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CYTOLOGY: DIAGNOSTIC PRINCIPLES AND ISBN: 978-1-4160-5329-3
CLINICAL CORRELATES
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Notice
Knowledge and best practice in this field are constantly changing. As new research and e xperience
broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or
appropriate. Readers are advised to check the most current information provided (i) on procedures
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dose or formula, the method and duration of administration, and contraindications. It is the
responsibility of the practitioner, relying on their own experience and knowledge of the patient,
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to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher
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The Publisher
Library of Congress Cataloging-in-Publication Data
Cibas, Edmund S.
Cytology : diagnostic principles and clinical correlates / Edmund S.
Cibas, Barbara S. Ducatman. — 3rd ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4160-5329-3
1. Cytodiagnosis. I. Ducatman, Barbara S. II. Title.
[DNLM: 1. Cytodiagnosis—methods. 2. Cytological Techniques. QY 95 C567c 2009]
RB43.C47 2009
616.07’582—dc22
2008027011
ISBN: 978-1-4160-5329-3
Publishing Director: Linda Belfus
Acquisitions Editor: William Schmitt
Developmental Editor: Katie DeFrancesco
Project Manager: Bryan Hayward
Design Direction: Ellen Zanolle
Printed in China
Last digit is the print number: 9 8 7 6 5 4
Dedicated to
Todd Bryant Stewart
and
Alan M. Ducatman
B978-1-4160-5329-3.00039-6
Preface to third edition
We hope this book will serve as a useful guide both for that lead to false-negative and false-positive diagnoses),
the pathologist in practice and for the trainee—resi- a description of normal elements, and finally, a how-to
dent or fellow—who is looking to obtain expertise in this guide for the diagnosis of benign and malignant lesions,
subspecialty. with an emphasis on differential diagnosis. We have
It has been 5 years since the publication of the second retained the bulleted “capsule summaries,” particularly
edition of Cytology: Diagnostic Principles and Clinical for summarizing cytomorphologic features and differ-
Correlates. Since then, cytology has continued to grow ential diagnoses. We have continued to emphasize clini-
and evolve as a subspecialty devoted to the diagnosis of cal correlation (hence the title). For example, Chapter 1
cellular tissue obtained by minimally invasive methods includes the recently revised algorithms of the American
(scraping, brushing, aspiration, etc.), and thus the need Society for Colposcopy and Cervical Pathology for man-
for this updated edition. But we have retained many of aging women with abnormal cervical cytologic diag-
the qualities of the prior editions. As did the first two, this noses. Good cytologists are those who understand the
edition aims to be concise yet comprehensive. We have clinical implications of their interpretations.
emphasized brevity and clarity. The text is grounded Once again, we hope we have succeeded in convey-
firmly in an understanding of surgical pathology and ing the beauty, strength, and challenge of cytology. With
current diagnostic terminology. Where relevant, we have this book we have tried to take some of the mystery out
illustrated the value of established ancillary studies (e.g., of cytology. But mysteries remain; their solutions still
flow cytometry and immunohistochemistry) as well as obscure. If this text inspires the reader to explore and
evolving techniques such as cytogenetics, which can be even solve some of them, we will consider ourselves
helpful in the diagnosis of certain lymphomas, soft tis- doubly rewarded.
sue tumors, renal neoplasms, and mesothelioma.
Although the book is multi-authored, the chapters fol- Edmund S. Cibas
low a similar format: indications, sample collection and
Barbara S. Ducatman
preparation methods, recommended terminology for
2008
reporting results, accuracy (including common pitfalls
vii
ContRiButoRS
Edmund S. Cibas, MD Jeffrey F. Krane, MD, PhD
Associate Professor Assistant Professor
Department of Pathology Department of Pathology
Harvard Medical School Harvard Medical School
Director, Division of Cytopathology Chief, Head and Neck Pathology Service
Brigham and Women’s Hospital Brigham and Women’s Hospital
Boston, Massachussetts Boston, Massachusetts
Barbara S. Ducatman, MD Xiaohua Qian, MD, PhD
Professor and Chair Instructor
Department of Pathology Department of Pathology
Associate Dean for Faculty Services Harvard Medical School
Director, West Virginia University Pathologist
National Center of Excellence Brigham and Women’s Hospital
in Women’s Health Boston, Massachusetts
West Virginia University School of Medicine
Morgantown, West Virginia Andrew A. Renshaw, MD
Pathologist
William C. Faquin, MD, PhD Baptist Hospital
Associate Professor Miami, Florida
Department of Pathology
Harvard Medical School Jian Shen, MD, PhD
Pathologist Instructor
Divisions of ENT Pathology Department of Pathology
and Cytopathology Harvard Medical School
Massachusetts General Hospital Pathologist
Boston, Massachusetts Massachusetts General Hospital
Boston, Massachusetts
Christopher A. French, MD
Assistant Professor Paul E. Wakely, Jr., MD
Department of Pathology Professor
Harvard Medical School Department of Pathology
Pathologist Ohio State University College of Medicine
Brigham and Women’s Hospital Columbus, Ohio
Boston, Massachusetts
Helen H. Wang, MD, DrPH
David W. Kindelberger, MD Associate Professor
Instructor Department of Pathology
Department of Pathology Harvard Medical School
Harvard Medical School Medical Director of Cytology
Pathologist Beth Israel Deaconess Medical Center
Brigham and Women’s Hospital Boston, Massachusetts
Boston, Massachusetts
ix
Acknowledgments
We owe a great debt to many individuals for their help allowing us to reproduce their clinical management
with this book. algorithms in Chapter 1.
