Exfoliative

Cytopathology

Ma. Minda Luz M. Manuguid, M.D.

 Exfoliative Cytopathology
• def : study of cells exfoliated (naturally or
artificially) or aspirated (FNAB) from body surfaces
or cavities
• staining: Papanicoulaou (Pap) method
• primary objective: detect neoplastic cells
• other purposes:
 identify infectious organisms &/or their products;
 detect inflammatory conditions;
 assess hormonal status;
 diagnose other disorders
 Exfoliative Cytopathology
• Advantages:
 Can assess large areas/tissues
 Non-invasive/ minimally invasive
 Can be done repeatedly
 Low cost
 Rapid processing & evaluation
• Disadvantages:
 Cannot localize lesion
 Needs to be confirmed by histopathology
 Inter-observer variation greater than with histopathology
 Cytopathology specimens
• Cervicovaginal smear – most common – “Pap” smear
• Buccal scrape
• Pleural fluid
• Pericardial fluid
• Peritoneal/Ascitic fluid
• GIT secretions: gastric juice, intestinal juice,
• RT secretions: bronchial lavage; pharyngeal;
• GUT secretions: bladder lavage; urethral secretions;
• Fine Needle Aspiration Biopsy
 Papanicoulaou staining
• 70% Ethanol 2 min
• Water 2 min
• Harris Hematoxylin 4 min
• HCl 0.05% 1min 30sec / Water 5 min
• 95% Ethanol 2 min
• Orange G 3 min
• 95% ethanol 2 min
• EA 50 4 min
• 95% ethanol 2 min
• Absolute Ethanol at least 2 min, 2 changes
Barr body
 Cytology specimens

Urine cytology
FNA, lymph node

Sputum cytology
bronchial lavage
Pleural effusion cytology
 Benign conditions

Thyroid aspirate
Liver aspirate

sialadenitis
fatty Liver aspirate
 Malignancies
Ascitic fluid Pleural fluid

Papillary adenoCA,
thyroid

Mucinous CA
from Ovary Malignant
mesothelioma

Mucinous adenoCA, breast

Papillary CA, thyroid

D
 Malignancies
Transitional
cell carcinoma

Small cell Lung CA

Gastric cancer

Lymphoma,
lymph node
aspirate
 CervicoVaginal Cytopathology
• specimen: smears from the vaginal canal (lateral
vaginal wall if cytohormonal index is required),
vaginal pool (posterior cul-de-sac), ectocervix,
transformation zone / endocervix
• purpose: detect Cancer or the cellular changes that
can lead to Cancer (“early detection”)
• recommended timing:
 Baseline smears at age 18 or age at first sexual intercourse
 Yearly during reproductive age plus when pregnant or with
gynecologic symptoms
 Slide prep (Conventional)

• 1 - Smearing of the exocervical sample with a wooden

spatula (Ayre's spatula). Some may have a longer bifid
extremity for a better endocervical sampling.
• 2 - Smearing of the endocervical sample taken with the
thinner extremity of the wooden spatula.
• 3 - Spray fixation: immediate, during a few seconds,
with a spray/slide distance around 20 cm.
 Cytobrushes

• Different types of brushes allowing to collect cells

from the ectocervix and endocervix. These brushes
can be used for conventional smears instead of the
Ayre’s spatula. They are mandatory for liquid based
preparation.
Liquid based (thin) prep
• Head of spatula, where cells are lodged, is
broken off into small glass vial containing
preservative fluid, or rinsed directly into
preservative fluid
• Sample is sent to lab, then spun and
treated to remove mucus, pus or other
obscuring material
• Random sample of remaining cells is
taken and deposited onto a slide
• Reduces number of inadequate smears
and need for repeat smears
• Thin-Prep appears to be superior to
convention Pap test in detecting SIL
 Reporting : Bethesda 2001
• Specimen
 Source:
• vaginal pool / lateral vaginal wall;
• ectocervical;
• transformation zone / endocervical
 Type:
• conventional;
• liquid-based (thin) prep;
• other : e.g. air-dried, diff quick
 Reporting: Bethesda 2001
• Specimen adequacy
 Satisfactory:
• Cellularity: conventional = 8000-12000, liquidbased = 5000;
• transformation zone component: 10 well-preserved
endocervical or metaplastic cells
 Unsatisfactory
• Specimen rejected/not processed: (not labeled; broken slide;
unacceptable method of transport;
• Specimen processed and examined, but unsatisfactory for
evaluation of epithelial abnormality: extensive (>75%)
obscuring inflammation / hemorrhage; cytolysis; drying
artifact;
 Adequate smear

