CHAPTER ONE: INTRODUCTION TO CYTOPATHOLOGY

Cytology
-

If pre-cancerous cell changes are found on smear women are

referred to Colopscopy

Study of microscopic appearance of cells

Cytologist

Conventional Smear

Trained to identify the type of cells that are normally present in

different areas of the body
Detect changes in the morphology of the cell
Detect presence of inappropriate cells

Cytology is used as:

1.
-

2.
-

Screening Tool
Used in healthy individual who are at risk of particular disease
o
Cervical Cytology

Detects CIN (Cervical Intraepithelial Neoplasia)

Pre-cancerous lesion that produces no symptoms

(invisible)

Primary screening tool for cervical cancer

Diagnostic Cytology
Used in symptomatic patients for diagnosis
Non-invasive/minimally invasive techniques
o
Non-invasive

Do not involve cutting/opening the body

Easy to collect (urine, sputum)

o
Minimally invasive

Procedures that cause least possible trauma

Bronchial wahings, FNAC

History
Anton van Leeuwenhoek (1632-1723)
o
Develop the microscope
o
First cytologist
o
Recorded the appearance of RBC, spermatozoa, bacteria,
yeast, diatoms
2.
Robert Hooke (1635-1703)
o
English inventor, improved the microscope
o
Coined the term cell
3.
Matthias Schleiden
o
Cell theory (with Schwann)
o
Proposed that all plants were made of cells
4.
Theodor Schwann
o
Cell theory
o
Proposed that all life begin as a single cell & all animal
tissues were composed of cells
5.
Donne
o
Published his work on cells found in respiratory tract
6.
Mueller
o
Published book containing microscopic appearances of
cancer cells
7.
Charles Spencer
o
Improved the design, magnification and clarity of CA cell
images
8.
Carl Zeiss
o
Produced excellent microscope
9.
August Kohler
o
Kohler illumination

Form of bright field microscopy that results in two major

advantages even illumination across the field of view &
elimination glare
10. George Papanicolaou
o
Researched oestrous cycle of animals and human menstrual
cycle
o
Used vaginal smears for the research
o
Developed the wet fixation method

Involves spreading the cells directly onto glass slide &

rapidly flooding w/ alcohol ether

Retains the fine cellular detail for morphological

evaluation
o
Papanicolaou Stain
o
Published atlas of of exfoliative cytology
11. Dudgeon & Wringley
o
Used wet fixation technique for the detection of neoplastic
cells in sputum samples

Liquid Based Cytology

-

Aim to reduce incidence of mortality of cervical CA

Screening interval: 3-5 years (Philippines: 5-7 yrs, age
recommended: 25-55 y/o)

Involves immediately dispersing cells in liquid medium

Automated or semi-automated
End result: monolayer
Reduces number of WBC and RBC on slide for better viewing of
epithelial cells
Does not require all cells present in original sample to be
deposited in slide

HPV Vaccination Programme

-

Girls under 13-18 years old

Protection against HPV 16 and 18

CHAPTER TWO: PREPARATION TECHNIQUES

Principles and Techniques of Specimen Collection
-

1.

Cervical Screening

Slides are prepared by spreading cells thinly but directly onto

glass prior to fixation

Main concern: presence of neoplastic cells

o
Tends to lose cohesiveness (less sticky)
o
Tend to exfoliate (shedding of loosely held cells from parent
cells)
Two main techniques
o
Exfoliative Cytology
o
Aspiration Cytology

Classification of Specimen Collection

1.
-

2.
-

Exfoliative Cytology
Collection of cells that have spontaneously shed from the surface
of the tissue
Malignant Cells: loss of cohesiveness from parent cells
Involves:
o
Scraping technique

Cervical sample (best known sample)

To collect a representative sample of cells from

transformation zone of the cervix

Area where transformation from columnar to

squamous epithelium occurs

Area where most cervical neoplasm originates

Rotate sampling device 360 degrees, clockwise motion

Sampling device:

Wooden/plastic spatulas (inflexible)

Cervical broom (flexible)

o
Brushing Technique

Sample: Bronchial brushing

Collect cells from epithelium of respiratory tract

Sample device:

Bronchial brush
o
Free nylon bristles
o
Gently rubbed over the surface of suspicious
lesions during bronchoscopy
o
Washing of body surfaces/cavities

Simpliest

Uses saline to rinse body surface or cavity in order to

dislodge cells that are loosely bound to their parent
tissue the rinse fluid is collected in water tight container

Sample: Peritoneal washing

Performed under general anesthesia

Initial diagnosis of cancer in abdominal region

Useful in staging cancer

o
Collection of body fluids (urine, sputum)
Aspiration Cytology
Force removal of cells from lesions or masses using needle (22-27
gauge) & syringe
o
FNAC (Fine Needle Aspiration Cytology)

Invasive technique

For lesion outside the reach of the exfoliative sampling

technique

Two imaging techniques:

Ultrasound

Computed Tomography (CT)

Palpable masses does not necessarily need imaging
techniques

Specimen Consideration
-

Contains cells that are representative of the site sampled

o
Cervical sample: must contain cells from transformation zone
o
Tumor sample: must contain tumor cells

Preparation Techniques
1.

