Eva M. Wojcik, Daniel F.I. Kurtycz, Dorothy L. Rosenthal - The Paris System For Reporting Urinary Cytology-Springer (2022)

The
Paris System
for Reporting
Urinary Cytology
Eva M. Wojcik
Daniel F.I. Kurtycz
Dorothy L. Rosenthal
Editors
Second Edition
123
The Paris System for Reporting Urinary
Cytology
Eva M. Wojcik • Daniel F.I. Kurtycz
Dorothy L. Rosenthal
Editors
The Paris System

for Reporting Urinary
Cytology
Second Edition
Editors
Eva M. Wojcik Daniel F.I. Kurtycz
Department of Pathology Department of Pathology and Laboratory
Loyola University Medical Center Medicine, University of Wisconsin School
Maywood, IL, USA of Medicine and Public Health, Wisconsin
State Laboratory of Hygiene
Dorothy L. Rosenthal Madison, WI, USA
Department of Pathology/Cytopathology
The Johns Hopkins University
Baltimore, MD, USA
ISBN 978-3-030-88685-1 ISBN 978-3-030-88686-8 (eBook)

https://doi.org/10.1007/978-3-030-88686-8
© Springer Nature Switzerland AG 2022

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This book is dedicated to the memory of
Stefan Pambuccian, MD (1957–2020), one of
the original members of The Paris Group.
From the beginning, Stefan was an integral
part of this project. His expertise and
commitment were always the guiding
principles for all of us to emulate.
Dr. Pambuccian was one of the greatest
pathologists our profession has ever
produced. He was a teacher and researcher
of the highest order. His work was
fundamental for The Paris System (TPS) and
his thoughtful, meticulous approach laid the
ground for the success of TPS.
Stefan was truly a gentle man. Despite his
monumental expertise, his humility and
dedication to work were exemplary. Stefan
left behind an army of his grateful mentees
who have been carrying on his legacy.
Preface
Almost a decade ago, members of two panels started preparing for the International
Congress of Cytology that took place in Paris in 2013. We all had the feeling that
maybe ours would be the last symposia on urine cytology. At that time, the rate of
atypia was raging and credibility with our clinical colleagues was dwindling. Urine
cytology was on the brink of losing clinical relevance, joining the fate of prostate
FNA and breast FNA, at least in the United States. Moreover, for many years, urine
cytology was one of the most frustrating types of cytology specimens, hated by
most cytology practitioners. Whatever we were doing, there was a very high prob-
ability that we were doing it wrong, at least in the eyes of our clinical colleagues.
They would have seen papillary lesions during cystoscopy, but cytology reports
were negative. On the other hand, they could not see any cystoscopic lesions and
cytology reports were positive. Of course, we knew why this happened. Low-grade
carcinomas do not exhibit enough morphologic changes to be distinguished from
normal and reactive urothelium, while flat, high-grade lesions (CIS, carcinoma in
situ), although cytologically detectable, are very difficult to visualize on cystoscopy.
Definitely, it was time for action to save urines!
Once we met in Paris, it was apparent that we were committed and ready to fix
the problem. That is how the original “Paris group” formed. During our first
impromptu meeting, we decided to start working on a standardized reporting system
that would be based on consensus and evidence. Of significance, during the very
first meeting we agreed that the new reporting system should concentrate on the
detection of a clinically significant disease, high-grade malignancy. That was a true
paradigm shift in urine cytology!
The rest is history! However, it did not happen overnight. It took a large group
of committed cytopathologists, surgical pathologists, and urologists who worked
very hard together to create, in record time, a new reporting system that was pub-
lished in 2016. We always knew that we had a good product, the system that we
truly believed in; however, the success of The Paris System (TPS) exceeded every-
one’s wildest imagination. Over the past 5 years, The Paris System has been trans-
lated into Japanese, Russian, and Chinese, and has been accepted throughout the
world as a standard of care. Our credibility in the eyes of clinicians has significantly
improved and, most importantly, now we are able to provide better patient care,
accurately screening for new or recurrent life-threatening high-grade bladder cancer.
vii
viii Preface
From the beginning, we realized that the first edition would only be a start. We
needed real data to confirm that the criteria of The Paris System are appropriate.
Most importantly, we needed prospective studies, based on TPS, to establish rates
of malignancy for each diagnostic category to guide our clinical colleagues.
This second edition fulfills these gaps. After the publication of TPS, a tsunami of
papers from all over the world confirmed the reliability of our established criteria.
Most significantly, the main goal of The Paris System was accomplished: lowering
the rates of the indeterminate categories, particularly diagnoses of “atypia.”
Moreover, after TPS had been published, we quickly realized that there were issues
not sufficiently covered in the first edition, particularly degeneration, subtypes of
high-grade urothelial carcinomas, and squamous dysplasias. TPS 2.0 extensively
discusses these topics. Although the overall concept of the first edition remains the
same, the second one is significantly expanded and much more richly illustrated.
With this continuously evolving project, we are confident that urine cytology is here
to stay. We hope you will agree!
Maywood, IL, USA Eva M. Wojcik MD

Madison, WI, USA Daniel F.I. Kurtycz MD
Baltimore, MD, USA Dorothy L. Rosenthal MD
Introduction
When The Paris System for Reporting Urinary Cytology (TPS) was introduced in 2016,
the new paradigm needed guidelines. Over the past 5 years, experience has provided
evidence that the proposed criteria are effective. As aids for microscopists when dealing
with a cytology slide, the authors of TPS 2.0 present the following in this section:

1. Decision Tree emphasizing changes in N/C ratios as morphologic find-
ings worsen.
The Paris System Approach to Diagnosis in Urinary Cytology
Is there increased
No N/C ratio ( 0.5)? Yes
(i.e., not intermediate
and basal urothelial cells)
Are any one of the following
features present in the atypical
cells?
Is there a reason for the 1. Hyperchromasia
2. Coarse Chromatin
“atypia”? 3. Irregular chromatinic rim
Polyomavirus?
Granuloma? At least 2 features
present and High (~≥ 0.7) N/C ratio
IIeal conduit?
Urolithiasis?
At least 1 feature How many cells with these
Reactive changes? present features?
“Few” ~ 5-10 cells “Many” ~ 10 cells
NEGATIVE FOR HGUC ATYPICAL SUSPICIOUS FOR HGUC HGUC
Graphic algorithm of the Paris System for Reporting Urinary Cytology decision tree.
The system emphasis is on the detection of High-Grade Urothelial Carcinoma (HGUC).
This snapshot conceptual flowchart illustrates the major points in the decision tree including
evaluation of the N/C ratio, nuclear features, and cell quantity. Ref: We’ll Always Have Paris:
The Paris System for Reporting Urinary Cytology 2022, Eva M. Wojcik, Daniel F.I. Kurtycz,
Dorothy L. Rosenthal. J Am Soc Cytopathol. online preprint, DOI: https://doi.org/1.01016/
jasc.2021.12.03
ix
x Introduction
2. Graphic illustration of visual differences in N/C ratios.
N:C
0.3
0.4
0.5
0.6
0.7
0.8
ML Zhang, Cancer Cytopathol 2016;124:669-77

Introduction xi
3. Essential data for each diagnostic category.

Diagnostic category Diagnostic criteria Example Frequency ROHM
Unsatisfactory Voided urine–volume (>30ml) 0 - 5% 0 - 16%

Instrumented urine-cellularity
Negative for High Benign urothelial, glandular, squamous cells, benign tissue 70 - 90% 8 - 24%
Grade Urothelial fragments, changes due to instrumentation, lithiasis,
Carcinoma (NHGUC) polyoma virus, therapy. Low Grade Urothelial Neoplasm
(LGUN)
Atypical Urothelial Required – increased N/C ratio ( > 0.5) and one of: 5 - 15% 24 - 53%
Cells (AUC) Hyperchromasia, Irregular clumpy chromatin or Irregular
nuclear contours
Suspicious for High Required – Few cells (< 5-10) with high N/C ratio (> 0.7) and 0.5 - 3% 59 - 94%
Grade Urothelial hyperchromasia, and/or Irregular clumpy chromatin,
Carcinoma (SHGUC) Irregular nuclear contours
Positive for High Required – Many cells (>10) with high N/C ratio (> 0.7) and 0.1 - 3% 76 - 100%
Grade Urothelial hyperchromasia, Irregular clumpy chromatin, Irregular
Carcinoma (HGUC) nuclear contours
ROHM –Risk of High Grade Malignancy
The authors of TPS will continue to monitor the utility of these criteria and will
rely upon our end users to publish their experience with TPS.
Abbreviations
AdCa Adenocarcinoma
AMACR Alpha-methylacyl-coA racemase
ASC American Society of Cytopathology
AUC Atypical Urothelial Cells
AUTF Atypical urothelial tissue fragments
BCG Bacillus Calmette-Guerin
BUTF Benign urothelial tissue fragments
CAP College of American Pathologists
CEP Chromosome enumeration probes
CIS Carcinoma in situ
CS Cytospin™
DAPI 4,6-Diamidino, 2-phenylindole dihydrochloride
DS Direct smear
ERG ETS-related gene
FDA U.S. Food and Drug Administration
Fig. Figure
FISH Fluorescence in situ hybridization
H&E Hematoxylin and eosin stain
HGUC High-grade urothelial carcinoma
HPV Human papillomavirus
IAC International Academy of Cytology
ISUP International Society of Urological Pathology
LBP Liquid-based preparations
LCNEC Large cell neuroendocrine carcinoma
LGPUC Low-grade papillary urothelial carcinoma
LGPUN Low-grade papillary urothelial neoplasm
LGUC Low-grade urothelial carcinoma
LGUN Low-grade urothelial neoplasia
LIS Laboratory Information System
LSI Locus-specific identifier
mag. Magnification
N/C Nuclear-to-cytoplasmic ratio
NE Neuroendocrine
NEC Neuroendocrine carcinoma
xiii
xiv Abbreviations
NHGUC Negative for high-grade urothelial carcinoma

non-UC Non-urothelial carcinoma
NUM Non-urothelial malignancy
NOS Not otherwise specified
Pap Papanicolaou
PSC Papanicolaou Society of Cytopathology
PUNLMP Papillary urothelial neoplasm of low malignant potential
RCC Renal cell carcinoma
SHGUC Suspicious for high-grade urothelial carcinoma
SMA Smooth muscle actin
SmCC Small cell carcinoma
SP SurePath™ LBP
SqCC Squamous cell carcinoma
The Paris System The Paris System for Reporting Urinary Cytology
TP ThinPrep™ process
TPS The Paris System
TTF-1 Thyroid transcription factor-1
U-FISH UroVysion™ fluorescence in situ hybridization
UC Urothelial carcinoma
UTF Urothelial tissue fragments
UUT Upper urinary tract
WHO World Health Organization
Contents
1 Pathogenesis of Urothelial Carcinoma �� 1

Kaitlin E. Sundling, Tatjana Antic, and Stefan E. Pambuccian
2 Adequacy of Urine Specimens (Adequacy)�� 7
Z. Laura Tabatabai, Güliz A. Barkan, Monique Courtade-Saïdi,
Daniel F.I. Kurtycz, Matthew T. Olson, Toyonori Tsuzuki,
Christopher J. VandenBussche, and Poonam Vohra
3 Negative for High-Grade Urothelial Carcinoma (NHGUC)�� 21
Christopher J. VandenBussche, Ashish Chandra, Jonas J. Heymann,
Zulfia McCroskey, Christopher L. Owens, Pawel T. Schubert, and
Yeh-Han Wang
4 Atypical Urothelial Cells (AUC) �� 63
Güliz A. Barkan, Margaret L. Compton, Tarik M. Elsheikh, Kim
A. Ely, Daniel F.I. Kurtycz, Merce Jorda, Zahra Maleki, Sachiko
Minamiguchi, Hiroshi Ohtani, Eric Piaton, Bo Ping, Spasenija Savic
Prince, Z. Laura Tabatabai, and Christopher J. VandenBussche
5 Suspicious for High-Grade Urothelial Carcinoma (SHGUC)�� 85
Fadi Brimo, Manon Auger, Ivan Chebib, Tarik M. Elsheikh, Mitsuru
Kinjo, Eric Piaton, Marc Pusztaszeri, and Tatsuro Shimokama
6 High-Grade Urothelial Carcinoma (HGUC)�� 97
Momin T. Siddiqui, Derek B. Allison, Guido Fadda, Jee-Young Han,
Patrick J. McIntire, Christopher L. Owens, Z. Laura Tabatabai,
Toyonori Tsuzuki, and Mingjuan Lisa Zhang
7 Cytopathology of the Upper Urinary Tract �� 115
Christopher J. VandenBussche, Jen-Fan Hang, Patrick J. McIntire,
Yurina Miki, Stephen Peyton, Poonam Vohra, and
Mingjuan Lisa Zhang
8 Non-Urothelial Malignancies and Other Miscellaneous Lesions�� 143
Tarik M. Elsheikh, Rana S. Hoda, Stefan E. Pambuccian, Jae Y. Ro,
and Sun Hee Sung
xv
xvi Contents
9 Ancillary Studies in Urinary Cytology�� 193

Lukas Bubendorf, Nancy P. Caraway, Andrew H. Fischer, Ruth
L. Katz, Fernando Schmitt, Margareta Strojan Fležar, Theodorus
H. Van der Kwast, and Philippe Vielh
10 Cytopreparatory Techniques�� 221
Donna K. Russell, Willam N. Crabtree, and Gary W. Gill
11 Risk of High-Grade Malignancy (ROHM)�� 249
Mauro Saieg, Güliz A. Barkan, Fadi Brimo, Ashish Chandra, Tarik
M. Elsheikh, Ricardo G. Pastorello, Marcus L. Quek, Jianyu Rao,
Momin T Siddiqui, Z. Laura Tabatabai, Christopher
J. VandenBussche, and Philippe Vielh
12 Clinical Management�� 257
Marcus L. Quek, Trinity J. Bivalacqua, Ashish M. Kamat, Mauro
Saieg, Alexander I. Sankin, Yuji Tokuda, and Bas WG van Rhijn
13 The History of Urinary Cytology: The Long and Winding Road to
Paris 2.0�� 267
Stefan E. Pambuccian
Afterword – Honoring the Past, Anticipating the Future�� 317

Future Clinical and Research Needs�� 318
Index�� 323
Contributors
Editors
Eva M. Wojcik, MD Department of Pathology and Laboratory Medicine, Loyola

University Medical Center, Maywood, IL, USA
Daniel F.I. Kurtycz, MD Department of Pathology and Laboratory Medicine,
University of Wisconsin School of Medicine and Public Health, Wisconsin State
Laboratory of Hygiene, Madison, WI, USA
Dorothy L. Rosenthal, MD Department of Pathology/Cytopathology, The Johns
Hopkins School of Medicine, Baltimore, MD, USA
Authors
Derek B. Allison, MD Department of Pathology & Laboratory Medicine,

University of Kentucky College of Medicine, Lexington, KY, USA
Tatjana Antic, MD Department of Pathology, The University of Chicago
Medicine, Chicago, IL, USA
Manon Auger, MD, FRCP(C) Department of Pathology, McGill University,
Montréal, QC, Canada
Güliz A. Barkan, MD Department of Pathology, Loyola University Healthcare
System, Maywood, IL, USA
Lukas Bubendorf, MD Institute of Molecular Genetics and Pathology, University
Hospital Basel, Basel, Switzerland
Trinity J. Bivalacqua, MD PHD Department of Urology, The Johns Hopkins
Hospital, The Johns Hopkins University, Baltimore, MD, USA
Fadi Brimo, MD, FRCP (C) Department of Pathology, McGill University,
Montréal, QC, Canada
Nancy P. Caraway, MD The University of Texas MD Anderson Cancer Center,
Houston, TX, USA
xvii
xviii Contributors
Ashish Chandra, MD FRCPath DipRCPath (Cytol) Department of Cellular