To Bill Schmitt, Kathryn DeFrancesco, Michael Troy, Thanks also to Sandy George and Deanna Reynolds at
and Kristin Saunders at Elsevier, who shepherded this West Virginia University, who were invaluable in provid-
book gently to completion: a thousand thank yous. You ing their assistance.
exemplified the spirit of teamwork, and we enjoyed We are indebted to many members of the staff of
working with all of you. the Brigham and Women’s Hospital and West Virginia
Paula Delgrosso’s administrative skills and hard work University School of Medicine and Hospital—the cyto-
contributed immeasurably to this edition. Edmund technologists, cytopathologists, and trainees—who
Carlevale heroically converted the previously unformat- inspire us with their devotion to cytopathology and
ted references of the prior edition into EndNote format, who continue to challenge us. In particular, we wish
saving us hours of tedious work. to acknowledge Dorothy Nappi, CT (ASCP), and Grace
We express our deep appreciation to Mr. Dennis Goffi, CT (ASCP) (IAC), who have helped us train so
Padget of Padget & Associates for his help with the com- many pathology residents and fellows over the years.
plexities of billing in Chapter 17. He lent us his watch- Without their help we would not have our extraordinary
ful eye through several versions of that section. We relied collections of cytology teaching cases from which so
extensively on his Pathology Service Coding Handbook many of the images in this book are derived.
for the information set forth in that chapter. Readers who Finally, to our friends, families, and loved ones, espe-
want more information on pathology coding questions cially Todd Stewart and Alan Ducatman, who tolerated
can contact Dennis Padget at EZPathCoding@bellsouth. the long evening and weekend hours that deprived them
net (502/722-8873) for information about subscribing to (temporarily!) of a large share of our time. This book
that comprehensive electronic text. would not exist without their love and strength.
We thank Drs. Robert Hasserjian and Tad Wieczorek
for their expertise and helpful comments on early drafts Edmund S. Cibas
of the Lymph Nodes chapter.
Barbara S. Ducatman
We are grateful to Kathleen Poole and the American
Society for Colposcopy and Cervical Pathology for
xi
1
Cervical and Vaginal Cytology
Edmund S. CibaS
THE HISTORY OF THE PAP TEST BENIGN AND REACTIVE CHANGES
Benign Squamous Changes
SAMPLING AND PREPARATION METHODS
Benign Endocervical Changes
Conventional Smears
Repair
Liquid-Based Cytology
Radiation Changes
ThinPrep Pap Test
Cellular Changes Associated with Intrauterine
SurePath Pap Test
Devices
MonoPrep Pap Test
Glandular Cells Status Post Hysterectomy
AUTOMATED SCREENING Other Benign Changes
Historical Overview
VAGINAL SPECIMENS IN “DES DAUGHTERS”
FocalPoint Slide Profiler
ThinPrep Imaging System SQUAMOUS ABNORMALITIES
Squamous Intraepithelial Lesions
ACCURACY AND REPRODUCIBILITY
Grading Squamous Intraepithelial Lesions
DIAGNOSTIC TERMINOLOGY AND Low-Grade Squamous Intraepithelial Lesion
REPORTING SYSTEMS High-Grade Squamous Intraepithelial Lesion
Problems in the Diagnosis of Squamous
THE BETHESDA SYSTEM
Intraepithelial Lesions
Specimen Adequacy
Squamous Cell Carcinoma
General Categorization
Atypical Squamous Cells
Interpretation and Results
Atypical Squamous Cells of Undetermined
THE NORMAL PAP Significance
Squamous Cells Atypical Squamous Cells, Cannot Exclude HSIL
Endocervical Cells
GLANDULAR ABNORMALITIES
Exfoliated Endometrial Cells
Endocervical Adenocarcinoma in Situ
Abraded Endometrial Cells and Lower Uterine
Adenocarcinoma
