The Bethesda System and Beyond

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The Bethesda System and Beyond.
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The Bethesda System and Beyond.
Atlas of Cervical Cytology using the BD SurePath™

Liquid-Based Cytology System
Cervical cancer is the second-most common cancer in women. It affects around half-a-million women and each year kills over
280,000 worldwide. The good news is that the majority of cases can be prevented through cervical cancer screening and vaccination
against the Human Papilloma Virus (HPV).
Today, cervical cytology is still the most important test to screen for cervical cancer. Together with the best choice of technologies,
the competencies and skills of all individuals in the process of smear taking, sample processing, screening and interpretation, are
equally important.
BD Diagnostics offers products for cervical cytology screening, including the BD SurePath™ Pap technology and the BD FocalPoint™
Slide Profiler system which, together, provide an integrated solution for sample preparation, processing, staining, and computer
assisted imaging of liquid-based Pap testing.
BD Diagnostics is engaged to train and support all practicing pathologists and cytotechnologists in the screening of BD SurePath™
slides. This atlas is both a bench top training manual and a reference guide. It is intended as an aid to all staff who screen and/
or report cervical samples. It is anticipated that it will supplement more detailed training delivered by BD Diagnostics and
contribute to improving the diagnostic standards of cervical cancer screening in the laboratory.
This atlas starts with an overview of the anatomy of the female reproductive system with insight in the histological and cytological
images of all different cell types that can be seen on a cervical cytology slide.
Different histologic types of lesions are also discussed to clarify the different manifestations of cytology lesions falling in the same
category. To encourage the understanding of the cytological image, histology pictures and descriptions are also provided.
This atlas is dedicated to all pathologists and cytotechnologists who spend so many hours at their microscopes, screening and
interpreting complex images, and in doing so, making a difference and helping all women live healthy lives.
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Authors
Maud Veselic - Charvat, M.D. cytopathologist
Maud Veselic - Charvat was born in the former republic of Czechoslovakia in 1951. She received her medical degree from the university
of Kosice in Slovakia in 1975 She completed her residency in anatomic and clinical pathology in Bratislava. She continued her pathology
specialization in Leiden, the Netherlands, where she finished in 1989. After 2 years of cytopathology specialization at the AMC university
hospital in Amsterdam, she became chief cytopathologist at the Leiden University Medical Center (LUMC) in the Netherlands, a function
she holds today. Maud is also responsible for cytology education of all LUMC pathology residents.
Klaas van der Ham, medical photographer
Klaas van der Ham (1944) finished the photographer education in 1966 and was employed as a medical photographer at the Leiden
University Medical Center in the Netherlands, a position he still holds today. During his career, Klaas specialized in digital microphotography
and has been involved in the development of teaching material for pathology courses for many years.
Anneke van Driel - Kulker, Ph.D. biologist / cytologist
Anneke van Driel - Kulker (1954) was trained as a cytotechnologist in Leiden, the Netherlands, where she was certified in 1974. She
practiced cytopathology and participated in a university study to develop liquid based cytology (LBC) and computer assisted screening
(CAS). That participation led to a Ph.D degree in 1986. Since 1995, Anneke works as an independent consultant to the cytopathology
industry and to cytopathology centers that apply LBC and/or CAS. For Becton Dickinson, Anneke has been involved in many of the
morphology training programs for BD SurePath™ LBC slides.
Acknowledgements
We would like to express our respect and gratitude to all those who have participated in the creation of this atlas.
The cytotechnologists from LUMC are greatly acknowledged for preparation of slides and diagnostic expertise: Annette Colijn, Ingrid
van der Linden-Narain, Petra Schreiner-Kok, Joke Moes, Belinda de Jong, Marijn Smit and Anouk Vink.
Special thanks go to BD's marketing and application teams involved in cervical cytology: Mieke Bamelis, Richard van der Biezen, Peggy
Verelst and Ryan Callaghan for their continuous support and positive feedback.
The authors are greatly indebted to Dr Maria Drijkoningen, cytopathologist at the Jessa hospital in Hasselt, Belgium, and to Dr Jason
Stone from Queensland Medical Laboratory in Brisbane, Australia, for their time and professional suggestions, which the atlas has
greatly profited from.
Acknowledgement from BD
BD Diagnostics would like to thank the Consultant Cytology Experts who have created this atlas on BD SurePathTM Cervical Cancer
cytology. From the structure of the book to the pictures of well documented cases, they have delivered an incredible amount of work
over many months that highlights the superior quality of BD SurePathTM Slides.
We hope sincerely that BD SurePathTM cytology users will enjoy using this high quality and well thought out Atlas as a tool to improve
their expertise in cervical cancer cytology.
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Contents
Chapter 1: Anatomy, Histology and Cytology of the Uterus 8
Chapter 2: Cytology Composition 16
Chapter 3: Micro-organisms, Inflammation and Repair 38
Chapter 4: Squamous Metaplasia, Glandular Metaplasia and Glandular Hyperplasia 52
Chapter 5: Atypical Squamous Cells of Undetermined Significance (ASC-US) 60
Atypical Squamous Cells, Cannot Exclude HSIL (ASC-H)
Chapter 6: Squamous Intraepithelial Lesions and Squamous Cell Carcinoma 66
Chapter 7: Endocervical Glandular Cells and Lesions 86
Chapter 8: Endometrial Cells, Atypical Endometrial Cells and Endometrial Adenocarcinoma 100
Chapter 9: Challenges 110
References: 124
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Introduction Chapter 1
Basic knowledge of anatomy and histology is needed to understand the different compartments of the female genital tract and the cells
and products these can shed in the cervical smear.
When we imagine the route of the 'ovum' from ovary to vagina, we pass the uterine tubes, the cavum uteri, the isthmus, the endocervix
and the ectocervix.
All physiological processes in either of these compartments, benign as well as malignant, can have an impact on the cytomorphology
of the cervical smear.
In chapter 1 we will look at the anatomy of the female genital tract and describe the subsequent histological and cytological cell images
that can be found.
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Chapter 1
Anatomy, Histology and Cytology of the Uterus
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Female Genital Organs - Anatomy
Cavum
Fundus Ampulla
Uterine (Fallopian) tube
Body (corpus) of uterus

Fimbriae Ovary
Isthmus
Infundibulum The World's Best Anatomical Charts
Endocervical canal Ostium ©2013 Wolters Kluwer Health
Lippincott Williams & Wilkins. All rights
reserved.
Cervix of uterus
Vagina
Anatomy
The uterus is a hollow muscular organ and is divided into a fundus, body, isthmus and cervix. Uterine tubes make connections between
the uterus and surface of both ovaries. They are open to the cavity of the uterus and to the peritoneal cavity. The eggs pass through
these oviducts to the uterus.
The body and the fundus are lined by endometrium, which undergoes changes during the menstrual cycle.
The isthmus is a short, narrowed portion between the body and the cervix. Here the endometrium passes over in endocervical mucosa.
The cervix is a rounded, distal part of the uterus with a central canal which projects into the vagina.
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Female Genital Organs - Histology
A B C
Ciliated tubal epithelium with Endometrium lines the cavity of Cortex of ovary with follicles and
secretory cells line the fallopian uterus and consists of glands with surface epithelium.
tubes. supporting stroma.
B
C
E The World's Best Anatomical Charts

©2013 Wolters Kluwer Health
reserved.
D F
Stratified squamous epithelium of Isthmus where corpus and cervical

the ectocervix. canal meet.
Columnar epithelium of the

endocervical canal.
Tuba, endometrium, isthmus, endocervix and ectocervix show very special mucosa with many physiological changes.
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Female Genital Organs - Histology
A B C
Tubal epithelium showing ciliated Glandular epithelium of endome- The surface of the ovary is covered
cells, scanty secretory cells and trium and supporting stroma. with cuboidal or low columnar
thin delicate stroma. epithelium. Benign calcification in
stroma can be noted
B
C
E
The World's Best Anatomical Charts
reserved.
D
D F
Epithelium of the ectocervix is Endometrium and columnar epithe-

non-keratinizing, stratified, lium of the endocervical canal meet
squamous and rich in glycogen. at the isthmus. Note highly cellular
endometrial stroma.
Mucin-secreting cells and ciliated

cells of the endocervical canal.
All mucosa details show cellular complexity. Knowledge of histology is essential for better understanding of cytological correlation.
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Architectural patterns in cytology and correlation with histology
A B C
Strips of tubal epithelial cells in When endometrial cells are found Cuboidal cells of the ovary surface
cytology show the same physiological in cytology, they appear as 3- epithelium. These cells are rarely
“atypia” as in histology. dimensional clusters of glandular seen in the cervical sample.
cells.
B
C
E
reserved.
D
D F
Cells of the ectocervix are Cells from the Lower Uterine Seg-
recognized as sheets of squamous ment (LUS) can be recognized by
epithelial cells, similar to the the pre-sence of endocervical
squamous cells of the vagina. columnar cells with endometrial
stromal cells, as found in the
Endocervical columnar cells are isthmus.
often seen in cytology in
honeycomb formation.
Cellular arrangemens reflect the architecture of the tissue.
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Different types of cells and cell arrangements in BD SurepathTM LBC

reserved.
Anatomical continuity from surface epithelium of the ovary to the cervix and vagina makes cervical cytology interpretation very complex.
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Squamocolumnar Junction And Transformation Zone
B. Normal Endocervical Mucosa: Shows

papillary projections and clefts lined by simple
columnar mucinous epithelium
A. Original/Native Squamocolumnar Junction