Pathology, Guy’s & St Thomas’ NHS Foundation Trust, London, UK
Ivan Chebib, MD, FRCPC Massachusetts General Hospital, Harvard Medical
School, Boston, MA, USA
Margaret L. Compton, MD Department of Pathology, Microbiology, and
Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
Department of Pathology and Laboratory Medicine, VA Tennessee Valley Healthcare
System, Nashville, TN, USA
Monique Courtade-Saïdi, MD, PhD Department of Cytology and Pathology,
Toulouse University Hospital, Toulouse, France
Willam N. Crabtree, PhD, SCT(ASCP) Department of Pathology and Laboratory
Medicine, Indiana University Pathology Laboratory, Indiana University School of
Medicine, Indianapolis, IN, USA
Tarik M. Elsheikh, MD Cleveland Clinic Laboratories, Pathology and Laboratory
Medicine Institute, Cleveland, OH, USA
Kim A. Ely, MD Department of Pathology, Microbiology and Immunology,
Vanderbilt University Medical Center, Nashville, TN, USA
Guido Fadda, MD, MIAC Department of Human Pathology of the Adulthood and
of the Developing Age, “Gaetano Barresi” of the University of Messina (Italy),
Rome, Italy
Consultant in Pathology of the Foundation “Agostino Gemelli” Hospital of the
Catholic University of Rome, Rome, Italy
Andrew H. Fischer, MD Department of Pathology and Cell Biology, University of
Massachusetts Memorial Medical Center, Worcester, MA, USA
Gary W. Gill, CT (ASCP) (Deceased) The John K. Frost Cytopathology
Laboratory, Johns Hopkins Hospital, Baltimore, MD, USA
Jee-Young Han, MD, PhD Department of Pathology, Inha University Hospital,
Incheon, South Korea
Jen-Fan Hang, MD, FIAC Department of Pathology and Laboratory Medicine,
Taipei Veterans General Hospital, School of Medicine, National Yang Ming Chiao
Tung University, Taipei, Taiwan
Jonas J. Heymann, MD Papanicolaou Cytopathology Laboratory, New York-
Presbyterian Hospital, Weill Cornell Medical College, New York, NY, USA
Rana S. Hoda, MD CBLPath/Sonic Healthcare USA, Inc., Rye Brook, NY, USA
Merce Jorda, MD, PhD, MBA Department of Pathology and Laboratory
Medicine, University of Miami Miller School of Medicine, Miami, FL, USA
Ruth L. Katz, MD Department of Pathology, Chaim Sheba Hospital, University of
Tel Aviv, Tel Aviv, Israel
Contributors xix
Ashish M. Kamat, MD, MBBS Department of Urologic Oncology (Surgery), The

University of Texas, MD Anderson Cancer Center, Houston, TX, USA
Mitsuru Kinjo, MD, PhD Department of Pathology and Laboratory Medicine,
Steel Memorial Yawata Hospital, Harunomachi, Yayata-higashiku, Kitakyushu-city,
Fukuoka, Japan
Zahra Maleki, MD, FCAP, MIAC Department of Pathology/Cytopathology, The
Johns Hopkins Hospital, Baltimore, MD, USA
Zulfia McCroskey, MD Florida Woman Care Laboratory, Tampa, FL, USA
Patrick J. McIntire, MD Department of Pathology, Cleveland Clinic,
Cleveland, OH, USA
Sachiko Minamiguchi, MD, PhD Department of Diagnostic Pathology, Kyoto
University Hospital, Kyoto, Japan
Yurina Miki, MBBS BSc FRCPath Guy's and St Thomas' NHS Foundation Trust,
London, UK
Hiroshi Ohtani, MD, PhD Department of Pathology, Hakujyuji Hospital,
Fukuoka, Japan
Matthew T. Olson, MD Roche Tissue Diagnostics, Tucson, AZ, USA
Christopher L. Owens, MD Quest Diagnostics, Marlborough, MA, USA
Stefan E. Pambuccian, MD (Deceased) Department of Pathology, Loyola School
of Medicine, Mayview, IL, USA
Ricardo G. Pastorello, MD Department of Pathology, Hospital Sírio Libanês,
São Paulo, SP, Brazil
Stephen Peyton, MBBS FRCPA(Australasia) Queensland Medical Laboratories,
Murarrie, QLD, Australia
Eric Piaton, MD, PhD Hospices Civils de Lyon, Université Claude Bernard Lyon,
Hôpital Femme-Mère-Enfant, Bron, France
Bo Ping Shanghai Cancer Center, Shanghai, China
Marc Pusztaszeri, MD Department of Pathology, Jewish General Hospital,
McGill University, Montréal, QC, Canada
Marcus L. Quek, MD, FACS Department of Urology, Loyola University Medical
Center, Maywood, IL, USA
Jianyu Rao, MD Department of Pathology and Laboratory Medicine, David
Geffen School of Medicine, University of California at Los Angeles, Los
Angeles, CA, USA
Jae Y. Ro, MD, PhD Department of Pathology and Genomic Medicine, Houston
Methodist Hospital, Weill Medical College of Cornell University, Houston, TX, USA
xx Contributors
Donna K. Russell, MEd, CT(ASCP)HT, CMIAC Department of Pathology and

Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
Mauro Saieg, MD, PhD, FIAC Cytopathology, AC Camargo Cancer Center,
Santa Casa Medical School, Sao Paulo, SP, Brazil
Alexander I. Sankin, MD, MS Department of Urology, Montefiore Medical
Center and Albert Einstein College of Medicine, Bronx, NY, USA
Spasenija Savic Prince, MD Institute of Medical Genetics and Pathology,
University Hospital Basel, Basel, Switzerland
Fernando Schmitt, MD, PhD, FIAC Department of Pathology, Medical Faculty
of Porto University, Unit of Molecular Pathology, Institute of Molecular Pathology
and Immunology, Health Research Network (RISE), Porto University, Porto,
Portugal
Pawel T. Schubert, MBChB, MMed, FCPath, MIAC Department of Pathology,
Stellenbosch University and National Health Laboratory Service, Cape Town,
Western Cape, South Africa
Tatsuro Shimokama, MD PhD Division of Pathology, Steel Memorial Yawata
Hospital, Kitakyushu, Japan
Momin T. Siddiqui, MD Department of Pathology and Laboratory Medicine,
Weill Cornell Medicine, New York, NY, USA
Margareta Strojan Fležar, MD, PhD, MIAC Institute of Pathology, Faculty of
Medicine, University of Ljubljana, Ljubljana, Slovenia
Kaitlin E. Sundling, MD, PhD Department of Pathology and Laboratory
Medicine, School of Medicine and Public Health, State Laboratory of Hygiene,
University of Wisconsin, Madison, WI, USA
Sun Hee Sung, MD, PhD Department of Pathology, Ewha Womans University,
MokDong Hospital, Seoul, South Korea
Yuji Tokuda, MD, PhD Department of Urology, Koga Hospital, Kurume,
Fukuoka, Japan
Toyonori Tsuzuki, MD, PhD Department of Pathology, Aichi Medical University
Hospital, Nagakute, Aichi, Japan
Z. Laura Tabatabai, MD Department of Pathology, University of California, San
Francisco (UCSF), San Francisco, CA, USA
Christopher J. VandenBussche, MD, PhD Department of Pathology/
Cytopathology, The Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Contributors xxi
Bas WG van Rhijn, MD, PhD, FEBU Department of Surgical Oncology
(Urology), Netherlands Cancer Institute – Antoni van Leeuwenhoek Hospital,
Amsterdam, The Netherlands
Theodorus H. Van der Kwast, MD, PhD Princess Margaret Cancer Center,
University Health Network, Toronto, ON, Canada
Philippe Vielh, MD, PhD, FIAC Department of Pathology, Medipath & American
Hospital of Paris, Paris, France
Poonam Vohra, MD Department of Pathology, University of California, San
Francisco (UCSF), San Francisco, CA, USA
Yeh-Han Wang, MD, MSS ACT Genomics Co., Ltd., Taipei, Taiwan
Mingjuan Lisa Zhang, MD Department of Pathology/Cytopathology,
Massachusetts General Hospital, Boston, MA, USA
Pathogenesis of Urothelial Carcinoma
1
Kaitlin E. Sundling, Tatjana Antic,
and Stefan E. Pambuccian
Summary of Changes in the Second Edition
• Incorporation of The Cancer Genome Atlas molecular characterization into the

pathogenesis of low-grade and high-grade urothelial carcinomas
Background
For any reporting system to be successfully applied in daily practice, it must be

based on evidence, consensus, inclusion, acceptance, and understanding [1].
Mechanistic understanding of the pathogenesis of disease underlies the accrual of a
body of evidence and scientific consensus supporting reproducible and clinically
relevant diagnostic categories. The main goal of urinary cytology is the detection of
high-grade urothelial carcinoma (HGUC). Therefore, the understanding of this dis-
ease, and particularly its pathogenesis, was crucial in the process of creating the
Paris System for Reporting Urinary Cytology (TPS).
Known risk factors for the development of both high-grade and low-grade uro-
thelial carcinomas include older age, male sex, tobacco smoking, and occupational
exposure to aromatic amines. Smoking may contribute to an increased risk of recur-
rence [2], and recent studies have sought to characterize molecular signatures asso-
ciated with risk factors [3].
The distinction of HGUC from benign changes and low-grade urothelial neo-
plasms (LGUNs) is central to the clinical utility of urine cytology. From a clinical
perspective, low-grade tumors comprise about 80% of urothelial carcinoma cases
and are almost always associated with a papillary appearance on cystoscopy, mak-
ing them possible to resect and follow clinically, even if cytologic detection is
limited.
Conversely, HGUC commonly presents as a flat lesion and may be cystoscopi-
cally indistinct. HGUC has high propensity for invasion into the muscle. Integral to
the detection of HGUC is the presence of the diagnostic nuclear features, including
© Springer Nature Switzerland AG 2022 1

E. M. Wojcik et al. (eds.), The Paris System for Reporting Urinary Cytology,
https://doi.org/10.1007/978-3-030-88686-8_1
2 K. E. Sundling et al.
an increased nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, coarse chroma-

tin, and nuclear membrane irregularities. The molecular basis of many of these hall-
marks of malignancy is not well understood but may center on the relationship
between the chromatin and the nuclear envelope via an intricate meshwork of pro-
teins that provide structural support as well as access to the chromatin [4].
This chapter establishes the molecular and pathophysiologic bases of HGUC and
LGUN, which provide a framework for the diagnostic categories established by TPS.
Molecular Pathways of Neoplastic Transformation of Urothelium
For many years it has been known that urothelial carcinoma has two distinct patho-
genetic pathways, a hyperplasia (low-grade urothelial neoplasms) and a dysplasia
(high-grade urothelial carcinoma) pathway [5–7]. More recently, The Cancer
Genome Atlas (TCGA) Research Network on molecular characterization of urothe-
lial bladder carcinoma revealed 302 mutations, 204 segmental copy number altera-
tions, and on average 22 rearrangements per tumor [8]. Thirty two genes showed
recurrent driver mutations. TCGA recognizes four molecular clusters based on dif-
ferential gene expression by RNA sequencing. TCGA also presents a pathway anal-
ysis based on mutation and copy number alterations (Fig. 1.1). It is not surprising,
given the rich and highly complex biological datasets studied, that the cluster and
pathway groupings have significant overlap. The Cluster I of TCGA corresponds to
commonly referred “luminal-type” in previously proposed systems [8]. The pre-
dominant pathway in “luminal-type” low-grade, non-muscle-invasive tumors is the
FGFR3/RAF/RAS signaling pathway with activating mutations in FGFR3 [9].
These tumors may progress from a precursor urothelial proliferation of unknown
significance (previously known as urothelial hyperplasia) with deletion of the gene
cyclin-dependent kinase inhibitor 2A (CDKN2A), located on the short arm of chro-
mosome 9, which encodes the p16INK4A protein. Papillary architecture is predomi-
nant in tumors with FGFR alterations. While these low-grade tumors uncommonly
progress to invasive carcinoma [10], if they do, they retain the molecular signature
involving FGFR3 alterations (about 20% of muscle-invasive bladder cancers). A
smaller group of low-grade urothelial carcinomas showed HRAS/KRAS/NRAS
mutations that are mutually exclusive with FGFR3.
The Cluster II of the TCGA is often referred to as “p53-like” and probably rep-
resents the less common dysplasia pathway. Initial dysplastic lesions harbor TP53
mutations and often progress to flat carcinoma in situ lesions, with concomitant RB
mutations. Lesions on this pathway have a high potential for muscle invasion, lymph
node metastases, and systemic metastases. This pathway has been found in 93% of
muscle-invasive carcinomas [8].
What is of significance is that the key molecular abnormalities associated with
the dysplasia pathway, especially the TP53 mutations, which are strongly associated
with high-grade and high-stage urothelial carcinomas, rarely co-occur with the
molecular abnormalities characterizing the FGFR3 pathway [8].
1 Pathogenesis of Urothelial Carcinoma 3
Normal
N orma
ormall Urothelium
ma Uro
roth
oth
the
thel
elliu
liu
um
Prec
Precursors
Prec
Precur
cur
urso
s rs
s
Hyperplasia Dysplasia
Urothelial Carcinoma, Non-Invasive
Low Grade High Grade Carcinoma

Carcinoma, Papillary Carcinoma, Papillary in situ
FGFR, RAS FGFR, TP53 TP53, RB
Urothelial Carcinoma, Muscle Invasive
RTK/RAS/PI3K Histone SWI/SNF

TP53/RB
Pathway (72%) Modification Complex
Pathway
FGFR3 alterations Pathway Alterations
(93%)
ERBB2 enrichment (89%) Pathway (64%)
Fig. 1.1 Schematic representation of the major histologic findings in urothelial carcinogenesis.
Key genetic alterations are noted in the noninvasive carcinomas, while pathway alterations from
TCGA analysis are noted in muscle-invasive urothelial carcinomas. Note that there is overlap
among the pathway and cluster designations, and the groupings are not mutually exclusive
4 K. E. Sundling et al.
The Clusters III and IV, which are referred to as “basal,” are tumors enriched in
squamous and sarcomatoid features that often have metastatic disease at presenta-
tion and express high levels of EGFR. About 12% of tumors in this cluster have
activating mutations in ERBB2 (Her2) [8].
The underlying pathways comprise four main groups, with significant overlap.
The RTK/RAS/PI3K pathway is altered in about 72% of urothelial carcinomas,
which contains a subset of tumors with FGFR3 alterations and/or ERBB2 enrich-
ment. The histone modification pathway includes changes in genes effecting meth-
ylation and acetylation and is altered in about 89% of urothelial carcinomas. The
SWI/SNF complex pathway involves nucleosome remodeling genes and is altered in
about 64% of urothelial carcinomas. Finally, the TP53/RB pathway includes many
well-known cell cycle regulatory genes and is altered in 93% of urothelial carci-
nomas [8].
The guiding principle for TPS is to detect HGUC. In line with the proven
strengths of urinary cytology, the negative category includes reactive changes,
infectious and nonneoplastic conditions, as well as cases that may have some cyto-
logic features of LGUN but are negative for HGUC. Therefore, the proposed diag-
nostic category is “Negative for High-Grade Urothelial Carcinoma” (NHGUC).
Despite the fact that we strive to detect all HGUC, we recognize that there will be
cases where a definite diagnosis cannot be made. Therefore, in TPS we include the
categories of “Atypical Urothelial Cells” (AUC) and “Suspicious for High-Grade
Urothelial Carcinoma” (SHGUC). Although the diagnosis of LGUC is not the main
goal of this system, a diagnostic option has been included to define those circum-
stances where cytologic features of HGUC are absent and LGUN are present (see
Chap. 3).
References
1. Sundling KE, Kurtycz DFI. Standardized terminology systems in cytopathology. Diagn
Cytopathol. 2019;47(1):53–63. https://doi.org/10.1002/dc.24103.
2. Lammers RJM, Witjes WPJ, Hendricksen K, Caris CTM, Janzing-Pastors MHC, Witjes
JA. Smoking status is a risk factor for recurrence after transurethral resection of non-
muscle- invasive bladder cancer. Eur Urol. 2011;60(4):713–20. https://doi.org/10.1016/j.
eururo.2011.07.010.
3. Koutros S, Rao N, Moore LE, et al. Targeted deep sequencing of bladder tumors reveals novel
associations between cancer gene mutations and mutational signatures with major risk fac-
tors. Clin Cancer Res. Published online April 13,. 2021; https://doi.org/10.1158/1078-0432.
CCR-20-4419.
4. Fischer EG. Nuclear morphology and the biology of cancer cells. Acta Cytol. 2020;64(6):511–9.
https://doi.org/10.1159/000508780.
5. Koss LG. Bladder cancer from a perspective of 40 years. J Cell Biochem Suppl. 1992;16I:23–9.
https://doi.org/10.1002/jcb.240501305.
6. Spruck CH, Ohneseit PF, Gonzalez-Zulueta M, et al. Two molecular pathways to transitional
cell carcinoma of the bladder. Cancer Res. 1994;54(3):784–8.
7. Lopez-Beltran A, Cimadamore A, Montironi R, Cheng L. Molecular pathology of urothelial
carcinoma. Hum Pathol. 2021;113:67–83. https://doi.org/10.1016/j.humpath.2021.04.001.
1 Pathogenesis of Urothelial Carcinoma 5
8. The Cancer Genome Atlas Research Network, Analysis Working Group: The University of
Texas MD Anderson Cancer Center. Comprehensive molecular characterization of urothelial
bladder carcinoma. Nature. 2014;507(7492):315–22. https://doi.org/10.1038/nature12965.
9. Kamoun A, de Reyniès A, Allory Y, et al. A consensus molecular classification of
muscle-invasive bladder cancer. Eur Urol. 2020;77(4):420–33. https://doi.org/10.1016/j.
eururo.2019.09.006.
10. Vollmer RT. A review of outcomes for stage Ta bladder tumors. Am J Clin Pathol.