Sampling Error (False Negative Cytology)

-

Due
o
o
Due
o

to collection error
Tumor present but not in the sample (Failure to collect)
Collect with insufficient quantity
to cytologist error
Tumor cells are present in the specimen but are not detected
or incorrectly interpreted

Specimen Containers
-

Vary in size & shape

May/ may not be prefilled
o
Anticoagulant: substance used to prevent clot formation
o
Preservative: chemicals used for cell preservation &
prevention of microorganism overgrowth
Should be fit for purpose
o
25 mL: cone shape, for immediate centrifugation
o
60 mL
Should be sufficiently robust
Should be properly sealed
Not sterile

Temperature for storage/transport

o
o

Room Temperature and above: promotes cellular

decomposition & overgrowth of MO
40C: slow down process of cellular degredation

2.

Transport Media
-

May contain cell nutrient/ fixative

o
Helps keep cells alive
Commercially available transport media:
o
CytoRich Red- promotes hemolysis
o
CytoLit- promotes hemolysis
o
Glacial acetic acid- aids in destroying RBC

3.

Fixation
-

Aim: prevent autolysis by inactivating enzymes & prevent

microbial attack
Preservation of cytoplasmic & nuclear morphology
Refers to the chemical bonding of soluble CHON to structural
CHON
Acts by disrupting chemical bonds which give CHON their 3D
dimension
Stabilization of nucleic acid & nuclear chromatin is the paramount
for successful staining

Ethyl Alcohol/ Ethanol

-

Most common cytological fixative

Precipitating/ denaturing/ coagulating fixative
o
Results in the product that is insoluble in water & thus
resistant to further chemical/structural changes
Preserve nuclear & cytoplasmic detail without excessive cell
shrinkage/ distortion
Fix within 10-15 mins
Not recommended for histology
o
Poor tissue penetration
Usually combined with water soluble waxy substance
o
Coats the cell
o
Acts as barrier to prevent cell damage
o
E.g. Carbowax (polyethylene glycol)

4.

5.

6.

Fixation by Air Drying

7.
-

Removes water from the cell

Rapid air drying: within 10 seconds

The effect of flattening cell to the glass slides results in cell

enlargement
Ideal for small cells in diagnostic cytology
o
Lymphocytes
o
Cells of small cell carcinoma
Only acceptable for Romanowsky staining
Ideal complement to wet fix smears stained with Papanicolaou

Direct Smear
o
Mucoid specimens (Mucus)

Constituents of mucus:

Mucin (glycoprotein)

Inorganic salts

Highly viscous

Produced by some types of epithelial surfaces

RT specimens: Sputum & bronchial washing

Pick and smear method

Specimen petri dish examined against black

background pick bloody/solid particles (Absence
of suspicious particles pick random)
immediate fixation (95% ethyl alcohol) stain
microscope

Poor diagnostic sensitivity

Mucolysis- breaks up mucus to maximize detection rate

Mechanical disruption methods

o
Permits homogenization of samples

Blender (Saccomanno technique)

Ultrasonic disintegration

Chemical disruption methods

o
Liquefaction of mucus to release admixed cell

Enzymes

Peptides

Dilute mineral acids

DTT (Dithiothreitol) most common

o
Fine Needle Aspiration Smears

Must be of optimal thickness

Must be immediately fixed

Less used than concentration techniques

Large Volume Centrifugation
o
Traditional method of fluid concentration
o
Fluid concentration facilitates the deposition of suspended
cells onto glass slide
o
25 mL
o
Select buffy layer (contains WBC & tumor cells) for smear
Small Volume Centrifugation (Cytocentrifugation)
o
<0.5 mL
o
Cytocentrifuge

Uses centrifugal force to spin cells in fluid suspension

directly on glass slide

Cytofunnel 0.5 mL

Megafunnel 6 mL (urine specimen)

Density Gradient Centrifugation
o
Sample aliquot dispense on top of density gradient fluid
centrifuge
o
Select buffy coat
o
Histopaque solution (1.077)
o
Density gradient fluid: conc. solution of sugars & other
substances
o
Different cell types are separated into layers according to
specific gravity
o
Epithelial cells
Gravity Sedimentation
o
Cells drift under their own weight to the base of the
container (specific gravity)
o
Decant fluid specimen settling chamber settle on glass
slide
o
Cells generally dont adhere very well to glass slides (unless
specimen is proteinaceous & sticky like serous fluid)
o
Albumin naturally occurring cell adhesive
o
Poly-L-Lysine commonly used potent cell adhesive

For urine and washings from FNA

Membrane Filtration
o
Uses circular filters with microscopic pores where sample can
be drawn
o
Dispersion mix negative pressure collect larger cells
on the filter glass slide
o
ThinPrep automated membrane filtration technology

Used for processing wide variety of specimen including

cervical sample
Cell Blocks
o
Paraffin embedded specimen derived from fluids, mucus or
sputum

Useful in immunocytochemical procedures

Multiple similar slides can be produced in a single block
Thrombin Cell Block Method

Fluid sample centrifuge cell pellet blood plasma

+ thrombin fibrin clot added to pot of formalin
processed
o
Heated Molted Agar

Alternative to TCB
Combination of Techniques
a.
Density Grandient Centrifugation followed by Gravity
Sedimentation

Basis of SurePath (semi-automated method)

For batch processing cervical samples

Cell enrichment process (proportion of neoplastic cells

is increased by reducing unwanted cells or debris)

Cervical epithelial cells occupies bottom layer of tube

( SG)

Example: Liquid Based Cytology

Involves immediate transfer of cells into a liquid

preservative as soon as specimen is collected
b.
Large Volume Centrifugation followed by Density Gradient
Centrifugation

For bloody serous fluid

o
o
o

8.