Segment
Endocervical Adenocarcinoma
Trophoblastic Cells and Decidual Cells
Endometrial Adenocarcinoma
Inflammatory Cells
Differential Diagnosis of Adenocarcinoma
Lactobacilli
Atypical Glandular Cells
Artifacts and Contaminants
Atypical Endocervical Cells
ORGANISMS AND INFECTIONS Atypical Endometrial Cells
Shift in Flora Suggestive of Bacterial
OTHER MALIGNANT NEOPLASMS
Vaginosis
Small Cell Carcinoma
Trichomonas Vaginalis
Malignant Melanoma
Candida
Malignant Lymphoma
Actinomyces
Malignant Mixed Mesodermal Tumors
Herpes Simplex
Metastatic Tumors
Cytomegalovirus
Chlamydia Trachomatis ENDOMETRIAL CELLS IN WOMEN OLDER
Rare Infections THAN 40 YEARS OF AGE
The 20th century witnessed a remarkable decline in the cancer was the most common cause of cancer deaths in
mortality from cervical cancer in many developed coun- women in the United States.1 Today, it is not even one of
tries. This achievement is directly attributable to the the top ten.2
implementation of the Papanicolaou (Pap) test. In the The incidence of cervical cancer in the United
1930s, before Pap screening was introduced, cervical States is approximately 11,000 cases, with 3670 deaths.2
1
2 CERVICAL AND VAGINAL CYTOLOGY
Worldwide, however, the cervical cancer incidence (over of negative smears she had had, by 48% with just one
500,000 cases annually) and mortality rates (288,000 negative smear, 69% with two to four negative smears,
deaths per year) are second only to those for breast and 100% with five or more smears.17
cancer.3 Screening programs, unfortunately, are rudimen- Screening guidelines differ around the world. Even in
tary or nonexistent in many parts of the world. Fewer than the United States, the recommendations of different orga-
5% of women in developing countries have ever had a Pap nizations vary in some of their details.18-20 The American
test.4 In contrast, 89% of women in the United States report Cancer Society (ACS) recommends the following:
having had a Pap test in the preceding 3 years. • Cervical cancer screening should begin approxi-
mately 3 years after a woman begins having vaginal
intercourse, but no later than 21 years of age.
The hisTory of The PAP TesT • Until age 30, cervical screening should be carried
out every year with conventional Pap tests or every
The Pap test is considered by many to be the most cost- 2 years using liquid-based Pap tests.
effective cancer reduction program ever devised.1 Credit • At or after age 30, a woman who has had three nor-
for its conception and development goes to George N. mal test results in a row may be screened every
Papanicolaou, an anatomist and Greek immigrant to the 2 to 3 years with a Pap test (smear or liquid-based)
United States. In 1928 he reported that malignant cells or every 3 years with a Pap plus human papilloma-
from the cervix can be identified in vaginal smears.5 virus (HPV) test.
Later, in collaboration with the gynecologist Herbert • A woman 70 years of age and older who has had
Traut, who provided him with a large number of clinical three or more normal Pap test results and no abnor-
samples, Papanicolaou published detailed descriptions mal results in the previous 10 years may choose to
of preinvasive cervical lesions.6,7 Pathologists and phy- stop cervical cancer screening.
sicians initially greeted this technique with skepticism, • A woman who has had a total hysterectomy may
but by the late 1940s Papanicolaou’s observations had choose to stop cervical cancer screening. (Exceptions
been confirmed by others. The Canadian gynecologist are women with a history of CIN 2,3, cervical cancer,
