BMS4004 Advanced Cellular Pathology

Biomedical Sciences, City University of Hong Kong

Cytology II:
Gynae and Non-gynae
Cytology
Dr Richard Wing-Cheuk Wong
Consultant Pathologist
Department of Pathology
United Christian Hospital
 Part 1:
Gynecologic Cytology
Reference: The Bethesda System for Reporting Cervical Cytology.
Nayar R, Wilbur DC, (Eds), 3rd Edition. Springer, 2015.
 Gynecologic cytology

• Primarily serve as screening test for HPV-related cervical malignant

and precursor lesions low risk cases
X distinguish high
•
分類

May be used as a triage test in primary HPV screening programme

• Occasionally detect some non-neoplastic conditions, endometrial
pathology or other neoplasms
• Includes cervical and vaginal cytology
• Preparations include conventional smear or liquid-based cytology
(ThinPrep / SurePath)
&rent
• morphology
in

Related techniques include HPV testing, automated screening and

some other biomarkers
Cimmunostain dual stain:P16, KS37
Normal histology of the cervix
(squamous epithelial)

Ectocervix

Endocervix

R. Sankaranarayanan, Ramani S. Wesley.

A practical manual on visual screening for cervical neoplasia. (2003) IARC, Lyons.
 Transformation zone

• The transformation zone is the area of the cervix where

endocervical glandular epithelium is being replaced by
metaplastic squamous epithelium
• This is where most HPV-associated cervical lesions develop,
probably related to the exposure of proliferative reserve cells
to viral particles
• Cervical cytology is thus usually obtained by sampling the
transformation zone
• The presence or absence of endocervical / transformation zone
components is routinely commented in cytology report
 Squamous metaplasia at the transformation zone

& basal layer (become cuboidal

Metaplastic
squamous
epithelium

R. Sankaranarayanan, Ramani S. Wesley.

A practical manual on visual screening for cervical neoplasia. (2003) IARC, Lyons.
 Squamous metaplasia

• In the cervix, endocervical

glandular epithelium may undergo
squamous metaplasia and
transform into squamous
epithelium
• Metaplastic squamous epithelium
①
may have smaller amount of
②
cytoplasm and higher N/C ratio
than mature squamous epithelium,
③
④
but the nuclei are uniform with
fine chromatin
HPV infection and cervical squamous
neoplasia ↓stromal invasion
just infection easily cleared
I
-

① ② I pre-malignant lesion)

incytoplasm of host cells

Cviral DNA
(viral DNA integrate to nucleus]

Crosbie EJ et al. Lancet 2013; 382: 889-99

 Integration of HPV DNA into host
genome is the critical step for
neoplastic transformation
• Circular HPV DNA becomes linear
when integrated into host genome

• Disruption of E2 gene during viral

DNA integration leads to
overexpression of E6 & E7
oncoproteins

• E6 and E7 together promotes cell

survival and proliferation

Crosbie EJ et al. Lancet 2013; 382: 889-99

 Histology of HPV-related cervical
squamous lesions

• Low-grade squamous intraepithelial lesion (LSIL)

• Squamous epithelium with episomal HPV DNA
• Histology shows morphology of koilocytosis (nuclear changes in superficial
squamous cells characteristic of HPV infection) or CIN I
• High-grade squamous intraepithelial lesion (HSIL)
• Squamous epithelium with HPV DNA integrated into host genome
• Histology shows morphology of CIN II or CIN III
• Invasive squamous cell carcinoma (SCC)
• Histology shows stromal invasion (invasion of neoplastic squamous cells
across the basement membrane)
 Low-grade squamous intraepithelial
lesion (LSIL) – histological features

• CIN 1
• Koilocytosis
• Diagnosis based on nuclear disarray
• Presence of koilocytes (superficial squamous in the parabasal layers
I basement membrane
cells with enlarged hyperchromatic nuclei and above

perinuclear halo) • Significant interobserver variability;

many cases are not premalignant
 lesion have false result(X mitosis.HSIL vs X mitosis.XHSCL)
1 pre-malignant & may

High-grade squamous intraepithelial

lesion (HSIL) – histological features

• CIN 2 ChighN:cratio • CIN 3 ChighN:cratio

• Diagnosis based on atypical squamous cells • Diagnosis based on full
involving the middle layers with surface thickness cytologic atypia in
maturation the squamous epithelium
 Squamous cell carcinoma – histological
features

• Squamous cell carcinoma

• Diagnosis based on presence of stromal invasion, usually more abundant cytoplasm than
CIN and⑭prominent nucleoli ⑬necroticbg Qabnormal cell
size