• Satisfactory Specimen: Adequate cellularity & transformation

zone component: superficial and intermediate squamous cells and
a cluster of columnar endocervical cells.
 Lactobacilli
• Lactobacilli and occasional cytolysis.
Bacteria get more dispersed in liquid
preparations; the background is thus
cleaner. (contrast with lower right inset
from a conventional smear).
• Lactobacilli (Doderlein bacilli) are
normal flora
• In determining specimen adequacy, Liquid-based
nuclear preservation and visualization Conventional
are of key importance. Changes such as
cytolysis and partial obscuring of
cytoplasmic detail may not necessarily
interfere with specimen evaluation.
Abundant cytolysis(>~50%) may be
mentioned as a quality indicator, but
most such specimens do not qualify as
unsatisfactory unless nearly all of the
nuclei are devoid of cytoplasm.
 Obscuring factors

hemorrhage /
RBCs

inflammation / WBCs

drying artifact
 “Cornflakes” artifact
• Also called “brown artifact”,
cornflaking
• Distinctive appearance is due
to evaporation of xylene
before cover slipping, with
deposition of air on
superficial squamous cells
• More common on
conventional than liquid
based preparations
 CytoHormonal Maturation Index
• specimen: middle third segment, lateral vaginal wall sample
• adequate cellularity: at least 300 cells
• purpose: assess hormonal status indirectly by determining the
differential of the squamous lining cells expressed as
percentages (ratio of P : I : S cells)
• P / I / S : parabasal/intermediate/superficial

P I S
Newborn (Maternal hormones present) 0 80 20
Childhood, pre-puberty 100 0 0
Pre-ovulatory (Estrogen dominant) 0 30 70
Post-ovulatory (Progesterone dominant) 0 60 40
Pregnancy (Progesterone dominant) 0 100 0
Menopause 100 0 0
 Bethesda 2001: Non-neoplastic
findings
• Organisms
 Trichomonas vaginalis (may be with Leptothrix)
 Fungal organisms morphologically consistent with Candida spp.
 Shift in flora suggestive of bacterial vaginosis (Gardnerella
vaginalis)
 Bacteria morphology consistent with Actinomyces spp.
 Cellular changes consistent with Herpes simplex virus
 Trichomonas
vaginalis
• pear-shaped, flagellated,
w/ blue-green cytoplasm,
ovoid eccentric nucleus
• Frequently associated with
Leptothrix (long slender
bacteria): “spaghetti &
meatballs”
• Squamous cell changes in
Trichomoniasis
 Minimal nuclear enlargement
 Cytoplasmic polychromasia
 Trichomonas and Leptothrix
• The finding of Trichomonas
and Leptothrix together has
been referred to as "spaghetti
and meatballs" When
Leptothrix are seen, one
should search for the possible
presence of trichomonads. In
liquid based preparations, the
leptothrix organisms may
tend to clump (arrow) as
opposed to conventional
smears
 Candida albicans
• Candida: yeasts & pseudohyphae formed by elongated
budding, with spearing of epithelial cells--"shish kebab"
effect.
• predisposing factors: diabetes mellitus; pregnancy;
change in pH of vaginal secretions / loss of normal flora
(Lactobacilli)
 Bacterial vaginosis
• Gardnerella vaginalis – coccobacilli – proliferate when vaginal
pH becomes less acidic & normal flora is eradicated
• Clue cells – individual squamous cells covered by a layer of
bacteria that obscures the cell membrane. Background is
usually clear in liquid based preparations.

Clue cell
 Actinomyces spp.
• Gram-positive, thin
filamentous bacilli in tangled
clumps, often with acute angle
branching, sometimes showing
irregular “wooly” appearance.
Swollen filaments may be seen
with clubs at periphery.
• A “cotton ball”- like acute
inflammatory response is
common.
• often associated with
intrauterine device (IUD)
usage. Organisms may alert
clinician to look for evidence
of pelvic infection.
 Herpes
• Multinucleation, Nuclei showing
"ground-glass" appearance due to
intranuclear viral particles
• nuclear Molding, and
• enhancement of nuclear envelope
caused by peripheral chromatin
Margination.
• Cowdry type inclusions –
intranuclear dense eosinophilic
amorphous or droplet-like bodies
surrounded by a halo
 Chlamydia trachomatis
• an obligate intracellular parasite with
elementary bodies (infectious but
incapable of cell division) and reticulate
bodies (multiply within cytoplasm, but not
infectious until they transfer back into
elementary bodies)
• Cytology: morphologic changes
(intracytoplasmic inclusions with central
small coccoid bodies) are not specific:
mixed flora and many neutrophils;
• If clue cells, yeasts and trichomonads, are
absent on a Gram stain and a wet
mount, Chlamydia should be suspected
• Presence of infection may not be
associated with symptoms
• Diagnosis is based on molecular tests
(PCR or ligase chain reaction)
 Non-neoplastic findings
• Atrophy
• Glandular cells status post hysterectomy
• Reactive cellular changes associated with
 Inflammation (including typical repair)
 Radiation
 Intrauterine contraceptive device
• Others
 Tubal metaplasia
 Keratotic cellular changes
 Lymphocytic (follicular) cervicitis
 other
 Atrophic vaginitis
• Parabasal cells with mostly bland nuclei (some
showing air drying). Some degenerated cells with
pyknosis also present. Basophilic granular
background with inflammation also present.
 Repair
• Repair is characterized by cohesive
cell groups “monolayer sheets” with
distinct cellular outlines, nuclei
oriented in the same direction
(streaming), increased nuclear size
and prominent nucleoli. There are few
or no single cells.
• Absence of single cells with nuclear
changes and lack of marked
anisonucleosis or irregularities in
chromatin distribution or variation in
size and shape of nuclei indicates this
is typical repair (as opposed to
"atypical repair")
 Non-neoplastic findings

