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The principles of diseases module; histopathology practical conducted in four practical sessions.
First practical session starts with demonstration of tissue processing and staining followed by explanation of
cytology procedures with more emphasis on papanicolaou smear. Rest of the three session has two parts 1.
Gross specimens & clinical image discussions & 2. Microscopic discussions in relation with basic principles of
cellular injury, adaptations, inflammation, tissue repair, hemodynamic disorders and neoplasia.
1. Gross specimen discussions:
- Demonstration of plastinated and wet (formalin fixed) specimens
- Descriptive discussions on standard macroscopic & clinical static images
- List of specimens & static images
Plastinated specimens Fatty liver
Acute appendicitis
Chronic pyelonephritis
Amoebic liver abscess
Cirrhosis liver
CVC Liver
Sub endocardial infarction
Infarct spleen
Infarct brain
Infarct lung
Lipoma
Mature teratoma Testes
Pleomorphic adenoma
Liposarcoma
Squamous cell carcinoma Skin
Carcinoma intestine
Carcinoma stomach
Metastatic tumour Brain
Formalinated (Wet) specimens Leiomyoma
Sarcoma Soft tissue
Cyst Skin
Polyp intestine
Carcinoma intestine
Standard clinical images Healthy & unhealthy wound
Pitting edema
Ascites
Anasarca
Periorbital edema
Gangrene Leg
Diabetic foot
Petechiae
Purpura
Hematoma
Papilloma Skin
Squamous cell carcinoma & basal cell carcinoma skin
Note: Students are permitted to take photograph or video of specimen demonstrations
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2. Microscopic discussions:
- Demonstration of robotic scanned digital microscopic slides with scaled microscopic magnification from 2x to
40x objective lens with 10x eye piece lens.
- Descriptive discussions on standard microscopic static images
- List of robotic digitally scanned microscopic slides
Robotic digital scanned microscopic slides Glandular metaplasia Esophagus
Monckeberg calcification
Fatty liver
Coagulative necrosis Kidney
Liquefactive necrosis Brain
Caseous necrosis Lymphnode
Fat necrosis
Acute appendicitis
Cirrhosis liver
Pulmonary edema
Coronary artery disease
Venous thrombosis
Pulmonary congestion
Dysplasia Cervix
Lipoma
Liposarcoma
Adenomatous polyp colon
Adenocarcinoma colon
Malignant melanoma
Squamous cell carcinoma Skin
Metastatic carcinoma Lymph node
Note: Students are permitted to take photograph or videos of slides. The manual contains photograph,
focused on the characteristic histopathological feature of that particular slide.
Mode of assessment:
- Station based objective structures practical evaluation (examination) - OSPE.
- 5 Stations
Station 1
[Observer]
Station 2
[Observer]
Station 3
[Non Observer]
Station 4
[Non Observer]
Station 5
[Observer]
Instruments
Students has to:
Identify
Indications
Specific features
Reagents & Stains
Students has to:
Identify
Indications
Specific features
Spotter
Spot diagnosis of
gross specimens
Spotter
Spot diagnosis of
microscopic slides
Objective reasoning
Students has to:
Describe & explain about
the changes happened in
the given scenario

- OSPE will be conducted in multiple batches. All the batch students should come prepared with all the practical
content, taught in the session. Students will be evaluated from the entire practical content.


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Pathology
Tissue processing & staining
















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Definition of pathology


The word Pathology is derived from two Greek wordspathos meaning suffering, and logos meaning
study. Pathology is the scientific study of structure and function of the body in disease; or in other
words, pathology consists of the abnormalities that occur in normal anatomy (including histology) and
physiology owing to disease. Another commonly used term to study of diseases is pathophysiology
comprised by two words: patho=suffering; physiology=study of normal function. Pathophysiology,
thus, includes study of disordered function or breakdown of homeostasis (homeostasis being in a
state of balance) in diseases.