• Traditionally classified into non-keratinizing and keratinizing types

① ②
• Now classified into HPV-associated and HPV-independent by WHO 2020
 Adenocarcinoma in situ (AIS), HPV-
associated
affect glandular epithelium
↓
• HPV-associated AIS is a premalignant lesion of endocervical glands (a form
of intraepithelial neoplasia as the glandular equivalent of HSIL)
① ② ③
• cenlongated
Recognized by nuclear enlargement with hyperchromasia, coarse chromatin,
④ nucleuss ③
nuclear pseudostratification, and mitosis in the endocervical glandular
⑥
epithelium, usually with abrupt transition with normal epithelium
 Histology of endocervical
adenocarcinoma
• There are many histologic types of HPV-associated adenoCA
endocervical adenocarcinoma,
now classified into HPV-
associated and HPV-independent
types (WHO 2020)
• HPV-associated adenocarcinoma
accounts for the majority (85-90%)
• About 10-15% of cervical XHPVscreening test
arise from adenocarcinomas are HPV- HPV-independent (gastric-type)
endocervical
gland
independent (predominantly adenoCA
(not surface gastric-type adenocarcinoma)
epithelium)
• By cytologic features alone,
distinction of endocervical
adenocarcinoma from AIS and
endometrial adenocarcinoma can
be difficult
Interpretation of gynecologic
cytology
Reporting of cervical smears
Cervical cytology Preparation and
specimen staining of
received by lab cytology slide

Screened slides Screening of

reviewed by cytology slide by
pathologist cytotechnologist

Reporting of Reporting of
positive & some most negative
negative smears smears by
by pathologist cytotechnologist
 The Bethesda System for Reporting
Cervical Cytology (2014)

• Online atlas
• https://bethesda.soc.wisc.edu

Nayar R, Wilbur DC. Acta Cytologica 2015; 59: 121-32

 Framework of the Bethesda System
Standardize reporting format)

• Specimen type (conventional / liquid-based)

• Specimen adequacy
• Satisfactory for evaluation
• Unsatisfactory for evaluation (specify reason)
• Interpretation / Result
• Negative for intraepithelial lesion or malignancy
• ① Non-neoplastic findings
• ②Organisms
③
• Other: Endometrial cells (≥ 45 years of age)
• Epithelial cell abnormalities
• Squamous cell
• Glandular cell
• Other malignant neoplasms
How do we make diagnosis on
cervical cytology?
 Normal cellular constituents in
gynecologic cytology
• Squamous cells from the ectocervix & vagina
• Squamous metaplastic cells from the transformation zone
• Columnar (glandular) cells from the endocervix
• (Occasionally) cells from the endometrial epithelium & stroma
• Leukocytes & erythrocytes
• Commensal organisms
• Mucus strands
• Contaminants
 Basic morphologic differences of
squamous and endocervical cells
Squamous cells
• Dense cytoplasm
• Pointed edges at the periphery of
cell clusters
(glandular cells)
Endocervical cells
• Foamy cytoplasm
• Smooth common borders at the
periphery of cell clusters
Superficial squamous cells CEosin) Intermediate squamous cells (light green)

Squamous metaplastic cells Parabasal cells (light green)

 Morphology of squamous cells
Superficial cells
• Polygonal cells
• Abundant cytoplasm, usually
eosinophilic
• Central, round, small & pyknotic
nuclei with condensed chromatin
Intermediate cells
• Polygonal cells
• Central, round to oval nuclei
slightly larger nucleus than superficial
• Intermediate cell nucleus serves
as size reference in cytology
• Fine chromatin
 Morphology of squamous cells
fine chromatin
normal nuclear morphology

Squamous
↓

Parabasal cells metaplastic cells

(normal nuclei, less cytoplasm)
• Oval shape • Variable cell size
• Round to oval nuclei with fine • Slightly enlarged round nuclei
chromatin with fine chromatin
• High N/C ratio • Variable increase in N/C ratio
 (glandular cells

Morphology of endocervical cells

• Monolayered sheets with a
characteristic honeycomb or
picket-fence arrangement
• Pale foamy cytoplasm
• Basally located, round nuclei with
fine chromatin and small nucleoli
• Variation in nuclear size possible
↓ uniform shape
(not severe variations
 Morphology of (spontaneously
exfoliated) endometrial cells
• Tight clusters
• Smaller than endocervical cells
• Nuclei similar to or smaller than
intermediate cell nucleus
• High N/C ratio
• Small nuclei with dense or
heterogeneous chromatin
• V

Poorly defined cytoplasmic border

• Scanty vacuolated cytoplasm
•
不显眼的
Nucleoli usually inconspicuous
 Morphology of (spontaneously
exfoliated) endometrial cells
外出

Exodus pattern
• Ball-like clusters of endometrial
stromal cells surrounded by cells
endometrial glandular cells at the landular
I
periphery
endometrial
stromal cells
 Directly sampled endometrial cells
in gynecologic cytology
• Large tissue fragments composed
of epithelial cells (center) and
stromal cells (periphery).
A
typical glandulan cell
•
vs