3.

4.

Demonstration Techniques
1.
a.

b.

2.

Stains for Wet-Fixed Preparations

Papanicolaou Technique
o
Hematoxylin

Natural dye extracted from Haematoxylin

campechianum haematin (when oxidized)

Stains the nuclei of cells (red color) when used with

proper mordant
Mordants: salts of aluminium, copper, iron, potassium

Used as regressive stain in Papanicolaou (added in

excess then wash out with acid solution)

Ehrlich Hematoxylin (histology H&E)

Harris Hematoxylin (cytology)
o
Acid Solution (Acid Alcohol)

Differentiator
o
Alkaline Solution

Blueing (converts red coloration of nuclei to blue)

o
Orange G (OG)-6

First counterstain

Stains keratin (normal component of squamous

epithelium)
o
Eosin Azure (EA)-50, 36, 65

Made up of Eosin and Light green

EA formulations are suffixed by a number denoting the

proportions of constituent dyes

EA 50- most common

Stains cytoplasm of cells (pink/green)

o
Dehydration- absolute alcohol
o
Clearing- xylene
H&E (Hematoxylin & Eosin) Technique
o
Most popular method for staining tissue sections in histology
o
Does not produce the range of cytoplasmic coloration

Dehydration

Removal of water from stain preparation

Absolute alcohol

Clearing

Rendering stained preparations optically cleared

Ensures that final preparation is transparent

Xylene (same refractive index as sample)

Mounting

Application of thin sheet of glass to stained

preparation
Stains for Air-Dried Preparations
o
Rapid drying introduces a controlled artefact as cells tend to
flatten against the slide, appearing larger than wet fixed
stain
o
Useful when theres high probability that sample contains
population of small cells (e.g. lymphocytes)
o
For serous fluids, FNA of lymph nodes, etc.
o
Do not stain well with papanicolaou technique
o
Stain with Romanowsky stains

Giemsa

May-Grunwald-Giemsa

Jenner

Wright

Leishman
o
Romanowsky effect

Purple nuclei, blue cytoplasm, pink RBC

pH 6.8-7.2

o
Rapi-Diff, Diff Quick Romanowsky commercial staining kits
Special Stains
o
Periodic Acid Schiff (PAS) method for carbohydrate
(glycogen)
o
Grocotts methenamine silver method for Pneumocystis
jiroveci
o
Replaced by immunocytochemistry
Immunocytochemistry
o
Cytological; detection of special cell constituents based on
antigenic structure
o
Uses antibodies that binds with samples antigen
o
For serous fluid and FNA diagnosis (determines primary
lesion)
a.
Direct Methods

Rely on direct visualization of ag-ab reaction

Uses primary antibodies conjugated with visible marker

(probe, tag or label)

Simple and quick

Poor sensitivity
b.
Indirect Methods

For ag concentration

Rely on signal amplification

Secondary antibody is applied after primary ag-ab

reaction

Secondary antibody is tagged

Several secondary antibodies can bind to single primary

antibody signal amplification

Tag is an enzyme (invisible, Only becomes visible upon

addition of Chromogen)

Biotin: used in labelling secondary antibody, low

molecular weight

ABC technique

Secondary ab: biotinylated

Avidin
o
Derived by streptavidin
o
High molecular weight glycoprotein which
reacts to biotin forming Avidin-biotin complex
ENZYME
Horseradis
h
peroxidase
Alkaline
Phosphatas
e

5.

CHROMOGEN
Hydrogen Peroxide
DAB (3,3diaminobenzidene)
New Fuchsin

END RESULT
Brown
reaction
Red color

Molecular Techniques
o
Molecular Pathology

Application of methods of molecular biology to patient

specimen for prediction of disease process

Prognosis

Diagnosis

Treatment response prediction

Treatment monitoring

Most commonly used techniques:

PCR (DNA amplification)

Southern blot (Detects DNA)

Northern blot (Detects RNA)

Cytogenitics

Hybridization techniques

Safety and Health

-

Hazard: situation with potential to cause harm

Risk: likelihood that harm from particular hazard is realized
Extent of Risk: number of people who might be affected by risk
(exposed)
Risk assessment: formal procedure for determining level of risk
from identified hazard
Risk control: practices, procedures, equipment and training that
are put in place to minimize or eliminate risk

Categories of Hazard
1.
2.
3.

Microbiological hazard
Chemical hazard
Physical hazard

CHAPTER THREE: THE CERVICAL SCREENING PROCESS

Principle
Condition must be relatively common and disabling
Treatment should be available
Natural history of target condition must have been studied
There must be a recognizable, treatable precursor or presymptomatic phase
Cost effective
The screening test must be reliable, valid and repeatable
High sensitivity and specificity
Target Population
Individuals at risk of having or developing the target condition
Prevalence
Proportion of population that has target condition
Coverage
Proportion of the target population that has been screened
Most important factor in influencing the effectiveness of screening
program
Low coverage = failure, 70-80% coverage = success
Dependent upon:
o
Target population that has access to screening program
o
Target population that actually participate (uptake)
Informed Consent
Sensitivity
Measure of ability of a screening test to identify the positives
(individuals within the target population that have the target
condition)

sensitivity=

true positives
100
true positives+ false negatives

3 years: 25 to 49 years old

5 years: 50-64 years old
POGS
3 years after the 1st
Yearly for sexually active
Colopscopy
Colopscone
Facilitated by the application of weak acetic acid (white=
cancerous area) and iodine solution to the surface of the tissue to
be examined to those with abnormal cells during Pap smear
CHAPTER FOUR: NORMAL CERVICAL CYTOLOGY
Normal Anatomy and Histology of the Female Genital Tract
Vulva
Protects openings of the vagina and urethra
Consists of two pairs of skin folds (lined by keratinizing stratified
squamous epithelium)
o
Labia majora
o
Labia minora
Vagina
Muscular canal leading from vulva to cervix
Lower part of the birth canal 10 cm long
Lined by stratified squamous non-keratinizing epithelium
Cervix
Neck of the uterus and forms the 1st part of the so-called birth
canal (4 cm long)
o
Endocervix