Pathologists are the diagnosticians of disease. Therefore, knowledge and understanding of pathology
is essential for all the doctors, general medical practitioners and specialists since unless they know the
causes (etiology study of causes), mechanisms, nature and type of disease and understand the
language spoken by pathologist in the form of laboratory reports, they would not be able to
treatment or advice patient. For the student of any system of medicine, the discipline of pathology
forms a vital bridge between initial learning phase of preclinical sciences and the final phase of clinical
subjects. It is best to quote an eminent founders of modern medicine Sir William Osler statement,
Your practice of medicine will be as good as your understanding of pathology.


Terminologies for beginner in pathology

It is important for students to be familiar with the language used in pathology:

1. Patient is the person affected by disease.
2. Lesions are the characteristic changes in tissues and cells produced by disease in an individual
or experimental animal.
3. Pathologic changes or morphology consist of examination of diseased tissues.
4. Pathologic changes can be recognized with the naked eye (gross or macroscopic changes) or
studied by microscopic examination of tissues.
5. Causal factors responsible for the lesions are included in etiology of disease (i.e. why of
disease).
6. Mechanism by which the lesions are produced is termed pathogenesis of disease (i.e. how of
disease).
7. Functional implications of the lesion felt by the patient are symptoms and those discovered by
the clinician are the physical signs.
8. Clinical significance of the morphologic and functional changes together with results of other
investigations help to arrive at an answer to what is wrong (diagnosis), what is going to happen
(prognosis), what can be done about it (treatment), and finally what should be done to avoid
complications and spread (prevention) (i.e. what of disease).




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Subdivisions in Pathology

Human pathology is the largest branch of pathology. It is conventionally divided into General
Pathology dealing with general principles of disease and Systemic Pathology that includes study of
diseases pertaining to the specific organs and body systems. With the advancement of diagnostic
tools, the specialty of pathology emerged, which include the following subspecialties:

1. Histopathology: Also called as anatomic pathology, pathologic anatomy, or morbid anatomy, is
the classic method of study and still most useful one which has stood the test of time. The
study includes structural changes observed by naked eye examination referred to as gross or
macroscopic changes and the changes detected by light and electron microscopy supported by
numerous special staining methods including histochemical and immunological techniques to
arrive at the most accurate diagnosis. Modern time anatomic pathology includes super
specialties such as cardiac pathology, pulmonary pathology, neuropathology, renal pathology,
gynecologic pathology, breast pathology, dermatopathology, gastrointestinal pathology, oral
pathology and so on. Anatomic pathology includes the following 3 main subdivisions:

a) Surgical pathology - It deals with the study of tissues removed from the living body. It
forms the bulk of tissue material for the pathologist and includes study of tissue by paraffin
embedding techniques and frozen section for rapid diagnosis.
b) Forensic pathology and autopsy work - This includes the study of organs and tissues
removed at postmortem for medico legal work and for determining the underlying
sequence and cause of death.
c) Cytopathology - It includes study of cells shed off from the lesions (exfoliative cytology)
and fine needle aspiration cytology (FNAC) of superficial and deep seated lesions for
diagnosis.

2. Hematology - Hematology deals with the diseases of blood. It includes laboratory hematology
and clinical hematology; the latter covers the management of patient as well.

3. Chemical pathology - Analysis of biochemical constituents of blood, urine, semen, CSF and
other body fluids is included in this branch of pathology.

4. Immunology - Detection of abnormalities in the immune system of the body comprises
immunology and immunopathology.

5. Experimental pathology - This is defined as production of disease in the experimental animal
and its study.

6. Geographic pathology - The study of differences in distribution of frequency and type of
diseases in populations in different parts of the world.

7. Medical genetics - This is the branch of human genetics that deals with the relationship
between heredity and disease.

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8. Molecular pathology - The detection and diagnosis of abnormalities at the level of DNA of the
cell is included in molecular pathology.

Even though many specialties exist; it is impossible to define their boundaries. Human body is an
integrated structure, so the branches evolved and evolving in pathology of diseases are also
integrated. Advancement in the field of pathology, expands the knowledge and keep everyone on
their life long journey of learning.