Directly sampled endometrium

(usually from lower uterine
segment) does not carry the same
significance as exfoliated
endometrial cells. They do not
need to be reported.
• When present as small fragments,
distinction from AIS can be
difficult
 LSIL HSIL
s
nuclearmorphology 7EE*
chromatic pattern Lt** *
*

hormochromatic/hypochromatic
LSIL has enlarged hyperchromatic nuclei, coarse HSIL has hyperchromatic nuclei, coarse
chromatin and LOW N/C ratio chromatin and HIGH N/C ratio

LSIL HSIL

condensation of
peripheral
&
cytoplasm

LSIL may contain koilocytes with abnormal HSIL may exist as isolated cells (especially in
nuclei surrounded by cavitating perinuclear halo liquid-based), which can be easily missed
 Low-grade squamous
intraepithelial lesion (LSIL)
• Squamous cells with enlarged
hyperchromatic nuclei
• Nuclear enlargement at least 3
times the size of intermediate
cell nucleus
• Coarse chromatin (or dense
smudgy chromatin) intermediatecell

• Low N/C ratio

• Nucleoli absent or inconspicuous
• Binucleation common
 Low-grade squamous
intraepithelial lesion (LSIL)

Koilocytes
• Sharply defined perinuclear halo
• Condensation of cytoplasm
around the periphery
• Nuclear abnormalities must be
present
 Atypical squamous cells of
undetermined significance (ASC-US)
• ASC-US is used for changes that
are suggestive of LSIL but
quantitatively or qualitatively ① coarse chromatin

insufficient for a definitive X & largenucleus

③ lowN:C
interpretation
• Usually this is used when small
number of squamous cells show
mildly enlarged nuclei (2.5 times
that of intermediate cell nucleus)
and coarse chromatin or irregular
nuclear outline
 High-grade squamous
intraepithelial lesion (HSIL)
Diagnostic
• Usually small cell size
• Occurs singly or in aggregates
①
• High N/C ratio
• Irregular nuclear outline
Birregular clumping of
• Coarse chromatin ↓ chromatin

&variable in size

• Nuclear enlargement variable

• Not necessarily larger than
intermediate cell nucleus
• Hyperchromatic or hypochromatic
 Atypical squamous cells, cannot
exclude HSIL (ASC-H)

• ASC-H is used for changes

suggestive of HSIL but Parabasal cell w/atrophy pattern
quantitatively or qualitatively atrophy
nuclear grooving
->

(miomicHSIL)
insufficient for a definitive &

interpretation
• Usually these cases have scanty
cells with high N/C ratio but the
nuclear abnormalities are not
florid enough
 Squamous cell carcinoma
(dirtybg)
characteristic of invasivetumor ->
Necroting bg:cell debris, fragments, degenerated cells

(majority)
Keratinizing squamous cell carcinoma
• Variation of cell size and shape, ↓
spindle cell

including “tadpole cells” and

Sinrasive
chromatin
dark staining

spindle cells with dense N

V

orangeophilic cytoplasm
• Nuclei often dark and opaque
nuclearde 555 lobes

• Chromatin coarse and vesicular

• Prominent nucleoli and tumour tadpole cell

diathesis may be seen

 Squamous cell carcinoma
mimic adenocarcinoma
&
Non-keratinizing squamous cell
carcinoma
• Single or aggregates of cells
• Features resembling HSIL
• Pleomorphic nuclei
①dark staining nuclei

• Chromatin coarse and vesicular

②
③ cytoplasmic
feature

• Prominent nucleoli sharp edges

dense

• rinbg cytoplasm
+

④
Tumour diathesis (necrotic debris
and degenerated RBCs) often
present in the background
 Mimics of HSIL and SCC
Atrophy vs HSIL HSIL vs SCC

fine chromatin
&
Parabasal cells in atrophy have high N/C ratio HSIL may contain occasional cells with
and may mimic HSIL elongated cytoplasm, which may mimic SCC
Adenocarcinoma in-situ (AIS) Endocervical adenocarcinoma

Adenocarcinoma in-situ (AIS) Endometrial adenocarcinoma

pseudostratified
strip
 Adenocarcinoma in-situ
• Glandular cells arranged in clusters
or pseudostratified strips (P.59pc) feathering (elongated nuclei)
&
•
⽊栅栏
Palisading nuclear arrangement
⑤
with “feathering” (nuclei
protruding from periphery)
• ① Hyperchromatic nuclei
• ④Coarse chromatin
• ④ Nucleoli usually inconspicuous
• Mitosis and apoptotic bodies may
be seen
 Adenocarcinoma
(endocervical vs endometrial)
General features of adenocarcinoma
• Glandular cells with vacuolated
cytoplasm and pleomorphic nuclei
• Irregular chromatin distribution,
chromatin clearing and nuclear
membrane irregularities
• Prominent nucleoli
• Tumour diathesis may be present
Endocervical (verynecroticbg)
• Usually some cells with features of
AIS, larger cell size & nuclear size
features of adenocar
+