2016;146(2):215–20. https://doi.org/10.1093/ajcp/aqw103.
Adequacy of Urine Specimens (Adequacy)
2
Z. Laura Tabatabai, Güliz A. Barkan,
Monique Courtade-Saïdi, Daniel F.I. Kurtycz,
Matthew T. Olson, Toyonori Tsuzuki,
Christopher J. VandenBussche, and Poonam Vohra
• More detailed discussions of urinary cytology adequacy

• Review of additional and updated references
• Updated figures
• Sample reports
• Quantitative volume recommendation for voided urine specimen adequacy
• Future directions
Background
Although adequacy criteria have been established in some areas of cytopathology to

prevent false-negative diagnoses, controversy has persisted in urinary tract cytol-
ogy. Accepting an inadequate sample as adequate for interpretation is one of the
most common causes of diagnostic discrepancies when two pathologists interpret
the same cytology specimen [1]. The overestimation of cellular or volume adequacy
is also recognized as a source of error and a reason for (1) failure to detect a lesion
due to an inadequate but accepted sample or for (2) overinterpreting cellular degen-
eration artifact as representing a significant lesion.
The analysis of urinary tract samples results from a complex interplay of mul-
tiple variables; at the pathologist sign out, these collapse into four concrete observ-
ables: collection type, cellularity, volume, and cytomorphological findings. While
the laboratory has a responsibility to validate standard procedures for ensuring the
maximum possible diagnostic uniformity, some pre-analytical variables are at
present too difficult to standardize. However, in the case of urine cytology, voided
urine stands as the only routinely processed cytological specimen collected with no
healthcare professional present as the specimen leaves the body. Thus, hygienic

https://doi.org/10.1007/978-3-030-88686-8_2
8 Z. L. Tabatabai et al.
collection policies can only be enforced partially. Similarly, the suggestion that the
void occur later than the first void of the morning is a detail not commonly covered
in lab collection systems. Other details including the physical and sexual activity
of the patient immediately prior to specimen collection are useful pieces of infor-
mation that often go unrecorded and therefore only partially appreciated in the
medical literature. The recommendations for adequacy evaluation per TPS stem
from the recognition that all of these variables are important and must be consid-
ered in the context of one another to properly address the questions pertaining to
adequacy.
Adequacy is an essential consideration for cytopathologists. It is worthwhile
repeating the obvious that diagnostic accuracy improves with adequacy.
Unfortunately, due to variable practices that existed before the formulation of
TPS, urine cytology specimens were commonly perceived to only infrequently
yield valuable clinical information. This perception is based on a reasonable
assumption that the limited sampling of cells exfoliating from the urothelial lin-
ing may not reflect the true biologic state of the patient, whether or not neopla-
sia exists.
To provide a quantitative context for sampling the urinary tract, the average
maximally distended healthy human bladder has an approximate volume of
600 mL. Given that a bladder has a roughly spherical shape, the inner surface
area of that bladder would be approximately 350 cm2 (350 × 10−4 m2). The aver-
age urothelial cell has an approximate diameter of 20 μm or a two-dimensional
surface area of 314 μm2 (314 × 10−12 m2), and the urothelium is approximately
five cells thick. Therefore, the total number of urothelial cells lining the bladder
can be estimated to be on the order of 108–109 cells. However, even a highly cel-
lular urine specimen contains only a minute fraction of the urothelial cells
derived from the urinary lining; the chance that a specimen will contain cells
from a clinically significant lesion is therefore a probabilistic event. The larger
the lesion, the greater the involvement of the urothelial surface; the more ana-
plastic and damaged the cellular adhesion structures, the more likely that diag-
nostic cells will be found in the sample. Alternatively, in the absence of disease,
a voided urine will at times contain so few cells that it can be technically consid-
ered “Inadequate for Diagnosis.” However, if the patient has no risk factors and
the volume is adequate, then a diagnosis of “Negative for High Grade Urothelial
Carcinoma (NHGUC)” may be more appropriate, with a limitation statement of
low cellularity.
A false-negative result can be the byproduct of a limited sampling size. Variation
in cellularity among urinary tract samples creates a desire for determining the mini-
mum number of benign cells available for examination that will provide confidence
that the remaining urothelium is benign as well.
2 Adequacy of Urine Specimens (Adequacy) 9
The Adequacy Algorithm
For the purposes of this chapter, the term “adequacy” refers to the usefulness of the
specimen to diagnose or raise suspicion for HGUC. As such, a specimen collected
for the diagnosis of acute bacterial cystitis that shows only neutrophils is inadequate
to detect urothelial carcinoma despite being perfectly useful to answer other specific
non-oncologic clinical questions. Adequacy of urine specimens for the diagnosis of
urothelial carcinoma is determined by the interplay of four specimen characteris-
tics: collection type, cellularity, volume, and cytomorphological findings. Of these
characteristics, cytomorphological findings should be considered first because the
presence of any atypical, suspicious, or malignant findings makes a specimen intrin-
sically adequate regardless of the collection type, cellularity, or volume. Thus, a
decision about specimen adequacy is most relevant when there are no findings
indicative of a disease process. If the cytomorphological findings are negative for
any markers of disease process, the other specimen characteristics should then be
factored into the classification of adequacy.
Since the publication of the first edition of TPS, studies on the role and specific
qualifiers of collection type, cellularity, and volume have provided additional
insights but remain limited, and the opinions expressed are variable. Therefore,
adequacy recommendations in TPS continue to be based on the adequacy algorithm
shown in Fig. 2.1. The purpose of this decision tree is threefold. The first is to com-
municate the relationship that exists among collection type, cellularity, and volume.
The second is to guide individual laboratories in validating appropriate choices for
their own practice settings at each branch in the adequacy algorithm. Finally, the
ultimate purpose is to provide a framework for future investigations dealing with
adequacy of urine specimens whereby each decision point will have clear, evidence-
based, and consensus-determined cutoff values for volume and cellularity in the
context of the collection method.
Due to the incomplete nature of the evidence to determine urine specimen ade-
quacy, the current algorithm does not take into consideration which method is used
for concentrating and preparing the urinary tract specimens. However, the generic
approach to adequacy determination should be uniform regardless of the method
used by a laboratory to prepare a specimen. As increasing data and evidence become
available in the literature, preparation-dependent cutoffs at each decision point in
the algorithm may become more apparent.
Volume and Adequacy
A common misperception is that cells in body fluids are solutes in a homogenous

solution; however, at times urine may be a heterogeneous mixture of non-solute
particulates such as crystals, microorganisms, decaying cell remnants, and viable
cells. Once collected, a urine sample is inherently heterogeneous because the par-
ticulates are not evenly distributed throughout the volume. Cells are denser than
most aqueous solutions and therefore sink. If a sample is not well mixed and the
paucicellular supernatant is examined, representative cells may not be found. If the
sample was decanted for cytology and the remaining denser material submitted for
biochemical or microbiologic studies, the cytologic picture may be skewed.
Collection itself may be an issue; in a voided sample the initial material will contain
more cells with a higher percentage being degenerated. Conversely, if only the latter
portion of the urine stream is captured in the specimen container for examination,
the results may be suboptimal due to lower cellularity.
Recent work shows that practices in determining adequacy are variable and often
not documented or existent. A survey performed prior to the publication of TPS
among 2116 laboratories revealed that adequacy criteria were established by 41.7%
and 34.5% of laboratories for voided urine and bladder washing/barbotage, respec-
tively. Adequacy was reported based on institutional criteria in voided urine speci-
mens and bladder washing specimens in 44.4% and 40.9% of laboratories,
respectively. For voided urine specimens, 38.9% used a volume of 10 mL, and
80.4% used a cellularity of 50 urothelial cells as a cutoff for adequacy [2].
More recently, a survey among 443 participants in the field showed that adequacy
criteria were employed by 59.9% and 57.5% for voided urine and instrumented uri-
nary specimens, respectively. For voided urine specimens, 21.7% and 29.9% used
volume and cellularity, respectively, for determining specimen adequacy. For instru-
mented urinary specimens, 10.6% and 38.8% used volume and cellularity, respec-
tively, for determining specimen adequacy. The reported volume used for determining
adequacy in voided and instrumented specimens ranged from 5–70 mL and 5–100 mL,
respectively. The reported cellularity used to determine adequacy ranged from 1 to
2600 cells for both specimen types. For voided and instrumented urinary specimens,
40.2% and 42.6% of participants, respectively, indicated it is not reasonable to use
either volume or cellularity for adequacy determination [3]. Volume factors only into
the adequacy of voided urine specimens for obvious reasons. A catheterized specimen
is instrumented, but the liquid is simply urine, requiring the same adequacy parame-
ters as voided urines. Washing specimens have added fluid volumes, so their adequacy
must be measured by the number and integrity of the cells therein. Conversely, urine
volume is important for voided urines for two main reasons (Fig. 2.2). First, a few
recent studies have shown a clear correlation between low-volume specimens and the
failure to diagnose malignancies [4–6], suggesting that some “benign” diagnoses in
low-volume specimens are due to the under-sampling of tumor by the low voided
urine volume rather than an absence of disease. This phenomenon is well-known in
effusion cytology, a field in which there can be a significant difference in malignancy
prevalence attributable to the volume submitted to the laboratory [7, 8]. Secondly, the
Atypical,
NO Suspicious YES
or
Malignant
YES Appropriate Benign

Instrumented
Urothelial Cellularity*
NO
NO YES
Non-Urothelial Features YES

Obscuring Urothelial Morphology
NO
YES Appropriate Benign

Urothelial Cellularity*
NO
NO
Adequate Volume
YES
Inadequate Adequate
Fig. 2.1 The adequacy algorithm shows The Paris System’s recommendation for the proper rela-
tionship between specimen source, cytological diagnosis, urine volume, urothelial cellularity, and
obscuring features. Obscuring features include non-urothelial cells such as lubricant, acute inflam-
mation, bacteria, vaginal contaminants, sperm, and crystals which, when copious, may obscure the
finding and characterization of urothelial cells. “*”: Cutoffs for appropriate benign urothelial cel-
lularity should be validated for both instrumented and non-instrumented sources
volume received by a laboratory can result from specimen manipulation at some point
in the collection, e.g., splitting the sample among diagnostic laboratories or discarding
some to fit into an available container.
Urine volume is logically an adequacy criterion, but data and studies remain
sparse. Assessing the role of urine volume as an adequacy criterion in clinical
practice remains challenging for the following reasons: the volume of urine
received in the cytology laboratory may not always be accurately documented,
6
Malignant
5 Suspicious for Malignancy
% Malignant or Suspicious
4
3
2
1
0
2.5 12.5 22.5 32.5 42.5 55 65 75 85 95 150

Volume Received (mL)
Fig. 2.2 Relationship of volume to the prevalence of malignant and suspicious diagnosis for
demographically comparable populations. There were no suspicious samples in the lowest volume
bin, and there was a nearly linear increase in the suspicious fraction up to the bin centered on
15 mL. A specimen was nearly twofold more likely to be suspicious when more than 15 mL was
received than when less than 5 mL was received. The global maximum for the malignancy fraction
was seen at 27.5 mL (range: 25–30 mL) which was also the location of a local maximum for the
suspicious fraction. The global maximum for the suspicious curve was centered at 85 mL, and the
difference between the global maximum (5.8, 95% CI: 5.6–5.9) and the local maximum at 27.5 mL
(5.7, 95% CI: 5.5–5.9) was not statistically significant. Based on this analysis, we concluded that
at least 30 mL is necessary to consider a urine fully adequate when processed with SurePath.
(From VandenBussche et al. [4] with kind permission of John Wiley & Sons)
and even if it is accurately documented, it may not reflect the true volume of the
original specimen. A variety of urine specimen types may be received including
voided, washing, and upper tract samples; the addition of a fixative to the sample
may be uncertain, even though it should be a regular part of each laboratory sam-
ple submission document; and the accurate amount of the added fixative is rarely
provided to the laboratory [9].
A recent study suggested that a minimum cutoff of 30 mL is appropriate in a
laboratory that uses SurePath® preparation performed on fresh, unfixed voided
urines [4]. A later, similar study recommended a minimum cutoff of 25 mL for
ThinPrep (Hologic, Inc., MA) preparations, also performed on fresh, unfixed voided
urines. Of note, in the second study, the cytology results were not confirmed by a
biopsy or surgical excision [5]. Yet another recent study justified urine volume as an
adequacy criterion in voided urine and reported that although 10 mL of fresh voided
urine might achieve sufficient cellularity, a higher volume (30 mL) is recommended