Microscope
Microscope is the most commonly used apparatus in the laboratory. It magnifies the minute objects
according to the desired calibration. It was first invented by Antony Von Leeuwenhoek (1632 1723)
and also introduced first histochemical staining. There are different types of microscopes, most
commonly used are light microscopy. A light microscope can be a simple or a compound microscope.
Simple microscope is a simple hand magnifying lens. The magnification power of hand lens is from 2x
to 200x. Compound microscope has a battery of lenses which are fitted in a complex instrument. One
type of lens remains near the object (objective lens with magnifying power of 4x, 10x, 20x, 40x and
100x) and another type of lens near the observers eye (eye piece lens with magnifying power of 5x,
10x or 15x). The eye piece and objective lenses have different magnification. The compound
microscope can be monocular having single eye piece (Fig. 1) or, binocular which has two eye pieces
(Fig. 2).





















Figure 1: Monocular microscope Figure 2: Binocular microscope



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Other microscope (specific condition) and its application:
No Microscope Application
01 Dark ground illumination Examination of unstained living microbes.
Example Trepenoma palladium
02 Polarizing microscope Demonstration of birefringence
Example - Amyloid
03 Fluorescent microscope Demonstration of naturally occurring fluorescent
material or substances stained with fluorescent dyes. UV
light is used for illumination.
Example Immunoglobulin, Amyloid, Mycobacterium
04 Electron microscope Study of ultra-structural details of the tissues and cells.
Example Glomerular basement membrane structure

Digital Pathology
Over the past few years, the evolution of information technology has vastly changed the practice of
pathology. New techniques have been implemented, primarily in the form of digital images and
telepathology. Digital pathology can be defined as the electronic capture, management, analysis and
distribution of gross or microscopic specimens. It encompasses the use of digital images.


The applications of digital pathology is wide and includes primary diagnosis, intraoperative diagnosis,
consultation, quality assurance, archiving, education, conferences, and research. Widespread
adoption of digital pathology applications could expedite second opinion consultation; digital
consultation can be performed within hours versus days to weeks for cases sent through the mail. It
can also help improve service quality to under serviced areas, facilitate immediate conferencing about
a specimen by individuals in different locations, reduce costs of slide storage, limit slide loss, expedite
slide retrieval and overcome the problem of slide fading.


There are, however, several challenges to the widespread implementation of digital pathology,
including cost, and a lack of standardization. Added to this is the potential negative attitude of
pathologists towards the change.


By The Grace and Mercy of The Almighty Allaah, with the help of our academic administration and
management, we overcome all hurdles and established the digital pathology teaching to benefit the
students to understand and apply the concepts of pathology in their future clinical life.



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Regular Histopathology Slide
(Step 1)















Digital Slide Scanner (Step 2) Viewed Through Software (Step 3)

Histopathology Technique

Histopathology is examination of tissues for presence or absence of changes in their structure due to
disease processes. Both are done by examining thin sections of tissues which are colored differently
by different dyes and stains. Total or selected representative part of tissue not more than 4 mm thick
is placed in steel or plastic capsules or cassettes and is subjected to the following sequential
processing (tissue processing):
Step 1: Fixation
Step 2: Processing Dehydration, Clearing, Impregnation
Step 3: Embedding and blocking
Step 4: Section cutting
Step 5: Routine staining

Fixation
Any tissue removed from the body starts decomposing immediately because of loss of blood supply
and oxygen, accumulation of products of metabolism, action of autolytic enzymes and putrefaction by
bacteria. This process of decomposition is prevented by fixation. Fixation is the method of preserving
cells and tissues in life like conditions as far as possible. Most fixatives act by denaturation or
precipitation of cell proteins or by making soluble components of cell insoluble.
Step 1: The regular histopathology slides were
scanned by using motic digital slice scanning
microscope.
Step 2: The digital images were collected and
stored in the computer database.
Step 3: By using motic digital microscopic
image software, the digital histopathology
images are visualized.




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Use of fixatives: Prevents putrefaction and autolysis, hardens the tissue which helps in section cutting,
makes cell insensitive to hypertonic or hypotonic solutions, acts as a mordant & induces optical
contrast for good morphologic examination.