Endometrial (not too necroticbg)

• Usually smaller clusters, smaller cell tumor cell
size & nuclear size, intracytoplasmic cluster have
2

neutrophils
neutrophils, watery diathesis
↓ not too
dirty by (X so necrotic
 Atypical glandular cells (AGC)
• Atypical glandular cells is the term
used for glandular cells with
nuclear abnormalities that are
indefinite for neoplasia
• Ideally they should be specified as
“atypical endocervical cells” or
“atypical endometrial cells” as far
as possible, but in daily practice
the diagnosis of AGC is usually
sufficient
• For cases with relatively
significant atypia, the diagnosis
“AGC, favour neoplastic” may be
used
 Candida Herpes (HSV)

①multi-nucleated
① hyphae
② ground glass nucleus
③ chromatin
margination

Actinomyces Trichomonas
② small nucleus

① cotton-ball apearshape flagella (microbes)

③ eosinophilic
granulated
cytoplasm
Strengths and limitations of cervical
cytology
Strengths Limitations
• Assessment of the whole • Sampling
transformation zone • Low sensitivity and more false
• instead of individual sites in biopsy negatives as compared to HPV
I
testing more sensitive

• Able to triage cases into different

risk categories
• as compared with HPV testing
• Occasionally pick up non-neoplastic
lesions and non-HPV-associated
tumours (cervical or non-cervical
origin)
• Performance affected by
competency of individual labs and
screeners ↓ hard to maintain
quality assurance
clots of mimicers)
• Interpretation difficulties
• E.g. Mimics of SIL, risks of under-
and over-diagnosis of malignancy,
typing of glandular abnormalities
 HPV testing – an alternative or
adjunct to gynecologic cytology
for cervical cancer screening
 HPV testing in gynecologic cytology
• HPV tests are molecular tests designed to detect specific
DNA or RNA sequences of HPV
• Many types of HPV tests are commercially available for
application in gynecologic cytology specimens
• DNA-based - most detect L1 gene of HPV
• RNA-based - most detect E6/E7 mRNA of HPV
• HPV tests can be used as a first-line test for primary
screening, or as an adjunct to gynecologic cytology
(reflex testing for triage of ASCUS cases; or co-testing
with cytology)
X
FDA-approved HPV tests
Digene HC2 Cervista Cobas Aptima BD Onclarity
Target DNA (full DNA (L1) DNA (L1) RNA (E6/E7 DNA (E6/E7)
molecule genome) mRNA)
Year approved 2001 2009 2011 2011 2018
for reflex
testing or co-
testing
Year approved N/A N/A 2014 N/A 2018
for primary (ThinPrep); (SurePath)
screening 2018
(SurePath)
Target HPV 13 types of 14 types of 14 types of 14 types of 14 types of
genotypes* hrHPV hrHPV hrHPV hrHPV hrHPV
HPV No Possible with Yes (16 & 18) Possible with Yes (16, 18, 45,
genotyping separate test separate test 51, 52, 33/58,
capacity (16 & 18) (16, 18/45) 56/59/66,
35/39/68)
Technology Hybridization Hybridization PCR-based PCR-based PCR-based
and signal and signal target target target
amplification amplification amplification amplification amplification
*The HPV genotypes covered by Cervista, Cobas, Aptima and BD Onclarity are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 & 68.
Digene HC2 covers all of these high-risk HPV types except for 66. Adapted from Salazar KL et al. J Am Soc Cytopathol 2019; 8: 284-292.
 * HPV testing – strengths and
limitations
Strengths
• Higher sensitivity than cytology
• May allow longer screening
interval for negative result
• Objective and standardized
performance
• Not require subjective
morphologic interpretation
• Easier to establish for developing
countries
• Compared to the training and
quality assurance for cytology
↑ test
still need for HIV
much easier
Limitations
• Sampling problem, false positive
and false negative still possible
• Different HPV tests may give
discrepant results (due to different
detection targets and threshold)
• A subset of cervical carcinomas
(especially adenocarcinoma) are
HPV-negative
• Triage test required for positive
result in primary screening
• By cytology, HPV genotyping or
biomarkers
I not used
but commonly
 Trends and challenges for
gynecologic cytology
cytology tragecostic) vs HPV test (screeninga

• HPV test is replacing cervical cytology as a first-line test

for cervical cancer screening worldwide
• The adoption of HPV testing and HPV vaccination will
have impact on gynecologic cytology services
• Despite the limitations of gynecologic cytology, its role as
a diagnostic test in hospital laboratories is likely to
continue
• Laboratories need to maintain adequate cytotechnologist
workforce and meet the quality assurance and other
requirements for both cytology and HPV testing
 Part 2:
Non-gynecologic
cytology
 Major areas of non-gynecologic
cytology