(SCJ): Is the meeting point of native squa-
mous epithelium of ectocervix and endocer-
vical columnar epithelium. This native SCJ
remains constant throughout life.
D. New Squamocolumnar Junction: This is a

junction between metaplastic epithelium and
glandular epithelium. This junction can vary in
location throughout the reproductive life.
C. Transformation Zone: This is an area of

endocervical mucosa where metaplastic
process occurs and new SCJ is formed. It is
also the most common place on the cervix for
abnormal cells to develop.
The transformation zone is the targeted area for cytology, colposcopy as well as histology.
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In Chapter 2, a description is given of all normal cell types that can be found in a cervical smear. There are epithelial cells from
ectocervix, transformation zone, endocervix and endometrium, as well as the different inflammatory cells.
With the improved sampling devices that are used in combination with the BD SurePath™ Liquid-Based Pap test, cells from the Isthmus
are seen more frequently in these preparations. These are also described in this chapter.
Cell morphology and the architectural patterns in cellular arrangements are described for all normal compartments as they occur in the
Female Genital Tract.
All cells described in this chapter are classified as Negative for Intraepithelial Lesion or Malignancy (NILM) within The Bethesda System
classification.
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Chapter 2
Cytology Composition
Knowledge and recognition of normal cells in a cytology
slide is essential in the diagnostic process.
Superficial Cells
Intermediate Cells
Parabasal Cells
Basal Cells
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Superficial cells
Large polygonal cells, 45-50 µm in diameter. Histological section showing fully mature
Cytoplasm is eosinophilic and stains pink. Nuclei squamous epithelium with surface of superficial
are small (2 µm in diameter), round and cells. Black line is enlarged in right upper circle
pyknotic. and indicates region of superficial cells.
The cytoplasm of less mature The shrunken nucleus can be sur-

superficial cells stains greenish- rounded by a small clear zone. The
blue. cytoplasm may contain kerato-
hyaline granules.
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Intermediate cells
Polygonal cells, 35-40 µm in diameter. Slightly Histological section showing fully mature
smaller than superficial cells. Cytoplasm is squamous epithelium. Black line is enlarged in
cyanophilic and stains greenish-blue. Nuclei are right upper circle and indicates region of
vesicular and about 8 µm in diameter. intermediate cells that are connected with
intercellular bridges.
Vesicular nuclei have finely Navicular cells (boat shaped) with

granular, evenly distributed thickened cell borders and eccen-
chromatin, with prominent tric nuclei are commonly seen
chromocenters and a smooth during pregnancy.
nuclear membrane.
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Parabasal and basal cells
Round-oval cells, 15-30 µm in diameter. Cyto- Histological section showing fully mature
plasm is cyanophilic and stains dense greenish- squamous epithelium. Black line is enlarged in
blue. The nucleus occupies about half of the cell. right upper circle and indicates region of para-
basal cells with hyperchromatic nuclei.
Sheet of parabasal cells with high Basal cells have slightly larger
N/C ratio. Nuclei have a smooth nuclei and increased N/C ratio.
nuclear membrane, uniform chro- The nuclear membrane is smooth.
matin distribution and small Basal cells are only sporadically
nucleoli. found in cervical smears.
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Columnar cells of the endocervix
Endocervical cells can be found in strips, sheets Histological section showing the endocervical
and single. Nuclei are basally located, but may canal lined by columnar cells with basally
be perceived centrally depending on the located nuclei.
orientation of the cell.
Endocervical cells are often Two types of endocervical cells

found in honeycomb sheets with can be distinguished: nonciliated
clearly defined cell borders and columnar shaped cells with large
rounded edges. amounts of mucus and ciliated
cells with tufts of cilia.
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Cells of squamous metaplasia
Metaplastic cells
Mature metaplastic cells are almost the same Mature metaplastic epithelium in the endocer-
size as intermediate cells, with cyanophilic cyto- vical canal can be indistinguishable from
plasm staining blue-green. The cells are often squamous intermediate cells.
rounded. Nuclei are vesicular and may vary in
size. They have finely granular chromatin and
nucleoli may be visible.
Immature metaplastic cells are Histological section showing the

the same size as parabasal cells endocervical canal with columnar
with cytoplasmic projections cells on the surface and squamous
(spider cells). metaplasia between the columnar cells
and the epithelial basal membrane.
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Endometrial cells
Endometrial cells can be found in loose groups Histological section of secretory endometrium.
with intact cytoplasm. Note intracytoplasmic vacuolization.
Endometrial cells are often found Histological section of prolifera-

in compact three-dimensional tive endometrium. The columnar
rounded clusters. Nuclei are shape of the cells in histology is
small (maximum the size of an often lost during the exfoliation
intermediate cell nucleus), bean process.
shaped, with evenly dispersed
chromatin and small nucleoli.
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Non-epithelial inflammatory cells
Firm sampling may result in the presence of Histological section showing endocervical
small tissue fragments, with endocervical cells epithelium with stromal lymphocytes.
and large amounts of stromal lymphocytes.
Inflammatory processes may Histological section showing ero-

lead to many granulocytes in the sion and many granulocytes in the
slide. upper layer of the squamous
epithelium.
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Lower Uterine Segment (LUS) / Isthmus
Histological section of isthmus, showing endo- Isthmus is composed of endocervical epithelium

cervical glands with endocervical stroma in the overlying endometrial glands. This can be seen
lower part and more densely nucleated, endo- in this Vimentin stained section where endo-
metrial stroma in the upper part of the picture. metrium cells stain positive. The image in the
Circle shows high magnification of typical circle shows the dense endometrial stroma in
endocervical stroma. H&E staining.
Samples of LUS are characteri- Strips of endocervical columnar

zed by the presence of biphasic cells with underlying endometrial
micro-biopsies in cytology, com- stroma cells can be identified in
bining well preserved columnar both histology as well as cytology.
endocervical cells with endo-
metrial stromal cells.
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Cytoplasmic features
Cytoplasmic staining ingredients

of the Papanicolaou staining are Eosin stains superficial cells pink
Eosin and Orange G
Eosin stains less mature cells light green
Orange G stains keratinizing cells orange
The cytoplasmic features of squamous epithelial cells
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Nuclear features
8µm
Absolute nuclear size: (diameter of measurement)

usually expressed in comparison to normal
intermediate cell nucleus.
Relative nuclear size (ratio of the size of nucleus to the size

of cytoplasm) = Nuclear to Cytoplasmic ratio = N/C ratio
Intensity of nuclear staining as compared to the

normal intermediate cell nucleus: normochromasia,
hyperchromasia, hypochromasia.
Chromatin distribution: uniform, irregular,

coarse, finely granular, speckled.
Nuclear shape: oval, round,

bean shaped etc.
Regularity of nuclear membrane: smooth, angular or wrinkled.
Presence and regularity of nucleoli.
General nuclear features
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Architectural patterns in cellular arrangements
Raspberries/mulberries Endometrial cells Morula formation
Corn Isthmus cells Papillary formation
Flower stem with insects Vascular structures Perivascular
Olive tree leaves Metaplastic cells Storiform
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Architectural patterns in cellular arrangements
Pavement Mature squamous cells Pavement
Honeycomb structure Endocervical cells Honeycomb structure
Strip/Palisade Endocervical cells Little soldiers
Acinary/rosette formation Columnar cells Flower
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Smear patterns
Proliferative phase (Day 4-14 of menstrual cycle)
Superficial cells with pink cytoplasm dominate the image.

Superficial cells that are not fully matured, stain light green/
grey. The nuclei are small and pyknotic.
Mid cycle or ovulation phase (Day 14-16 of menstrual cycle)
A mixture of superficial and intermediate cells are seen.

Intermediate cells stain light green/grey and display
vesicular nuclei.
Luteal phase (Day 16-28 of menstrual cycle)
Intermediate cells with light green/grey cytoplasm dominate

the image. The nuclei are uniform with round-oval shapes.
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Smear patterns
Cytolysis
At the end of the luteal phase, clustered intermediate cells

dominate the image. This is due to an increase of Döderlein
bacilli that lyse the cytoplasm of the epithelial cells (cytolysis)
and cause this pattern.
Menstruation
Clumps of left over old blood can be found in combination

with endometrial cells. All cells show normal morphology.
No necrosis. Due to the "cell enrichment" process used in
preparation, BD SurePathTM slides will have minor portions
of old blood present, with little or no overlap with epithelial
cells.
Postpartum
Increased numbers of parabasal cells, often showing high

amounts of glycogen and somewhat angular shapes.
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Many faces of atrophy
Single parabasal cells with karyopyknosis. Degenerated

cells in the background. The so called 'blue blobs' can in
fact have multiple stain uptakes and be dark red or
grey.
Single pseudoparakeratotic cells with small pyknotic

nuclei are often seen in atrophic cervicitis.
Cytomorphological details such as pyknosis are very clear.
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Many faces of atrophy
Deep atrophy.
Large cohesive sheets of parabasal cells can dominate in

deep atrophy. Nuclei are small, round-oval and normo- or
hyperchromatic with even distribution of chromatin. The
pattern of cells within the group is very monotonous.
Some oral contraceptives can induce atrophic changes.

Typically navicular-like cells can be observed with
intense cytoplasm staining of cell periphery resulting in
'pseudo-koilocytic' changes. Nuclear features
are normal.
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Manual screening of a BD SurePath™ LBC slide.
Use 10x objective
10x
Stepwise screening through the entire slide.
Overlap microscopy fields with 30% in X and Y direction.
20x
Frequently increase to 20x and 40x magnification
40x
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Screening challenges
Abnormal cells may be adjacent to

groups of normal cells. The human eye is
easily distracted by groups, thereby
overlooking (single) abnormal cells.
Single abnormal cells may be small in size.

Meticulous searching will warrant detection.
Abnormal cells are not always

hyperchromatic. Pale cells are easily
overlooked.
Hyperchromatic crowded groups should

always be inspected at higher magni-
fication.
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Discussion.
The vast majority of cellular elements in a cervical sample is benign. In cervical cytology we do not know exactly where the cells are
collected; neither do we know if these cells are directly sampled or exfoliated. Hence the complexity in recognizing all the different cell
types.
In the conventional smears, different cells from the same population often stick together, entangled with mucus. In a LBC slide, this
pattern is no longer present and the individual cells should be recognized using the individual cell features.
Typical smear patterns, such as cytolysis and atrophy, have slightly different appearances in LBC as compared to the conventional
smear. Cytolysis is typically accompanied by many naked nuclei in a mass of Döderlein bacilli. In the BD SurePathTM LBC specimens, the
background becomes more clean and the groupings of squamous cells dominate.
The most apparent change in atrophic LBC cases is the presence of single cells and the lack of red color and swollen cells that are
common in atrophic conventional smears, often suffering from air-drying. The fact that fixation and staining of the cells in a BD
SurePathTM LBC slide is standardized, helps us to use the cellular features reproducibly.
In this chapter the different normal cells and cell patterns have been described and nuclear and cytoplasmic features have been
documented. The guidelines for screening of a BD SurePathTM LBC slide have been discussed.
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Note!
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Most frequent micro-organisms of the female genital tract, both commensal as well as pathogenic, will be in this chapter.
Inflammatory processes, degeneration and regeneration, can all cause specific cytomorphologic changes that mimic neoplastic cells.
Correct recognition of these changes prevents false positive diagnoses.
All cellular changes from processes mentioned in this chapter are categorized as Negative for Intraepithelial Lesion or Malignancy
(NILM) in The Bethesda System, unless mentioned otherwise.
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Chapter 3
Micro-organisms, Inflammation and Repair
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Micro-organisms
Lactobacillus (Döderlein's bacillus)
Lactobacilli are rod-shaped bacteria, 3-6 µm in size.

In BD SurePath™ slides they are visible in small
colonies on the surface of squamous cells.
Actinomyces-like organisms
Gram positive bacteria known to colonize an IUD.

'Cotton balls' of filamentous organisms are recognizable
at low power. Many leukocytes can be seen in the
background.
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Micro-organisms
Trichomonas vaginalis
A pear shaped organism, 15-30 µm in diameter.