Fixatives may be simple or compound: Simple fixative consists of one substance (e.g. formalin).
Compound fixative has two or more substances (e.g. Bouins).

Commonly used fixatives and its application:

No Fixative Composition & action Application
01 Formalin It is a saturated solution of formaldehyde
gas in water, 40% by weight/volume (w/v).
For all practical purposes, this 40% solution
is considered as 100% formalin. For fixation
of tissues, a 10% solution is used which is
prepared by dissolving 10 ml of
commercially available formalin in 90 ml of
water.
It takes 6-8 hours for fixation of a thin piece
of tissue 4 mm thick at room temperature.
The amount of fixative required is 15 to 20
times the volume of the specimen.
Formalin acts by polymerization of cellular
proteins by forming methylene bridges
between protein molecules.
Commonly used for
all the tissues
02 Glutaraldehyde It is used as 4% solution at 40 degree Celsius
for 4 hours for fixation of tissues.
Electron microscopy
03 Bouins fixative Picric acid , Formaldehyde, Glacial acetic
acid
Testes & renal tissues
04 Carnoys fixative Alcohol fixative (Methyl and ethyl alcohol) Cytology examination
05 Osmium
tetroxide
2% solution of osmium tetroxide Electron microscopy
CNS tissues & lipids

Dehydration
This is a process in which water from cells and tissues is removed so that this space is subsequently
taken up by wax. Dehydration is carried out by passing the tissues through a series of ascending
grades of alcohol: 70%, 80%, 95% and absolute alcohol. If ethyl alcohol is not available then methyl
alcohol, isopropyl alcohol or acetone can be used.

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Clearing
This is the process in which alcohol from tissues and cells is removed and is replaced by a fluid in
which wax is soluble and it also makes the tissue transparent. Xylene is the most commonly used
clearing agent. Toluene, benzene (it is carcinogenic), chloroform (it is poisonous) and cedar wood oil
(it is expensive and very viscous) can also be used as clearing agent.

Impregnation
This is the process in which empty spaces in the tissue and cells after removal of water are taken up
by paraffin wax. This hardens the tissue which helps in section cutting. Impregnation is done in molten
paraffin wax which has the melting point of 56
o
C (54-62
o
C).

Tissue processors
Nowadays all the processes of fixation, dehydration, clearing
and impregnation are carried out in a special equipment
which is known as automated tissue processor. Tissue
moves automatically by hydraulic mechanism from one jar
to another after fixed time schedule and the whole process
takes 16-22 hours

Embedding and blocking
Embedding of tissue is done in molten wax. Wax blocks are
conventionally prepared using metallic L (Leuckharts
mould); nowadays plastic moulds. Molten wax is poured in
the cavity in the moulds. The processed tissue pieces are put
into wax with number tag and examining surface facing
downward. Wax is allowed to solidify. After solidification, if
L-moulds are used they are removed while plastic mould
remains with the wax block. In either case, each block
contains a tissue piece carrying an identification label.
Embedding and blocking can also be performed in a special
instrument called embedding centre.

Section cutting (microtomy)
Microtome is an equipment for cutting sections. There are 5
types of microtomes:
1. Rotary,
2. Sliding,
3. Freezing,
4. Rocking,
5. Base sledge.
Most commonly used microtome is rotary microtome.
The section is cut with a thickness of 5 - 6 m (microns).




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Routine staining (Hematoxylin and Eosin H & E Staining)

Hematoxylin - Natural dye obtained from log wood tree, Haematoxylon campechianum. The natural
extract from the stem of this tree is haematoxylin which is an inactive product. This product is
oxidized to an active ingredient which is haematein. This process of oxidation is known as ripening
which can be done naturally in sunlight, or chemically by addition of oxidant like sodium iodate,
KMnO4 or mercuric oxide. Hematoxylin will not directly stain tissues, but needs a mordant or link to
the tissues. So mordant is added to it (e.g. potash alum) which helps in attaching the stain particles to
the tissue. Variety of hematoxylin available, most commonly used is Harris hematoxylin. They vary in
intensity or hue. Hematoxylin is a basic dye gives blue color, has an affinity for the nucleic acids of the
cell nucleus.