• Exfoliative cytology
• Examination of cells that are spontaneously shed into body fluid or
scraped/removed from body surface
• E.g. urine cytology, sputum / bronchial cytology, effusion fluid
cytology, biliary cytology, CSF cytology
• Fine needle aspiration cytology (FNAC)
• Examination of cells that are aspirated from a mass through a
needle
• E.g. breast, thyroid, salivary gland, lymph node, lung, pancreas
Non-gynecologic Exfoliative
Cytology
 Clinical utility of non-gynecologic
exfoliative cytology

• Most specimens serve as screening tests for malignancy

• Urine cytology for (high grade) urothelial carcinoma
• Sputum / bronchial cytology for lung carcinoma
• Effusion fluid cytology for malignant cells in pleural / pericardial /
peritoneal fluid
• Although the primary aim is to detect carcinoma, sometimes
other tumour types or non-neoplastic diseases may be
diagnosed in exfoliative cytology
 Clinical utility of non-gynecologic
exfoliative cytology

• Some specimens are submitted to detect specific non-

neoplastic diseases
• Synovial fluid for crystals
• Urate crystals (gout)
• Calcium pyrophosphate crystals (pseudogout)
• Sputum or bronchial lavage for “PCP”
(pneumonia)
• Pneumocystis jirovecii (formerly known as PneumocysticS carinii)
infection
•
virally infected epithelial cells
Urine for decoy cells
• Polyomavirus inclusion
I patients wirenal transplant are susceptible
 Role of cytotechnologist in
non-gynecologic exfoliative
cytology
•
as •
Preparation and staining of smears
effusion • Most specimens employ Pap stain (+/- H&E)
Cell block preparation if necessary
• Not routinely performed for most exfoliative cytology specimens; only upon
request from pathologist
• Screening of cytology slides to identify abnormal cells
• Drafting of cytology report with opinion on diagnosis
• NOT responsible for sign-out
• All non-GYN exfoliative cytology reports (positive or negative) are signed
out by pathologist
 Diagnostic terminology in
non-gynecologic exfoliative
cytology
• Unsatisfactory
• No malignant cell seen / Negative for malignancy / No
evidence of malignancy
• Atypical cells seen
•
)
Suspicious of malignancy
high degree of abnormalities
• Malignant cells seen
• The specific tumour type would be named if sufficient to make the
diagnosis (e.g. adenocarcinoma / squamous cell carcinoma)
General principles of interpretation
for non-gynecologic exfoliative
cytology
• Awareness of normal cell types in different types of specimen
• Recognition of abnormal cells with nuclear features that may
represent malignancy
• Cytoplasmic features may give some clue to the lineage of the
abnormal cells
•
(scanty amount:atypical suspicious)
Assessment of the overall cellularity of the abnormal cells, and
any other clues to the diagnosis
• Beware of benign cellular changes that may mimic malignancy
General principles of interpretation
for non-gynecologic exfoliative
cytology
• The nuclear chromatin pattern is an important cytologic
parameter.
* • “Fine” (or open) chromatin means evenly distributed, homogeneous
nuclear material, while “coarse” chromatin refers to uneven
distribution and irregular clumping of nuclear material.
• Nuclear atypia (or cytologic atypia) refers to abnormal
appearance of the nuclei. The exact criteria for “atypia” differs
between different cell types.
• Use of the term “atypia” does not imply malignancy, instead it
means that benign reactive changes (reactive atypia) and
malignancy are both possible.
 General cytologic features of
malignant cells
• Nuclear features commonly observed in malignant cells:
• Nuclear pleomorphism (variation in nuclear size and shape)
• Hyperchromasia (dark-staining nuclei)
• Coarse chromatin (uneven distribution and clumping of nuclear material)
• Enlarged nucleoli
• Increased nuclear-to-cytoplasmic (N:C) ratio
• Irregular nuclear membrane
• Please remember that malignant cells may not necessarily have all
the above features and no single feature is totally specific
 Basic Interpretation of
Non-gynecologic Exfoliative
Cytology
 Urine Cytology

• Normal constituents I superficial urothelial cells)

• Urothelial cells (umbrella cells, ↓
urothelial cells

intermediate cells, +/- basal cells)

• Squamous cells
• Common non-neoplastic findings
• White blood cells, especially
neutrophils para et
• Red blood cells
• Crystals
 multi-nucleated
M
↓

Urothelial cells Umbrella cell (left) and

squamous cell (right)

Neutrophils
 Urine Cytology

• Morphological features of high grade urothelial

carcinoma (transitional cell carcinoma / TCC):
*
• High N/C ratio
• Nuclear hyperchromasia
• Coarse chromatin
• Irregular nuclear outline
• +/- Prominent nucleoli
High grade urothelial carcinoma