Nucleus is small, pale and vesicular.
Small, internal structures (red granules) and flagella
are often better identified in BD SurePath™.
Note the typical bicolor cytoplasm in squamous cells in

the background. Nucleus is ghost-like with a small
perinuclear halo.
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Micro-organisms
Candida species
Budding yeasts. Pseudohyphae are eosinophilic and

often stain bright red. Spearing of squamous cells with
hyphae ('kebab structures') is typically seen at low
power. Squamous cells may show degenerative
vacuolization of the cytoplasm.
Hyphae in a degenerative state may stain gray.
Fungal spores have the tendency to clump together in

groups of epithelial cells. The background of the slide is
usually clean.
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Micro-organisms
Herpes Simplex Virus (HSV)
Presence of multinucleated cells. Nuclei have a ground

glass appearance due to accumulation of viral particles.
Clear margination of nuclear chromatin is also noted.
Intranuclear viral inclusions can be visible as large dark
bodies in the center of the nucleus.
Multinucleated giant cells.
Think of 3 x M for HSV

- Margination of chromatin
- Molding
- Multinucleation
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Micro-organisms
Leptothrix vaginalis
Hair-like unbranching filaments approximately 80 µm

in length that typically stain pale grey. Often seen in
conjunction with Trichomonas infection.
Bacterial vaginosis (usually caused by Gardnerella

vaginalis)
Small, rod-shaped, gram negative cocco-bacillus. These

bacteria stain blue in Papanicolaou. Squamous cells are
covered by several layers of these cocco-bacilli, forming
so called 'clue cells'. The background is typically clean
without granulocytes.
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Micro-organisms
Human Papilloma Virus (HPV)
The presence of koilocytes is typical for a HPV infection.

Squamous cells show large, well defined, perinuclear
halo's and a thick cytoplasmic rim. Nuclei are finely
granular or pyknotic. Binucleation is often present.
The presence of koilocytes leads to a

diagnosis of LSIL in The Bethesda System.
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Inflammation
Acute cervicitis
In acute cervicitis, the inflammatory cells consist

predominantly of neutrophils. The epithelial cells show
specific inflammatory changes, both degenerative as
well as regenerative. The background may show
sheets of mucus with entangled granulocytes.
A sheet of endocervical cells with many neutrophils

outside as well as inside. The nuclei will show finely
dispersed chromatin.
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Inflammation
Degenerative changes / Cell injury
Nuclei:
- Enlargement
- Vacuolization
- Condensation of chromatin at nuclear membrane
- Wrinkling of nuclear membrane
- Nuclear fragmentation or karyorrhexis
- Nuclear shrinkage or karyopyknosis
- Nuclear lysis or karyolysis
Cytoplasm:
- Vacuolization
- Leucophagocytosis
- Perinuclear halo
- Eosinophilia
Regenerative changes / Repair
Nuclei:
- Enlargement
- Multinucleation
- Hyperchromasia
- Evenly distributed chromatin
- Enlarged nucleoli
Cytoplasm:
- Vacuolization
- Syncytial formation
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Inflammation
Follicular cervicitis
Follicular Cervicitis (FC) in BD SurePath™ may be

slightly different from FC seen in conventional smears.
Small fragments of tissue with lymphocytes, plasma
cells and macrophages, in combination with stromal
cells and endocervical cells on the periphery, can be
seen. Look for tingible body macrophages.
Atrophic cervicitis
Parabasal cells showing degenerative changes.

(so called 'blue blobs'). Clumping of bare nuclei.
Cytoplasmic changes show a staining spectrum from
green to orange. Neutrophils are noted in
the background.
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Reactive changes
Radiation and chemotherapy
- Marked nuclear and cellular enlargement

- Cytoplasmic vacuolization
- Nuclear wrinkling, and hyperchromasia
- Multinucleation
- Bizarre cell shape
Bizarre cell shapes can be seen with strong cytoplasmic

vacuolization and nuclear hyperchromasia, but without
increase in N/C ratio.
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Discussion
Different micro-organisms can be recognized in the BD SurePathTM slide. Their morphology is similar to those in conventional smears.
Inflammatory and regenerative processes can result in cellular changes that mimic (pre-)neoplastic changes. Care should be taken
to recognize and correctly interpret these.
Physical, chemical, infectious agents and drugs can lead to cell injury with typical morphological manifestation such as nuclear
enlargement, chromatin condensation and wrinkling of nuclear membrane with cytoplasmic vacuolization and membrane blebs. This
can lead to 'atypical' appearances.
After damage follows regeneration and repair with replacement of lost structures and re-epithelialization. The process of repair
starts by the proliferation of adjacent epithelium and is very apparent in cytology. Sheets of immature metaplastic cells and sheets
of columnar cells show large nuclei and prominent nucleoli.
Degenerative changes with inflammation and regenerative changes associated with repair can be found in the same smear with
possibility for misinterpretation.
Degenerative changes Regenerative changes
Nuclei: Nuclei:
- Enlargement - Enlargement
- Vacuolization - Multinucleation
- Condensation of chromatin at nuclear membrane - Hyperchromasia
- Wrinkling of nuclear membrane - Evenly distributed chromatin
- Nuclear fragmentation or karyorrhexis - Smooth nuclear membrane
- Nuclear shrinkage or karyopyknosis - Enlarged nucleoli
- Nuclear lysis or karyolysis
Cytoplasm: Cytoplasm:
- Vacuolization - Vacuolization
- Leucophagocytosis - Syncytium formation
- Perinuclear halo
- Eosinophilia
A typical non-specific inflammatory background consists of many granulocytes. The quantity of these granulocytes in a BD SurePathTM
slide is considerably different to that of a matching conventional slide. As a rule of thumb we can assume that the BD SurePathTM slide
has at least 50% less leukocytes than the conventional slide. Variations exist however, mainly depending on whether these leukocytes
are loose granulocytes or strongly entangled in mucus, as is often the case in chronic inflammation.
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Note!
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The definition of metaplasia is a process in which one differentiated cell type is replaced by another cell type. Metaplasia with found in
association with inflammation, tissue damage, repair and regeneration. Replacement of columnar with squamous epithelium is the
most common epithelial metaplasia and occurs in the cervical transformation zone.
Metaplasia and hyperplasia are common processes in the transformation zone.
Using histology pictures to explain the observed cellular features, this chapter will attempt to clarify and support a correct
interpretation of these benign processes.
All cells mentioned in this chapter are categorized as Negative for Intraepithelial Lesion or Malignancy (NILM) in The Bethesda System,
unless mentioned otherwise.
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Chapter 4
Squamous Metaplasia, Glandular Metaplasia

and Glandular Hyperplasia
Primary SCJ
Ectocervix Endocervix
Normal cervix
Endocervical cells
Reserve cell hyperplasia

and immature metaplasia
Reserve cell hyperplasia

and immature metaplastic
Transitional cells
zone
Secondary SJC
Mature metaplasia
Mature metaplastic cells
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Metaplastic process - Various stages
Tissue section showing reserve cell hyper- Immature squamous metaplasia with
plasia with proliferation of reserve cells columnar cells on the surface and proli-
underneath the columnar cells. feration of reserve cells in the basal
layers. Cytology: metaplastic cells with
spider shape cytoplasm, sharp cell bor-
ders and small intracytoplasmic vacuoles.
The nuclei are enlarged, round, and,
hypochromatic, with smooth nuclear
membranes.
Tissue section showing sharp Mature metaplasia is almost

demarcation between imma- identical to native ectocer-
ture and fully mature meta- vical epithelium. Cytology:
plastic epithelium. Cytology: sheets of intermediate-type
cells with polygonal shape, cells. The cytoplasm is den-
dense cytoplasm and long sely stained and less
cytoplasmic endings, which is angulated. Metaplastic
caused by disruption of the nature can still be
intercellular bridges. recognized.
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Metaplastic process - Various stages
Hyperkeratosis with a thick layer of Parakeratosis with a thin layer of flat,

keratin on the surface. Cytology: keratinized cells on the surface.
orangeophilic cells without nuclei. Cytology: orangeophilic cells with small
pyknotic, slightly elongated nuclei.
A small sheet of immature Immature metaplasia in a

metaplasia, showing large flat sheet of angulated
'spidery', densely stained cells of varying sizes.
cytoplasm and variation in Metaplastic cells have large
nuclear size. The nuclear amounts of cytoplasm and
membrane is slightly bland nuclei. Granulocytes
wrinkled and nucleoli and are present in the group.
chromocenters are present.
55
Microglandular hyperplasia
Tissue section showing numerous cribri- At high power, the small lumina can be
form glands, that are lined with endocer- appreciated, with many reserve cells with
vical cells with proliferation of reserve cells high N/C ratio. The composition of 3 cell
and immature metaplastic cells. The same types often present in tight cell groups in
composition can be seen in cytology in the cytology specimen, makes interpretation
crowded groups. difficult. Always look at the nuclear detail of
the individual cells in the group. This will be
monotonous and lack specific malignancy
features, in spite of the high N/C ratio.
Histology shows intestinal Mucus-secreting goblet cells

metaplasia with typical of colon type in histology
goblet cells. Compare and cytology.
goblet-form with the narrow
form of normal columnar
endocervical cells.
56
Tubal metaplasia
Tubal metaplasia in histology and cytology. Larger sheets can also be found, with
Groups of columnar cells mimicking normal ciliated columnar cells noted at the
tubal epithelium. The presence of pseudo- periphery.
stratification, anisokaryosis and
hyperchromasia is typical for tubal
metaplasia. The cells have visible cilia.
Strip of columnar cells with In cytology we find strips of

pseudo-stratification. Nuclei columnar cells with pseudo-
show finely distributed stratification. Nuclei are round
chromatin and a smooth to oval. Chromatin is hyper-
nuclear membrane. The chromatic and evenly distri-
strip is well defined at both buted. All cells are ciliated both
lateral ends and mirrors in cytology and histology.
tubal epithelium in histology
and cytology.
57
Discussion
The complexity of metaplastic and hyperplastic processes in the endocervical canal, may lead to unusual cell type combinations and
uncertainty in diagnosis.
Understanding the metaplastic process will support the correct identification of these benign conditions.
Criteria that differentiate squamous metaplasia from intermediate and superficial squamous cells include:
- Cells of squamous metaplasia lack intracytoplasmic glycogen

- Metaplastic cells are smaller and have a higher N/C ratio
- Cells are less polygonal
- Cytoplasm is dense and cyanophylic
- Nuclei are centrally located
- Nuclear membrane is smooth
Squamous metaplasia is dominated by single pleomorphic Tubal metaplasia is characterized by the presence of cell
cells with spidery protrusions. groups in glandular strip formation.
58
Note!
59
Within The Bethesda System a diagnostic 'crossroad' has been created between 'normal' and 'abnormal', the category ASC.
Atypical Squamous Cells of Undetermined Significance (ASC-US) is mainly used to categorize those samples that fall between NILM and
Low Grade Squamous Intraepithelial Lesion (LSIL): cells do not fully display features of LSIL, but are too abnormal to be called 'NILM'.
Atypical Squamous Cells - cannot rule out High Grade Intraepithelial Lesion (ASC-H) is used mainly for those smears that are suspicious
for HSIL, but the cytology features or the number of abnormal cells are not sufficient to support a HSIL diagnosis.
Examples are given of both categories.
60
Chapter 5
Atypical Squamous Cells of Undetermined