Eosin Synthetic dye derived from the action of bromine on fluorescein. There are variety of eosin
based on their pH. Eosin is an acidic dye gives pink color, with an affinity for cytoplasmic components
of the cell and extra cellular components include collagen, elastin, muscle fibers and red blood cells.
Perinuclear areas look bluish to pink because of mixture of protein and RNA.

There are two types of staining methods "regressive" or "progressive". With a regressive stain, the
slides are over stained and excess stain is removed by acid alcohol. This is good for staining large
batches of slides. With a progressive stain the slide is dipped in the hematoxylin until the desired
intensity of staining is achieved. This is good for small batches of slide (frozen section)

Procedure for Staining

Step 1: Removing the wax (hydrophobic) deparaffinization. Place the slide in a jar of xylene
(hydrocarbon solvent) for 10-15 minutes.
Step 2: Hydration. As haematoxylin is a water-based dye, the sections before staining are rehydrated
which is done by passing the sections in a series of descending grades of alcohol and finally bringing
the section to water.
Step 3: Hematoxylin staining. Place the slide in haematoxylin stain (contains oxidized hematoxylin +
Aluminum salt) for 8-10 minutes. The section become reddish purple
Step 4: Blueing. Rinse in water. Dip the section in Scotts tap water (containing sodium bicarbonate
and magnesium sulfate) or saturated solution of lithium carbonate for 2-10 minutes to bring required
blue color to the section.
Step 5: Differentiation. Dip the section in a in a solution of 1% acid alcohol for 10 seconds to
selectively remove excess dye from the section. Rinse in water. [Differentiation is used only in
regressive staining method]
Step 6: Counterstain with eosin. 1% aqueous solution of eosin for 1- 3 minutes.
Step 7: Rinse, dehydrate, clear and mount. Rinse in tap water. Before mounting, the sections have to
be dehydrated which is done by passing the sections in a series of ascending grades of alcohol and
finally cleared in xylene. 2 - 3 dips in each solution. Mount in DPX (dextrene polystyrene xylene) or
Canada balsam.

Results: Nuclei stain blue (basophilic, hematoxyphilic). Cytoplasm, muscle, collagen & keratin stain
pink (acidophilic, eosinophilic). Red blood cells stain red.

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Changes seen in each steps:


Unstained section in
glass slides.



Step 1
Removing the wax



Step 2
Hydration



Step 3
Hematoxylin staining



Step 4
Blueing



Step 5
Differentiation



Step 6
Eosin Staining



Step 7
Dehydrate, clear, mount
In the end of staining, section clearly demonstrate the tissue characteristics













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Hematoxylin and Eosin staining in Liver Section:













































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Special Histopathology Technique

During the tissue examination, some diseases or certain tissues need other than routine technique
and staining. Such procedures are grouped under special histopathology technique which includes
frozen section to Immunophenotyping. Here only basic and essential special methods are discussed.

Decalcification
Calcified tissues or bone are extremely hard to cut using microtome. Chemical substances are used to
remove the calcium such process is called as decalcification. Mineral acids, organic acids, EDTA and
electrolysis are used to remove calcium from the tissues.
Mineral acids (nitric and hydrochloric acids) - Used for dense cortical bone because they will remove
large quantities of calcium at a rapid rate but these strong acids also damage cellular morphology, are
not recommended for delicate tissues such as bone marrow.
Organic acids (acetic and formic acid) Use for bone marrow, since they are not as harsh. Formic acid
in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that
combine formic acid with formalin to fix and decalcify tissues at the same time.
EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and works
slowly and is expensive in large amounts. Electrolysis has been tried in experimental situations where
calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily
use.
Frozen section
At times during performance of surgical procedures, it is necessary
to get a rapid diagnosis of a pathologic process. During such
situation frozen sections are performed. The piece(s) of tissue to be
studied are snap frozen in a cold liquid or cold environment (-20 to -
70 Celsius). Freezing makes the tissue solid enough to section with a
microtome. It is done by using cryostat. The cryostat is just a
refrigerated box containing a microtome. The temperature inside
cryostat is about -20 to -30 Celsius. The tissue sections are cut and
picked up on a glass slide. The sections are then ready for staining.
Frozen sections are commonly done for;
- To confirm the margins of resection of a malignant neoplasm are
clear before closing.
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- Unexpected disease process may be found and require diagnosis to decide what to do next, or it may
be necessary to determine if the appropriate tissue has been obtained for further workup of a disease
process and to demonstrate some special substances in cell or tissue such as fat, enzymes etc.
Special stains