Inner canal lined by single layer of columnar epithelial

cells derived from reserve cells
Reserve cells

Undifferentiated cells characterized by their ability

to divide and differentiate into number of different
cell types

Usually differentiate to endocervical cells

Squamous metaplasia- differentiate to squamous

epithelial cells

True Positives
Individuals within the target population that have the target
condition and test positive
False Negatives
Individuals within the target population that have the target
condition and test negatives
Specificity
Measure of ability of a screening test to identify the
negatives(individuals within the target population that do NOT
have the target condition)

specificity=

true negatives
100
true negatives+ false positives

True Negatives
Individuals within the target population that do not have the
target condition and test negative
False Positives
Individuals within the target population that do not have the
target condition and test positive
Positive Predictive Value
Measure of the accuracy of a positive result
clinicians gold standard

PPV =

true positives
100
true positives+false positives

Negative Predictive Value

Measure of the accuracy of a negative result

NPV =

truenegatives
100
true negatives +false negatives

Cervical Screening
Paps Smear
Commonly used screening tool
Developed by George Papanicolaou
Precursor lesion: CIN/SIL (Cervical Intraepithelial
Neoplasia/Squamous Intraepithelial Lesion)
CGIN (Cervical Glandular Intraepithelial Neoplasia)
Exclusion Criteria
Men
Total hysterectomy
Female virgins
Women >65 years old with 3 negative smears for previous 10
years

Screening Interval
NHS

Types of columnar cells found in endocervical canal

1. Glandular cells
o
Secrete mucus

Forms physical barrier protecting uterus

Becomes thin & watery to optimize

conditions for fertilization
2. Ciliated cells
o
Have fine hair appendages (cilia)
o
Help waft mucus along the canal towards the
opening with the vagina
Ectocervix

Outer portion lined by SSNKE

Divided into
o
Basal cellsLeast mature
o
Divides and move gradually outwards as they
undergo morphological changes

Parabasal cells

Intermediate cells

Superficial cells
o
Most mature
o
Largest
o
Most rigid
o
For protection of underlying tissue
Squamocolumnar Junction

Transition between columnar epithelium lining the

endocervical canal and squamous epithelium of the
ectocervix

Changes position constantly according to age and

hormonal status

Before puberty & after menopause: deep within

endocervical canal

Reproductive years: shifts according to shape &

size of cervix
Basement Membrane

Proteinaceous sheet separating columnar epithelium of

endocervix and squamous epithelium of ectocervix

Differentiation or maturation
Process of cell specialization following cell division in both
ectocervix and endocervix
Uterus
Pear shaped hallow muscular organ (7.5 cm long, 5 cm wide in
non-pregnant state)
House and protects fetus during gestation

Located between bladder and rectum, connected to the

abdominal wall by ligaments
o
Endometrium

Epithelial lining of uterus

Single layer of cuboidal cells

Altered during menstrual cycle

o
Connective Tissue

Located beneath the endometrium

Supplies blood
o
Myometrium

Coat of smooth muscle

Fallopian Tubes
Thin walled cylinders (12 cm in length)
Transport egg released at ovulation from ovary to uterus
Opening of tube is expanded and split into fringes or fimbrae
Cilia on fimbrae creates currents in abdominal cavity so after
ovulation, egg is wafted towards the opening of fallopian tube
Ovaries
Located within the abdomen at the end of fallopian tube
Attached to the pelvic wall and uterus by ligaments
3-4 cm long
At birth: contains about 2million primordial follicles
o
Primary oocyte
o
Granulosa cells
Follicles are lost by degeneration
Each cycle, several follicles begin to mature, becoming primary
follicles, but usually only one follicle completes maturation and
releases ovum

5-13

14

Same

Proliferative
Phase

Endocervix
secretes mucus

Endometrium
regrows

Superficial cells
proliferate

Ovulatory Phase

Mucus becomes
thin and watery

LH surge

Dominance of
Superficial cells

Increased Estrogen
secretion

15-28

Release of egg
from Graafian
follicle
Luteal Phase
Corpus luteum
secrets Estrogen
and Progesterone

Secretory Phase
Endometrium
secretes uterine
milk (thick fluid rich
in sugar, amino
acids, GP that
nourishes early
embryo in the
event of
fertilization)

Hormonal Control of Female Genital System

-

Negative and Positive feedback control (homeostatic control)

Gonadotropin Releasing Hormone (GnRH)