Significance (ASC-US)
Atypical Squamous Cells - cannot rule
out HSIL (ASC-H)
61
Atypical Squamous Cells of Undetermined Significance (ASC-US)
Atypical parakeratosis ASC-US
A sheet of slightly elongated cells with A sheet of atypical squamous cells with
dense orangeophilic cytoplasm and oval slightly irregular nuclei and perinuclear
nuclei. Nuclei can be hypo- as well as cytoplasmic clearing. These findings are
hyperchromatic, and show minimal insufficient for a diagnosis of LSIL and
irregularity of nuclear membrane. should be categorized ASC-US.
Atypical repair Atypical repair
Metaplastic cells with en- Enlarged nuclei in metaplastic

larged nuclei and slightly cells with slightly coarse
irregular nuclear mem- chromatin and nucleoli.
brane. Anisokaryosis,
abundant cytoplasm and
nucleoli are present.
62
Atypical Squamous Cells - Cannot Exclude HSIL (ASC-H)
Sparse small cells with high N/C ratio. Small groups and single abnormal cells
Note irregularity in the nuclear membrane. with large hyperchromatic nuclei and a
The background is clean. high N/C ratio. Note the irregularity of
the nuclear membrane.
A sheet of metaplastic cells A sheet with metaplastic

with high N/C ratio and type cells and leukocytes.
mildly hyperchromatic Nuclei are enlarged and
nuclei. Chromatin is slightly hypochromatic with nucleoli.
coarse. The nuclear mem- The N/C ratio is high. Irre-
brane is even. gularity in the nuclear mem-
brane and variation of
nuclear size is noted.
63
Discussion
Atypical Squamous Cell (ASC)
Definition:
Squamous cells showing more nuclear changes than expected in benign reactive processes, but not fulfilling the criteria for
Squamous Intraepithelial Lesion (SIL) should be classified as Atypical Squamous Cells (ASC).
2 categories can be distinguished:

ASC-US ASC-H
Atypical Squamous Cells of Undetermined Atypical Squamous Cells - Cannot Exclude

Significance. HSIL.
Microscopy: superficial or intermediate Microscopy: small parabasal cells with
cells with mildly enlarged nuclei and high N/C ratio and nuclear abnormalities
chromatin abnormalities insufficient for not sufficient for a HSIL diagnosis.
LSIL diagnosis. For an ASC-H diagnosis, the specific
For an ASC-US diagnosis, the specific categories of NILM (reactive changes)
categories of NILM (reactive changes) and and HSIL (moderate/severe cervical
LSIL must first be excluded. intraepithelial neoplasia or carcinoma in
situ) must first be excluded
HPV detection with or without phenotyping can be used to guide management of ASC patients.
64
Note!
65
In this chapter, we focus on the cytomorphological features of the cells that lead to a Squamous Intraepithelial Lesion (SIL) /
Cervical Intraepithelial Neoplasia (CIN) or cancer diagnosis.
We follow The Bethesda System but images as well as terminology will be linked to other classification schemes that are used
worldwide.
In the next table we compare the different classification schemes for SIL or CIN lesions.
WHO classification Papanicolaou classification TBS
CIN I Mild Dysplasia LSIL

CIN II Moderate Dysplasia HSIL
CIN III/CIS Severe Dysplasia HSIL
The HSIL category comprises different types of CIN lesions, such as keratinizing CIN, large cell non-keratinizing CIN etc. In terms of
patient management there is no difference for these lesions. Treatment is identical. This explains the fact that all these lesions fall
under the same HSIL category. However, to understand the full range of cytological manifestations of HSIL, the different CIN categories
and their respective cytology appearances, are discussed.
The graphics of the atlas has been designed to easily explain the different morphological features without using too much text; the
pictures should be self-explanatory. In those pages where the left and right page refer to the same category, the left page will focus
on group and 'cells in group' features, whereas on the right page, the single cell features will dominate.
Intermediate cell LSIL HSIL

The nucleus of normal intermediate squamous cell can always be used as a ruler.
66
Chapter 6
Squamous Intraepithelial Lesions and

Squamous Cell Carcinoma
Histology
LSIL / Mild dysplasia / CIN I
- Dysplastic cells occupy lower third of the epithelium

- Cells of upper 2/3 show differentiation and orientation
- The transition between these two layers is well demarcated
- Mitotic figures are infrequent in the lower third
- Nuclear abnormality is present in all levels
- Koilocytic changes are present
HSIL / Moderate dysplasia / CIN II
- Dysplastic cells occupy lower 2/3 of the epithelium

- Cells of upper 1/3 show differentiation and orientation
- Mitotic activity can be found in lower 2/3
- Nuclear abnormality is more marked than in CIN I
- Koilocytic changes are present
HSIL / Severe dysplasia / Carcinoma in situ / CIN III
- Dysplastic cells occupy total thickness of epithelium

- Loss of polarity and nuclear crowding
- Mitotic figures can be found at all levels
- The nuclei throughout the epithelium show marked abnormalities
67
Sheets of intermediate cells with anisokaryosis and koilocytic halos.
Koilocytes can occur singly.
Koilocytes are often observed in

angulated clusters.
Small group of koilocytes with sharply delineated

koilocytic halos and dense cytoplasmic rim.
Sheet of hyperkeratotic cells showing multinucleation and variable

degree of hyperchromasia.
At low power, solitary koilocytes,
as well as those in groups, can
easily be picked up.
68
Large superficial cells with binucleation are commonly seen.

Nuclei are enlarged and hyperchromatic.
Koilocytic changes in a single squamous cell

showing distinct cytoplasmic clearing and well
defined border, sometimes referred to as 'wire
loop' appearance.
Nuclear enlargement with slightly increased

N/C ratio. The coarsely granulated chromatin
is evenly distributed throughout the nucleus.
Nuclear membrane irregularities are mild but can

be clearly observed. Cytonuclear details are
excellently preserved.
Sheet of koilocytes with variability in nuclear size, shape (angulation)

and intensity.
Histology demonstrates mild

dysplasia (CIN I) with koilocytes in
upper layers of epithelium.
69
Sheet of dysplastic cells with large nuclei and moderate amounts of

cytoplasm. N/C ratio is higher than cells observed in LSIL.
Syncytial group of abnormal cells showing

anisokaryosis and hyperchromasia. Nuclear
size is larger than observed in LSIL.
Cytoplasm keratinization and koilocytic

clearing can also be observed in HSIL.
Parabasal cell with large elongated nucleus.

Nuclear invaginations result in typical 'coffee
bean' shape and can often be seen in HSIL.
Small group of parabasal-type cells with large irregular and

hyperchromatic nuclei.
At low power, the dysplastic cell

groups, as well as the single
dysplastic cells, can be easily
picked up in the clean back-
ground. Further examination at
high power will reveal the abnor-
mality criteria as described.
70
Intermediate-type cells with enlarged nuclei and increased N/C ratio.

Nuclear chromatin is fine with several chromocenters.
Abnormal cell with slightly hyperchromatic

nucleus and clearly irregular nuclear membrane.
Indentations and nuclear grooves are present.
Koilocyte with large hyperchromatic

nucleus. Nuclear chromatin shows
abnormal clumping.
Binucleation is also seen in HSIL with large

hyperchromatic nuclei and koilocytic changes.
Koilocytes are sampled from the upper layers of the dysplastic

epithelium (see histology picture). Koilocytes in moderate dysplasia
generally show more pronounced nuclear enlargement and
hyperchromasia than those in low grade dysplasia.
Note: criteria overlap with those mentioned in LSIL. Look specifically

The upper layers show for the presence of HSIL cells when LSIL cells are present in
koilocytotic atypia, suggesting abundance.
LSIL. There is also proliferation of
atypical parabasal cells up to 2/3
of the epithelium.The lesion
therefore qualifies as CIN II/HSIL.
71
HSIL / Severe dysplasia - classic / CIN III
Sheet of hyperchromatic cells with high N/C ratios that may imitate
sheets of squamous metaplasia at low power.
Syncytial group of abnormal cells with enlarged

hyperchromatic nuclei and increased N/C ratios.
Hyperchromatic crowded groups (HCG)

with anisokaryosis are frequently obser-
ved in HSIL lesions. All HCG's and cell
clusters should be examined at high
power.
Small group of dysplastic cells with high N/C

ratio and overlapping, enlarged nuclei.
Dysplastic cells can occur singly with enlarged nuclei, membrane

irregularities and abnormal N/C ratios.
Nuclear abnormality is picked up

by comparison with normal inter-
mediate cells. All suspect cells
and groups of cells should be
further examined at high power.
72
HSIL / Severe dysplasia - classic / CIN III
Abnormal parabasal-type cells display a very high N/C ratio with

large nuclei and small amounts of cytoplasm.
Chromatin is coarsely granular and evenly

distributed. Comparison with nuclei of
surrounding intermediate cells is used to
describe nuclear size as well as nuclear
chromasia.
Abnormal cells show large variation in

size and shape of the nuclei. Nuclear
angulation and membrane irregularities
are common.
Cytoplasm is delicate.
Hyperchromasia can be very prominent. In this cell the hyperchromatic

nucleus also shows severe nuclear membrane invaginations.
Histological picture showing

abnormal cells with large hyper-
chromatic and angulated nuclei
throughout the full thickness of the
epithelium.
73
HSIL / Keratinizing dysplasia / CIN III
Three-dimensional clusters, so called 'pearls', with dyskeratotic and

pleomorphic single cells can be observed in a clean background.
Clusters of atypical parakeratotic cells and

anucleate squamous cells with orangeophilic
cytoplasm are typical for keratinization.
Note: excessive hyperkeratosis and

parakeratosis may obscure underlying dysplastic
changes and can be ASC(US/-H) categorized.
Always look specifically for enlarged irregular
nuclei.
Abnormal cells with relatively a low N/C

ratio and large amount of cytoplasm.
Nuclei show anisonucleosis and bland
chromatin pattern.
Nuclei of large pleomorphic cells display irregular

nuclear membranes. Nuclei of fiber cells and
small keratinizing cells can be hyperchromatic or
pyknotic.
Sheet of polygonal keratinizing dysplastic cells in a clean background.
Histology section showing

abnormal keratinizing surface
epithelium with abnormal cells
and mitotic activity up to the
upper layers of the epithelium.
74
HSIL / Small cell non-keratinizing dysplasia / CIN III
Parabasal-type cells with poor cohesion, high N/C ratio,

hyperchromatic nuclei and dense cytoplasm.
Isolated small dysplastic cells may mimic

histiocytes and can easily be overlooked.
However, the nucleus is enlarged and
hyperchromatic and the cytoplasm is too dense
for histiocytic origin.
Cluster of very small abnormal cells with

hyperchromatic enlarged nuclei when
compared to normal intermediate cells.
Nuclei have a coarse irregular chromatin
pattern and display nuclear membrane
irregularities.
Groups of abnormal hyperchromatic cells may