↑hyperchromasiais
① high N:2
High grade urothelial carcinoma
Note the coarse
chromatin and
nuclear
pleomorphism
Mitotic
figure
7
 Urine Cytology
• In clinical practice, the cellularity of abnormal cells should
be high when we make a cytologic diagnosis of carcinoma
• If the abnormal cells are present in small numbers, or if
some of the morphologic features are lacking, we usually
diagnose such cases as “atypical cells” or “suspicious of
malignancy”
Atypical cells
with enlarged
hyperchromatic
nuclei but just
mildly increased
N/C ratio
 Urine Cytology

• Benign cellular changes that may mimic urothelial

carcinoma:
appears dark
2
• Degenerated urothelial cells
enlarged nuclear size but fine chromatin, prominentnucleoli
• Reactive atypia related to inflammation or injury of urothelium
(especially associated with urinary stone)
• Treatment effect related to chemotherapy or radiotherapy
nuclear chromatin
↓> ground glass
• Polyomavirus inclusion
Reactive urothelial cells
Reactive urothelial cells
 Other malignant tumours in urine
cytology

• Squamous cell carcinoma

• Adenocarcinoma
• Small cell carcinoma
• Metastatic carcinoma

• In practice, we rarely diagnose these tumour types in urine cytology

specimen, because high-grade urothelial carcinoma can harbour
tumour cells that resemble other types of carcinoma
(adenocarcinoma, squamous cell carcinoma, or small cell carcinoma).
These diagnoses are preferably diagnosed on biopsy.
The Paris System for Reporting Urinary
Cytology

Barkan GA et al. Acta Cytologica 2016; 60(3): 185-97.

 Sputum and Bronchial Cytology

cells
2 squamous
• Normal constituents in sputum and
bronchial
↑
cytology:
bronchial cells

• Ciliated columnar cells (bronchial cells)

Scarbonated)

• Alveolar macrophages (indicates

adequacy of sample)
• Squamous cells (usually from oral
cavity, more common in sputum)
• White blood cells (including neutrophils,
lymphocytes and eosinophils)
 Alveolar
macrophage

Macrophage
small nucleus
#
Bronchial cells &terminal bartcilia

Bronchial cells
 Sputum and Bronchial Cytology

• The major types of lung carcinoma include adenocarcinoma,

squamous cell carcinoma and small cell carcinoma
• Definitive diagnosis is often difficult to make in cytology specimens
alone, unless the tumour cells are abundant with clear-cut features of
malignancy (maybe supported by IHC on cell block)
• Reactive atypia in bronchial cells or pneumocytes may also mimic
malignancy
•
不⾜的

If the abnormal cells are scanty or the cytologic features are

不確定的 equivocal, we usually make the diagnosis of “atypical cells” or
“suspicious of malignancy”
Adenocarcinoma
Squamous cell carcinoma

Small cell carcinoma

 Morphologic features of
adenocarcinoma
• Architecture: three-dimensional clusters, honeycomb-like
sheets, acini, papillae, isolated cells
• Eccentrically placed, round or irregular nuclei, variable
①

nuclear pleomorphism
• Chromatin may be fine or coarse
• Prominent nucleoli
②

• Foamy or vacuolated cytoplasm

Adenocarcinoma
 Adenocarcinoma typically
presents as three-dimensional
ball-like clusters on cytology
Note the foamy vacuolated cytoplasm

↓
typical of adenocarcinoma

① variation in nuclear size

② large nucleoli
Pleomorphic nuclei with
prominent nucleoli
Cell block histology
(H&E section)

Cadeno:trevs scci-ve)

TTF-1 immunohistochemistry
Morphologic features of squamous
cell carcinoma

• For well-differentiated squamous cell carcinoma, usually discohesive

cells with various shapes (polygonal, spindle, tadpole-shaped) and
pyknotic hyperchromatic nuclei with indistinct nucleoli
• Dense orangeophilic cytoplasm (on Pap stain) for keratinized cells
• For moderately to poorly differentiated squamous cell carcinoma,
usually large cohesive clusters of cells with large pleomorphic nuclei,
coarse chromatin and prominent nucleoli.
• Keratinization may be absent, in which case the diagnosis of “non-small cell
carcinoma” is preferable.
Squamous cell carcinoma

X densecytoplasm
 I
pointed edges

Note the dense cytoplasm, pointed

cytoplasmic edges, hyperchromatic
nuclei and coarse chromatin of
squamous cell carcinoma
Squamous cell carcinoma