Stains are applied for demonstration of certain specific substances/constituents in cells/tissues.
Special stains Tissue Sections Comments
Sudan black /
Oil red stain



Muscle with adipose tissue
Used to demonstrate of fat.
Fat stains black (Sudan black) & red
(oil red)
Van Gieson stain



Muscle, adipose & fibrous tissue
Used to demonstrate collagen.
Muscle stains yellow and collagen
stains red
Massons
Trichrome stain




Muscle and fibrous tissue with vessel
Used to demonstrate muscle
Muscle stains red, collagen stains
blue and nuclei blue
Reticulin
(Silver stain)
silver methanamine
periodic acid Schiff
stain




Glomerulus & renal tubules
Used to demonstrate reticular
fibers.
Reticulin fibers stain black.
Especially used in renal, liver and
lymphnode biopsies and in vascular
tumours
Verhoeff (Elastin)
stain



Muscular artery (elastic fibers)
Used to demonstrate elastic fibres.
The section is counterstained with
van Gieson stain. Elastic fibres stain
black, collagen stain red, cytoplasm
& muscle fibres stain yellow


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Periodic - acid
Schiff stain




Goblet cells of colon
Used to demonstrate glycogen and
mucopolysaccharides. PAS positive
substances are glycogen, amyloid,
colloid, neutral mucin and hyaline
cast, stains bright pink.
Congo red stain


Glomerulus with amyloid
Used to demonstrate amyloid, an
extra cellular fibrillar proteinaceous
substance. It gives green
birefringence when viewed by
polarized light and stain red

Cytology Technique

Cytology is the study of body cells that are either exfoliated spontaneously from epithelial surfaces or
are obtained from various body tissues and organs by different techniques. Currently, cytology has
following branches: A. Exfoliative cytology, B. Aspiration cytology & C. Imprint cytology.

Exfoliative cytology: This is the study of cells which are spontaneously shed off from epithelial
surfaces into body cavities or fluid. The cells can also be obtained by scraping, brushing or wash of
body surfaces. The principle of this technique is that in diseased states, rate of exfoliation of cells is
increased.

Applications of Exfoliative Cytology

Exfoliative cytology is applied in diagnosing diseases of the following:

1. Female genital tract
2. Respiratory tract
3. Gastrointestinal tract
4. Urinary tract
5. Body fluids (pleural, peritoneal, pericardial, CSF and semen)
6. Buccal smears for sex chromatin

Female Genital Tract

Smears from female genital tract are known as Pap smears. These smears are prepared by different
methods depending upon the purpose for which they are intended:



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1. Cervical smear: It is obtained by Ayres spatula from portio
of the cervix by rotating the spatula through 360o to sample
the entire cervix. The scraped material is placed on a clean
glass slide and smear prepared. It is ideal for detection of
cervical carcinoma.

2. Lateral vaginal smear (LVS): It is obtained by scraping upper
third of lateral walls of the vagina and is ideal for
cytohormonal assessment.

3. Vaginal pool smear: It is obtained by aspirating material
from posterior fornix of vagina and is done for detecting
endometrial and ovarian carcinoma.



Method of obtaining cervical
material for fast smears

Respiratory Tract

Material from respiratory tract may be obtained during bronchoscopic procedures as expectorant
(sputum) or by brushing (BB), washing (BW) and bronchioalveolar lavage (BAL). Sputum examination is
advantageous as samples are easily obtained and cellular content is representative of entire
respiratory tract. At least three samples of sputum, preferably early morning samples, should be
examined.