Secreted by the Hypothalamus
Stimulates the release of Gonadotropins from Anterior Pituitary
gland
Gonadotropins
Follicle Stimulating Hormone (FSH)
Stimulates follicle maturation in the ovary
Luteinizing Hormone (LH)
Causes a mature follicle (Graffian follicle) to ovulate and
transform into a corpus luteum
Estrogen
Released by the maturing follicle in the ovary
Effects:
o
Growth of the endometrium (1st half), external genital,
internal sexual organs, breasts
o
Establishes female pattern of body fat deposition, bone
growth and body hair
o
Growth and maturation of cervical and vaginal epithelium
Progesterone
Secreted by the corpus luteum (2nd half of menstrual cycle)
Functions:
o
Thickening of the endometrium
o
Relaxes the smooth muscle of the myometrium during
gestation
Menstrual Cycle
Begins at around 12 years of age until around 50 years of age
28-day cycle
Days
Ovary
Uterus
Cervix
1-4
Follicular Phase
Menstrual Phase
Blood and
Menstrual Debris
Follicles mature
Endometrium
appear in cervical
under the influence
sheds after
sample
of FSH
estrogen &
progesterone levels
Follicles secrete
fall
ESTROGEN

Mucus secretion
declines, becomes
thick, impenetrable
to sperm
Maturation of
squamous
epithelium to
intermediate cells
Intermediate cells
dominate the
sample with
lactobacilli &
cytolysis

Cytology During:
Menstrual cycle
Menstrual Phase (Day 1-4)
Menstrual debris
Proliferative phase (5-14)
Superficial cells
Few intermediate cells
Less menstrual debris
Secretory phase (15-28)
High intermediate cells
Numerous lactobacilli and cytolysis
Pregnancy
Navicular cells boat cells
Post-partum
Lactating Parabasal cells
Non-lactating same with reproductive women
Menopause (Cessation of Menstrual cycle)
Early menopause
Intermediate cells
Mild menopause
Parabasal cells
Late menopause
Deep parabasal cells singly or in sheets (post-menopausal
atrophic pattern)
Blue blobs (degenerative Parabasal cells
Non Epithelial Cells in Cervical Samples
RBC
7-8 um, bincocave disc
Lysis: pink, granular appearance
Responsible for oxygen supply
Normal physiological process: menstruation
Pathological conditions: inflammation/ cancer
PMNs
12 um, granular cytoplasm, multi-lobed nuclei
Plenty during acute stages of infection/inflammation, Extremely
numerous during malignant disease
Inflammatory exudate: term describing large infiltrate of
polymorphs in cervical sample
Macrophages (Histiocytes)
Variable in size, foamy cytoplasm, eccentric bean-shaped nucleus
Presence of ingested particulate material
Component of inflammatory exudate
Giant Macrophage: (>100um) seen in post-menopausal women

Lymphocytes
Small round cells with rounded nuclei & narrow rim cytoplasm
Minor component of inflammatory exudate
Increase when inflammatory conditions become long-standing
Other Inflammatory Cells
Eosinophils
Pink granular cytoplasm, bilobed nucleus
Plasma Cells
Activated lymphocytes
Chromatin pattern: clockface/ cartwheel chromatin
Stromal Cells
Elongated & spindle shaped w/ round to oval nuclei & wispy
cytoplasm
Originate from connective tissue underlying basement membrane
Rarely found in cervical sample except during menstruation
Mimic neoplastic epithelial cells
Microorganisms in Cervical Samples
Bacteria
Lactobacillus
3-5 um, rod shaped
Utilizes glycogen to lactic acid (maintains acidic pH of vagina)
Glycogen: stored within intermediate squamous epithelial cells
Actinomyces-like organisms
Colonize intrauterine contraceptive device without causing
infection
Cause ascending infection & pelvic inflammatory disease
Characterized by tangled mass of hematoxyphilic filaments in
Papanicolaous stain
Bacterial vaginosis
Inflammatory condition of vagina
Cause: Anaerobic coccobacilli (Gardnerella vaginalis)
o
Part of normal bacterial population of vagina
o
Causes fishy smelling vaginal discharge
o
Clue cells: Hazy blue appearance of squamous epithelial
cells
Fungi
Candida
Commonly found in lower genital tract
Usually exists as spores but can elongate to form psuedohyphae
(eosinophilic tangled filaments with septa)
Protozoa
Single-celled eukaryotic organisms
Trichomonas vaginalis
o
Obligate pathogen in lower female genital tract
o
Sexually transmitted
o
With flagella
o
With smudgy grey nucleus & tiny pink granules within
cytoplasm
Virus
HSV (Herpes Simplex Virus)
Multinucleated giant cells with nuclei appear moulded together
(empty ballooned appearance)
Ground glass appearance of chromatin
With intranuclear viral inclusions (large round bodies in center of
nucleus)
HSV Type 1
Cause ulcerating lesions of the mouth, eyes, and skin
HSV Type 2
Sexually transmitted
Infects genital and anal regions
Causes meningitis and skin lesions
HPV (Human Papilloma Virus)
HPV Types 16, 18, 31, 33, and 45
High-risk types
Associated with high-grade CIN, invasive squamous cell
carcinoma of the cervix, and cervical adenocarcinoma
HPV Types 6, 11, 42, 43, and 44
Low-risk category
Associated with benign warts and low-grade cervical
intraepithelial neoplasia.
Cytological Diagnosis
Koilocytes
o
Squamous cells (usually superficial cells) with a large
perinuclear clear space
o
Thickened uneven rim of dense cytoplasm (wire loop
appearance)