mimic clusters of endometrial cells. High power
evaluation is needed to correctly interpret the
enlarged hyperchromatic nuclei and squamous
differentiation of the cytoplasm.
Isolated small dysplastic cell with high N/C ratio and enlarged
irregular nucleus. To pick up these cells at low power, the process
of screening a slide should be followed correctly.
Histology section showing
anaplastic-like cells with large,
hyperchromatic and very irregular
nuclei with small amount of
cytoplasm.
75
HSIL / Pale cell dysplasia - hypochromatic / CIN III
Flat rounded sheet of intermediate-type cells with large

hypochromatic nuclei and high N/C ratio.
Sheet of cells with crowded hypochromatic nuclei

and some leukocytes. Cells at the group
periphery can be used to estimate the increase in
N/C ratio and to correctly differentiate these cells
from tissue repair.
Squamous cells with large round nuclei

and anisonucleosis of hypochromatic
nuclei with some clumping of chromatin.
Sheet of abnormal cells with vesicular nuclei with

irregular membrane and abnormal, but
hypochromatic nuclear texture.
Single cells can be found with enlarged round nuclei and coarsely
granular, but evenly distributed chromatin.
Note: the dominant population of abnormal cells will have

hypochromatic nuclei. Few small hyperchromatic cells can also be
found. Histological section showing
large, irregular and empty-
looking nuclei throughout most
of the epithelium.
76
HSIL / Cylindrocellular-like dysplasia / CIN III
Sheet of abnormal cells with nuclear 'streaming'. All nuclei show

the same polarity.
Abnormal sheet of elongated cells, with

enlarged 'streaming' oval shaped nuclei,
mimicking abnormal cylindrical cells.
Elongated abnormal nuclei with slightly

irregular membrane and high N/C ratio.
Some sheets of abnormal cells show clear

squamous differentiation of the cytoplasm.
Isolated abnormal cells may also mimic abnormal cylindrical cells

due to the elongated shape of both the nucleus and the cytoplasm.
The nucleus however is centrally located and the cytoplasm shows
squamous differentiation. Cylindrical cells display columnar form,
Histological section showing basal placed nuclei and more fragile cytoplasm.
elongated cells with elongated
nuclei throughout full thickness of
the epithelium.
77
HSIL / Large cell non-keratinizing dysplasia / CIN III
Atypical metaplastic-type cells with enlarged nuclei and mild nuclear

hyperchromasia.
Flat sheets of dysplastic cells with relatively low

N/C ratio.
Orangeophilic cytoplasm and koilocytic

changes can be seen in the sheets of
dysplastic cells.
Population of large dysplastic cells can show

enlarged nuclei and chromatin clumping as
well as irregular nuclear membranes.
Groups of small HSIL cells with hyperchromatic irregular nuclei

and a high N/C ratio.
Histology showing pleomorphic
dysplastic cells without
maturation.
78
HSIL / Metaplastic dysplasia / CIN III
Sheets of metaplastic-type cells with relatively low N/C ratio but

marked variation in nuclear size and chromasia.
Sheets are cohesive and can be very large

resembling normal squamous metaplasia. The
nuclear detail should be well examined under
high power, showing irregularity of the nuclear
membrane and hyperchromasia.
Single cells are seen with spider-like,

densely stained cytoplasm. The nuclei are
enlarged and round oval with hyper-
chromasia and membrane irregularities.
Suspicious looking cells with enlarged irregular

nuclei, densely stained cytoplasm and increased
N/C ratio.
Enlarged irregular hyperchromatic nuclei with 'spidery'

cytoplasm are typical for this type of lesion.
Also histology can resemble the
benign metaplastic process but
there is loss of polarity and mitotic
activity in the upper layers of the
epithelium.
79
Squamous Cell Carcinoma - keratinizing
Large sheet of dysplastic cells with relatively low N/C ratio and
dense orangeophylic cytoplasm.
Cluster of parakeratotic cells with elongated

pyknotic nuclei.
Pleomorphic cells with large,

hyperchromatic and irregular nuclei.
Presence of fiber cells and small dyskeratotic cells.
‘Pearl' formations with atypical nuclei are easy to find.
At low power, the enormous

variation can be picked up, in
groups and solitary cells, as well
as ghost cells and necrotic
elements.
80
Squamous Cell Carcinoma - keratinizing
Pleomorphic cell with large hyperchromatic nucleus, irregular

distribution of chromatin and relatively large amount of dense
cytoplasm.
Marked variation in size and shape of nuclei.

Chromatin pattern is coarsely granular and
irregularly distributed.
Fiber cells with tiny elongated nuclei.
Large elongated cell with hyperchromatic nucleus.
Tumor diathesis in the background is essential for the diagnosis of

invasive carcinoma but is less pronounced than in non-keratinizing
carcinoma.
Histology section showing invasive
keratinizing carcinoma. White
circles indicate the presence of
laminated keratin whirls and
hyperkeratinized surface.
81
Squamous Cell Carcinoma - non keratinizing
Cells occur in compact rounded groups with hyperchromatic

abnormal nuclei.
Cells occur in sheets with large variation in

nuclei; large hyperchromatic, as well as irregular
but hypochromatic nuclei can be identified.
Nucleoli can be large and easily visualized.
Small sheet of metaplastic looking cells

with large, slightly hyperchromatic
nuclei.
Single tumor cells are scattered in the

background. Nuclei are irregular with large
nucleoli.
Fibrillar and granulated material in the background (remains of

from old blood and protein debris), combined with tumor necrosis,
form the typical tumor diathesis.
The low power image shows highly
abnormal cells, single as well as in
groups, in a background that is
highly suspicious: fibrillar
proteinous material and necrotic
elements.
82
Squamous Cell Carcinoma - non keratinizing
Single neoplastic cells with high N/C ratio.
Chromatin is coarsely granular, with chromatin

clumping and irregular distribution.
Highly abnormal cell with irregular

nuclear membrane. The abnormal macro
nucleoli are visible.
Large variation in nuclear size and shape. Squamous

differentiation of the cytoplasm is still visible and
nuclei are centrally placed.
Detail of tumor necrosis: delicate, finely granular material and nuclear

debris.
Histology section showing the

surface epithelium of a non-
keratinized squamous carcinoma.
The pleomorphic nuclei are well
visible.
83
Discussion
In this chapter, an attempt has been made to present different manifestations of squamous abnormalities in BD SurePath™ LBC
specimens. The different histological appearances, combined with concurrent cytology images, show a large range of lesions that lead
to a SIL classification.
Summarizing the cytology criteria that lead to SIL, the following features apply:
- Increased nuclear size (compared to a normal intermediate cell nucleus)

- Increased N/C ratio
- Abnormal nuclear texture (hyperchromasia, coarse chromatin, increased number of chromocenters)
- Nuclear membrane irregularities
- Nuclear pleomorphism
Koilocytosis or koilocytotic atypia is a common feature of SIL and, when strictly defined, is indicative of the cytopathologic effect of HPV
on the squamous epithelium in the lower female genital tract.
LSIL HSIL
vs.
The main criteria to differentiate between LSIL and HSIL are N/C ratio and nuclear abnormality. It should be noted that in many HSIL
lesions, LSIL cells predominate. Cytology classification should be based on the most abnormal cells present in the slide. Searching for
HSIL cells must occur when LSIL cells are found.
HSIL SCC
vs.
Differentiating SCC from HSIL on the basis of cytology alone requires the presence of numerous highly abnormal cells in combination
with tumor necrosis.
84
Note!
85
Endocervical Glandular Cells and Lesions.
The sampling method of BD SurePathTM , where the tip of the device is placed in the vial, leads to a presence of endocervical component
in the vast majority of the slides. Smears contain well preserved, large sheets of endocervical columnar cells and small tissue fragments
of endocervical mucosa (microbiopsies) containing epithelial cells with underlying stroma.
In Chapter 2, the morphology of columnar cells and architectural patterns have been described in detail. Reactive changes have been
highlighted (Chapter 3) and criteria of tubal metaplasia and microglandular hyperplasia have been discussed (Chapter 4).
In this chapter, benign endocervical cells are described in more detail, followed by an elaborate description of a variety of glandular
abnormalities that fall under different categories of The Bethesda System as there are: Atypical Endocervical Cells: Not Otherwise
Specified (NOS), Atypical Endocervical Cells - Favor Neoplastic, Adenocarcinoma In Situ (AIS) and Invasive Adenocarcinoma.
The diagnosis Atypical Glandular Cells - NOS has been defined by TBS as: 'endocervical-type cells display nuclear atypia that exceeds
obvious reactive or reparative changes but that lack unequivocal features of endocervical adenocarcinoma in situ or invasive
adenocarcinoma'. All reactive or reparative changes should be excluded when a diagnosis of “Atypical Endocervical Cells” is made.
For the diagnosis Atypical Endocervical Cells - Favor Neoplastic, TBS adds: 'Cell morphology, either quantitatively or qualitatively falls
just short of an interpretation of endocervical adenocarcinoma in situ or invasive adenocarcinoma'.
The diagnosis Endocervical Adenocarcinoma In Situ - AIS has been described as: 'High-grade endocervical glandular lesion that is
characterized by nuclear enlargement, hyperchromasia and mitotic activity, but without invasion'.
Histology images are used to explain the observed cytological images.
86
Chapter 7
Endocervical Glandular Cells and Lesions
87
Normal Endocervical Cells
Sheet of columnar cells with honeycomb formation.
Strip of columnar cells in a palisading formation.

The nuclei are slightly elongated and positioned
at the basal level of the cell.
Sheet with honeycomb pattern and row of

palisading cells at the periphery.
Cells may show tendency for semi-circular

formation, but nuclear membranes remain
smooth.
Small sheets of columnar cells with mildly overlapping, smooth,

round-oval nuclei can be present.
At low power, we can find many

single cells and groups of
columnar cells that look slightly
hyperchromatic.
88
Normal Endocervical Cells
Single columnar cell with well defined terminal plate and cilia. The
cell is tall and slender. The nucleus is basally placed and round with
finely granular chromatin.
Tall columnar cell with intracytoplasmic mucin.

The nucleus is round-oval, hypochromatic, with
nucleolus.
When looked at 'on end', nuclei of

endocervical cells are centrally located.
Nuclear shape is round. Nuclear mem-
brane is smooth with vesicular chromatin.
Cytoplasmic borders are fuzzy. Nucleoli
can be present, but are not always easy
to differentiate from chromocenters.
From a lateral perspective, nuclei are basally

located. They are round-oval with smooth
nuclear membranes and small nucleoli.
'Microbiopsy' with stromal cells in the center and columnar cells at

the periphery.
Endocervical glands are not true

glands but cleft like infoldings of the
surface epithelium with numerous
blind, tunnel-like channels.
89
Atypical Endocervical Cells - Not Otherwise Specified (NOS)
Small compact group of columnar cells showing loss of polarity and

overlapping nuclei.
Short strip of columnar cells showing

anisokaryosis. Nuclei have slightly irregular
membrane and small nucleoli. Minimal deposition
of chromatin at the nuclear periphery is noted.
Hyperchromatic crowded group showing

overlapping nuclei and loss of polarity.
Some cells at the periphery of the group
can be identified as columnar.
Nuclear crowding and nuclear overlap in a group

of cells with columnar shape.
Hyperchromasia, increased N/C ratio and some variation in nuclear

shape can be present. Cytoplasm is dense with some vacuolization.
BD SurePath™ slides show many

sheets, 'disordered' hyperchro-
matic crowded groups and single
cells. Background is usually clean.
90
Atypical Endocervical Cells - Favor Neoplastic
Sheet of columnar cells with hyperchromasia, nuclear crowding

and overlapping.
Small rosette-like formations with peripheral

abnormal nuclei. Nucleoli may be present.
Small strips with pseudo-stratification.