Gastrointestinal Tract

Lesions in the oral cavity can be sampled by scraping the surface
with a metallic or wooden spatula. Samples can be obtained from
the oesophagus, stomach, small and large intestine either by
brushing or lavage during fibreoptic endoscopy.

Urinary Tract

Samples from lesions in the urinary tract are either urinary
sediment examined from voided urine/ catheterised urine or
washings of the urinary bladder obtained at cystoscopy.

Cytospin
Body Fluids
Fluid from pleural, peritoneal or pericardial cavity is obtained by paracentesis. At least 50-100 ml of
fluid is aspirated. The sample is examined fresh but if delay is anticipated then fluid should be
anticoagulated either in EDTA 1 mg/ml or 3.8% sodium citrate 1ml/10ml. Fluid should be centrifuged
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and smears are prepared from the sediment. If amount of fluid is less (less than 1 ml), then it can be
subjected to cytospin centrifuged smear preparation.

Buccal Smears for Sex Chromatin

Smears are prepared from the oral cavity after cleaning the area. Vaginal smears can also be used. In
normal females, Barr bodies are present in 4-20% nuclei. In males their count is in less than 2% nuclei.

Fixation of Smears in Exfoliative Cytology

Methods of fixation depend upon type of staining employed. Pap smears are wet-fixed (i.e. smears
are immersed in fixative without allowing them to dry). Smears to be stained by Romanowsky stains
are air-dried as fixation is affected during the staining procedure. Fixative used is either equal parts of
ether and 95% ethanol, or 95% ethanol alone, 100% methanol, or 85% isopropyl alcohol. Fixation time
of 10-15 minutes at room temperature is adequate. Smears may be left in fixative for 24 hours or
more. Smears should be transported to the laboratory in fixative solution in coplin jars.

Staining of Smears in Exfoliative Cytology

Three staining procedures are commonly employed: Papanicolaou and H and E stains are used for
wet-fixed smears while Romanowsky stains are used for air-dried smears.

Papanicolaou Stain

This is the best stain for routine cytodiagnostic studies. In this, haematoxylin gives nuclear stain while
OG-6 and EA-50 are two cytoplasmic counterstains.

H & E Stain

This is the same as that used for histological sections. In this, haematoxylin is nuclear stain and eosin
is cytoplasmic counterstain.

Romanowsky Stain

Leishmans stain, Giemsa and May-Grunwald-Giemsa (MGG) are usually used; the last one is most
commonly used.

Aspiration cytology: In this study, samples are obtained from diseased tissue by fine needle aspiration
(FNA) or aspiration biopsy cytology (ABC).

Applications of FNA

FNA or ABC is applied for diagnosis of palpable as well as non-palpable lesions.
I. Palpable Mass Lesions in: 1. Lymph nodes, 2. Breast, 3. Thyroid, 4. Salivary glands, 5. Soft tissue
masses & 6. Bones

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II. Non-Palpable Mass Lesions in: 1. abdominal cavity, 2. Thoracic cavity & 3. Retroperitoneum

Procedure for FNA

Materials for performing FNA, a Franzens handle, syringe with needles, clean glass slides and suitable
fixative are required.
Equipment required for transcutaneous FNAC.

Method

- No anaesthesia is required.
- Ask the patient to lie down in comfortable position exposing the target area.
- Palpate the target area.
- Clean the overlying skin with spirit.
- Fix 10/20 ml disposable syringe in Franzens handle. Insert 20-25 gauge disposable needle into
syringe.
- Fix the mass by palpating hand and insert needle into target area. Apply suction while moving
needle back and forth within the lesion and change the direction of the needle.
- Terminate the aspiration when aspirated material or blood is visible at the base/hub of the
needle.
- Release the suction before withdrawing the needle to equalize pressure within the syringe.
- After withdrawal of needle, apply pressure for 2-3 minutes at the site of puncture to arrest
bleeding and prevent hematoma formation.
- Aspirated material from the needle is expressed on to clean glass slides by first detaching the
needle and filling the syringe with air and expressing it with pressure.
- Smears are prepared as for blood smears. If the material is semi-solid, it is first crushed by
gentle pressure with a glass slide and smears prepared.
- Fixation and staining are the same as for exfoliative cytology.