Cytology of Inflammation

Acute Cervicitis
Initial response to tissue injury
Presence of exudate is often
Main cells involved: neutrophils
Characteristics:
o
Marked increase in inflammatory cells (neutrophils)
o
Epithelial cells are covered by exudate of neutrophils, some
accompanied with leucophagocytosis
o
Lymphocytes & plasma cells if inflammation persists
o
Specific morphological changes in epithelial cells
Results in complete resolution & regeneration if cause of
inflammation is removed
Chronic Cervicitis
Occurs if cause of inflammation persists
Exudates with inflammatory cells (macrophage)
Characteristics
o
Heavy infiltrate of lymphocytes and plasma cells
o
Fragile columnar epithelium may react undergoing squamous
metaplasia
o
Hyperkeratosis/ parakeratosis
Hyperkeratosis
Squamous epithelium has
thick layer
Absence of nuclei
Cytoplasm is densely
orange
o

Parakeratosis
Pyknotic nucleus is visible
in deep orange

Individual cell keratinization

Special Types of Cervicitis

Follicular Cervicitis
Form of chronic cervicitis in which subepithelial lymphoid follicles
develop
Associated with Chlamydia trachomatis infection
Tingible body macrophages
Atrophic Cervicitis
Inflammation of atrophic cervical epithelium
Most commonly occurred in post-menopausal women
Squamous Metaplasia
Due to changing position of SCJ under the influence of ovarian
hormones
o
Before puberty: SCJ within endocervical canal
o
Onset of menstruation/ first pregnancy: sex steroids
cervix volume = eversion of columnar epithelium

Ectopy/Ectropion- everted portion of endocervical canal

where squamous metaplasia occurs
o
Menopause- recedes to endocervical canal
Change from columnar to non-keratinizing stratified squamous
epithelium in response to the acid pH of the vagina
Acid environment initiates reserve cell hyperplasia
o
proliferation of the single layer of subcolumnar reserve cells
to produce two or more layers of cells in the ectopic
columnar epithelium
o
Further division leads to immature squamous metaplasia
o
mature metaplastic cells (CT may contain endocervical
glands) cannot be distinguished from native squamous
epithelial cells in a cervical sample
Iatrogenic Changes of the Cervix
Inadvertent tissue damage or disease that can be caused by
medical treatment or any other form of medical intervention
Physical Intervention
laser therapy, cryotherapy, electrodiathermy, cold coagulation
Tubo-endometrioid metaplasia (TEM)
Cervix becomes lined by epithelium resembling fallopian tube or
endometrium
o
Hyperchromatic crowded groups of small cells resembling
endometrial cells
o
Lack central core of stromal cells typical of physiological
shedding
o
Border of ciliated cells
o
Uniform chromatin pattern/distribution.
IUCD (Itrauterine Contraceptive Device)
May cause inflammatory response to the surface of the
endometrium, resulting to exfoliation of cell mistaken as
Glandular or Squamous
IUCD Cells
o
High NCR, prominent nucleoli, bi/multinucleated
Bubble Gum Cells
o
Cells with vacuolated cytoplasm and displaced nuclei

Colonization with Acantinomyces spp.

o
Coexistent Entamoeba gingivalis

Changes Associated with Exogenous Hormones

Oral Contraceptives
o
Cytolysis and lactobacilli (progesterone-only pill)
o
Atrophic changes
o
Increased risk of candida infection
Microglandular hyperplasia & Arias-Stella reaction- noted in users
of oral contraceptives
Hormonal Replacement Therapy
Used to treat menopausal symptoms
Protect from long-term risks of postmenopausal osteoporosis &
heart disease
Based on estrogen and progesterone
May induce maturation of a previously atrophic epithelium

Used in the treatment and control of malignant disease

o
Marked nuclear and cellular enlargement
o
Cytoplasmic vacuolatin
o
Nuclear wrinkling, vacuolation, and Hyperchromasia
o
Multinucleation
o
Leucophagocytosis
o
Bizarre cell shape
o
Risk of infections by HPV, HSV

Irradiation and Chemotherapy

Cytology of Normal Epithelial Cells

PARABASAL
INTERMEDIATE
CELLS
CELLS
Picture

SUPERFICAL
CELLS

ENDOCERVICAL
CELLS

METAPLASTIC
CELLS

ENDOMETRIAL
CELLS

Shape

Polygonal

Honeycomb sheets
Palisaded strips
Loosely associated
single columnar cells

Depends on degree
of maturation

Round/oval cell
clusters with dense
core of stromal
cells & periphery of
larger epithelial
cells

Size
Cytoplas
m
Nucleus

Round/oval

12-30um
diameter
Dense green

Round/oval
8um diameter
Occupies half of
the cell

Polygonal
Sometimes w/ folded
edge

30-40um diameter

35-45um diameter

Cyanophilic
Sometimes
eosinophilic

Eosinophilic

Round/ oval
8um diameter

Cilia visible at one

end
Small, pyknotic
5um diameter

Low N:C Ratio

Low N:C Ratio

Chromati
Evenly distributed
Fine vesicular
n
Vesicular
CHAPTER FIVE: ABNORMAL CERVICAL CYTOLOGY
Two main types of cervical cancer
1.
Squamous cell carcinoma
75% of all cervical cancer
Associated with HPV type 16
2.
Adenocarcinoma
Usually affects women under age of 35
Associated with HPV type 18
Cofactors
Smoking
Immunosuppression
Hormones
Genetics
Mechanism of HPV
Virus contact with basal epithelial cells
Capsid is shed, viral DNA enters one or more basal cells
Viral DNA remains in episomal form, replicates in tandem with
host cells
LATENT PHASE
o
No new viral particles are produced
o
No clinical/cytological manifestation
PRODUCTIVE PHASE
o
DNA replication
o
Intermediate/superficaial layers
o
Cytopathic effect koilocytosis