/
1515A
Ex specific)
 Note the orangeophilic cytoplasm (on
Pap stain) with hyperchromatic enlarged
nuclei of squamous cell carcinoma
Spindle or tadpole-shaped cells
are occasionally seen in
squamous cell carcinoma
 Morphologic features of small cell
carcinoma
• Loose aggregates of round to fusiform cells
• High N/C ratio and scant cytoplasm
• Nuclear molding
①
• Hyperchromatic nuclei with stippled (“salt-and-pepper”) chromatin
②
• ⑨Indistinct nucleoli (occasional small nucleoli possible)
• Apoptotic bodies and/or mitosis
④
• Crushing and smearing artifact common
• Cell size usually small but larger cells possible
 nuclei
push against
each other

/
Small cell carcinoma
- Note the nuclear molding
Small cell carcinoma is characterized
by stippled or “salt-and-pepper”
chromatin pattern, typical of
neuroendocrine differentiation

Note the apoptotic bodies in

the background
Small cell carcinoma

nuclear
molding
 Benign cellular changes that may
mimic lung carcinoma

• Reactive bronchial cells (nuclear size enlargement with

distinct nucleoli; preserved cilia)
• Goblet cell hyperplasia
• Basal cell hyperplasia (reserve cell hyperplasia)
• Repair
• Type II pneumocyte hyperplasia
 Cirregular nucleibut fine chromatin)
can see cilia:normal cells

Reactive bronchial cells

 Effusion Fluid Cytology

• Normal constituents in pleural /

peritoneal / pericardial fluid
cytology:
• Mesothelial cells
• Histiocytes (macrophages)
• Lymphocytes
• Red blood cells and neutrophils may
be present
 loval-shaped nuclei, small nucleoli)

Mesothelial cells Lymphocytes

Macrophages

Mesothelial cells
 Effusion Fluid Cytology
• Metastatic carcinoma (usually adenocarcinoma) is the most common
positive diagnosis in effusion fluid cytology
• The presence of two populations of cells (mesothelial cells and
carcinoma cells) is an important clue to recognizing metastatic
carcinoma in effusion fluid
• Adenocarcinoma typically presents as three-dimensional ball-like
clusters and isolated cells with pleomorphic nuclei and prominent
nucleoli
• Cell block with immunohistochemistry could help confirm the
tumour type and predict the likely site of origin for metastatic
carcinoma
• Other tumour types may also be detected in effusion fluid cytology
(e.g. mesothelioma, lymphoma)
Metastatic adenocarcinoma in
effusion fluid cytology
Carcinoma cells
Carcinoma cells
 Mesothelial cells
↳ fine chromatin
Carcinoma cells
Carcinoma cell (recognized
by nuclear atypia in this
isolated cell)
 Effusion Fluid Cytology

• Benign cellular changes that may mimic malignancy:

• Reactive mesothelial cells (due to inflammation or other stimuli)
may show nuclear enlargement, slightly more granular
chromatin, irregular nuclear contour, prominent nucleoli.
However, they usually form a spectrum of atypia instead of 2
populations
Reactive mesothelial cells ① fine chromatin

② distinct nucleoli
Reactive mesothelial cells
 BerEP4 immunohistochemistry
(epithelial marker) - negative

Cell block histology (H&E section)

Calretinin immunohistochemistry
(mesothelial marker) - positive
 Non-neoplastic findings in
non-gynecologic exfoliative
cytology

• Viral inclusion
• E.g. Herpes simplex virus inclusion
• Fungal organism
• E.g. Pneumocystis jirovecii
Herpes simplex virus
(HSV) inclusion
#jEziE Fg!

Fine Needle Aspiration

Cytology (FNAC)
 Clinical utility of fine needle
aspiration cytology (FNAC)
• Generally used for diagnosis of mass lesions
• The aspiration procedure may be performed by clinician
or pathologist on superficial palpable masses (e.g. breast,
lymph node, thyroid)
• Alternatively, it may be performed by radiologist on
deep-seated or non-palpable imaging-detected masses
(e.g. lung, pancreas, non-palpable thyroid nodules)
 Role of cytotechnologist in fine
needle aspiration cytology (FNAC)
• Preparation and staining of smears
• Pap / H&E / Giemsa, depending on individual centres
• Preparation of cell block for most cases if possible
• Cell block preparation is routinely performed for FNAC to allow histologic
assessment (like a small biopsy) and ancillary studies (IHC and molecular studies)
• Assisting pathologist in performance of FNAC (in some centres)
• On-site assessment of specimen adequacy may be performed for
imaging-guided FNAC (in some centres)
• Screening and interpretation of FNAC slides is NOT required for most
centres
• All FNAC reports are signed out by pathologists
 Thyroid FNA cytology

• Diagnostic evaluation of thyroid nodules is an important

clinical application of fine needle aspiration
• The FNAC result affects the management of thyroid
nodules detected by clinical examination or ultrasound
• The Bethesda system for reporting thyroid cytopathology
is a set of terminology and interpretation criteria (with
associated malignancy risk for each category) that has
been widely adopted
 The Bethesda System for Reporting
Thyroid Cytopathology (2017)