to maximize the chance of detecting a high-risk diagnosis [9]. Probabilistically, the
more sample the lab receives, the more likely the result will reflect the true biology.
Patients with a history of an inadequate or suboptimal voided urine specimen are
highly likely to yield an adequate specimen if they return for a repeat specimen
within 6 months of the original specimen and provide a higher volume of urine
(>30 mL) [4].
Conversely, another study by Renshaw and Gould demonstrated that whereas the
number of urothelial cells in a voided urine specimen was significantly associated
with the sensitivity for detecting HGUC, the specimen volume was not [10]. In
comparison to instrumented specimens, another study by the same authors found
that both the cellularity and sensitivity of voided urine specimens were lower [6].
While volume is an important factor in the adequacy algorithm for voided urines,
it is clearly not a variable that should solely disqualify a specimen from analysis.
Disqualification of a sample based simply on its lower volume may lead to labora-
tories discarding specimens in which diagnostic findings can still be present. A
lower volume may be proportional to a lower chance of finding diagnostic features,
but that chance is not zero. Two microscope-dependent decision points precede vol-
ume in the adequacy algorithm: the finding of atypical, suspicious, or malignant
cells and the finding of an adequate number of benign urothelial cells, the numeric
range of which needs to be more rigorously determined and agreed upon. Because
the urothelial cell content is unknown prior to processing and microscopic examina-
tion, laboratories should not reject urine specimens based on volume alone.
Cellularity and Adequacy
Instrumentation of the urinary tract results in cellular samples in which cells and
tissue fragments are forcibly exfoliated from the urothelium; these include bladder
washings (also called “barbotage” specimens). Other instrumented urinary speci-
mens include urine collected after cystoscopy, radiologic procedures, catheteriza-
tion, washings and brushings from the urethra, ureters, and renal pelvises. Of these,
the specimen type most commonly processed is bladder washing, which is often
part of a cystoscopic procedure [11].
The cellularity of instrumented urinary tract specimens obtained via cystoscopy
is affected by various technical factors including those related to the proceduralist’s
skill, method of performing the washing, amount and type of fluid with which the
bladder is washed, and the distance of the cystoscope to the mucosa. Although the
laboratory cannot control most of these factors, setting adequacy criteria will alert
clinicians when the specimen has insufficient cellularity and may be more prone to
a false-negative diagnosis. One factor that the laboratory can influence is the type of
irrigation fluid used. See Chap. 10 (Cytopreparatory Techniques) for a discussion of
optimal cellular preservation during washings.
Compared to voided urine specimens, bladder and upper tract washing speci-
mens have a volume that is dependent on the amount of fluid used during the proce-
dure, generally lack contamination by cells originating outside of the urinary tract,
and should have a higher cellularity. In addition, such samples are more often
received fresh or admixed with fixatives. These features may contribute to the higher
sensitivity of bladder washing specimens [6, 12] but also suggest the need for
volume-independent adequacy criteria. A recent study suggested that all instru-
mented specimens should be considered adequate because it is unlikely that the
invasive procedure necessary for a repeat sample will be based only on nondiagnos-
tic cytology [6]. However, since the decision to repeat is based on the overall clini-
cal findings, the urologist has to know that the procedure did not yield diagnostic
material.
Another recent study suggested that for instrumented urine specimens, cases
with no urothelial cells should be considered inadequate, whereas cases with <20
cells per 10 high-power fields (HPF) should be considered as “limited” because they
have a significantly lower sensitivity than samples with higher cell counts. The sen-
sitivities reported for Cytospin preparations and ThinPrep preparations are similar,
suggesting that cell density rather than total cells may be the preferred measure of
cellularity in the setting of urinary cytology [10].
Quantitative adequacy criteria for specimen cellularity have been established
for Pap tests based on evidence obtained by studies using serial dilutions and for
thyroid aspirates based on retrospective review of cytology specimens with histo-
logic follow-up [13, 14]. For urinary cytology, an arbitrary cutoff value of 15
well-preserved and well-visualized urothelial cells has been proposed by some
authors [15, 16].
To date, only a single study has applied both the serial dilution and surgical exci-
sion methods to bladder washing specimens to establish the cell count adequacy
criteria for specimens processed using the ThinPrep method [17]. In this study, an
adequate bladder wash specimen was determined to have at least 20 well-preserved,
well-visualized urothelial cells per 10 HPF. This cutoff was only viable in the
absence of obscuring features. These results were further supported by a more
recent study performed on Cytospin preparations of instrumented urine cytology
specimens which showed that less than 10 urothelial cells per 10 HPF is associated
with significantly increased false-negative rates compared to cases with 10–49 uro-
thelial cells per 10 HPF [10]. Interestingly, this study also noted that cases with 50
or more urothelial cells per 10 HPF also had an increased false-negative rate. In
these high cellularity cases, the neoplastic cells may be diluted by the many benign
urothelial cells in the specimen. In such cases, it is not known whether repeat testing
would resolve this problem.
Another recent study, using voided urine samples from patients who were either
positive or negative for HGUC, compared ThinPrep and Cytospin processing
devices. Specimens from a total of ten individuals positive for HGUC and nine
individuals negative for HGUC were collected and split to compare the two prepara-
tion methods. This study showed that the morphological features of HGUC and
normal cells were comparable in specimens prepared using either the ThinPrep or
Cytospin systems. The authors concluded that TPS can be applied reliably in diag-
nostic laboratories that use either of these methods [18].
These studies support the observations that instrumented urine cytology cases
where there are 10–20 well-preserved, well-visualized urothelial cells per 10 HPF
are “satisfactory but limited by low cellularity” and cases where there are less than
10 well-preserved, well-visualized urothelial cells per 10 HPF are “unsatisfactory/
nondiagnostic.” Given that these thresholds were determined using ThinPrep and
Cytospin preparations, additional studies are required to determine the thresholds
for other specimen preparation methods. Similarly, since the majority of studies
have been performed on bladder washing specimens, the adequacy criteria for upper
tract and voided specimens are not as rigorously defined. The presence of excessive
lubricant, inflammatory cells, or red blood cells, either as the sole finding or obscur-
ing the urothelial cells, must be interpreted with caution (Fig. 2.3) (see sample
reports). As further data become available for upper tract and voided specimens, the
criteria will reflect the evidence, and this evidence will provide hard quantitative
data to the adequacy algorithm.
a b
Fig. 2.3 Unsatisfactory/nondiagnostic specimen. (a) Acellular specimen showing only lubricant
without the presence of urothelial cells (Bladder wash, TP, medium mag.). (b) Scantly cellular
specimen with obscuring blood (voided urine, TP, medium mag.). (c) Scantly cellular specimen
with obscuring inflammation in a patient with a papillary lesion observed on cystoscopy (bladder
wash, TP, medium mag.)
The Less Than Optimal Adequacy Category
The most uncertain feature of the adequacy algorithm is the classification of voided
urine specimens that have an insufficient or low number of benign urothelial cells but
have an adequate volume. There is currently no evidence that demonstrates the require-
ment for benign urothelial cells in voided urine specimens. In many practices, speci-
mens that meet every adequacy criterion except for urothelial cellularity are assigned a
“less-than-optimal” adequacy. However, there is not a precise cellular metric for when
a voided urine specimen should be classified as “nondiagnostic” or “less than optimal”
[6]. The sensitivity of low cellularity specimens is still relatively high, and in these
cases, the cytology specimen is often one part of an examination that includes clinical
observation and biopsy. As a result, laboratories may prefer to diagnose such cases as
benign/negative with an explanatory note. Alternatively, if sufficient volume were
available, laboratories could make additional preparations in the face of low cellularity
samples which may increase the number of cells found to adequate levels and avoid the
expense and invasiveness of obtaining an instrumented urine specimen.
Previous studies of ThinPrep preparations of urinary bladder wash cytology sug-
gest that specimens with 10–20 cells per 10 HPF should be diagnosed as “less than
satisfactory,” and a more recent study reported that cases with less than 20 cells per
HPF should be considered as “limited” because of significantly lower sensitivity [6,
17]. While the available data are small and limited to a few institutional experiences,
the addition of this category appears to be useful: patients who returned to provide
a repeat voided urine sample usually yielded fully adequate specimens, some of
which resulted in diagnostic findings [4].
Recommendations
Adequacy in urine specimens is a topic for which there remains limited concrete,
quality data in the context of the number of variables at play. As such, the recom-
mendations for adequacy in TPS continue to center on the adequacy algorithm.
When properly validated for each decision point, this algorithm will increase stan-
dardization and quality in reporting across laboratories. As more quantitative results
of validation become available in the literature, we expect this algorithm to adopt
more defined prescriptive qualities that may lead to uniform practice in all urine
adequacy determinations. For now, TPS recommends adequacy as at least 30 mL
voided urine volume when evaluating specimens in the context of the adequacy
algorithm. (Figs. 2.1 and 2.2).
Future Directions
Additional studies are needed to evaluate the correlation between volume and ade-
quacy for voided urines as well as cellularity and adequacy for bladder and upper
urinary tract washings in laboratories that use different cytopreparatory techniques.
Another focus for future studies is the assessment and definition of “less than
optimal” in urinary cytology specimens. Furthermore, it may be beneficial to incor-

porate the patient’s medical history in addition to using urine volume cutoff values
to assess specimen adequacy with more collaborative efforts among the cytopathol-
ogy and urology communities [11, 19].
Sample Reports
Example 1
Voided urine:
Adequacy: Satisfactory for evaluation
Interpretation: Atypical urothelial cells present
Example 2
Voided urine:
Adequacy: Less than optimal
Interpretation: Negative for high-grade urothelial carcinoma. See
note.
Note: The interpretation is limited by low urothelial cellularity/obscuring inflam-

mation/blood/bacteria, etc. Clinical correlation is advised with consideration for
repeat sampling if clinically indicated.
Example 3
Voided urine:
Adequacy: Unsatisfactory for evaluation
Interpretation: Nondiagnostic sample; see note.
Note: The sample shows only rare benign urothelial/inflammatory/other cells in

a low-volume voided specimen (<30 mL). Clinical correlation is advised with con-
sideration for repeat sampling if clinically indicated.
Example 4
Urine, bladder wash:
Adequacy: Less than optimal
Interpretation: Negative for high-grade urothelial carcinoma. See
note.
Note: The interpretation is limited by scant cellularity/obscuring inflammation/

lubricant/blood/bacteria, etc. Clinical correlation is advised with consideration for
repeat sampling if clinically indicated.
Example 5
Urine, bladder wash:
Adequacy: Unsatisfactory
Interpretation: Nondiagnostic sample. See note.
Note: The specimen is acellular or consists of only lubricant/blood/bacteria, etc.

without the presence of urothelial cells. Consideration for repeat sampling is
recommended.
References
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center’s experience with second review of 3885 thyroid cytopathology specimens. J Clin
Endocrinol Metab. 2013;98:1450–7.
2. Barkan GA, Tabatabai ZL, Kurtycz DFI, Padmanabhan V, Souers RJ, Nayar R, Sturgis
CD. Practice patterns in urinary cytopathology prior to The Paris System for Reporting Urinary
Cytology. Arch Pathol Lab Med. 2020;144(2):172–6.
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The Paris System for Reporting Urinary Cytology 2.0 (TPS 2.0). J Am Soc Cytopathol.
2021;10(5):S4 (PL06).
4. VandenBussche CJ, Rosenthal DL, Olson MT. Adequacy in voided urine cytology specimens:
the role of volume and a repeat void upon predictive values for high-grade urothelial carci-
noma. Cancer Cytopathol. 2016;124:174–80.
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Negative for High-Grade Urothelial
Carcinoma (NHGUC) 3
Christopher J. VandenBussche, Ashish Chandra,
Jonas J. Heymann, Zulfia McCroskey, Christopher L. Owens,
Pawel T. Schubert, and Yeh-Han Wang
The following major changes have been made to this chapter:
• Clarification and streamlined discussion of atypical vs. benign-appearing urothe-

lial tissue fragments
• Integration of discussion regarding low-grade urothelial neoplasms
• Further characterization of degenerative changes in benign specimens
• Updated performance data for the NHGUC category
• Updated and increased number of representative photographs
Background
Many cytology reporting systems regard the “negative” category as containing only
normal cells from a body site without any evidence of neoplasia. Mild indeterminate
changes are placed in the “atypical” category. If TPS followed that premise, the
“atypical” category would be so large that it would serve no clinical usefulness [1].
Following the lead of The Bethesda System for Reporting Gynecologic Cytology,
TPS includes in the negative category all entities that pose no significant risk to the
patient for developing high-grade urothelial carcinoma (HGUC) based upon current
knowledge. Therefore, if a specific cause of a particular morphologic alteration in
urothelial cells is recognized and the cause is not associated with malignancy, e.g.,
radiation-induced “atypia,” those cases are best classified as “Negative for HGUC”
(NHGUC) and not atypical, unless there are other cellular alterations in the sample
that warrant the “Atypical Urothelial Cells” (AUC) designation. Furthermore, the
goal of TPS is to highlight those cases that are at risk for HGUC. Therefore, the
negative category emphasizes that goal by stating NHGUC.

https://doi.org/10.1007/978-3-030-88686-8_3
22 C. J. VandenBussche et al.
Many conditions can cause discernible reactive and reparative cytologic changes
but pose no threat to the patient of developing HGUC [2], and they are included in
this chapter. Those entities in which there may be an association with neoplasia will
be so noted and included in both the NHGUC and AUC categories with appropriate
codicils.
efinition of Negative for High-Grade Urothelial

D
Carcinoma (NHGUC)
A sample of urine, either voided or instrumented (including washing specimens),

may be categorized as NHGUC if it is diagnostically adequate (see Chap. 2,
Adequacy) and lacks any cytomorphologic findings shown to be significantly asso-
ciated with HGUC.
Criteria of Components of NHGUC
Benign Superficial (Umbrella) Urothelial Cells
Superficial cells are large, shaped like the canopy of an umbrella, with rounded
(convex) luminal surfaces and scalloped (concave) borders onto which the underly-
ing intermediate cells are sometimes attached (Fig. 3.1). The cytoplasm is abundant
and vacuolated or foamy, not to be mistaken for virally induced koilocytes. They are
often bi- or multinucleated or may contain a single large nucleus. The nuclei are
centrally located, round to oval, with smooth nuclear membranes. The chromatin is
fine and an occasionally prominent chromocenter/nucleolus is present.
Characteristically, the nuclear/cytoplasmic (N/C) ratio is low.
In contrast, intermediate (“parabasal-like”) urothelial cells have nuclei with basi-
cally the same size and character as superficial cells, but have less cytoplasm,
imparting a higher N/C ratio (Fig. 3.2). Since they are less mature than superficial
cells, their nuclear chromatin may be a bit coarser than the ubiquitous superficial
cells, and they may have shallow grooves oriented along the long axis of the nucleus;
but otherwise thin, even nuclear membranes and uniform chromatin distribution
will support their totally benign condition. Cytoplasm will not be as vacuolated as
superficial cells but is not completely opaque (homogeneous), the latter feature
being cited as a characteristic of low-grade urothelial neoplasms in some studies
[3–6]. Because of their increased N/C ratios, they are commonly misinterpreted and
placed in the AUC category. Identification of their bland nuclear features can help
avoid that pitfall.
Deep urothelial cells (basal cells) are small cells that form small-to-intermediate
sized fragments (Fig. 3.3). They may appear hyperchromatic at low magnification
due to their elevated N/C ratios. However, the nuclei are uniform and regularly
3 Negative for High-Grade Urothelial Carcinoma (NHGUC) 23
Fig. 3.1 Superficial
urothelial (umbrella) cells. a
(a) The cytoplasm of large
superficial cells is frothy
and abundant, resulting in
a low N/C ratio. Nuclei
have pale, finely granular
chromatin and prominent
nucleoli. Multinucleation
is common, especially in
instrumented samples.
(Washing, TP, medium
mag.). (b) In addition to
superficial (umbrella) cells,
clusters of smaller cells are
seen (arrows). The nuclei
are darker and slightly
smaller than the superficial
cells, but the nuclear b
shapes are round, nuclear
membranes are smooth,
and architecture is
uniform. N/C ratios are
high but, in the context of
the other criteria, should
not be considered atypical.
(Washing, TP, medium
mag.). (c) Umbrella cells
are the most superficial
cells in the bladder,
creating an “umbrella”
over all other urothelial
cells. Their nuclear and
cytoplasmic character is
the same as other
superficial cells, but c
additionally, they possess a
thickened cytoplasmic
edge that doesn’t go all
around the cell. (Washing,
CS, medium mag.)
Fig. 3.2 Intermediate urothelial cells. These cells have a “fried egg” appearance and resemble the
parabasal squamous cells seen in Pap test specimens, causing them to also be described as
“parabasal-type” or “parabasal-like” cells. They are usually found singly or in small, loose clusters
(as seen here). Note the low N/C ratios, oval-shaped nuclei with regular borders, and bland chro-
matin pattern. The nuclei also are uniform in size. (Voided, SP, high mag.)
Fig. 3.3 Deep urothelial

cells. This sheet is essentially
a monolayer of uniform cells
with round nuclei and
uniformly pale chromatin.
N/C ratios are high, a
reflection of the deep, more
basal position of these cells
in the urothelial layer.
(Washing, TP, medium mag.)
arranged within their tissue fragments. The chromatin lacks the coarse, clumpy
quality associated with HGUC. Although they may form papillary fragments, they
lack fibrovascular cores and thus are not “true” papillary fragments. This helps dis-
tinguish fragments of basal cells from low grade urothelial neoplasms.
Squamous Epithelial Cells, Both Superficial and Intermediate
Both men and women can be expected to have benign squamous cells (Fig. 3.4) in
their urine, although they are more common in women. In voided urine from a
woman, the origin may be the urethra but also may be a contaminant arising from
the vagina or perineum. The bladder trigone is subject to estrogenic effect and can
produce squamous cells similar to those found in a cervical sample. Instrumentation
can liberate squamous cells from the urethra and from areas of squamous metapla-
sia in both men and women. Of note, chronic irritation, particularly due to stones, is
often responsible for squamous metaplasia. Although keratinizing squamous meta-
plasia may be a precursor of squamous neoplasia, TPS does not require specifically
reporting the presence of squamous epithelial cells, including parakeratotic or anu-
cleate forms.
Fig. 3.4 Benign squamous

cells. Two benign
squamous cells line up
below an umbrella cell
with three nuclei. The
presence of squamous cells
in voided urine may come
from external genitalia,
including the vagina. In a
catheterized patient, their
origin is usually in an area
of metaplasia in the
trigone. (VU, TP, medium
mag.)
Glandular Cells
In voided urine from women, benign glandular cells (Fig. 3.5) may be derived from
the uterine cervix or corpus and are usually few and degenerated. Endometrial-type
cells are characterized as cohesive, three-dimensional aggregates of small glandular
cells with scant cytoplasm, slightly irregular nuclei with vesicular fine chromatin,
and visible small nucleoli. A few clusters in a menstruating woman are of no conse-
quence. However, when the patient is menopausal, and endometrial cells are present
in her voided urine, this is of concern, and clinicians should be alerted (see Chap. 8,
“Non-Urothelial Malignancies and Other Miscellaneous Lesions”).
Glandular-type cells from the urinary tract have small nuclei and vacuolated
cytoplasm. Most often glandular groups are multilayered true tissue fragments but
also can be seen as single cells. There are many potential sites of origin for glandu-
lar cells in urologic samples. Glandular epithelium may arise in the dome of the
bladder (urachal remnant) or trigone as a developmental rest. These changes are not
metaplastic. Endometriosis may involve the urinary tract and present an unexpected
cellular picture of very small hyperchromatic cells, either in voided urine or brush-
ings from the ureter (Fig. 3.5) Cells from Mullerian rests (Mullerianosis) are also
native, albeit rare and may have endometrial, endosalpingial, or endocervical fea-
tures. Conversely, cystitis cystica/glandularis and intestinal metaplasia (Fig. 3.6) are
both metaplastic processes of the urothelium resulting from chronic inflammation.
Although the metaplasia is benign, patients with extensive intestinal metaplasia
involving the urinary tract are at risk of developing subsequent bladder adenocarci-
noma [12]. Therefore, if definitively identified, such metaplasia may be reported as
an additional finding in the NHGUC category.
Renal tubular epithelial cells (RTCs) (Fig. 3.7) will usually appear as degener-
ated, small cells with mildly irregular nuclear contours. When derived from the
distal tubules, the cells will have high N/C ratios; if examined at high magnification,
Fig. 3.5 Benign glandular cells – endometriosis. Glandular cells in a urinary tract specimen may
be native to the urinary collecting system, or external to it. The larger cells are urothelial or squa-
mous. The cluster of small dark cells originated in an endometriosis of the ureter. The patient was
presenting with hematuria and pain coincident with her menstrual periods. (Ureteral brushing, CS,
high mag.)
Fig. 3.6 Cystitis cystica/glandularis. (a) Glandular cells from the lining of the bladder can origi-
nate from a focus native to the urothelium, metaplasia from an inflammatory focus (cystitis cystica/
glandularis), or from a glandular neoplasm, either primary or secondary. Unless the cytomorphol-
ogy suggests a neoplasm, glandular cells are considered benign. (Washing, TP, high mag.). (b)
Benign glandular cells. The nuclei are uniform in size and shape, with a bland chromatin pattern,
and the cells maintain a columnar architecture. (Washing, SP, high mag.). (c) Cystitis cystica may
appear as a single layer of glandular cells. They closely resemble endocervical cells and could be
from a case of endocervicosis. This mucosal strip was from a focus of cystitis cystica. (Washing,
TP, high mag.). (d) Another tight glandular group, a BUTF, demonstrates nuclear compression by
relatively large cytoplasmic vacuoles. Regardless of their origin, these cells fulfill the criteria of
benignity, making the diagnosis of NHGUC appropriate. (Washing, TP, high mag.). (e) Benign
glandular cells. The glandular cells are numerous in this field, and the formation of a hyperchro-
matic crowded group may be alarming. It is easiest to identify their columnar morphology at the
edges or in the singly dispersed cells. Note how the nuclei are all relatively similar in size, in addi-
tion to maintaining their apical cytoplasm. (Washing, SP, high mag.). (f) Benign glandular cells. In
this separate field, the glandular cells are mostly individually dispersed and some appear more
cercariform than columnar. The cells have similarly sized, oval-shaped nuclei with bland chroma-
tin. This cytomorphologic pattern has significant overlap with that of a LGUN but without a fibro-
vascular core. Fortunately, there are no features of HGUC, so a diagnosis of NHGUC is not
challenging to make. (Washing, SP, intermediate mag.)
Fig. 3.6 (continued)
c
f
Fig. 3.7 Renal tubular