Nuclei show variation in size and shape,
irregular membranes and hyperchromasia.
Nuclei can be enlarged. The N/C ratio is

increased. Note coarse granulation of chromatin.
Cell borders are ill-defined. Nuclear abnormalities in combination

with columnar morphology of cells are important diagnostic clues.
Columnar origin can be identified by cell shape as well as by basal
location of nuclei.
This histological section shows a
very small Adenocarcinoma In Situ
lesion (dark staining glands). The
majority of glands are benign.
91
Adenocarcinoma In Situ
Sheets, strips and part of rosettes can be present in one group of

columnar cells.
Typical rosette with peripheral hyperchromatic

nuclei that show overlapping and crowding.
Rosette-like formations with crowding of

hyperchromatic nuclei. Note columnar
shape of cells on edge.
Short strips with pseudo-stratification of

elongated irregular nuclei.
Strip with elongated cigar-shaped nuclei showing pseudo-

stratification. These are common findings for these lesions
in BD SurePathTM specimens and is often referred to as
'bird-tail' like strips. The low magnification image is
dominated by columnar cells in
strips, sheets, rosettes as well as
single cells. Background is clean.
Dominance of glandular cells is
characteristic.
92
Adenocarcinoma In Situ
Columnar shaped cells with high N/C ratio and overlapping nuclei.
No cilia are present.
Dense group of small nuclei showing some size

and shape variation. Small nucleoli are visible.
Nuclear membrane is irregular and can be

angular. Chromatin is coarse, which is
characteristic for AIS cells.
Mitotic activity can be seen in groups.
Single cells with elongated abnormal nuclei and indistinct

cytoplasm with ill-defined cell borders. Nipple protrusion of
Adenocarcinoma In Situ in nuclei (in circle) can be seen as found in cells of glandular
histological section: Glandular origin.
architecture is preserved, but
normal endocervical epithelium is
replaced by neoplastic columnar
cells.
93
Endocervical Adenocarcinoma
Sheet of tumor cells with abnormal hyperchromatic nuclei. Nuclear

shape can be angulated. Due to nuclear overlap, the monomorphic
honeycomb pattern is lost.
Berry-like group with large crowded nuclei.

Group is more rounded than in conventional
slides.
Small tissue fragment of gland structure

with small crowded nuclei with prominent
nucleoli. Note cylindrical cells at the lower
edge of the group.
Feathering formation of columnar shaped cells

with elongated, cigar-shaped nuclei and some
surrounding single cells.
Small rosettes can be present with peripheral location of

abnormal nuclei.
The low magnification image is
dominated by abnormal glan-
dular cells with surrounding tumor
diathesis and necrotic ele-ments
in the background.
94
Endocervical Adenocarcinoma
Single cells with columnar shape and irregular nuclear outline.
Nuclei overlap and are enlarged with irregular and

angulated shapes. Chromatin is coarsely granulated
and varies within the group.
Nuclei can also be hypochromatic with

macro-nucleoli.
Cytoplasm can be vacuolated.
Remains of tumor diathesis and necrosis should be

identified and is essential for differentiation from AIS.
Histological section of endo-

cervical adenocarcinoma showing
atypical, irregular glands, that
are lined with neoplastic
columnar cells.
95
Villoglandular Adenocarcinoma
Large chunks of fibrovasculair stroma covered with a layer of

relatively small cuboidal cells showing mild atypia.
Part of papillae with fibrovasculair stroma,

covered with one layer of mildly atypical
glandular cells on both sides.
Small strips with pseudo-stratification. Note

the high N/C ratio of the cells and the
apoptotic bodies present in the strip.
Small groups of glandular cells with

hypochromatic nuclei showing overlap and
crowding. Mitotic figures and apoptotic bodies are
present. Nuclei are oval with irregular membrane.
Strip with crowding of small, oval nuclei with granular chromatin and
prominent nucleoli.
At low power, large tissue

fragments are presented with
tumor necrosis in the back-
ground. Low power architectural
abnormalities are very important
for recognition of this rare tumor.
96
Villoglandular Adenocarcinoma
High power view of histology of papillae showing stroma with

surrounding, small columnar cells, with high N/C ratio. Image is very
similar to cytology depicted on the left page.
Small strip with strong pseudo-stratification.

Group contains some pyknotic nuclei as well as
well-preserved atypical nuclei.
Staining of tissue section with P16 shows

the clear palisading of the tumor cells.
Small disorganized group of columnar cells with

mild atypia and prominent nucleoli. Very high
N/C ratio is the diagnostic clue to this specific
variant of cervical adenocarcinoma.
KI 67 staining in this tissue section shows the proliferative status

of the tumor cells as well as the abnormal nuclear morphology.
In the histology section, a

villoglandular tumor is a malig-
nant papillary process with broad
fibrovascular stroma. The papillae
are lined with small columnar cells
with mild atypia.
97
Discussion
In this chapter, Adenocarcinoma In Situ and Adenocarcinoma have been described.
In summary, the following changes can be observed in these lesions:
Architectural features Cellular morphology
- Honeycomb sheets with hyperchromatic and crowded nuclei - Columnar shape is retained
- Pseudo-stratified strips - Nuclei are enlarged, oval or elongated
- Rosette formations - N/C ratio is high
- Compact crowded clusters - Nuclear membrane is irregular
- 'Bird-tail' like short strips - Chromatin is coarse or finely granular
- Nucleoli can be prominent (especially in invasive lesions)
- Cytoplasm is diminished (in honeycomb sheets, cell
borders are still visible)
- Mitotic activity can be seen in groups
Against the clean background, cell groups have more three-dimensional appearances and look more dense. Peripheral feathering is
more subtle than in conventional smears. Cases with many single abnormal cells are known.
Invasive adenocarcinoma cases show similar architectural patterns to AIS but with tumor diathesis in the background. The cytonuclear
atypia can be more pronounced. Macro-nucleoli are typical in invasive lesions.
The most recent WHO classification of Glandular Tumors and Precursors of The Female Genital Tract (2003) recognizes a variety (13) of
histological types. These have not been described individually in this chapter as the differences are not easily recognized in cytology.
One exception is made for the villoglandular adenocarcinoma. This well differentiated adenocarcinoma is highlighted because of its
specific cytological patterns. The villoglandular adenocarcinoma consists of large fragments of stroma, that are lined by very small, low
columnar pseudo-stratified epithelium with only mild cytological atypia that can be easily mistaken for normal glandular cells. However
N/C ratio is extremely high.
In general, well differentiated adenocarcinomas retain glandular morphology. In cytology, poorly differentiated adenocarcinomas can
be difficult to differentiate from poorly differentiated squamous carcinomas.
98
Note!
99
In this chapter, we focus on the morphology of normal and abnormal endometrial cells as we find them in the BD SurePathTM slide.
Clinical information is important when interpreting the presence of endometrial cells in the cervical sample. Normal endometrial cells
can be found in the cervical sample in the first 2 weeks of the menstrual cycle or in association with benign conditions such as polyps,
immediate post partum, abortion, IUD use, leiomyoma, endometritis and also in hyperplasia, with and without atypia, and adenocarcinoma.
Most malignant endometrial conditions will also have abnormal endometrial cells in the sample.
In a cervical sample, a major difference between endocervical and endometrial glandular cells, benign as well as malignant, is the
effect of the collection method; endocervical cells are directly sampled and therefore well preserved and present in relatively large
numbers. Endometrial cells are not reached by the sampling device. These cells spontaneously exfoliate and are seen in cytology in a
natural state of degeneration and usually in low numbers.
Apart from reporting the presence of endometrial cells, The Bethesda System recognizes the classification of 'Atypical Endometrial
Cells' without further sub classification, and 'Endometrial Adenocarcinoma'. Examples of these categories will be presented, together
with histological images.
100
Chapter 8
Endometrial Cells
Atypical Endometrial Cells
Endometrial Adenocarcinoma
101
Benign Endometrium
Rounded, three-dimensional, berry-like cluster of endometrial cells.

The cells have scant cytoplasm as seen during the proliferative
phase. Although the nuclei can be quite irregular in shape, the size
will not exceed the size of an intermediate cell nucleus.
Rather loose group of endometrial cells with

more cytoplasm and some vacuolization as can
be picked up in cytology during the early
secretory phase.
Compact group with small hyperchromatic

nuclei. Upper interior of group shows some
degenerated stromal cells. At the left lower
periphery, the cuboidal origin of the cells
can still be appreciated.
Groups vary in size and shape. Individual

nuclei vary in shape, but not in size and do
not exceed the size of normal intermediate
cell nuclei.
Small three dimensional group with strong degenerative features,

including nuclear shape irregularity and vacuolization. Similar features
are also often seen in the reactive processes of women carrying an
Intra Uterine Device (IUD).
The combination of endometrial
cells and a clean background is
specific for BD SurePath™ slides,
as most of the blood and
leukocytes are removed during the
preparation process.
102
Benign Endometrium
In case of doubt, Vimentin staining can be used to correctly identify

the endometrial origin of the cells. Vimentin staining can be done on
extra BD SurePath™ slides prepared from the same sample.
Histological section showing Vimentin positive

endometrial cells with underlying stroma.
Small sheet of endometrial cells with oval

nuclei, small distinct nucleoli and small
amount of cytoplasm. This in contrast to
endocervical cells with large amount of
cytoplasm.
Small papillary group with spontaneously

exfoliated endometrial cells with central stromal
cells.
A group showing the classic exodus pattern, with a dark core of

stromal cells surrounded by endometrial epithelial cells.
The upper image shows histology

of the endometrium during the
proliferative stage, whereas the
lower image represents endome-
trium during the secretory phase.
Note the difference in stroma
compactness.
103
Small fragments of a gland with rounded cytoplasmic degeneration

and leukocyte inclusion.
Berry-like group with slightly enlarged

eccentrically placed nuclei and vacuolization.
Cytoplasm is generally much better retained in
BD SurePath™ slides than in conventional slides.
Small three-dimensional cluster. Nuclei are

not larger than intermediate cell nuclei.
Small group of cuboidal cells with enlarged

eccentrically placed nuclei and vacuolated
cytoplasm. Note the intracytoplasmic
granulocytes.
Small fragment of tissue with interior stroma cells and

rim of epithelial cells.
Low power view showing atrophic

cells with small hyperchromatic
berry-like groups. Because of the
clean background, the three
dimensionality of the groups are
easily picked up at low power.
104
Endometrial epithelial cells are cuboidal and smaller than columnar

endocervical cells. Nuclei are slightly enlarged with small nucleoli.
Small group with scalloped borders. Nuclei are

mildly hyperchromatic.
Overlap of nuclei can be seen in some

groups. Prominent nucleoli are present.
Cytoplasm is quite dense and vacuolated
and is generally much better retained in
BD SurePath™ slides than in conventional
slides due to the direct fixation in
preservative liquid.
Three-dimensional group with scalloped borders

and crowded nuclei with coarse hypochromatic
chromatin and small but clearly visible nucleoli.
Group with rounded cells exhibiting variation in nuclear size and shape,
nuclear hyperchromasia and intracytoplasmic vacuoles. The category
'Atypical endometrium cells' is also used for the presence of highly
abnormal endometrium cells, in very low quantities or with unmatching
Histological section of hyperplasia clinical data.
with atypia, showing pseudo-
stratified epithelium with nuclear
crowding, mild nuclear atypia and
distinct nucleoli.
105
Papillary group with branching and crowded nuclei.