20



Procedure of FNAC of palpable masses

Needle is introduced into the mass (A). Plunger
is retracted after needle enters the mass (B).
Suction is maintained while needle is moved
back and forth within the mass (C). Suction is
released and plunger returned to original
position before needle is withdrawn (D).



Preparation of smears
Semisolid aspirates are crush-smeared by flat pressure with cover slip or glass slide (A). Fluid or blood
droplet is collected along edge of spreader (B), and pulled as for peripheral blood films (C). Particles at
the end of the smear are crush-smeared (D).

Advantages of FNA over Surgical Biopsy
- Outdoor procedure
- No anaesthesia required
- Results obtained within hours
- Procedure can be repeated
- Low cost procedure

Imprint cytology: This is a touch preparations from cut surfaces of fresh unfixed surgically excised
tissue are prepared on clean glass slides. These are fixed, stained and examined immediately. It is
considered complementary to frozen section.


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PAP smear
A normal Pap smear shows the following cells
1. Superficial cells, 2. Intermediate cells and 3. Parabasal cells


Inflammatory smear:

In inflammatory smear, the superficial and intermediate cells show features as under: Cytoplasmic
eosinophilia, cytoplasmic vacuolisation, large
number of leucocytes in the background,
perinuclear halos, clumping of chromatin, nuclear
enlargement and pyknosis, increase in the number
of parabasal cells.


The specific inflammation could be by the following
microbial infections: Trichomonas vaginalis,
candida albicans & human papilloma virus

Dysplastic and malignant smear:

It is one of the key screening procedures for
diagnosing precursor lesions as well as for
carcinoma of the cervix. The cytological diagnosis
of cervical lesions is based commonly on the
Bethesda system which divides them into precursor
(1. LSIL - low grade squamous intraepithelial lesions
& 2. HSIL - high grade squamous intraepithelial
lesions) and malignant lesions.


22








Macroscopic Specimens & Clinical Images

















23

Macroscopic Specimens Work sheet
Plastination Specimen
Description
Fatty liver Acute appendicitis











Chronic pyelonephritis

















Amoebic liver abscess















24

Macroscopic Specimens Work sheet
Plastination Specimen
Description
Cirrhosis liver

















Chronic venous congestion Liver









Sub endocardial infarction



Infarct spleen




25

Macroscopic Specimens Work sheet
Plastination Specimen
Description
Infarct brain Infarct lung











Lipoma


















Mature teratoma - Testes




26

Macroscopic Specimens Work sheet
Plastination Specimen
Description
Pleomorphic adenoma

















Squamous cell carcinoma









Carcinoma intestine


















Carcinoma stomach



27

Macroscopic Specimens Work sheet
Plastination Specimen
Description
Liposarcoma Metastatic tumour - Brain










Formalinated (Wet) Specimen
Description
Leiomyoma Sarcoma Soft tissue



28

Macroscopic Specimens Work sheet
Formalinated (Wet) Specimen
Description
Cyst Skin Polyp Intestine










Carcinoma Intestine



















Others



29

Clinical Images Work sheet
Clinical Images
Healthy wound healing













Unhealthy wound healing















30

Clinical Images Work sheet
Clinical Images
Gangrene leg











Diabetic ulcer
















31

Clinical Images Work sheet
Clinical Images
Anasarca








Periorbital edema








Pitting edema




32

Clinical Images Work sheet
Clinical Images
Petechiae









Purpura









Hematoma with ecchymosis




33

Clinical Images Work sheet
Clinical Images
Papilloma - Skin









Squamous cell carcinoma - Skin









Basal cell carcinoma - Face




34








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Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis


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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

40

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Microscopic Features
Histopathological Diagnosis

41

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Microscopic Features
Histopathological Diagnosis

42

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Microscopic Features
Histopathological Diagnosis

43

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Microscopic Features
Histopathological Diagnosis

44

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Microscopic Features
Histopathological Diagnosis

45

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Microscopic Features
Histopathological Diagnosis


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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis

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Microscopic Features
Histopathological Diagnosis