cells (RTC). (a) RTCs can a
be very small, the size of
histiocytes. When
aggregated as in this group,
a cast should be
considered. There are
usually variable amounts
of cellular degeneration
(voided, SP, high mag.). (b)
This larger fragment
represents a tubular cast.
The renal tubular cells vary
from small with scant
cytoplasm to larger and
vacuolated. The patient
was in renal failure.
(Voided, SP, high mag.). (c)
RTC within this cast
b
demonstrate small nuclei
with relatively abundant
cytoplasm. The material
supporting the RTC in the
cast is protein. The patient
was in renal failure.
(Voided, CS, high mag.)
c
they may be incorrectly interpreted as atypical urothelial cells. Cells from the proxi-
mal tubules, less commonly seen, have lower N/C ratios due to abundant, granular
cytoplasm, resembling degenerated histiocytes. RTCs are usually found in voided
urine specimens as rare, small fragments of cells with scalloped edges that appear
loosely attached at low magnification. The fragments typically contain five to ten
cells. The recognition of this pattern at low magnification can prevent over interpre-
tation at higher magnifications. Depending on the site from which they arise, the
morphology of these cells may vary slightly, and they may also be seen to form casts.
Degenerative Changes
Most voided urine specimens contain urothelial cells that have undergone some
degree of degeneration. These cells have naturally exfoliated into the urinary stream
and remained in the bladder at body temperature until specimen collection. Urine is
a poor cellular preservative, and once a specimen is collected, it may sit at room
temperature for some time until it is refrigerated, processed, or placed into a preser-
vative. Certain conditions may contribute to cellular degeneration, such as immuno-
therapy (described below), urolithiasis, and bacterial infection. Urine specimens
collected following the relief of a urinary tract obstruction may contain numerous
degenerated cells (Fig. 3.8).
Degenerated benign intermediate urothelial cells are often smaller than well-
preserved urothelial cells and have shrunken (condensed), dark nuclei with mildly
irregular nuclear borders. The N/C ratio of these cells typically remains below 0.5
(Fig. 3.9). Characteristic of degeneration are large eosinophilic and/or cyanophilic
Fig. 3.8 Cellular degeneration secondary to stasis. When cells sit in the urinary tract for long
periods of time, they begin to degenerate. Many of the cells here have small, dark pyknotic nuclei
but retain a large amount of cytoplasm. The cells have granular cytoplasm and some cells have
irregular cytoplasmic vacuoles. More severely degenerated cells have lost their nuclear-cytoplasmic
interface. The dark nuclei may be concerning for urothelial carcinoma; however, specimens with
degenerated HGUC cells will often contain some larger cells, and the nuclei will be larger in size
than those seen here. (Catheterized, SP, intermediate mag.)
Fig. 3.9 Degeneration. (a) Specimens may contain a large number of degenerated cells, which can
give the impression of a proliferative or neoplastic process. Examination of each individual cell in
this field is reassuring, as the cells contain dark yet very small nuclei and maintain their N/C ratios.
Many are RTCs (voided urine, SP, high mag.). (b) This degenerated benign cell has chromatin that
appears smudged and homogenous. The N/C ratio is low and the cytoplasm is vacuolated (voided
urine, SP, high mag.). (c) These degenerated cells are enlarged and dark, but the nuclear-cytoplasmic
interface is becoming lost in the cell on the right. The nuclei of both cells contain a condensed ball
of chromatin. These cells are too far degenerated for assessment. (Voided urine, SP, high mag.). (d)
At low magnification, this cell may appear to have a high N/C with a prominent nucleolus, but the
cytoplasm is vacuolated and the nuclear-cytoplasmic interface cannot be clearly demarcated. The
specimen diagnosis should not include consideration of this cell, given the severity of its degenera-
tion. (Voided urine, SP, high mag.)
d
intracytoplasmic inclusions, so-called Melamed-Wolinska bodies (Fig. 3.10a).

Degenerated HGUC cells may also have decreased N/C ratios due to nuclear con-
densation, but the cells and their nuclei are usually larger than degenerated interme-
diate cells (Fig. 3.10b). Degenerated umbrella cells tend to have reduced cytoplasm
and a corresponding increase in N/C ratio (Fig. 3.10c). The cells maintain their
granular cytoplasm and distinctive chromatin pattern.
Severely degenerated cells often have abundant, vacuolated, and/or granular
cytoplasm that may also be fragmented. The interface between the nucleus and
cytoplasm is indistinct. At low magnification, these cells may appear dark and may
give the impression of large, hyperchromatic nuclei. If a cell has degenerated such
that the cytoplasmic and/or nuclear boundaries are ill-defined, it should not be con-
sidered when rendering a specimen diagnosis, acknowledging that interpretive
errors increase in proportion to inadequacy and deterioration.
Fig. 3.10 Degeneration of

benign cells compared to a
high-grade urothelial cells.
(a) Melamed-Wolinska
bodies. These
intracytoplasmic round red
inclusions are not
indicative of malignancy.
(Voided, TP, high mag.).
(b) HGUC cells are often
degenerated, limiting a
definitive diagnosis of
malignancy. Here, two
cells have pyknotic nuclei
which cause the cells to
have decreased N/C ratios.
However, the chromatin is
very dark, and the nuclei
are still larger than the
nuclei of adjacent benign
cells – an indication that
these cells may have an
abnormally high content of
DNA. The nuclear borders b
are highly irregular, but the
chromatin pattern cannot
be assessed due to the
hyperchromasia caused by
degeneration. (Voided, SP,
high mag.). (c) These
umbrella cells are
relatively well-preserved
but appear smaller than
usually well-preserved
versions. Smaller forms
have N/C ratios that
approach 0.5 and can be
visually striking at low
magnifications. However,
they maintain their c
granular cytoplasm,
distinctive nucleoli, and
marginated chromatin,
reassuring features that
help identify them as
umbrella cells. (Voided, SP,
high mag.)
Explanatory Note Degeneration is nonspecific. It can be found in samples derived

from patients with malignancy as well as benign conditions. HGUC cells can, and
often do, undergo degeneration, a phenomenon discussed in subsequent chapters.
As benign and malignant cells degenerate, they begin to lose their defining
characteristics. Thus, it can be especially difficult to determine whether a highly
degenerated cell was once a benign cell or a malignant cell [13]. A diagnosis cannot
be based on the assessment of only highly degenerated cells. However, because
some amount of cellular degeneration is unavoidable, degenerated benign cells
must be distinguished from degenerated HGUC cells whenever possible. When it is
not possible, indeterminate categories (AUC and Suspicious for HGUC [SHGUC])
may be used.
Urothelial Tissue Fragments
Benign Urothelial Tissue Fragments
Benign-appearing urothelial tissue fragments (BUTFs) are tissue fragments con-

taining cells that lack cytologic atypia concerning for HGUC. If the cells within a
tissue fragment meet the criteria of the AUC category, the fragment would be con-
sidered an atypical urothelial tissue fragment (AUTF) and not a BUTF (see below).
Due to cellular overlap and the forces exerted between neighboring cells, the cyto-
morphologic features seen in tissue fragments can be difficult to assess. Benign
cells may have, or appear to have, irregular nuclear contours, irregular nuclear
shapes, and increased N/C ratios. It is more difficult for light to penetrate a three-
dimensional group than a flat sheet, and thus such groups may exhibit hyperchroma-
sia (Figs. 3.11 and 3.12). Cells at the edges of tissue fragments have less overlap
with neighboring cells and may provide a more accurate cytomorphologic
assessment.
Fig. 3.11 Benign urothelial tissue

fragment (BUTF). (a) Voided. a
BUTF can be seen in voided
urines and do not mandate a
diagnosis of AUC. In this
fragment, nuclei are uniform in
size and shape, evenly spaced, and
with finely granular chromatin.
(Voided, SP, high mag.). (b)
Instrumented from renal pelvis.
Cell fragments from the renal
pelvis should be cautiously
considered. In this case, the
diagnosis rendered was
“suspicious for low-grade
neoplasm.” The excision of the
kidney revealed only urothelial b
hyperplasia overlying a
subepithelial hemangioma.
Retrospective review recognized
the uniform nuclear size and
round shape. The resemblance to
a papillary lesion was likely the
result of instrumentation. (Renal
pelvic washing, CS, high mag.).
(c) BUTF. This fragment contains
cells with oval-shaped nuclei and
mildly irregular nuclear contours.
However, the nuclei are similar in
size, and the chromatin pattern is c
bland, with the nuclei containing
one to two small chromocenters
rather than coarse/clumpy
chromatin. (Bladder washing, SP,
high mag.). (d) BUTF. While the
N/C ratios in these papillary
fragments may seem elevated,
examination of cells lying flat at
the edges reveals a reassuring
amount of cytoplasm. This
architecture is not concerning
unless a fibrovascular core is
present. (Bladder washing, SP,
high mag.)
d
Fig. 3.12 BUTF in voided

urine. (a) This patient was a
found to have a bladder
mass which was
subsequently biopsied and
identified as a leiomyoma.
It is uncertain whether the
mass may have contributed
to the natural exfoliation of
the BUTF seen in this
initial voided urine
specimen. While the nuclei
are oval and have mildly
irregular nuclear contours,
the nuclei are similar in
size and have a bland
chromatin pattern. (Voided
urine, SP, high mag.). (b) b
Additional field containing
a BUTF in the same
patient. (Voided urine, SP,
high mag.)
Explanatory Note The use of the phrases “tissue fragments,” “cell clusters,” and
“cellular aggregates” has not been consistent in the literature. According to Frost,
clusters consist of cells that associate together in the fluid, whereas true tissue frag-
ments contain cells that arise and grow together in vivo [14]. Moreover, in a cluster,
cells are loosely arranged with “windows” (spaces) between cells, and nuclei are
round, most likely due to the absence of forces exerted by neighboring cells. This
section refers to “tissue fragments” with the understanding that small tissue frag-
ments meeting the definition of Frost may be referred to as “clusters” by some.
Atypical Urothelial Tissue Fragments (AUTFs)
Atypical urothelial tissue fragments (AUTFs) are tissue fragments that contain atyp-
ical cytomorphologic features (Fig. 3.13). While the level of atypia seen may merit
classification of these fragments into the AUC category, an increased threshold is
recommended for making an AUC diagnosis when only AUTFs are seen. Because
HGUC cells tend to be discohesive, it is generally more helpful to examine the sin-
gly dispersed cells in a specimen for atypia rather than tissue fragments. If the only
atypical cells in a specimen are in tissue fragments, one should strongly consider the
possibility that they are from a benign entity (“negative”). The type of specimen,
voided vs. instrumented, is also a factor in the evaluation.
Fig. 3.13 Atypical urothelial tissue fragment (AUTF) adjacent to HGUC cell. The cells in this
urothelial tissue fragment display atypical features: irregular nuclear membranes, increased N/C
ratios, coarse chromatin, and anisonucleosis. It is difficult to assess N/C ratios and chromatin pat-
tern in a three-dimensional tissue fragment; thus, one may have an increased threshold to make a
diagnosis of AUC or SHGUC based on a tissue fragment alone. The more concerning cell is to the
bottom right of the AUTF, which has all the qualitative features of HGUC. This patient had HGUC
on follow-up biopsy. (Bladder washing, SP, high mag.)
Low-Grade Urothelial Neoplasia (LGUN)
In keeping with the 2004/2016 World Health Organization/International Society of

Urologic Pathologists (WHO/ISUP) terminology, LGUN is regarded as a combined
cytologic term for low-grade papillary urothelial neoplasms, which includes urothe-
lial papilloma, urothelial proliferation of unknown malignant potential (UPUMP),
papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade
papillary urothelial carcinoma (LGPUC), and flat, low-grade intraurothelial neopla-
sia. We support the view, which represents the current consensus in the field of
cytopathology, that we should not try to differentiate these entities in urinary tract
cytologic specimens [15–17]. It is crucial to separate these entities from HGUC
including CIS, which are discussed in the corresponding chapter (Chap. 6). We also
recognize that cytologic distinction between low-grade lesions and normal urothe-
lium is extremely difficult. Therefore, the only circumstances in which we can make
a definitive diagnosis are described below.
Low-grade urothelial neoplasms (LGUN) may exfoliate into voided urine speci-
mens, though the degree to which this occurs is unknown. LGUN is more fre-
quently seen in washing/barbotage specimens in which neoplastic cells and
fragments are forcibly exfoliated during procurement [18]. The cytologic diagno-
sis of LGUN should be highly restricted and only include specimens containing
tissue fragments with innocuous cells as well as fibrovascular cores (Figs. 3.14 and
3.15). Because LGUN cells often have bland cytomorphology, LGUN tissue frag-
ments without cores may be interpreted as BUTFs and/or AUTFs in urinary tract
specimens and have morphologic overlap with other nonneoplastic entities. Since
cytologically benign fragments from LGUN are appropriately assigned to the
NHGUC category, determining whether UTFs represent LGUN or nonneoplastic
UTFs is unimportant.
Fig. 3.14 Low-grade urothelial neoplasm (LGUN). (a) A monotonous population of neoplastic
cells fills the field, almost forming a sheet. However, the presence of vascular structures containing
flattened endothelial cell nuclei indicates that this is a papillary lesion. The neoplastic cells have
bland chromatin and are small in size. This finding falls under the NHGUC diagnostic category
(renal pelvic washing, Cytospin, medium mag.). (b) At higher magnification, one can appreciate
the bland chromatin pattern of the neoplastic cells. The nuclei are oval-shaped and have minimal
irregularity in their nuclear contours. These features are not compatible with HGUC category
(renal pelvis washing, Cytospin, high mag.). (c) This papillary fragment contains an endothelial-
lined fibrovascular core. Numerous monomorphic neoplastic cells line the core, and detached neo-
plastic cells can also be seen nearby in the background. The N/C ratio of these cells (which is less
than 0.5) can be more clearly assessed using the detached cells rather than the cells within the
three-dimensional papillary fragment (renal pelvis washing, Cytospin, high mag.). (d) In a separate
field, small fragments of neoplastic cells can be seen which lack the fibrovascular core. They lack
features of HGUC and have significant overlap with benign urothelial tissue fragments. Thus, the
cytomorphologic features of individual LGUN cells and tissue fragments are nonspecific (renal
pelvis washing, Cytospin, high mag.)
c
Fig. 3.15 LGUN. A true

papillary tissue fragment
a
contains a fibrovascular core, as
seen in this field, and indicates
the presence of a papillary
urothelial neoplasm. Neoplastic
cells line the fibrovascular core.
Many cells can also be seen in
the background, which have been
forcibly removed from the
papillary structure during the
washing procedure. The cells
have monomorphic nuclei and
N/C ratios that approach 0.5. b
They lack the irregular nuclear
contours, coarse chromatin, and
hyperchromasia that would be
seen in HGUC (bladder washing,
Cytospin, medium mag. [a] and
high mag. [b])
Explanatory Note 1 Considering that the histologic definition of LGUC includes