• Online atlas
• https://www.papsociety.org/image-
atlas/

Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid
Cytopathology. J Am Soc Cytopathol. 2017 Nov-Dec;6(6):217-222.
 The Bethesda System for Reporting
Thyroid Cytopathology (2017)
• 6 categories
• Nondiagnostic / Unsatisfactory
• Benign
• Atypia of undetermined
significance (AUS) / Follicular lesion
of undetermined significance (FLUS)
• Follicular neoplasm / Suspicious for
a follicular neoplasm
• Suspicious for malignancy
• Malignant
Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid
Cytopathology. J Am Soc Cytopathol. 2017 Nov-Dec;6(6):217-222.
The Bethesda System for Reporting
Thyroid Cytopathology (2017)

Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid
Cytopathology. J Am Soc Cytopathol. 2017 Nov-Dec;6(6):217-222.
 Thyroid FNAC – Nondiagnostic

• Includes cases reported as cyst fluid only (e.g. only hemosiderin-laden

histiocytes), specimens with only blood cells, or those with very scanty
follicular cells (less than 6 groups with 10 follicular cells each)
 Thyroid FNAC – Benign follicular nodule

• Presence of macrofollicular fragments of follicular cells with uniform

round nuclei, without the nuclear features of papillary thyroid
carcinoma (some may have a background of colloid material)
Thyroid FNAC – Atypia of undetermined significance /
Follicular lesion of undetermined significance

• This category may be used in a variety of situations, for example:

• Focal or mild nuclear atypia which has some suggestion of papillary
thyroid carcinoma (e.g. some nuclei showing nuclear grooves or nuclear
pseudoinclusions in a background of non-atypical follicular cells)
• Sparsely cellular samples with mostly microfollicles
• Sparsely cellular samples with mostly Hurthle cells (abundant
eosinophilic cytoplasm)
Thyroid FNAC – Follicular neoplasm / Suspicious for a
follicular neoplasm

• Cellular specimens often with crowded follicular cells, microfollicles

and scant colloid
• Some cases may have mild nuclear atypia resembling those of
papillary thyroid carcinoma, but true papillae or nuclear
pseudoinclusions should be absent
Thyroid FNAC – Suspicious for malignancy (suspicious for
papillary carcinoma)

• Sheets of follicular cells with features suspicious of papillary

carcinoma, such as nuclear enlargement, nuclear contour irregularity,
nuclear grooves and chromatin clearing, but quantitatively or
qualitatively insufficient for a definitive diagnosis of malignancy
Thyroid FNAC – Malignant (papillary thyroid carcinoma)

• Apart from nuclear enlargement, nuclear contour irregularity,

nuclear grooves and chromatin clearing, these cases also display
‘classical’ features of papillary thyroid carcinoma, such as true
papillae, psammoma bodies and nuclear pseudoinclusions
Other applications of FNA cytology
• Lymph node FNAC
• Commonly used for diagnosis of metastatic carcinoma, also occasionally
identify specific infections or lymphoma
• Cell block important for immunohistochemistry and molecular studies (to
verify the site of origin and/or determine treatment)
• Breast FNAC
• Diagnosis of breast lumps but limited by difficulties such as distinguishing in
situ from invasive malignancy, or typing of papillary lesions and
fibroepithelial lesions
• Salivary gland FNAC
• A challenging area of FNA cytology due to the wide range of lesions with
overlapping morphological features
• For many types of salivary gland neoplasms, the nuclear features are not
very useful to distinguish between benign and malignant lesions, and
different tumour types may share similar cytoarchitectural features
 The Milan System for Reporting
Salivary Gland Cytopathology (2017)
• 7 categories:
• Non-diagnostic
• Non-neoplastic
• Atypia or undetermined
significance
• Neoplasm: benign
• Neoplasm: salivary
gland neoplasm of
uncertain malignant
potential
• Suspicious for
malignancy
• Malignant
Pusztaszeri M, Rossi ED, Baloch ZW, Faquin WC. Salivary Gland Fine Needle Aspiration and
Introduction of the Milan Reporting System. Adv Anat Pathol. 2019 Mar;26(2):84-92.
 Trends and challenges for
non-gynecologic cytology
• Standardized reporting terminology has been developed for
many types of specimens (e.g. urinary cytology, thyroid FNA
cytology), but not all systems are commonly used
• The role of exfoliative cytology in cancer screening is limited
by its relatively low sensitivity (despite high specificity), but it
serves as an important piece of data which guides further
specialized testing
• Non-gynecologic cytology specimens serve as an important
source of material for molecular studies, which may help to
establish a specific diagnosis or to guide therapeutic decisions
 Thank you!
Happy to answer your questions!
Email: wwc091@ha.org.hk