Radiation effects IUD effects

parakeratosis

Glandular cells post-hysterectomy

Tubal metaplasia hyperkeratosis
 Squamous
metaplasia
• Normal polygonal squamous
metaplastic cells with round to
oval nuclei and bland chromatin
pattern.
• Change from columnar to
squamous lining cells –
signifies chronic cervicitis
• The presence of squamous
metaplastic cells indicates that
the transformation zone has
been sampled (a minimum of 10
well-preserved endocervical or
metaplastic cells is required for
this quality indicator).
 Reporting: Bethesda 2001
• Endometrial cells in a woman aged 40 years or older
• Atypical Squamous cells
 Of undetermined significance (ASCUS)
 Cannot exclude HSIL (ASC-H)
• Epithelial abnormalities (squamous)
 Low-grade squamous intra-epithelial lesion (LSIL) encompassing
HPV, mild dysplasia, CIN 1
 High-grade squamous intraepithelial lesion (HSIL) encompassing
moderate & severe dysplasia, CIN 2, CIN 3, CIS
• With features suspicious for invasion
 Endometrial cells

Endometrial cells

Menstrual smear

• Endometrial cells after age 40, particularly out of phase or after

menopause may be associated with benign endometrium,
hormonal alterations and less commonly, endometrial /uterine
abnormalities (rarely, Endometrial CA)
• Because of this association, all glandular endometrial cells are
reported in women 40 years and over.
 ASCUS

• mild nuclear enlargement, binucleation

• hyperchromasia, fine chromatin
• smooth nuclear & cytoplasmic membranes
• no clear-cut evidence for diagnosis of an
intraepithelial lesion
 ASC – H

• Less mature squamous cells/metaplastic cells with

polygonal shape, and slightly enlarged nuclei with
occasional nuclear contour irregularities.
• Atypical multinucleated cells.
 LSIL - HPV

• Mature squamous cells displaying enlarged nuclei (3

to 4 times the size of normal intermediate cell nuclei)
with granular chromatin
• Binucleation and koilocytosis are consistent with
HPV cytopathic effect.
 HSIL

• Metaplastic cells with increased N:C ratios and

nuclear contour irregularities.
• Hyperchromasia, abnormally distributed chromatin.
• dense / "metaplastic" cytoplasm
 HSIL r/o Invasion

• Numerous abnormal cells are present in varying sizes and shapes.

Nuclei vary from round to oval to spindle shaped. This type of
pleomorphism suggests invasive carcinoma. However, the absence
of nucleoli and necrosis are consistent with CIS/HSIL
• Keratinized dysplastic cells with nucleoli, and angulated or carrot-
shaped nuclei that may raise suspicion for invasion and qualify for
an interpretation of HSIL, cannot rule out invasion.
 
 Reporting: Bethesda 2001
• Epithelial abnormalities • Epithelial abnormalities
(Squamous) (Glandular)
 Squamous cell carcinoma  Atypical, NOS / specify in
 Others comments
• Keratinizing lesions • Endocervical / Endometrial /
• Squamous intraepithelial lesions Glandular
(SIL) – borderline  Atypical, favor Neoplastic
• SIL with Glandular involvement • Endocervical / Endometrial /
Glandular
 Endocervical adenoCA in situ
 Adenocarcinoma
• Endocervical / Endometrial /
Extrauterine
• Not otherwise specified (NOS)
 Invasive Squamous Cell Carcinoma

• Tumor diathesis (lysed blood and stripped nuclei); variation in

cell size and shape; evidence of keratinization; scant cytoplasm;
nuclei with irregularly distributed, coarsely granular chromatin
and prominent nucleoli; ragged cellular borders
• Dysplastic squamous cells with anisocytosis and anisonucleosis
including keratinization and tadpole cells are diagnostic of
invasive squamous cell carcinoma.
• centrally located nuclei and flat arrangement of cells is consistent
with squamous cell carcinoma.
 Epithelial Abnormalities
•Irregular chromatin
distribution and
prominent or
macronucleoli are
classic findings in
invasive endocervical
adenocarcinoma.
HSIL & Endocervical AdenoCA Endocervical adenoCA

Loose aggregate of small epithelial cells

with slightly enlarged nuclei, small
nucleoli, and vacuolated cytoplasm;
Endometrial adenoCarcinoma "watery diathesis" and histiocytes
 Extrauterine (Metastatic)
Malignancies

• Colonic adenoCA: atypical • Malignant Melanoma: brown

glandular cells with nuclear cytoplasmic pigment consistent
pleomorphism, hyperchromasia, with melanin in the malignant
and cellular overlap; tall columnar cells - Large single or loosely
cells and scattered goblet cells cohesive cells with round
with distended mucin-filled hyperchromatic nuclei, irregular
vacuoles nuclear membranes, coarsely
clumped irregularly distributed
chromatin and prominent nucleoli
Thank You !