This is a fragment of an atypical gland.
Small part of an atypical gland in a typical three

dimensional group with scalloped borders.
Berry-like group with atypical cells and

overlapping nuclei with high N/C ratio.
Some berry-like groups are more complex.
Some three-dimensional formations in gland-like structures can

also be present.
Low power shows small, hyper-

chromatic, berry-like groups with
remains of diathesis and necrosis
in the background.
106
Individual cells have enlarged angulated nuclei and high N/C ratio.
Nuclei are hyperchromatic with chromatin clearing.
Cytoplasm is cyanophilic and may be

vacuolated.
Strong anisonucleosis can be seen in combination

with nuclear membrane irregularity.
The presence of tumor necrosis is essential for the diagnosis of

adenocarcinoma in cytology. Single abnormal endometrium cells may
be present, but are usually very difficult to identify. The architectural
features of the groups, even very small groups, are most helpful for
the correct identification.
Wel differentiated endometrioid
adenocarcinoma shows back-to-back
glands. Note nuclear crowding,
nuclear enlargement and some
intracellular granulocytes.
107
Discussion.
Endometrial cells are usually present in the first 2 weeks of the menstrual cycle. According to The Bethesda System, their presence
should be reported in women over 40 years of age. This refers only to the presence of endometrial epithelial cells; histiocytes and
stromal cells should not be taken into account. The differentiation between endometrial epithelial cells and stromal cells is thus
important and has been outlined in this chapter.
Although cervical cytology screening programs are not intended to identify endometrial pathology, the detection of lesions are considered
an additional positive benefit. In a clinical context, cytology can be a helpful tool in the detection of these lesions.
The diagnosis of endometrial adenocarcinoma and its precursors can be challenging in a cervical smear, mainly because the number of
endometrial cells can be limited. Often the cells show marked degeneration due to their pathway from uterus to cervix or vagina where
they are collected.
The diagnosis of endometrial hyperplasia, with or without atypia, cannot be made reliably or reproducibly on a cervical smear. These
precursor lesions are placed in the same category as all endometrial abnormalities that cannot be clearly identified as carcinoma:
Atypical Endometrial Cells.
When we compare endometrial cells in conventional slides (CS) to those in BD SurePathTM slides, several differences should be noted:
1. in CS, the presence of endometrial cells is often accompanied by blood. In BD SurePathTM slides, much of the blood is removed and
the endometrial cells are often found in a clean background.
2. As the fragile and degenerated endometrial cells are spread on a glass slide for the CS, much of the delicate nuclei and cytoplasm is
further destroyed. In BD SurePathTM slides, the cells are fixed in suspension and processed by slowly sedimenting the cells onto the
glass slide. This explains why morphological detail of endometrial cells, benign as well as malignant, can be better observed in
BD SurePathTM slides than in the CS.
Typical features for endometrial adenocarcinoma in BD SurePathTM slides include:
- Berry-like three-dimensional cell groups

- Scalloped borders
- Papillary groups
- Single abnormal, rounded cells can be present
- Cytoplasmic vacuolization
- Prominent nucleoli
- Intracytoplasmic granulocytes
- Tumor diathesis in background is not always present
108
Note!
109
In this chapter we will look at some of the challenges that we meet in diagnosing a cervical sample.
Extraordinary compositions of slides will be discussed, as well as some rare findings.
Several examples of ‘look-alikes’ will be given, where the criteria for the differential diagnostic categories are discussed in detail.
Examples of additional staining techniques are given that can be used on extra slides made from the BD SurePathTM vial.
Alternatively, leftover cell material can be blocked in paraffin and immuno staining can be applied on sections cut from these blocks.
110
Note! Chapter 9
Challenges
111
Special Compositions
Few metaplastic-like cells
Solitary and small groups of pleomorphic metaplastic

cells with dense cytoplasm in a clean background.
No inflammation. The slide should be screened at
reduced speed or at higher magnification to pick up
these isolated abnormal cells. Note high N/C ratio, large
hyperchromatic nuclei and irregular nuclear membranes.
Blood and only few dysplastic cells
It is rare to see only red blood cells in BD SurePathTM

and no epithelial cells. This can be an indicator of an
underlying lesion. At high magnification, few cells show
large hyperchromatic nuclei, irregular nuclear
membranes and scant cytoplasm.
112
Solitary dysplastic cells and cytolysis
Many large sheets of intermediate cells. Look specifically

in the white spaces between these cells to find single
abnormal cells: small basal-type cells with raisin-like
hyperchromatic nuclei.
Groups of hypochromatic dysplastic cells
Groups of hypochromatic cells with leukocytes can easily

be misinterpreted as repair cells on low magnification.
At high magnification however, the nuclear irregularity
and crowding is clearly visible.
113
Hyperchromatic Crowded Groups (HCG) in HSIL
Some HSIL cases are dominated by compact cell groups

imitating endometrial cells. Look at high magnification for
nuclear pleomorphism and squamous differentiation of the
cells.
HSIL and endocervical stromal cells
Microbiopsies of endocervical stroma. At the periphery

and in the background, we find not only columnar cells,
but also small HSIL cells. These 'busy' groups should be
carefully screened for cytomorphological detail at high
magnification.
114
Hyperchromatic Crowded Groups and Cell Block Cytology
Paraffin blocking of left-over cells is sometimes applied in

pathology labs and can be useful to categorize lesions that
otherwise may cause problems in interpretation.
In this case, the cytology showed large hyperchromatic

crowded groups, which were transformed to the histological
two-dimensional tissue fragment of a HSIL.
Several tissue sections can be cut and immuno-

histochemical staining procedures can be easily applied.
In this case, the left part of the square image shows

positive P16 staining and the right part shows positive
KI 67 staining of a HSIL (different glass slides but
combined in picture). H&E staining of the same lesion
can be seen in the circle.
115
Look-alikes
Typical for an HPV infection is the presence of koilocytes with large, well defined, perinuclear
halo's and a thickened rim of cytoplasm ('wired-loop' appearance). Nuclei are enlarged and
have finely granular, often hyperchromatic chromatin. Nuclear membrane irregularities may
be present.
Nuclei of koilocytes are enlarged and have finely granular, often

hyperchromatic chromatin. Nuclear membrane irregularities may
be present.
Not all HPV infections have clear koilocytes. Sometimes the cytoplasmic rim is not well
defined, but the nucleus is clearly enlarged and hyperchromatic. These changes are generally
categorized as ASC-US.
Endometrial cells
Compact cluster of endometrial cells with a less common honey-comb like

formation. These cells are smaller than endocervical cells and show some
degree of molding.
LUS
Samples of LUS are characterized by the presence of biphasic micro-biopsies in cytology,

combining well preserved columnar endocervical cells, with endometrial stromal cells.
116
Look-alikes
Intracytoplasmic glycogen
The presence of intracytoplasmic glycogen in parabasal and intermediate squamous cells can
cause a thickened cytoplasmic rim and clearing around the nuclei, thereby imitating
koilocytes. However, nuclei are not enlarged, chromatin is fine, membrane is smooth and
clearance area is not empty but finely fibrillar.
Perinuclear halos in inflammation
Small perinuclear halos are often seen in inflammation.

Nuclei show normal morphology of mature squamous cells.
White Sponge Naevus
Squamous cells of patients with White Sponge Naevus show perinuclear halos
suggestive of HPV.
Endocervical cells
Endocervical cells are often found in honeycomb formation.

Note clearly defined cell borders in the flat sheet.
Tubal metaplasia
Strips of columnar cells with pseudo-stratification. All cells are

ciliated. No biphasic morphology.
117
Look-alikes
Keratinizing dysplasia
Three-dimensional clusters-pearls show dyskeratosis and atypical nuclei. Pleomorphic

single cells are present in the background.
Pale cell dysplasia
Group of cells with crowded hypochromatic nuclei. Clue is irregular nuclear

membranes and the abnormal pale chromatin pattern. No nucleoli.
Cylindrocellular dysplasia
Sheet of dysplastic cells with nuclear streaming. Cells are

elongated with oval shaped nuclei but they are not columnar.
Always look for single cells.
Small cell dysplasia
Compact group of very small and very hyperchromatic cells. Nuclei are
enlarged. Always compare size of nuclei with the nuclei of intermediate
squamous epithelial cells. Evaluation must be done at high power.
Metaplastic dysplasia
These cohesive sheets of dysplastic cells can be very large and resemble normal metaplasia.
Always look for nuclear details at high power.
118
Look-alikes
Parakeratosis
Squamous pearls are a common finding. Cytoplasm is orangeophilic and nuclei

are small, pyknotic and show no atypia. The background is clean.
Repair
Flat sheet of squamous cells with distinct cytoplasmic borders. Nuclei are
bland with smooth nuclear membranes. Nucleoli are prominent.
AIS
Strips of columnar cells showing pseudo-stratification. Clue is columnar shape of cells with
basally placed nuclei.
Endometrial cells
Three-dimensional group of small cells.

Compare size of nuclei of endometrial cells with nuclei of intermediate
squamous cells. Size of both is the same.
Metaplasia
Sheet of metaplastic cells with regular nuclei and dense homogenous cytoplasm.
119
Uncommon Primary Tumor: Small Cell Carcinoma
At low magnification, small cells resembling leukocytes are seen.
At high magnification, the small cells have minimal cytoplasm and

high N/C ratio. Mitotic figures can be easily found.
Nuclei are hyperchromatic with salt and pepper chromatin; a stippled texture that is
typical for neuroendocrine tumors. Nucleoli are not observed.
Histology picture showing endocervical gland with subepithelial infiltrate of

small cell carcinoma.
As in most invasive carcinomas, some tumor diathesis is present.
120
Secondary or Metastatic Tumors
Presence of atypical lymphoid cells in a diathetic background.

Patient is known with lymphoma.
Berry-like three-dimensional clusters of large abnormal cells. Psammoma

bodies can be seen. No necrotic background. Patient is known with serous
adenocarcinoma of ovary.
Large quantity of bizarre pleomorphic cells in a background with tumor diathesis.