Cyanophilic

wreath/ top hat

formation
Individual cell: 810um
Little

w/ projections
Spider cells
Vesicular
Variable in size
Small nucleoli

Crumpled nuclei
Hyperchromatic
8um diameter

30-45um diameter
Cyanophilic
Finely vacuolated
Sometimes filled
with mucus
Oval
Sometimes with
nipple like
protrusions
(occasionally with
one or more small
nucleoli)
Fine
o
o

Malignant transformation
Malignant tumor formation

CIN/ SIL
Precancerous lesion of squamous cell carcinoma
Characterized by replacement of normal cervical squamous
epithelium with neoplastic cells
Introduced by Richart in 1967
Slow growing lesion usually taking around 10 years
CIN lesions can progress, regress, or persist
Progression from CIN3 to invasive cancer can take a further 812
years
Approximately one-third of cases of CIN1 will progress to CIN3 if
left untreated
About two-thirds of cases of CIN3 will progress to invasive cancer
if left untreated
Diagnosis of CIN/SIL is based on nuclear atypia characterized by:
1.
Nuclear enlargement
2.
Hyperchromasia
3.
Presence of chromatin granules
4.
Variation of nuclear size and shape
3 Grades of CIN/SIL are diagnosed by:

1.
2.

3.

Proportion of epithelial thickness occupied by undifferentiated

cells
Level within the epithelium at which mitotic figures are found
o
Mitotic figure is a snapshot of cell undergoing mitosis
o
The more frequent the mitosis, the greater chance of
neoplasia
Presence of abnormal mitosis

Undifferentiat
ed neoplastic
cells
Cytoplasmic
differentiation
Mitotic Figures
(appearance)
Low
-

CIN 1
Lower 1/3

CIN 2
Middle 3nd

CIN 3
Full thickness

Mid & Upper

3rd
Infrequent
(Normal)

Upper 3rd

No sign

Lower 2/3
(Abnormal)

Frequent
(Abnormal)

Grade Squamous Intraepithelial Lesion

Associated with productive HPV infection
Most regress spontaneously
Does not progress to invasive carcinoma
CIN 1

High Grade SIL

Characterized by progressive deregulation of cell cycle by HPV
Increase cellular proliferation
Lower rate of viral replication
CIN 2 & 3
Atypical Squamous Cells of Undetermined Significance (ASCUS)
Suggestive of low grade SIL
Some are associated with underlying CIN
CIN 2 & 3
Atypical Squamous Cells Cannot Excede HSIL (ASC-H)
Suggestive of high grade SIL
Associated with higher PPV for detecting CIN 2 & 3
Natural History of SIL with Approximately 2-year Follow Up
Lesion
Regress
Persist
Progress
LSIL
60%
30%
10% to HSIL
HSIL
30%
60%
10% to
carcinoma
-

More than 80% of LSIL and 100% of HSIL associated with high
oncogenic risk HPV
Progression to invasive carcinoma may take place on few months
to more than a decade
Persistent infection with high risk HPV is necessary cause for CIN

Dyskaryosis
Nuclear
enlargeme
nt
NCR
Chromatin
pattern
Nucleus
Cytoplasm

Mild
<1/2 diameter
of the cell

Moderate
1/2 2/3 of the
cell diameter

Severe
> 2/3 of the
cell diameter

<0.5
Uneven

0.5 0.67
Uneven

> 0.67
Uneven

Hyperchromasi
a
Multinucleation
Plentiful
With angular
cell border

Nuclear Hyperchromasia
Multinucleation
Irregular nuclear membrane
Reduced
Abnormal
Borders:
cellular
angular/
maturation
rounded
(keratinization)
Dense cell
clusters
(hyperchromati
c crowded cell
groups

Moderately Differentiated Squamous Cell Carcinoma

Show some evidence of squamous origin but less keratinization
Cells bearing a moderate resemblance to their normal
counterparts
Poorly Differentiated Squamous Cell Carcinoma
No definite squamous features and require special stains to
confirm their origin
Poor resemblance to their normal counterparts
Cytological features that suggest a strong possibility of invasive
Carcinoma:
1.
Numerous dyskaryotic cells
2.
Cellular pleomorphism
o
bizarre-shaped
o
fibre-shaped
o
tadpoleshaped,
3.
Very coarse aggregates of nuclear chromatin
4.
Large, irregular, sometimes multiple nucleoli
5.
Cytoplasmic keratinization
6.
Tissue fragments (microbiopsies) composed of dyskaryotic cells
7.
Tumour diathesis
Why Invasive carcinoma cannot be diagnosed reliably from a cervical
sample
1.
Cells from CIN 3 are morphologically indistinguishable from those
shed from an invasive cancer
2.
Surface of an invasive tumour is often covered with debris with no
or only few malignant cells present
Endocervical Carcinoma
Adenocarcinoma in situ or Cervical Glandular Intraepithelial
Neoplasia (CGIN) is the risk lesion
Risks:
o
number of sex partners
o
Earlier age of 1st sexual contact
o
Obesity
o
Infection with high risk HPV
Second most common tumor
Main symptoms:
o
Abnormal vaginal bleeding in 75% cases
o
Asymptomatic: 10-20% cases
Prognosis: 5 years survival rate is 48-56%
Histological features of CGIN
Replacement of normal endocervical epithelium by neoplastic
cells.
Abrupt transition
Focal and may be found adjacent to an area of CIN.
Abnormal glands are lined by cells with enlarged hyperchromatic
nuclei with coarse chromatin.
Pseudostratification
Loss of polarity.
Mitotic figures are frequent
Basement membrane may be intact or ill-defined
Cytological features of CGIN
Architectural features
Numerous endocervical cells, singly and in small groups.
Nuclear crowding and overlapping
Little or no pleomorphism.
Feathering or fraying of the edges of cell sheets
Rosettes
Pseudostratification
Nuclear features
Nuclei are often oval in shape with smooth nuclear membranes
Nuclear enlargement
Enlarged nucleus often bulges the otherwise parallel cell edges.
Nuclei are hyperchromatic and have a coarsely granular
chromatin pattern
Some cases of CGIN have nuclei with finely granular and only
slightly hyperchromatic chromatin.
Nucleoli are not a conspicuous feature of CGIN but when present
they are usually small and multiple.
Mitoses can be seen within sheets of cells