J. Ernest Ayre suggested taking samples directly from the or in utero diethylstilbestrol [DES] exposure.)
cervix with a wooden spatula rather than from the vagina Women with a history of cervical cancer, in utero DES
with a pipette as originally described by Papanicolaou.8 exposure, and who are immunocompromised (organ
Eventually, cytologic smears were embraced as an ideal transplantation, chemotherapy, chronic corticosteroid
screening test for preinvasive lesions, which, if treated, treatment, or positive for human immunodeficiency
would be prevented from developing into invasive virus [HIV]) may benefit from more frequent screening.19
cancer. Adherence to these guidelines is critical for cervical can-
The first cervical cancer screening clinics were estab- cer prevention. In the United States, more than 50% of
lished in the 1940s.9 The Pap test was never evaluated women who develop cervical cancer have not had a Pap
in a controlled, prospective study, but several pieces of test in the 3 years before their cancer diagnosis.21
evidence link it to the prevention of cervical cancer. The recent development of two prophylactic HPV
First, the mortality rate from cervical cancer fell dramat- vaccines provides a new opportunity for cervical can-
ically after screening was introduced, by 72% in British cer prevention.3 Both vaccines consist of empty protein
Columbia10 and 70% in Kentucky.11 Second, there was a shells called virus-like particles that are made up of the
direct correlation between the intensity of screening and major HPV capsid protein L1. They contain no DNA and
the decrease in mortality. Among Scandinavian countries, are not infectious. One of the vaccines, Gardasil (Merck
the death rate fell by 80% in Iceland, where screening was & Co., Inc.), is a quadrivalent vaccine against HPV
greatest; in Norway, where screening was lowest, the types 6, 11, 16, and 18. The other is the bivalent vaccine
death rate fell by only 10%.12 A similar correlation was Cervarix (GlaxoSmithKline) that protects against HPV 16
observed in high and low screening regions of Scotland13 and 18. They have shown extraordinary efficacy in pre-
and Canada.14 In the United States, the decrease in venting type-specific histologic CIN 2,3 lesions, with no
deaths from cervical cancer was proportional to the difference in serious adverse effects compared to pla-
screening rates in various states.15 Finally, women who cebo.22 The vaccines are administered in three doses to
do not develop invasive cancer are more likely to have females ages 9 to 26 years before the initiation of sexual
had a Pap test than women with cancer. In a Canadian activity. Continued Pap screening will remain important
study, the relative risk for women who had not had a for many decades, however, because these vaccines do
Pap test for 5 years was 2.7,16 and screening history was not protect against 30% of cervical cancers (i.e., those
a highly significant risk factor independent of other not related to HPV 16 or 18); the duration of protection
factors such as age, income, education, sexual history, is unknown; they are not effective in treating prevalent
and smoking. In Denmark, a woman’s risk of developing HPV infections; and the cost of the vaccines might limit
cervical cancer decreased in proportion to the number their use in some populations.3
SAMPLING AND PREPARATION METHODS 3
As seen in the aforementioned ACS recommenda- plastic spatula is recommended because wooden fibers
tions, the combination of a Pap test plus HPV test is trap diagnostic material. The spatula is rotated at least
included as an option for screening women 30 years of 360 degrees. The sample can be smeared on one half of
age or older. The rationale is to combine the superior sen- a slide and spray fixed (the other half should be covered
sitivity of HPV testing with the superior specificity of the to avoid coating it with fixative before the endocervi-
Pap test. This recommendation is controversial because cal sample is applied). Alternatively, one may set aside
it increases screening costs. Moreover, questions remain the spatula sample momentarily while the endocervical
regarding the ideal management of women with discrep- brush sample is obtained.
ant results (e.g., HPV test positive and Pap negative). The After the brush is inserted in the endocervical canal,
search for the best screening algorithm will undoubtedly some bristles should still be visible. If inserted too
continue, particularly as molecular diagnostic methods far, there may be inadvertent sampling of the lower
become more readily available. uterine segment (LUS), which causes diagnostic diffi-
culties because its epithelium resembles a high-grade
squamous intraepithelial lesion (HSIL) and adenocarci-
sAmPling And PrePArATion
noma in situ (AIS). The brush should be rotated gently
meThods only one-quarter turn. A larger rotation is unnecessary
because the circumferential bristles are in contact with
To obtain an ideal Pap specimen, the following guidelines the entire surface the moment the brush is inserted.
have been established by the Clinical and Laboratory The spatula sample, if not already applied and fixed,
Standards Institute.23 should be applied to the slide, then the brush sample
rolled over the slide, followed by immediate fixation. The
Patient instructions: two samples can be placed in quick succession on two
separate halves of the slide, or the endocervical sam-
• Schedule the examination 2 weeks after the first
ple can be rolled directly over the spatula sample, both
day of the last menstrual period. (It is preferable to
covering the entire slide. Immediate fixation (within
avoid examination during menses because blood
seconds) is critical to prevent air-drying artifact, which
may obscure significant findings.)
distorts the cells and hinders interpretation.