only minimal variation in cytologic features, mainly mild nuclear enlargement and
irregularity of the nuclear contours, the recognition of LGUC separate from urothe-
lial papilloma, UPUMP, and PUNLMP in urine cytology is practically impossible.
Relatively few studies have been done on cytopathology specimens to define the
cytologic features of LGUC in urine specimens. Although earlier reports [13, 14,
17] listed three key morphologic features based on which the diagnosis of LGUC
could be made (nuclear enlargement, slight nuclear contour irregularity, and cyto-
plasmic homogeneity), the reported sensitivity and inter-observer agreement for
cytologic diagnosis of LGUC remained low [9, 18, 19]. Those studies were based
on highly selected populations of only lower urinary tract specimens, with a very
high index of suspicion, retrospective reviews of the morphologic features, and long
term follow-up after the initial positive cytologic diagnosis [13, 15]. Most impor-
tantly, in some of those studies, grade 2 tumors (transitional cell carcinoma, grade
2) were included in the group of low-grade tumors. Since the introduction of the
2004 WHO/ISUP classification, there has been a significant shift in grading of uro-
thelial neoplasms. Tumors previously classified as grade 2 are now more often cat-
egorized as high grade [5, 6, 20].
Explanatory Note 2 Similar to earlier reports [11, 18], a recent study [21] found
that the majority of the features described previously as diagnostic for LGUC were
observed almost equally in patients with or without biopsy-proven LGUC, regard-
less of whether the specimens were from the upper or the lower urinary tract.
Specifically, mild nuclear membrane irregularity was present in 48% of LGUC and
47.2% of negative controls (p = 0.93); mild nuclear enlargement was observed in
42.9% of LGUC patients and 49.1% negative controls (p = 0.26). Although homo-
geneous cytoplasm and three-dimensional papillary structures with fibrovascular
cores were found only in LGUC, there were many that did not show these features.
Hence, these criteria were not statistically significant in this study. The only time a
definitive diagnosis of LGUN can be rendered in instrumented urine is when well-
defined fibrovascular cores (with capillaries) are present [9]; this finding, however,
is exceedingly rare (Figs. 3.16 and 3.17). In these instances, LGUN can be diag-
nosed as a second line diagnosis under a top line diagnosis of NHGUC, or the find-
ing can be described in a note with a top line diagnosis of NHGUC.
Explanatory Note 3 Occasionally the washing (barbotage) specimens from patients

with LGUN will be very cellular and composed of very uniform, mostly singly
arranged cells (Fig. 3.17). In these cases, umbrella cells are usually lacking or they
are rare. Individual cells have minimal cytologic atypia. Corresponding tumors are
usually large and easily visualized during cystoscopy. These cases should still be
categorized as NHGUC without mention or suggestion of LGUN.
Explanatory Note 4 The presence of fibrovascular cores by themselves is not diag-

nostic of LGUN. These may be seen in papillary HGUC. Due to poor cell cohesion,
papillary HGUC is more likely than LGUN to be associated with dispersed cells in
the background that display features of HGUC. Such cases should be placed in the
category that accounts for the highest degree of cellular abnormality in the same
way as they would be in histological diagnosis of urothelial tumors.
Fig. 3.16 LGUN. In this

field, the neoplastic cells
are predominantly attached
to the fibrovascular stalk.
The cells have oval-shaped
nuclei with mild nuclear
contour irregularities and
hypochromasia; they lack
coarse chromatin (bladder
washing, Cytospin,
high mag)
Fig. 3.17 LGUN. The

N/C ratios of these
dispersed single cells are
clearly below 0.5. The cells
possess a few small
chromocenters and lack the
“clumpy” chromatin seen
in HGUC (bladder
washing, Cytospin,
high mag)
Urothelial Tissue Fragments (UTFs) in Voided Urine
Although cytologists often regard UTFs in voided urine specimens as abnormal, a

recent review of such samples has found them commonplace and benign [19].
Causes of BUTFs in voided urines are multifold and include prostate/rectal manipu-
lation prior to collection of the sample, jogging, abdominal palpation, etc. Most
often BUTFs are of no clinical importance. Table 3.1 demonstrates the results of a
large slide review of noninstrumented voided urine specimens that contained BUTFs
and AUTFs and their clinical follow-up. In most cases, no etiology was identified
for the presence of BUTFs. The presence of AUTFs in voided urine specimens was
associated with an increased risk of HGUC (8.8% of specimens) and much more
likely to be associated with urolithiasis (28.8% of specimens) [20]. However, as
with BUTFs, the etiology of AUTFs could not be determined in most cases. These
Table 3.1 Significance of benign urothelial tissue fragments (BUTFs) and atypical urothelial tis-
sue fragments (AUTFs) in voided urines (Onur I, Rosenthal DL, VandenBussche CJ. Atypical
urothelial tissue fragments in noninstrumented voided urine specimens are associated with low but
significantly higher rates of urothelial neoplasia than benign-appearing urothelial tissue frag-
ments [20]
No. (%) No. (%)
Follow-up diagnosis BUTF AUTF
Histopathologic 29 (10.6) 24 (14.1)
Benign 17 (6.2) 6 (3.5)
Urothelial neoplasia 12 (4.4) 17 (10.0)
PUNLMP 1 (0.4) 0 (0.0)
LGUC 9 (3.3) 2 (1.2)
HGUC 2 (0.7) 15 (8.8)
Cytopathology 45 (16.4) 25 (14.7)
Benign/AUC
Urolithiasis (clinical/radiologic/gross) 45 (16.4) 49 (28.8)
No follow-up 25 (9.1) 10 (5.9)
data support using a higher threshold for classifying AUTFs in the AUC category, as
the ROHM for specimens containing AUTFs is much lower than what has been seen
for the AUC category (8.8% vs. 12.3–69%); (see Chap. 11, Risk of High-Grade
Malignancy).
Benign Urothelial Tissue Fragments (BUTFs) in Instrumented Urine
Instrumented urine specimens are often cellular, consisting of numerous benign-

appearing cells arranged in groups that resemble papillary clusters with smooth
(community) borders, and that lack fibrovascular cores. They have been detached
from the bladder wall by mechanical forces. Urothelial cells within clusters may be
either round or slightly elongated, but they maintain N/C ratios of <0.5. If atypia is
seen in UTFs, assessment regarding the degree of atypia and subsequent classifica-
tion into a diagnostic category does not differ from that used in voided urine speci-
mens [21, 22].
Urolithiasis (“Stone Atypia”)
Clusters/groups or sheets of benign urothelial cells (Fig. 3.18), visually different

from BUTF because of reactive changes (Fig. 3.11), are commonly found in benign
samples and have no significance, so long as the nuclei bear benign characteristics.
Urothelial cells exfoliated in the presence of lithiasis may demonstrate a mild degree
of nuclear hyperchromasia. However, they will maintain low N/C ratios and smooth
nuclear contours and, therefore, may be confidently considered NHGUC.
Fig. 3.18 Urothelium with

nephrolithiasis (BUTF). A
BUTF in a voided urine
may be the result of one or
several among numerous
causes. In this patient,
nephrolithiasis was the
reason. Cellular changes
are mild when compared to
those in the photos to
follow. The absence of any
fibrovascular stalk
eliminates a diagnosis of
LGUN. (Voided, SP, high
mag.)
Explanatory Note Instrumented urine specimens include any specimen that was
obtained by any instrument or whenever force was applied to dislodge individual
cells from the lining urothelium. These include catheterized urines, bladder wash-
ings (barbotage), brushings, or upper urinary tract urine specimens obtained by uri-
nary catheterization. In addition, any immediately post-cystoscopy urine specimen
is also considered instrumented. In general, these specimens are cellular, and the
presence of BUTF in instrumented urine specimens is a normal finding; therefore,
we should avoid the term “atypia” in these types of specimens so long as the nuclear
and architectural features of the UTF do not warrant another diagnosis.
Voided urine specimens from patients who present with hematuria and/or filling
defect on imaging studies are often cellular and consist of three-dimensional urothe-
lial fragments composed of cells that may exhibit significant pleomorphism
(Fig. 3.19). Urinary stones usually abrade the lining causing morphologic changes
to liberated cells and tissue fragments. These three-dimensional urothelial frag-
ments display smooth (community) borders and cytoplasmic collars/collarets, i.e., a
rim of cytoplasm surrounding the nuclei as in BUTF. If a cause of the atypia is
found, such as a stone, then the diagnostic category should be NHGUC.
There may also be sheets or clusters of cells that are not true tissue fragments
(see Fig. 3.11). The cells in the fragments or clusters may also exhibit nuclear
enlargement and atypia, slightly increased N/C ratios, nuclear and cytoplasmic
degeneration, and/or squamous metaplasia (Fig. 3.19). With a confident diagnosis
of lithiasis, and without single HGUC cells in the sample, then the NHGUC cate-
gory is appropriate. If there are any truly atypical well-preserved single cells, the
sample needs to be considered either AUC or SHGUC based upon the criteria for
those categories. Occasionally, crystalline material from the stone will support the
diagnosis (Fig. 3.19c).
Fig. 3.19 Urothelium with

nephrolithiasis (AUTF). (a) A a
sheet of urothelium consists of
relatively uniform cells with
moderately hyperchromatic
nuclei. Even though the nuclear
chromatin is darker than normal,
the presence of a bladder stone is
reason enough for the changes.
Because of the history and
presence of only mild atypia,
this sample was placed in the
NHGUC category. (Voided, SP,
medium mag.). (b) Compare the
cells in the center of the field
with those to the right, especially
considering the nuclear
chromatin and nuclear shapes.
The central cells are
b
hyperchromatic and the shapes
vary. Inflammation is seen in the
background. Without the history
of nephrolithiasis, these cells
would indicate a diagnosis of
AUC. If there were any
consideration of a urothelial
lesion in addition to lithiasis, a
note or a diagnosis of AUC is
appropriate. (Washing, CS,
medium mag.). (c) Most often,
direct evidence of stones is not
so dramatic as in this
photograph. Variation in cells in
the background can be c
appreciated. (Washing, SP, low
mag.). (d) A small fragment of
hyperchromatic urothelial cells
is adjacent to an X-shaped
crystalloid. The nuclei of the
cells in the fragment have
slightly irregular borders, and
the N/C ratios are difficult to
assess within a tissue fragment.
The presence of crystals and/or
crystalloids can account for mild
atypia in urothelial tissue
fragments, as seen here, and
prevent classification into the
AUC category. (Voided urine,
SP, high mag.)
d
Explanatory Note Urolithiasis was first noted as a cause of false-positive diagno-

ses by Papanicolaou. The clinical history, if available, is valuable to avoid a false-
positive diagnosis. As described above, BUTF may be seen in instrumented urines
and sometimes in voided urine from patients with stones. Urothelial carcinoma and
squamous cell carcinoma have been associated with renal calculi with and without
associated infection [23, 24]. Whether this association represents a causal relation-
ship is not clear.
Urothelial Changes Characteristic of Infectious Processes
Acute Bacterial Infections
In the urinary bladder, these infections may cause generalized reactive changes in
the urothelium similar to those that have been discussed above. Urine specimens
from acute bacterial infections are sometimes cellular, consisting of reactive urothe-
lial cells with slightly enlarged nuclei and prominent nucleoli, but with fine, evenly
distributed chromatin and thin nuclear membranes. The presence of infiltrating neu-
trophils admixed with reactive urothelial cells supports a reactive, benign process,
as do clusters of bacteria in the background. In the absence of a significant number
of neutrophils, clusters of bacteria may also represent specimen contamination dur-
ing collection. If the predominant cells are neutrophils, with rare urothelial cells,
then the specimen should be considered inadequate, with the reason for that desig-
nation included in the report.
Characteristic Viral Cytopathic Effects
In urine specimens the most commonly recognized characteristic agents include

polyomavirus; herpes simplex virus (HSV), usually type II but also type I; cytomega-
lovirus (CMV); and human papillomavirus (HPV) (Fig. 3.20). Over the last few
Fig. 3.20 Herpes simplex

virus (HSV). Herpes
criteria are similar to those
seen in other cytologic
preparations, with the three
Ms: (nuclear) molding,
(chromatin) margination,
and multinucleation. HSV
rarely infects the urinary
tract and when seen in
urinary tract specimens
may arise from
contaminating extra-
urinary sites (voided urine,
SP, high mag.)
decades, one of the most important and most identified viruses in urine specimens is
polyomavirus (Fig. 3.21). Human polyomaviruses are small, non-enveloped double-
stranded DNA viruses that are classified into two main strains that may infect the
urinary tract, named after the initials of the patients from whom they were first identi-
fied (BK and JC) [25]. Polyoma virus infected cells are enlarged with single, usually
homogeneous basophilic inclusions occupying most of the enlarged nuclear area
(Fig. 3.21). Nuclear membranes of those cells are smooth and regular in shape as
compared to the irregular nuclear membranes in high-grade malignant cells, which
they often mimic (“decoy” cells). When these cells degenerate, the basophilia clears
as the chromatin extrudes, leaving a framework or spider web. Usually, only a few
infected cells are found (Fig. 3.21), unless the patient is immunosuppressed.
The presence of cells with well-recognized viral changes should not lead to a
diagnosis of atypia. Primary polyomavirus infections occur during childhood and
are usually subclinical. Over 90% of adults are seropositive for viral antibodies. The
virus generally remains latent in the renal tubular epithelium, but intermittent viru-
ria can be detected in 0.3% of healthy adults. The infection is reactivated in indi-
viduals with various degrees of immunological deficit. In renal transplant recipients,
polyomavirus nephropathy occurs in 3–5% of patients, and loss of transplant occurs
in 50% of those affected [25]. Urines from such patients may contain many cells
with polyoma cytopathic effect.
The infected cells can easily be misclassified as malignant. Therefore, they were
referred to as “decoy cells,” analogous to “decoy ducks” used in hunting wild ducks,
by Andrew Ricci, a cytotechnologist working in the cytology laboratory of Memorial
Sloan Kettering Cancer Center. The term was coined and popularized by Leopold
Koss in the second edition of his influential textbook, Diagnostic Cytology and Its
Histopathologic Bases [26, 27]. In addition, urothelial cells infected by polyomavi-
rus can be aneuploid and, thus, be a potential pitfall for any DNA-based tests,
including FISH [28–30].
Once polyoma-infected cells are identified in a specimen, one should be alert and
consider whether other atypical cells in the specimen may simply be artifact induced
by polyomavirus-infected cells as well. Because degenerated HGUC cells may
b c
Fig. 3.21 Polyomavirus – classic (a), spider web (b–c), benign case (d–f). (a) Classic polyoma
(BK) cytopathic effect includes enlargement of the nucleus and nuclear chromatin homogenization
and/or chromatin margination. The shape of the nucleus is always round or oval with a very smooth
outline. The cell cytoplasm may be absent or, in the case of these cells, form degenerating tails
(“comet cells”). In this case, the chromatin appears somewhat “clumpy” which may initially cause
concern for HGUC. However, all the other features suggest this is simply BK cytopathic effect
(voided, SP, high mag.). (b) Dissolution of the nuclear chromatin is also a characteristic of poly-
omavirus infection. The size and shape of the nucleus are the same as the classic features. (Voided,
SP, high mag.). (c) If the focal plane is changed, then a spider web of the cytoskeleton comes into
view. (Voided, SP, high mag.). (d) The assortment of pale and darker cells is striking on low mag-
nification. (Washing, TP, low mag.). (e) Closer view will demonstrate the reasons for the dark cells
observed on low magnification. Almost all the cells display glassy nuclear inclusions diagnostic of
polyomavirus (washing, TP, high mag.). (f) The BK-infected cells in this field demonstrate positive
nuclear staining for SV40 by immunocytochemistry. In most instances, a morphologic assessment
is sufficient for the diagnosis of BK infection; furthermore, infection with BK (and SV40 positiv-
ity) does not exclude HGUC. (Voided urine, SV40 immunocytochemistry, high mag.)
d e
sometimes resemble decoy cells, polyomavirus-infected cells should be examined

closely to exclude this possibility. Galed-Placed et al. reported a rare case of decoy
and malignant cells coexisting, and, hence, identification of decoy cells does not
exclude the existence of carcinoma [31, 32]. Even the utilization of IHC with SV40
antibody to prove the presence of the virus does not confirm or exclude the possibil-
ity that HGUC is also present. Two studies examined the risk of HGUC in speci-
mens containing cells with polyomavirus-like changes. One study found a slight
increase in HGUC on follow-up when atypical cells were found in addition to poly-
omavirus compared to those lacking atypical cells (10% vs. 6%, respectively) [33,
34], whereas the second study found the rates to be 12.5% vs. 6.8%. These data
support a careful search of the specimen to find diagnostic cells of malignancy if
present, even in the presence of polyomavirus.
Parasites
Rarely, parasites may be seen in urinary tract specimens. These may be extra-
urinary contaminants, such as pinworm (Enterobius vermicularis) ova, or may
arise from within the urinary tract (Schistosoma haematobium) (Fig. 3.22). While
Fig. 3.22 Schistosoma haematobium (image courtesy of Greta Neethling, Tygerberg Academic

