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The Bethesda System and Beyond.
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The Bethesda System and Beyond.
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.
5
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 (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 (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 8: Endometrial Cells, Atypical Endometrial Cells and Endometrial Adenocarcinoma 100
References: 124
7
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.
8
Chapter 1
9
Female Genital Organs - Anatomy
Cavum
Fundus Ampulla
Uterine (Fallopian) tube
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.
10
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
D F
Tuba, endometrium, isthmus, endocervix and ectocervix show very special mucosa with many physiological changes.
11
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
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D
D F
All mucosa details show cellular complexity. Knowledge of histology is essential for better understanding of cytological correlation.
12
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
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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.
13
Different types of cells and cell arrangements in BD SurepathTM LBC
Anatomical continuity from surface epithelium of the ovary to the cervix and vagina makes cervical cytology interpretation very complex.
14
Squamocolumnar Junction And Transformation Zone
The transformation zone is the targeted area for cytology, colposcopy as well as histology.
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Introduction Chapter 2
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.
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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.
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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.
21
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.
22
Endometrial cells
Endometrial cells can be found in loose groups Histological section of secretory endometrium.
with intact cytoplasm. Note intracytoplasmic vacuolization.
23
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.
24
Lower Uterine Segment (LUS) / Isthmus
25
Cytoplasmic features
26
Nuclear features
8µm
27
Architectural patterns in cellular arrangements
28
Architectural patterns in cellular arrangements
29
Smear patterns
30
Smear patterns
Cytolysis
Menstruation
Postpartum
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Many faces of atrophy
32
Many faces of atrophy
Deep atrophy.
33
Manual screening of a BD SurePath™ LBC slide.
10x
20x
40x
34
Screening challenges
35
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.
36
Note!
37
Introduction Chapter 3
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
39
Micro-organisms
Actinomyces-like organisms
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Micro-organisms
Trichomonas vaginalis
41
Micro-organisms
Candida species
42
Micro-organisms
43
Micro-organisms
Leptothrix vaginalis
44
Micro-organisms
45
Inflammation
Acute cervicitis
46
Inflammation
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
Nuclei:
- Enlargement
- Multinucleation
- Hyperchromasia
- Evenly distributed chromatin
- Enlarged nucleoli
Cytoplasm:
- Vacuolization
- Syncytial formation
47
Inflammation
Follicular cervicitis
Atrophic cervicitis
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Reactive changes
49
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.
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.
50
Note!
51
Introduction Chapter 4
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.
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.
52
Chapter 4
Primary SCJ
Ectocervix Endocervix
Normal cervix
Endocervical cells
Secondary SJC
Mature metaplasia
53
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.
54
Metaplastic process - Various stages
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.
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.
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:
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
Introduction Chapter 5
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.
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Chapter 5
61
Atypical Squamous Cells of Undetermined Significance (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.
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.
63
Discussion
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).
HPV detection with or without phenotyping can be used to guide management of ASC patients.
64
Note!
65
Introduction Chapter 6
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.
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.
66
Chapter 6
Histology
67
LSIL / Mild dysplasia / CIN I
68
LSIL / Mild dysplasia / CIN I
69
HSIL / Moderate dysplasia / CIN II
70
HSIL / Moderate dysplasia / CIN II
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.
72
HSIL / Severe dysplasia - classic / CIN III
Cytoplasm is delicate.
73
HSIL / Keratinizing dysplasia / CIN III
74
HSIL / Small cell non-keratinizing dysplasia / CIN III
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
Single cells can be found with enlarged round nuclei and coarsely
granular, but evenly distributed chromatin.
76
HSIL / Cylindrocellular-like dysplasia / CIN III
77
HSIL / Large cell non-keratinizing dysplasia / CIN III
78
HSIL / Metaplastic dysplasia / CIN III
79
Squamous Cell Carcinoma - keratinizing
Large sheet of dysplastic cells with relatively low N/C ratio and
dense orangeophylic cytoplasm.
80
Squamous Cell Carcinoma - keratinizing
81
Squamous Cell Carcinoma - non keratinizing
82
Squamous Cell Carcinoma - non keratinizing
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:
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
Introduction Chapter 7
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'.
86
Chapter 7
87
Normal Endocervical Cells
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.
89
Atypical Endocervical Cells - Not Otherwise Specified (NOS)
90
Atypical Endocervical Cells - Favor Neoplastic
91
Adenocarcinoma In Situ
92
Adenocarcinoma In Situ
Columnar shaped cells with high N/C ratio and overlapping nuclei.
No cilia are present.
93
Endocervical Adenocarcinoma
94
Endocervical Adenocarcinoma
95
Villoglandular Adenocarcinoma
Strip with crowding of small, oval nuclei with granular chromatin and
prominent nucleoli.
96
Villoglandular Adenocarcinoma
97
Discussion
- 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
Introduction Chapter 8
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
102
Benign Endometrium
103
Atypical Endometrial Cells
104
Atypical Endometrial Cells
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
Endometrial Adenocarcinoma
106
Endometrial Adenocarcinoma
Individual cells have enlarged angulated nuclei and high N/C ratio.
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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.
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Note!
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Introduction Chapter 9
In this chapter we will look at some of the challenges that we meet in diagnosing a cervical sample.
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.
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Note! Chapter 9
Challenges
111
Special Compositions
112
Special Compositions
113
Special Compositions
114
Hyperchromatic Crowded Groups and Cell Block Cytology
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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.
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
LUS
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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.
Squamous cells of patients with White Sponge Naevus show perinuclear halos
suggestive of HPV.
Endocervical cells
Tubal metaplasia
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Look-alikes
Keratinizing dysplasia
Cylindrocellular 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.
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Look-alikes
Parakeratosis
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
Metaplasia
Sheet of metaplastic cells with regular nuclei and dense homogenous cytoplasm.
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Uncommon Primary Tumor: Small Cell Carcinoma
Nuclei are hyperchromatic with salt and pepper chromatin; a stippled texture that is
typical for neuroendocrine tumors. Nucleoli are not observed.
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Secondary or Metastatic Tumors
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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.
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).
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Note!
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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.
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Chapter 2
The Bethesda System for Reporting Cervical Cytology. 2nd Edition. 2004. Editors Solomon D, Nayar R. Springer.
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& Wilkins.
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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.
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The Bethesda System for Reporting Cervical Cytology. 2nd Edition. 2004. Editors Solomon D, Nayar R. Springer.
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.
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