• Do not use vaginal medication, vaginal con-
The broomlike brush (“broom”) has a flat array of
traceptives, or douches for 48 hours before the
plastic strips contoured to conform to the cervix, with
appointment.
longer strips in the middle. This design allows simultane-
• Intercourse is not recommended the night before
ous sampling of the endocervix and ectocervix. The long
the appointment.
middle strips are inserted into the os until the shorter
outer strips bend against the ectocervix. The broom is
Specimen collection: rotated three to five times. To transfer the material, each
side of the broom is stroked once across the slide in a
• Specimens should be obtained after a nonlubri-
painting motion.
cated speculum (moistened only with warm water
The cotton swab moistened with saline is no longer
if needed) is inserted.
recommended because its fibers trap cells, reducing the
• Excess mucus or other discharge should be
efficiency of cell transfer onto slides.
removed gently with ring forceps holding a folded
There are two options for smear fixation. Coating
gauze pad.
fixatives contain alcohol and polyethylene glycol and
• The sample should be obtained before the application
are applied by pump sprays, by droppers from dropper
of acetic acid or Lugol iodine.
bottles, or by pouring from an individual envelope
• An optimal sample includes cells from the ectocervix
included as part of a slide preparation kit. Alternatively,
and endocervix.
the smear can be immersed directly into a container
filled with 95% ethanol.
Samples for liquid-based cytology (LBC) are obtained
Recent studies have challenged the prohibition as described except that, instead of smearing the cells on
against a lubricated speculum and suggest that water- a slide, the collection device is rinsed in a vial containing
based lubricants may be acceptable.24 a liquid fixative. In the United States, the LBC Pap test is
more common than the smear.
Conventional smears
liquid-Based Cytology
Conventional smears are often obtained using the com-
bination of a spatula and brush. The spatula is used first. An important landmark in the history of the Pap
Although a wooden or plastic spatula is acceptable, the test occurred in 1996 when the U.S. Food and Drug
4 CERVICAL AND VAGINAL CYTOLOGY
Administration (FDA) approved the ThinPrep™ (Hologic, increases in HSIL detection after the implementation of
Marlborough, Mass.) as an alternative to the conven- the ThinPrep.30–35
tional cervicovaginal smear. This was followed 3 years The ThinPrep is equivalent to the conventional
later by approval of the AutoCyte Prep™ (now known smear in the detection of endocervical AIS.36 Data also
as SurePath™; BD TriPath, Burlington, NC). The newest show comparable results between ThinPrep slides and
LBC is the MonoPrep™ (MonoGen, Inc., Lincolnshire, conventional smears for the detection of endometrial
Ill.), which was approved in 2006. LBCs were an impor- pathology.37
tant step in the development of automated Pap screening The ThinPrep collection vial has been approved by
devices—an improved preparation was needed to min- the FDA for direct testing for HPV, which is particularly
imize cell overlap so that automated screeners would useful for managing women whose Pap tests show atypi-
perform better in identifying abnormal cells. But LBC cal squamous cells (ASC).38,39
performed so well in clinical trials against conventional
smears that it found a market independent of auto-
SurePath Pap Test
mated screening. Although there are exceptions,25 the
great majority of peer-reviewed studies, some of them TriPath Imaging (acquired by Becton Dickinson in 2006)
detailed in this chapter, show an increased detection developed the SurePath Pap test (formerly AutoCyte Prep
of low-grade squamous intraepithelial lesions (LSILs) and CytoRich) for samples collected in an ethanol-based
or HSILs with LBC.26 The debate over increased disease transport medium. The process is shown in Figure 1.1B.
detection with LBC continues, however, and the stud- In contrast to the ThinPrep and MonoPrep methods, the
ies comparing LBC to smears have come under criticism practitioner snips off the tip of the collection device and
for allegedly sacrificing methodologic purity in their includes it in the sample vial. The equipment to pre-
design.26 Nevertheless, LBC offers several clear advan- pare slides includes a Hettich centrifuge and a PrepStain
tages over conventional smears: the opportunity to pre- robotic sample processer with computer and monitor.
pare duplicate slides and even cell block preparations The PrepMate™ is an optional accessory that automates
from the residual sample;27,28 the option of “out-of-vial” mixing the sample and dispensing it onto the density
aliquoting for HPV, chlamydia, and gonorrhea testing; reagent. Red blood cells and some leukocytes are elimi-
an improved substrate for automated screening devices; nated by density centrifugation. In addition to preparing
and a thinner cell preparation that most pathologists and an evenly distributed deposit of cells in a circle 13 mm in
cytotechnologists find less tiring to review than smears. diameter, the method incorporates a final staining step
that discretely stains each individual slide.