Hospital, South Africa). (a) Intact Schistosoma haematobium ovum. The ovum has the character-
istic terminal spine, which distinguishes it from other schistosomes. However, S. haematobium is
the only schistosome that involves the urinary tract. The intact ovum contains the schistosome
miracidium. The faint-staining nuclei within the miracidium is one way to distinguish the ova from
similarly shaped crystals. (Voided urine, conventional smear, high mag.). (b) Hatching Schistosoma
haematobium. The ovum hatches, releasing the miracidium which is infectious to snails. The
hatching can occur after excretion or within the urinary tract. (Voided urine, conventional smear,
high mag.). (c) Hatching Schistosoma haematobium. Alternative view. (Voided urine, conventional
smear, high mag.). (d) Naked Schistosoma haematobium miracidium. Note the internal nuclear
staining and cilia lining the edges of the miracidium. (Voided urine, conventional smear, high
mag.). (e) Empty Schistosoma haematobium ova. The only structures that may be seen are the
empty ova in a background of inflammation and granular debris. These may be more difficult to
distinguish from vegetable material or crystals. The presence of the terminal spine and inflamma-
tory response, together with patient history of being from an endemic area, prompts the diagnosis.
(Voided urine, conventional smear, high mag.)
c
e
S. haematobium is classically associated with the development of primary squa-

mous cell carcinoma of the bladder, the organisms may be seen in patients from
endemic regions in a setting of inflammation and prior to the development of car-
cinoma. While the identification of a parasitic organism is clinically important and
should be reported, one must exclude the possibility of more common elements,
such as crystals and contaminating vegetable material that closely resemble para-
site ova [35]. Owing to the vagaries of sample collection, urinary specimens may
also be contaminated by material from the elsewhere in the GU tract and GI tract,
as well as the vagina.
Urothelial Changes Associated with Treatment Effects
Radiation
Radiation-induced cytomorphologic changes will display significant cytomegaly

and nucleomegaly and a preserved N/C ratio (Fig. 3.23). Multinucleation may be
seen, and nuclear and cytoplasmic vacuoles are often demonstrated. In addition,
characteristic cytoplasmic polychromasia can be appreciated. Chromatin is gener-
ally finely granular. All of these changes define the sample as NHGUC if there are
no other features of atypia or malignancy.
Fig. 3.23 Radiation changes. Patients may receive radiation treatment to the urinary tract to treat
a primary malignancy or areas of the urinary tract may become radiated due to treatment of extra-
urinary malignancies, such as cervical cancer. The changes seen are similar to those seen at other
anatomic sites: nucleomegaly with a corresponding increase in cytoplasm, multinucleation, and
cytoplasmic vacuolization. (Voided urine, SP, high mag.)
Immunotherapy
Certain therapeutic compounds instilled intravesically are associated with recogniz-

able changes in urine specimens. Intravesical BCG immunotherapy can cause gran-
ulomatous inflammation that may be seen in urine specimens (Fig. 3.24). Granulomas
are composed of epithelioid histiocytes admixed with lymphocytes. Occasionally,
multinucleated histiocytic giant cells are also seen. Once again, the presence of
granulomata in an appropriate clinical setting should not trigger the diagnosis of
atypia in urine specimens. Granulomatous change caused by BCG or another etiol-
ogy should be noted in the report, but not interpreted as belonging to AUC, SHGUC,
or worse unless there are characteristic changes for those entities in the urothelial
Fig. 3.24 Granulomatous
reaction following BCG a
immunotherapy. (a)
Multinucleation in
superficial urothelial cells
is common. In contrast,
Langhans-type giant cells
resulting from fused
macrophages are
multinucleated but have
their smaller and slightly
hyperchromatic nuclei
clustered at one pole of the
cytoplasm. Clinical history
revealed recent BCG
instillation following
diagnosis of bladder
cancer. (Voided, CS, high
b
mag.). (b) In addition to
Langhans giant cells,
granulomata can be found
in urine following BCG
immunotherapy. These
granulomata are no
different from those in any
other body site, complete
with monocytes,
lymphocytes, and
histiocytes in a tight
mélange. (Washing, TP,
high mag.)
cells present. Specimens may contain residual malignant cells (usually degenerated)
in patients who have recently received or have failed therapy. Six weeks should
elapse between the last of six BCG instillations and a urine sample to allow malig-
nant cells to die, thus avoiding a mistaken interpretation of “failed therapy.” Other
nonspecific findings include reactive umbrella cells, seen with large forms or in
increased numbers and background granular debris.
Chemotherapy
Intravesical mitomycin and thiotepa usually affect superficial cells and cause
nuclear enlargement, multinucleation, and hyperchromasia of those cells, all of
which are nonspecific but may be worrisome. History of therapy is important to
anticipate and interpret these changes. Systemic cyclophosphamide (Cytoxan),
given for reasons other than urothelial malignancy, often causes hemorrhagic cysti-
tis and has been reported to be associated with urothelial hyperchromasia and
degeneration, plus the presence of large nuclei and increased N/C ratios. In addition
to mimicking HGUC, these changes may indeed reflect the presence of HGUC
caused by systemic cyclophosphamide, albeit rarely [36, 37].
Chemotherapy may be combined with hyperthermia (chemohyperthermia) or
electric currents (electromotive drug administration) to increase drug penetration.
One study has shown cells in these specimens can have increased N/C ratios, hyper-
chromasia, and irregular nuclear membranes, which can simulate HGUC [38].
Seminal Vesicle Cells
Sporadically occurring and scarce, degenerated seminal vesicle cells (Fig. 3.25a)

can be seen in urine specimens, particularly from older patients, especially after a
digital rectal examination or prostatic massage. Seminal vesicle cells in urine speci-
mens may have a bizarre appearance (“monster cells”) with greatly enlarged nuclei
and minimal cytoplasm (Fig. 3.25b) [39, 40]. The chromatin is hyperchromatic,
degenerated, and smudgy. In contrast, the chromatin of malignant cells is coarse. As
in prostatic specimens, seminal vesicle cells may be distinguished from cancer cells
by their golden-brown lipofuscin pigment. This pigment may not always be present
or easy to identify. In most circumstances, numerous spermatozoa will be present in
the background and/or associated with the cells.
Fig. 3.25 Seminal vesicle

cells. Seminal vesicle cells
are unusual and may
provide confusion with
HGUC cells because of
their large size and nuclear
hyperchromasia. Two clues
to their identity include
intracytoplasmic yellow
lipofuscin pigment (arrow)
and accompanying sperm.
(Washing, TP, high mag.)
Explanatory Note Seminal vesicle cells have an abnormal DNA content and poten-
tially are a pitfall for DNA-based adjuvant tests [41]. When seminal vesicle cells are
recognized by the presence of yellow pigment and mature spermatozoa in the back-
ground, there is no need to call the urine specimen AUC.
Bladder Diversion Specimens
Urinary diversion specimens are urines obtained from patients who underwent cys-
tectomy and one of the surgical procedures designed to reroute the urine flow (ileal
conduit, Indiana pouch, or neobladder). All of these procedures use a portion of
small bowel (ileum) that is anastomosed to the ureters and/or urethra (Fig. 3.26).
Urine specimens from these neobladders are very cellular and composed mainly
of degenerated enteric glandular cells, either singly or in small clusters, resembling
histiocytes, in a dirty background with mucus and bacteria (Fig. 3.27). In some
specimens, these components may obscure exfoliated urothelial cells, but they
should not distract from examination for HGUC. Often, urothelial cells from upper
tracts are present and show marked degeneration. These degenerated glandular and/
or urothelial cells may lead to an inappropriately increased rate of AUC diagnoses
for these specimens, with one study demonstrating a significantly lower positive
predictive value for the atypical category in patients with ileal neobladders com-
pared to those with native bladders (5.9% vs. 12%, respectively) [42].
Fig. 3.26 Enteric cells

from a urinary diversion a
post-cystectomy. (a) One
superficial urothelial cell is
present to conveniently
compare with the small
round cells in the figure.
All are of the same size
and have small punctate
nuclei. These are typical of
degenerated enteric cells.
(Catheterized, TP, medium
mag.). (b) Following a
cystectomy, a diversionary
pouch is constructed, lined
by cells from the portion of
the intestine used. They
usually are single and
closely resemble b
histiocytes. Sometimes
they cluster, which can
present a diagnostic
dilemma. Careful focusing
will reveal the small
nuclei, dissimilar to
HGUC. (Catheterized, SP,
medium mag.)
Fig. 3.27 Urinary diversion specimen. (a) Urinary diversion specimens usually contain numerous
small degenerated cells in a background of granular debris. The degenerated cells arise from the
intestinal segment used for diversion. The cells have dark, yet very small, nuclei and granular
cytoplasm. (Catheterized, SP, low mag.). (b) A separate field shows the benign degenerated cells
with apoptotic bodies. These patients usually have a history of aggressive HGUC and are at high
risk for recurrence. HGUC cells would be larger than the small benign degenerated cells seen in
diversion specimens. This field contains strictly benign degenerated cells, though some three-
dimensional areas of cellular piling could appear atypical at lower magnification (catheterized, SP,
high mag.)
Visually striking vegetable material may be seen in patients with ostomies

(Fig. 3.28). This material arises from the use of adhesive materials containing guar
gum at the ostomy site [43]. Such material should not be misinterpreted to represent
squamous dysplasia, urothelial neoplasia, viral cytopathic effect, or, most impor-
tantly, parasite ova.
Fig. 3.28 Guar bean. (a)

The vegetable cells that a
comprise this structure
have a distinctive
appearance, with
pink-staining geometrically
shaped nuclei. Guar bean
is a component of adhesive
material used on skin
around a urostomy stoma.
(Catheterized, SP, high
mag.). (b) An additional
view of vegetable material
derived from guar bean in
a patient with a urostomy.
While the material is
visually striking, its
appearance is unlikely to
be confused for a b
malignant process.
(Catheterized, SP, medium
mag.)
Explanatory Note The purpose of cytologic evaluation of urinary diversion speci-

mens is to monitor the upper tract in patients with a history of urothelial carcinoma.
For all practical purposes, all of these specimens appear different from normal or
“atypical” due to marked degeneration. The diagnosis of malignancy should be
made only if clear criteria of malignancy, usually HGUC, have been met. Fortunately
for the microscopist, the large, single cells of HGUC are easy to detect even among
a myriad of small degenerated cells. Otherwise, these specimens should be catego-
rized as NHGUC. Rarely, patients will develop adenocarcinoma in their diversion
“bladder”; cytologic changes are consistent with gastrointestinal adenocarcino-
mas [44].
The Rate of Negative Samples in a Usual Laboratory Population
The rate of each diagnostic category depends upon the population served by the
laboratory. Referral centers, with oncologic urologists and a greater proportion of
follow-up specimens after diagnosis of malignancy compared to primary screening
specimens, will undoubtedly have much higher rates of SHGUC and outright
HGUC than reference laboratories serving general practitioners and internists.
Having established a baseline figure for each category, every laboratory is wise to
watch for “diagnostic drift,” wherein the indeterminate categories become waste-
baskets, influencing the rate of the adjacent categories; in urinary cytology, AUC
catches the overflow of difficult cases from the negative and suspicious categories.
Careful cytohistologic correlation is strongly recommended to monitor for and pre-
vent “diagnostic drift”.
Category Performance
The NHGUC category has a risk of high-grade malignancy (ROHM) of 8.7–36.7%

(see Chap. 11) in studies from laboratories following the implementation of TPS as
well as in studies in which slides were retrospectively classified according to
TPS. These numbers, however, do not accurately reflect the true performance of the
NHGUC category, since the vast majority of patients with NGHUC diagnoses do
not have corresponding or follow-up biopsy specimens; the published rate of fol-
low-up biopsy after negative or benign urine cytology ranges from 3.4% to 6.2% [1,
45, 46]. Because most patients without biopsy are likely to have benign follow-up,
the number of falsely negative diagnoses is likely to be quite low in most laborato-
ries. One large study, which only included patients with well-documented, extended
(6 months) clinical follow-up, found an overall false-negative rate of 3.3%, which
translated to a negative predictive value (NPV) of 96.7%. In patients with a history
of urothelial carcinoma, the NPV for the NHGUC category was 93.9%; for patients
presenting with hematuria and no history of urothelial carcinoma, the NPV was
98.9% [47, 48].
In addition to the indication for specimen procurement, the specimen type has
been shown to impact category performance. In pre-Paris studies, the false-negative
rate of ileal conduit and neobladder urinary diversion was 5.7–8.7% [49, 50].
Careful examination of such specimens, including in areas of obscuring factors, is
prudent.
Sample Reports
1. Urine cytology (voided): Satisfactory for evaluation
Negative for high-grade urothelial carcinoma

Low-grade urothelial neoplasm (LGUN)
2. Urine cytology (voided): Satisfactory for evaluation
Negative for high-grade urothelial carcinoma. (See note)

Note: The sample contains singly dispersed and groups of urothelial cells includ-
ing an occasional group with a fibrovascular core. The urothelial cells in the groups
as well as those singly dispersed show bland nuclear features with no hyperchroma-
sia, chromatin, or membrane abnormalities to suggest high-grade urothelial carci-
noma. The findings may represent a low-grade urothelial neoplasm.
3. Bladder washings: Satisfactory for evaluation
NEGATIVE FOR HIGH-GRADE UROTHELIAL CARCINOMA

Note: Numerous groups include pseudopapillary structures lacking fibrovascular
cores. Urothelial cells show bland nuclear features with no hyperchromasia, chro-
matin, or membrane abnormalities to suggest high-grade urothelial carcinoma.
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Atypical Urothelial Cells (AUC)
4
Güliz A. Barkan, Margaret L. Compton, Tarik M. Elsheikh,
Kim A. Ely, Daniel F.I. Kurtycz, Merce Jorda,
Zahra Maleki, Sachiko Minamiguchi, Hiroshi Ohtani,
Eric Piaton, Bo Ping, Spasenija Savic Prince,
Z. Laura Tabatabai, and Christopher J. VandenBussche
• Discussion of cytomorphologic criteria for this category