Patient is known with large cell carcinoma of the lung.
Presence of papillary clusters with large irregular nuclei. Squamous cells

are well matured and background is clean. Patient is known with
adenocarcinoma of Fallopian tube.
Presence of pleomorphic malignant cells with dark staining of cytoplasmic granules in

necrotic background. Patient is known with malignant melanoma.
121
Discussion
This chapter elaborates on some of the challenges that cervical cytology is facing.
Special compositions of slides should be recognized by the screener / cytologist and the screening procedure should be adapted when
needed. Generally the squamous cells dominate and limited numbers of glandular cells with slightly darker nuclear staining complete
the cellular picture against a clean background (as described in Chapter 2). Unusual compositions should be observed and interpreted
correctly. E.g. a bloody slide is highly unusual for a BD SurePathTM slide and must be screened carefully.
Cytomorphology alone has its limits; some large hyperchromatic crowded groups and thick tissue fragments can be difficult to inter-
pret. Special staining procedures may be useful to further differentiate the lesion. E.g. P16 and KI67 are used to discriminate between
(pre)neoplastic changes and benign reactive changes; Vimentin staining can be used to positively identify cells of endometrial origin.
These staining procedures can be carried out on extra cytology slides made from the left-over cell material of BD SurePath™. Another
method often used in the pathology laboratory, is the concentration and embedding of remaining material in paraffin to prepare cell
blocks. Tissue sections can then be cut and used for numerous immunohistochemical staining procedures.
Some of the typical morphology pitfalls have been highlighted in this chapter to assist correct classification. Through all chapters,
observations and descriptions of the architectural patterns and cellular features have been described from benign to (pre)malignant
lesions. As there exist no single criterion for malignancy confirmation, the cytologist must use architectural patterns as well as individual
cell morphology. In the decision making process, all options from normal to abnormal, should be checked.
The general abnormality criteria for cell morphology are:
- Enlargement of the nucleus

- Increase of N/C ratio
- Nuclear hyperchromasia
- Clumping of chromatin
- Nuclear membrane irregularities
- Increase in size and number of nucleoli
- Multinucleation and multilobulation
- Abnormal mitoses
- Variations in size and shape of nucleus and cytoplasm
A B C
Compact rounded sheet of HSIL cells with columnar cells on edge should not
be mistaken for AIS (A). Squamous origin is confirmed by immunostaining
on histology showing normal columnar epithelium in PAS+ mucus staining
(B) with underlying dysplastic squamous cells in KI 67 staining (C).
122
Note!
123
ATLAS SUMMARY
Since the 1960's, cytology has been used worldwide as the primary screening method for cervical cancer. The conventional slide, in
combination with staining and morphological parameters, described in detail by George Papanicolaou, has set the standard for many
years. In the last decennium, one of the major changes in cytology has been the adaption of Liquid Based Cytology (LBC).
Although there are quite some differences between conventional slides and LBC, many of the known morphology parameters still apply.
The nuclear morphology reflects the state of proliferation and reproductive capacity of the cell and the cytoplasm generally provides an
indication of origin, functional state and degree of differentiation.
Each LBC method has its own specifics, depending on the sampling, the preservative solution, the technical manipulations and the
staining. The authors have chosen BD SurePath™ as the highest quality LBC method on the market, with the lowest unsatisfactory rate
and the highest detection rate of abnormalities in combination with a standardized Papanicolaou staining. An additional benefit of LBC
is the possibility to add other diagnostic methods, such as immunocyto- and histochemistry or molecular biology tests (like HPV), to
morphology. This has extended the diagnostic boundaries of the Pap test.
Love of cytology in combination with the excellent quality of BD SurePath™ has inspired the authors to make this atlas. It is their sincere
hope that this atlas will find its way to users of the BD SurePath™ technology, to support and facilitate the interpretation of cell images.
124
REFERENCES
Chapter 1
Fertility. In: Diseases and Disorders. 2nd Edition. The World's Best Anatomical Charts. 2005 Lippincott Williams & Wilkins.
Human Histology. Stevens A, Lowe JS. 3rd Edition. 2012 Elsevier Mosby.
Papanicolaou GN. Atlas of Exfoliative Cytology, 1954 Harvard University Press, Cambridge, Mass.
Chapter 2
The Bethesda System for Reporting Cervical Cytology. 2nd Edition. 2004. Editors Solomon D, Nayar R. Springer.
Advanced Techniques in Diagnostic Cellular Pathology. Ed: Hannon-Fletcher M, Maxwell P. 2009 John Wiley & Sons Ltd.
Cytopathology. Ed: Shambayati B. 2011 Oxford University Press.
Coleman DV, Evans DMD. Biopsy Pathology and Cytology of the Cervix. 2nd Edition. 1999. Oxford University Press.
Takahashi M. Color Atlas of Cancer Cytology. 3rd Edition. 2000 IGAKU-SHOIN Ltd.
Kumar V, Abbas AK, Fausto N, Aster J. Robbins and Cotran Pathologic Basis of Disease. Metaplasia. Page 10-14. 8th Edition. 2010
Saunders Elsevier.
Koss' Diagnostic Cytology and its Histopathologic Bases. 5th Edition. 2005. Volume 1. Editors Koss LG, Melamed MM.
Chapter 3
Uterine Pathology. 1st Edition. 2012. Editors: Soslow RA, Longacre TA.
Cytologic Atypia Associated With Microglandular Hyperplasia. Valente PT, Schantz H, Schultz. In Diagnostic Cytopathology, 1993. Vol
10, No 4, page 326-331.
Takahashi M. Color Atlas of Cancer Cytology. 3rd Edition. 2000 IGAKU-SHOIN Ltd.
Chapter 5.
Chapter 6.
Pathology and Genetics of Tumours of the Breast and Female Genital Organs. By Tavassoli FA and Devilee P. 2003. From World
Health Organization Classification of Tumours.
Tumors of the Cervix, Vagina, and Vulva. By Kurman RJ, Ronnett BM, Sherman ME, Wilkinson EJ. 4th Series. 2010. From AFIP Atlas
of Tumor Pathology.
Cervical Intraepithelial Neoplasia. Diagnostic Criteria and Pitfalls. By Maud Veselic. 2009. Veldhuizen course. Leiden University
Medical Centre. In house press.
Chapter 7.
Cytologic Features of Endocervical Glandular Lesions: Comparison of SurePath, ThinPrep and Conventional Smear Specimen
Preparations. Belsley NA, Tambouret RH, Misdraji J, Muzikansky A, Russell DK, Wilbur DC. In Diagnostic Cytopathology 2007 Vol 36,
No 4, page 232-237.
Chapter 8.
Differential Diagnosis in Exfoliative and Aspiration Cytopathology. 2nd Edition, 2011. By Kini SR. Wolters Kluwer Lippincott Williams
& Wilkins.
Gynecologic Pathology. By Nucci MR, Oliva E. and Goldblum JR. 2009. Elsevier Churchill Livingstone.
Endometrial Cells in Cervical Cytology: Review of Cytological Features and Clinical Assessment. Greenspan DL, Cardillo M, Davey
DD, Heller DS, Moriarty AT. Journal of Lower Genital Tract Disease 2006, Vol 10, Nr 2, page 111-122.
Chapter 9
Weeding Atypical Glandular Cell Look-Alikes From the True Atypical Lesions in Liquid-Based Pap Tests: A Review. Wood MD, Horst JA,
Bibbo M. in Diagnostic Cytopathology 2006, Vol 35 No1 page 12-20.
125
126
127
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Germany
BD, BD logo and BD SurePath are property of Becton, Dickinson and Company. c 2013 BD Tel : (49) 6221 305 0
Becton Dickinson GmbH General Manager: Roland Pfleger Registered Office: Heidelberg Commercial Register: Mannheim HRB 330 707 Fax : (49) 6221 305 604
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1

Atlas of Cervical Cytology using the BD SurePath™

Klaas van der Ham, medical photographer

Anneke van Driel - Kulker, Ph.D. biologist / cytologist

Chapter 1: Anatomy, Histology and Cytology of the Uterus 8

Chapter 2: Cytology Composition 16

Chapter 3: Micro-organisms, Inflammation and Repair 38

Chapter 4: Squamous Metaplasia, Glandular Metaplasia and Glandular Hyperplasia 52

Chapter 5: Atypical Squamous Cells of Undetermined Significance (ASC-US) 60

Atypical Squamous Cells, Cannot Exclude HSIL (ASC-H)

Chapter 6: Squamous Intraepithelial Lesions and Squamous Cell Carcinoma 66

Chapter 7: Endocervical Glandular Cells and Lesions 86

Chapter 9: Challenges 110

Anatomy, Histology and Cytology of the Uterus

Body (corpus) of uterus

E The World's Best Anatomical Charts

Stratified squamous epithelium of Isthmus where corpus and cervical

Columnar epithelium of the

Epithelium of the ectocervix is Endometrium and columnar epithe-

Mucin-secreting cells and ciliated

Cellular arrangemens reflect the architecture of the tissue.

The World's Best Anatomical Charts

B. Normal Endocervical Mucosa: Shows

A. Original/Native Squamocolumnar Junction

D. New Squamocolumnar Junction: This is a

C. Transformation Zone: This is an area of

The cytoplasm of less mature The shrunken nucleus can be sur-

Vesicular nuclei have finely Navicular cells (boat shaped) with

Endocervical cells are often Two types of endocervical cells

Immature metaplastic cells are Histological section showing the

Endometrial cells are often found Histological section of prolifera-

Inflammatory processes may Histological section showing ero-

Histological section of isthmus, showing endo- Isthmus is composed of endocervical epithelium

Samples of LUS are characteri- Strips of endocervical columnar

Cytoplasmic staining ingredients

Eosin stains less mature cells light green

Orange G stains keratinizing cells orange

The cytoplasmic features of squamous epithelial cells

Absolute nuclear size: (diameter of measurement)

Relative nuclear size (ratio of the size of nucleus to the size

Intensity of nuclear staining as compared to the

Chromatin distribution: uniform, irregular,

Nuclear shape: oval, round,

Regularity of nuclear membrane: smooth, angular or wrinkled.

Presence and regularity of nucleoli.

General nuclear features

Raspberries/mulberries Endometrial cells Morula formation

Corn Isthmus cells Papillary formation

Flower stem with insects Vascular structures Perivascular

Olive tree leaves Metaplastic cells Storiform

Pavement Mature squamous cells Pavement

Honeycomb structure Endocervical cells Honeycomb structure

Strip/Palisade Endocervical cells Little soldiers

Acinary/rosette formation Columnar cells Flower

Proliferative phase (Day 4-14 of menstrual cycle)

Superficial cells with pink cytoplasm dominate the image.

Mid cycle or ovulation phase (Day 14-16 of menstrual cycle)

A mixture of superficial and intermediate cells are seen.

Luteal phase (Day 16-28 of menstrual cycle)

Intermediate cells with light green/grey cytoplasm dominate

At the end of the luteal phase, clustered intermediate cells

Clumps of left over old blood can be found in combination

Increased numbers of parabasal cells, often showing high

Single parabasal cells with karyopyknosis. Degenerated