A multicenter, split-sample clinical trial showed a
ThinPrep Pap Test
7.2% increase in the detection of LSILs and more serious
The practitioner obtains the ThinPrep Pap sample with lesions and a significant decrease in the percentage of
either a broom-type device or a plastic spatula/endocer- unsatisfactory specimens.40
vical brush combination. The sampling device is swirled
or rinsed in a methanol-based preservative solution
MonoPrep Pap Test
(PreservCyt) for transport to the cytology laboratory and
then discarded. Red blood cells are lysed by the trans- The practitioner obtains the MonoPrep sample with
port medium. The vials are placed one at a time on the standard collection devices that are swirled or rinsed
ThinPrep 2000 instrument. The entire procedure (Fig. in a preservative-filled collection vial, after which the
1.1A) takes about 70 seconds per slide and results in a sampling device is discarded. As with the ThinPrep, red
thin deposit of cells in a circle 20 mm in diameter (con- blood cells are lysed by the transport medium. The vials
trast with cytospin: diameter = 6 mm). A batch-process- are delivered to the laboratory where slides are prepared
ing version (the ThinPrep 3000) is also available. It uses using the MonoPrep Processor, a fully automated, batch-
the same consumables (filters and solutions) but allows processing instrument capable of processing 40 samples
automated processing of 80 samples at one time. In most per hour, with a throughput capacity of 324 samples per
cases, only a fraction of the sample is used to prepare the 8-hour run. The process is shown in Figure 1.1C. In a
slide used for diagnosis. If needed, the residual sample is split-sample clinical trial similar in design to the ThinPrep
available for additional ThinPrep slide preparation, cell and SurePath trials, slides prepared by the MonoPrep
block preparation, or molecular diagnostic testing (e.g., method showed a 26% increase in the detection of LSILs
high risk HPV, chlamydia, gonorrhea). and more serious lesions, with no significant difference
A multicenter, split-sample study found that the in relative specificity.41 MonoPrep provided a signifi-
ThinPrep detected 18% more cases of LSILs and more cant reduction in unsatisfactory slides, and there was no
serious lesions as compared to conventional smears, difference in the presentation of endocervical or trans-
with no significant difference in the detection of organ- formation zone component or the detection of benign
isms.29 A number of studies have shown significant conditions.
SAMPLING AND PREPARATION METHODS 5
1. Dispersion 2. Cell collection 3. Cell transfer
A
1 2 3 4 5
Vortexing Disaggregation Transfer Sedimentation Cell deposition
to sedimentation 2 and staining
tube
B
1 2 3 4
High-speed Turbidity Aspiration Cell deposition
mixing check
C
figure 1.1 Liquid-based slide preparation methods. A, ThinPrep method: 1. The sample vial sits on a stage and a hollow plastic
cylinder with a 20-mm diameter polycarbonate filter bonded to its lower surface is inserted into the vial. A rotor spins the cylinder for
a few seconds, homogeneously dispersing the cells. 2. A vacuum is applied to the cylinder, trapping cells on the filter. The instrument
monitors cell density. 3. With continued application of vacuum, the cylinder (with cells attached to the filter) is inverted 180 degrees,
and the filter is pressed against a glass slide. The slide is immediately dropped into an alcohol bath. B, SurePath method: 1. The
sample is quickly vortexed. 2. A proprietary device, the Cyringe™, disaggregates large clusters by syringing the sample through a
small orifice. 3. The sample is poured into a centrifuge tube filled with a density gradient reagent. 4. Sedimentation is performed in
a centrifuge. A pellet is obtained and resuspended, and the sedimentation is repeated. 5. The tubes are transferred to the PrepStain
instrument, where a robotic arm transfers the fluid into a cylinder. Cells settle by gravity onto a cationic polyelectrolyte-coated slide.
The same robotic arm also dispenses sequential stains to individual cylinders. C, MonoPrep method: 1. An integrated stirrer mixes
the specimen briefly to disperse mucus and aggregates. 2. The specimen is aspirated into the hollow stirrer and dual-flow technol-
ogy captures a representative sample on a frit-backed filter. 3. The filter is pressed against the slide to transfer the cells onto a 20-mm
diameter circular area. 4. After cell transfer, the instrument applies a premeasured amount of alcohol fixative directly onto the slide.
