquids encountered in

tissue processing
Properties Preserve tissue volume
of an Ideal Avoid excessive hardness of fixed tissue
Fixatives
Allow enhanced staining of tissues

Should be non-toxic & non-allergic

Should be economic
 • The fixation can be carried out at room
temperature.
Temperature • Tissue should not be frozen once it has been
placed in the fixation to prevent ice crystal
distortion
 • The speed of fixation of most fixative is
Speed of almost 1mm/hr.
fixation • Fixation time of several hours is needed for
most specimens
 • This should be approximately 10-20 times
the volume of the specimen.
Amount of
fixative • Fixative should surround the specimen in all
sides
 • Size and thickness of piece of tissue
• Tissue covered by large amount of
mucous fix slowly
• Tissue covered by blood or organ
containing very large amount of blood
Factors affecting also fix slowly.
• Fatty and lipomatous tissue fix slowly.
fixation • Fixation is acceleratred by agitation
• Fixation is accelerated by maintaining
temp around 60 degrees celsius
 Tissue fixatives

Classification of
Cytological fixatives
Fixatives

Histochemical
fixatives
 There are many tissue fixatives

• Buffered formalin
Tissue fixatives • Buffered glutaraldehyde
• Zenker’s formal saline
• Bowen’s fluid
 Ethanol

Cytological Methanol
fixatives

Ether
 Formalin saline
Histochemical
fixatives
Cold acetone

Absolute alcohol
Formalin

• 10% neutral buffered form

• Most commonly used fixative
• Prepared by mixing 40% formaldehyde gas in 100 w/v of distilled water
→ results mixture: 100% formalin
• Routinely, 10% is used which is prepared by mixing 10ml of 100%
formalin in 90ml distilled water
Fixation: Mechanism of action
• It forms cross links between amino acids of proteins thereby making
them insoluble
• It fixes 4mm thick tissue in 8 hours
• The fixative should be 10 times more in volume then the specimen
TISSUE PROCESSING
• Describes the steps required to take animal or human tissue from
fixation to the state where it is completely infiltrated with a suitable
histological wax and can be embedded ready for section cutting on the
microtome
• Steps
1. Dehydration
2. Clearing
3. Impregnating
 1. Manual tissue processing
• Tissue specimen is transferred from one
container of reagent to another by hand
Types of
tissue 2. Mechanical tissue processing
• Tissue is moved from one jar to another by
processing mechanical device and timing is controlled by a
timer which can be adjusted in respect to hours
and minutes. Temp is also maintained around 60
degrees Celsius
• Tissues are dehydrated by using
increasing strength of alcohol; e.g. 50%,
70%, 90% and 100%

• Duration of dehydration depends upon

the size of tissue, fixative used and type Dehydration
of tissue

• Volume of alcohol should be 50-100

times that of the tissue
 • Process of removal of dehydrant with a
substance that will be miscible with the
embedding medium (paraffin)

Clearing agents
Clearing 1. Xylene – most commonly used
2. Chlorform
3. Benzene
4. Carbon tetrachloride
5. toluene
Impregnation
• The process wherein the spaces in tissues and cells emptied after
removal of clearing agent is filled with wax

• This allows hardening of tissue which helps in section cutting

• Occurs at melting point of paraffin wax which is 54-60 degrees celsius

 • Involves the enclosing of properly processed,
correctly oriented specimen in a support
medium that provide external support
during microtomy.
• It should provide elasticity and resist
Tissue distortion during sectioning
• It is carried out using an “embedding
Embedding centre” where a mould is filled with molten
wax and the specimen is placed into it
keeping in mind that it has to be very
carefully orientated in the mould because its
placement will determine the “plane of
section”
Embedding
Center
Embedding process
Tissue Sectioning
• Microtomy is the process by which
tissue is sectioned and attached on the
surface for microscopic examination
• Microtome – instrument used in
microtomy

Types of microtomes
1. Rotary – most commonly used
2. Sliding
3. Freezing
4. Rocking
5. Base-sledge
 Microtome knives
1. Glass knife – used to cut 4-6 um-thick
tissue sections which are mounted on a
glass microscope slide used for light
microscopy
Tissue 2. Diamond knife – used to cut 50nm-thick
Sectioning tissue sections which are mounted on a
3mm diameter copper grid. Mounted
section is then treated with appropriate
stain
3. Cryostat – used to cut frozen tissue
embedded in a freezing medium
• Water bath is used for floating out tissue ribbons
after sectioning.
• The temperature of the water in the bath should be
10°C below the melting point of the paraffin.
• Alcohol or a small drop of detergent may be added
to the water allowing the section to flatten out with
greater ease
• 30 seconds are long enough for a ribbon to flatten
• After tissue sectioning, the remaining section will now look like a
tissue specimen embedded in a thin layer of paraffin wax mounted in
microscope slide

Slide
Tissue specimen Sliced paraffin wax
block

• The section will now go through staining in order to visualize tissue

morphology. However, most staining solutions are aqueous, so to
stain the sections, the wax has to be dissolved and replaced with
water → DEPARAFFINIZATION AND REHYDRATION
Deparaffinization
and Rehydration
• Deparaffinization is the
removal of paraffin wax from
which the specimen is
embedded in and also the
paraffin wax infiltrated in the
tissue.
• Wax is removed with the use
of xylene and later on, xylene
is removed with the use of
100% alcohol.
 • Rehydration is the process of submerging tissue
specimen in decreasing concentrations of alcohol to
allow absorption and retaining of water.
Rehydration will allow staining as most stains are
miscible with water.

Deparaffinization
and Rehydration
Slide Staining
• Artificial coloration of a substance
with the use of colored organic
molecules called dye

Purpose of staining
• Outline the tissue and cellular
components
• Identify tissue
• Establish presence or absence of
disease processes
 • Staining provides visual contrast between
the constituent parts of a tissue section
• The depth of coloration is affected by
chemical affinity, density and permeability
Slide Staining
Classification of Stains
1. Acid stains - eosin
2. Basic stains - haematoxylin
3. Neutral stains - Leishman stain
Precautions
• Rapid fixation of fresh specimen is essential as post-mortem
degeneration markedly alters the appearance of various cell types in
tissue making interpretation unrewarding

• Quality of nuclear staining eventually deteriorates and precipitate

may form in stored stains after a few months. At this stage, stain
should be filtered and staining time should be increased.

• Prepare a fresh batch of stain every month