Invasive Squamous Cell Carcinoma

Note that squamous cell carcinoma is the most common
malignant tumor of the cervix
HSIL is the immediate precursor

Invasive endocervical adenocarcinoma

occurs at a mean age of 56 years
Main symptom: abnormal vaginal bleeding (75%)

Microinvasive Carcinoma
Pre-clinical stage of invasive carcinoma
<5mm deep lesion up to 7mm wide
Well-differentiated Squamous Cell Carcinoma
Large islands of tumour cells with intercellular bridges, epithelial
pearls, and keratinization
Close resemblance to their normal counterparts

Cytological features of invasive endocervical adenocarcinoma

Numerous small groups of crowded endocervical cells
Many discrete malignant cells due to loss of cell cohesion
Nuclear enlargement and pleomorphism will be quite obvious in
the poorly differentiated lesions.
Enlarged and prominent nucleoli.
Cytoplasm may be vacuolated as a result of mucus secretion
Tumor diathesis.

Endometrial adenocarcinoma
Fifth leading cancer in women
cervical cytology is an inappropriate test for endometrial cancer
o
Endometrial cells are a normal finding in cervical samples
taken from pre-menopausal women (12th day to 25th-28th
day)
o
May also be shed at any time during the cycle due to benign
endometrial polyps, fibroids, intrauterine contraceptive
device
o
Cells shed from a well-differentiated endometrial cancer may
appear indistinguishable from normal endometrial cell
Cytological features of endometrial carcinoma
Influenced by
Degree of differentiation of the tumour
Histological subtype
Extent of the neoplasm
State of preservation of the malignant cells
Features:
Scant 3D balls of malignant cells with scalloped edges.
Nuclei larger than normal endometrial cells, and frequently
hypochromatic
Prominent and multiple eosinophilic nucleoli.
Cytoplasmic vacuoles of mucin. Large vacuoles may push the
nucleus to one edge of the cell (signet-ring formation.)
Leucophagocytosis)
Tumour diathesis
Degenerate tumour cells may appear squamoid, with pyknotic
nuclei and eosinophilic/orangeophilic cytoplasm.
Adenocarcinomas from the fallopian tubes and ovaries
Very rare
Not morphologically distinguishable from endometrial
adenocarcinoma
Clean background
Psammoma bodies
o
Rounded calcified protein deposits that have a weak
association with ovarian cancer
Paps Numerical System
Class I
Absence of atypical or abnormal cells
Class II
Atypical but no evidence of malignancy
Class
Cells suggestive of but not conclusive to malignancy
III
Class
Cells strongly suggestive to malignancy
IV
Class V
Cells conclusive to malignancy
CHAPTER SEVEN: DIAGNOSTIC CYTOPATHOLOGY
Underlying principles of diagnostic cytology

1. Cancer cells lose their cohesive properties early in the disease

process
2. Specimen collection is normally simple, painless, and rapid.
3. Cytological diagnosis can remove the need for exploratory surgery.
4. Clinical and cost effective
6. Can also be used for diagnosing infections and other non-cancerous
conditions
Fundamental properties of malignant tumours
1. Cell immortalization
2. Evasion of apoptosis
3. Resistance to growth inhibition
4. Independence from mitotic control
5. Angiogenesis

NON GYNECOLOGICAL SPECIMEN

Respiratory Tract Specimen
Obtained to exclude the possibility of malignancy or infectious
agents especially from patients with immunodeficiency syndrome
o
Sputum
o
Bronchoalveolar lavage/ bronchial washing
o
Bronchial brushing
Peritoneal, Pleural, and Pericardial Fluid
Malignant cells in serous effusions usually indicate metastatic
involvement (higher stage of cancer)
Breast Secretions
Nipple Discharge
Extremely low diagnostic yield for diagnosis of breast CA
Due to benign breast lesion such as duct ectasia and papilloma
Due to endocrine problem (prolactin)
Spontaneous nipple discharge is usually a result of hormonal
imbalance in young patient
Bloody secretions: benign intraductal papilloma should be
considered
Urine
Major goal is the diagnosis of malignancy usually urothelial origin
Types of specimen:
o
Voided urine
o
Catheterized urine (previous contamination with vulvar cells)
o
50-100 mL
Bloody Cavity Effusions
Important diagnostic value in patients with a known history of CA
Positive effusion for malignancy is the first presentation of CA of
unknown origin
Types of specimen
o
Pleural fluid
o
Ascetic/abdominal fluid
o
Peritoneal washings
o
Pericardial fluid

CSF