• Morphologic appearances seen in different cytopreparatory techniques
• Updated pictures, figures, and tables
• Review of studies published after the first edition of The Paris System, with a
focus on reported atypia rates and the risk of high-grade malignancy
• Description and clarification of issues since the first edition of The Paris System
Background
In addition to standardizing the reporting of urine cytology, one of the main objec-
tives of The Paris System (TPS) was to reduce the reported atypia rate, improve
clarity of communication, and thereby aid clinicians in patient management. Prior
to TPS the reported “indeterminant or atypical” rate of urine cytology was quite
variable and often facility dependent. There are reports of excessive atypia rates
reaching as high as 50% [1]. Obviously, such performance characteristics impair the
utility and reputation of urine cytology as a screening and/or a diagnostic tool [2].
The attempt to unify reporting and apply restraint to the reporting of “atypia” has
had some effect. Since the publication of TPS in 2016 [3, 4], there have been numer-
ous publications on urine cytology supporting the concepts behind the system [5–
40]. These studies have shown that the standardized criteria provided by TPS have
reduced the “atypia” rate when comparing the post-TPS to the pre-TPS rates (see
Table 4.1). Additionally, TPS has caused the cases that are interpreted as atypical to
be more meaningful, in that the risk of high-grade malignancy (ROHM) is signifi-
cantly increased in the TPS atypical urothelial cells (AUC) category. Early meta-
analyses exhibited ROHM ranging from approximately 32% in retrospective

https://doi.org/10.1007/978-3-030-88686-8_4
64 G. A. Barkan et al.
Table 4.1 Published reporting rates of the “atypia” and “atypical urothelial cells” category before
and after The Paris System
Pre-TPS % (n) Post-TPS % (n)
Author Year Study location Atypia AUC
Vosoughi et al. [7] 2021 The USA (Miami) 16 (249) 9 (56)
Compton et al. [8] 2021 The USA (Nashville) N/A 12.5 (199)
Stanzione et al. [6] 2020 The USA (Los 59.8 (52) 41.5 (122)
Angeles)
Anbardar et al. [5] 2020 Iran 26 1.2 (22)
Rai et al. [60] 2019 India 16.7 (15) 11.1 (10)
Bakkar et al. [31] 2019 The USA (Los 44 (44) 23 (23)
Angeles)
Vallamreddy et al. [61] 2019 India 21.6 (16) 9.5 (7)
Wang et al. [15] 2018 Canada 18.6 (442) 14.4 (345)
Meilleroux et al. [25] 2018 France 6.1 (100) 5.2 (94)
VandenBussche et al. 2018 The USA (Baltimore) 23.9 (568) 23.0 (589)
[24]
Xing et al. [27] 2018 The USA (Pittsburgh) 34 (52) 24 (37)
Rohilla et al. [22] 2018 India 54.3 (73) 8.5 (114)
Zare et al. [23] 2018 The USA (San Diego) 24.2 (47) 11.9 (23)
Torous et al. [20] 2017 The USA (Boston) 29.5 (328) 21.8 (302)
Roy et al. [62] 2017 India 41.2 (40) 11.3 (11)
Granados et al. [9] 2017 Spain 4.7(7) 20.1 (30)
Suh et al. [17] 2017 Korea 25.4 (36) 14.8 (21)
Hassan et al. [12] 2016 Canada 38.7 (48) 25.8 (32)
AUC atypical urothelial cells, TPS The Paris System, N/A not applicable
(pre-TPS) studies to 43% in prospective (post-TBS) studies. From these works the
estimated ROHM for AUC is placed at 38.5% (±14.3%) as of this edition of TPS
(see Chap. 11). In addition, the use of TPS has resulted in an increase in specificity
and positive predictive values as well as an improved diagnostic concordance of
benign and malignant categories with histologic results [29].
Almost all diagnostic testing has a range of imprecision where values of diseased
and non-diseased states overlap. In clinical chemistry one may see the overlap in
analyte values like glucose or enzyme activity studies; in anatomic pathology it is
the range of increasing morphologic abnormality. There are values that are clearly
derived from a normal or benign state and others that are unmistakably derived from
disease, but the middle values are often a problem and do not yield a clear answer.
This is well explained in the classic 1975 clinical laboratory text Beyond Normality
by Galen and Gambino [41]. In the ideal testing situation there would be a clear set
of features or values that would be discrete for normal and abnormal situations
(Fig. 4.1); however, that is only occasionally the case.
More frequently we are presented with a range of values or morphologic features
that overlap (Fig. 4.2). We can confidently interpret the values or morphologies
without overlap, but at the places where both curves are present, we have a problem.
These overlapping values represent the domain of atypia (Fig. 4.3). In cytology,
cellular degeneration, poor preparations, reparative phenomena, and other artifact
4 Atypical Urothelial Cells (AUC) 65
Fig. 4.1 Diagnostic ideal: in the ideal testing situation, the values derived from a test, be they
numeric or a morphologic impression, would clearly separate the non-diseased population from a
diseased population
Fig. 4.2 Diagnostic reality: in the usual testing situation, some benign states yield values or fea-
tures that overlap with those found in a diseased population
Fig. 4.3 Diagnostic reality: the area of overlap between benign states and diseased states consti-
tutes a region of diagnostic uncertainty and provides a conceptual representation of the domain
of atypia
inducing situations can increase the overlap making interpretation more difficult.
On the other hand, well-fixed excellent preparations will tend to lessen the artifact.
Atypical cytology interpretations are frequently criticized as lacking specificity
and reproducibility, leaving clinicians without a clear course of action. Generic des-
ignations of “atypia” are not diagnostic entities but rather interpretations that reflect
the inability to make a definitive diagnosis due to a lack of reliable morphologic
features. Historically, the term “atypia” was introduced to cytopathology by Dr.
George N. Papanicolaou, to convey a very low suspicion of malignancy [42], but the
category leaves clinicians in a quandary and uncertainty in the minds of patients
whose samples generate such a result. Too frequent use of the term will stimulate a
search for another method which will almost certainly be more expensive and may
be more invasive.
Unfortunately, “atypia” is an unavoidable reality in our morphologic interpreta-
tions of cytology.
To reiterate, it fills the gap in the spectrum between what we can recognize as
entirely normal and what we can recognize as being clearly abnormal. Countless
papers lament that one cannot strictly define “atypia,” but these articles are missing
the point. Cytologic atypia is an amorphous concept of not being “typical” or “nor-
mal.” It is the negation of two terms which are themselves difficult to define. It may
be better to think of “atypia” in terms of probability, as Fig. 4.3 above; it represents
an x-axis potential for being more than normal. In other diagnostic systems, atypical
cytologic features are those which are more than can be explained by benign states
and reactive change but less than those which lead to a definitive diagnosis. It is
more than normal, but the cytologist cannot make the call. If a distinct definition
were possible, would the diagnosis still be atypia and not something else? One can
try to squeeze the edges of definition, but like an electron’s orbit, one cannot be
precise. An alternative approach would be to define the boundaries within which
atypia exists. Atypia is chaotic and capable of doing damage; we can set criteria and
keep it constrained. In TPS, the mandate is identifying life-threatening lesions,
especially HGUC; because of the bland nondiagnostic cytology associated with
low-grade, noninvasive neoplasms, AUC has been chosen to indicate worrisome
cells that may be associated with a high-grade invasive urothelial carcinoma
(HGUC). In effect AUC shifts the diagnostic point for atypia to the right on Fig. 4.3
and improves specificity, helping to ensure that HGUC is the target for TPS.
Definition
The general diagnostic category AUC is reserved for specimens that contain urothe-
lial cells with mild to moderate cytologic (not architectural) change that is concern-
ing for HGUC. Non-basal urothelial cells need to exhibit a nuclear to cytoplasmic
(N/C) ratio equal to or greater than 0.5. This feature serves as a warning that the
sample needs special attention and a diligent search for other features, including
nuclear hyperchromasia, coarse chromatin, and an irregular chromatic rim. In AUC
only one of these features needs to be present. Cytologic changes in AUC have to
fall short of a diagnosis of suspicious for high-grade urothelial carcinoma (SHGUC)
or high-grade urothelial carcinoma (HGUC) both of which demonstrate at least two
of the aforementioned features (see Chaps. 5 and 6). An interpretation of AUC

requires exclusion of nonneoplastic changes and conditions in which the reason for
“atypia” is known and the morphology recognized, such as changes caused by poly-
omavirus and other infections, reactive umbrella cells, and seminal vesicle cells.
Recognized reactive changes due to stones, instrumentation, and therapy are like-
wise excluded from AUC [43]. Such cases should be assigned to the NHGUC cat-
egory (see Chap. 3). AUC also does not include urothelial clusters (tissue fragments)
without cytologic abnormalities or with reactive changes. Again, samples with those
features belong in NHGUC. Figures 4.4 and 4.5 depict normal benign/reactive uro-
thelial cells compared to Figs. 4.6, 4.7, 4.8, 4.9, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15,
4.16, and 4.17, which depict AUC or potentially more advanced categories.
Fig. 4.4 Benign urothelial cells. The top left corner shows benign superficial urothelial cells
(umbrella cells), and the bottom right corner has benign intermediate/basal type urothelial cells.
Although the non-superficial urothelial cells have a high N/C ratio, they have a smooth nuclear
contour and do not show nuclear enlargement, placing them in the “negative” category. The follow-
up diagnosis was benign. (Bladder washing, TP, high mag.)
Fig. 4.5 Benign urothelial

cells. Intermediate
urothelial cells display
slight nuclear enlargement
and prominent
chromocenters. There is no
nuclear hyperchromasia,
clumped chromatin or
nuclear contour
irregularity. These changes
are consistent with the
“negative” category. The
follow-up diagnosis was
benign. (Bladder washing,
TP, high mag.)
Fig. 4.6 Atypical urothelial cells (AUC). Two groups of urothelial cells are shown. The group on
the top left is orderly, composed of intermediate type urothelial cells with smooth nuclear contours,
and no features of atypia. The group on the bottom right is disorganized; urothelial cells have high
N/C ratio and nuclear contour irregularity. Nuclear chromasia is similar in both groups. Due to the
cytologic atypia seen in the group on the bottom, this case should be categorized as AUC. (Voided
urine, TP, medium mag.)
Fig. 4.7 Atypical
urothelial cells (AUC). A
group of atypical urothelial
cells with variable N/C
ratios and nuclear contour
irregularity. The absence of
hyperchromasia and the
presence of degenerated
clumped chromatin
preclude a diagnosis of
SHGUC. (Bladder
washing, TP, high mag.)
Fig. 4.8 Atypical
urothelial cells (AUC).
Atypical urothelial cells
with high N/C ratio,
enlarged nuclei (compared
to the neighboring benign
urothelial cells), and mild
nuclear contour
irregularities. The
chromatin is uniform and
hypochromatic, precluding
a diagnosis of
SHGUC. (Bladder
Washing, TP, high mag.)
a b
Fig. 4.9 Atypical urothelial cells (AUC). (a) Atypical urothelial cells with high N/C ratio, enlarged
nuclei (compared to the neighboring benign urothelial cells), and mild nuclear contour irregulari-
ties. (Bladder washing, TP, medium mag.). (b) Another image of the same case shows irregular
nuclear contours in a group of urothelial cells that have degenerative cellular changes. The cellular
changes are worrisome, but the degree of degeneration and lack of marked atypia precludes a
definitive diagnosis. (Bladder washing, TP, medium mag.)
Fig. 4.10 Atypical
urothelial cells (AUC).
Urothelial cells display
high N/C ratio,
anisonucleosis, nuclear
contour irregularities, and
alteration of nuclear
polarity. The chromatin is
finely granular with
minimal hyperchromasia.
This patient had renal
urolithiasis on follow-up.
These are reactive changes
and retrospectively
categorize this sample as
NHGUC. (Renal pelvis
a b
Fig. 4.11 Atypical urothelial cells (AUC). (a) Urothelial cells display increased N/C ratio, nuclear
enlargement, and mild hyperchromasia. (Renal pelvis washing, TP, high mag.). (b) The follow-up
nephrectomy showed a low-grade urothelial carcinoma by histology, causing a filling defect on
imaging. (Nephrectomy surgical sample, H&E, medium mag)
Fig. 4.12 Atypical urothelial cells (AUC). Urothelial cells have increased N/C ratio, enlarged
nuclei, and conspicuous nuclear contour irregularities. The chromatin is coarse and clumped. In
some of the urothelial cells, the N/C ratio is <0.5, and there are degenerative changes (such as fray-
ing of the cytoplasm), but due to the hyperchromasia and coarse chromatin pattern, a diagnosis of
AUC was rendered. Follow-up revealed a high-grade urothelial carcinoma of the kidney. (Bladder
a c
Fig. 4.13 Atypical urothelial cells (AUC). Urothelial cells with high N/C ratio and nuclear hyper-
chromasia. These three figures display all the atypical urothelial cells that are present in the speci-
men. (a) Atypical urothelial cells (AUC) (upper left). Urothelial cells display irregular nuclear
contours and cytoplasmic vacuolization, consistent with degenerative changes. (b) Aggregate of
cells (lower left) have markedly irregular nuclear contours and variation in nuclear size. In com-
parison to the neighboring squamous cells, there is mild nuclear hyperchromasia. There are cellu-
lar degenerative changes, such as partial loss of the cytoplasm and loss of crisp nuclear detail. (c)
Small aggregate of atypical cells adjacent to squamous cells (right). The urothelial cell nuclei also
show degeneration, but the one cell with the high N/C ratio is worrisome. The patient is a 36-year-
old woman with recurrent urolithiasis and no history of urothelial carcinoma. Her age and history
are low-risk factors for bladder cancer; however, without a history of renal calculi, these cytologic
features warrant the diagnosis AUC. (Voided, TP, high mag.). The follow-up was benign
Fig. 4.14 Atypical urothelial cells (AUC). The urothelial cell shown here has a high N/C ratio,
nuclear enlargement, and prominent nucleoli. Chromatin is pale and slightly coarse, but the chro-
matinic rim is thin and uniform. Compared to the nucleus of the neighboring inflammatory cells,
the nucleus of the atypical urothelial cell is much larger. Follow-up with cystoscopy and urinary
bladder biopsy within 6 months showed urothelial mucosa with reactive changes, likely due to
inflammation. (Bladder washing, TP, high mag.)
Fig. 4.15 Atypical urothelial cells (AUC). Urothelial cells show high N/C ratio, nuclear enlarge-
ment, and hyperchromasia. Some have prominent nucleoli. The nuclear contours are relatively
regular. The paucity of the atypical cells precluded a HGUC diagnosis. There are more than five
abnormal urothelial cells, and while they do not all show a N/C ratio >0.7, due to the presence of
the other criteria mentioned above, a diagnosis of SHGUC may also be appropriate for this case.
The follow-up showed high-grade urothelial carcinoma in the urinary bladder. (Bladder washing,
TP, high mag.)
Fig. 4.16 Atypical urothelial cells (AUC). Urothelial cells with high N/C ratio, mild nuclear
enlargement, and hyperchromasia with some degenerative changes in the cytoplasm. The follow-
up showed high-grade urothelial carcinoma of the bladder. (Bladder washing, TP, high mag.)
Fig. 4.17 Atypical urothelial cells (AUC). A single urothelial cell with high N/C ratio, nuclear
enlargement, hyperchromasia, and irregular nuclear contours with some degenerative cytoplasmic
changes. Compare this cell to the surrounding benign urothelial cells with low N/C ratio and regu-
lar chromatin pattern. Since this was the only atypical urothelial cell in the case, either an AUC or
a SHGUC diagnosis would be appropriate for this case. The patient was lost to follow-up. (Bladder
larification of Issues Unresolved by the First Edition of TPS

C
Regarding Atypia
Urothelial Degeneration: Does Degeneration Mean Atypia?
Cellular degeneration has been well described in earlier publications and reflects
forms of both reversible and irreversible cellular injury. Cytoplasmic borders may
be irregular, have a moth-eaten appearance, be variably lost, or display cytoplasmic
vacuolization. The chromatin can be clumped, hazy, smudged, or indistinct; the
chromatin rim may be interrupted or may exhibit variable thickness and irregular
contours but rarely possesses the sharp angles that are often seen in malignancy.
Multinucleated umbrella cells may contain numerous small, degenerate, and
pyknotic nuclei of variable sizes that may mimic the hyperchromasia of malignancy.
Urothelial cells, especially in voided urine, may show involutional changes with
poorly preserved urothelial cells possessing single and/or multiple cytoplasmic
eosinophilic inclusions, “Melamed-Wolinska bodies,” were named after Dr. Myron
R. Melamed and Ms. Wanda H. Wolinska CT (ASCP), who first described these
findings [43, 44]. These structures are apparently eosinophilic aggregates of degen-
erate cytoplasmic organelles and other cytoplasmic debris. Recent studies have
documented a reduction in the degenerative features mentioned above with the use
of collection fluids/fixatives [45, 46] (see Chap. 10).
The AUC category also includes specimens where, due to poor preservation and
degeneration, the nature and degree of change in the urothelial cells cannot be well
characterized and there is concern about HGUC; however, the mere presence of
degeneration does not warrant the diagnosis of AUC. Degeneration is an expected
finding in voided urine samples, especially after delayed processing and in urinary
diversion specimens. These changes should be interpretable given a sufficient level
of experience. If one is going to invoke AUC, there has to be a considered reason for
the interpretation. Alternatively, if a few well-preserved atypical cells, worrisome
for but not completely fulfilling criteria for SHGUC, are found in an otherwise
degenerated sample, AUC may be appropriate. It should be reiterated that all inter-
pretations may be modified by history and clinical setting.
Atypical Squamous Cells (ASC) and Categorization
For a discussion of this topic, the reader is referred to Chap. 8 (section “Atypical
Squamous Cells”). As stated there, the presence of atypical squamous cells (ASC)
in urine is a rare finding, and its categorization has proven problematic. The cells are
not clearly urothelial and so placement under AUC is inappropriate. They are not
clearly malignant, so inclusion in “non-urothelial malignancy” (NUM) also does
not fit. The TPS solution is to place specimens exhibiting atypical squamous cells in
a free-text (“other”) category with an explanatory note indicating the presence of
ASC and its implications. Sample reports are provided in Chap. 8; example 4 spe-
cifically addresses atypical squamous cells.
Nuclear Cytoplasmic (N/C) Ratio as a Criterion
One of the significant and as yet unresolved issues regards an important cytomor-
phologic criterion to determine TPS categories, namely, the N/C ratio. This is a
number calculated by dividing the cross-sectional nuclear area by the cross-sectional
area of nucleus
cytoplasmic area of the cell ( ). Typically, this ratio is estimated
area of cytoplasm
by microscopists and is exposed to some degree of subjectivity. An N/C ratio of 0.5
was chosen by the authors of TPS as a discrete value to signal the possibility of a
significant lesion. It is well recognized that the estimation and value chosen is only
semiquantitative but does provide an observer with a mark at which to aim. A study
by Zhang, Guo, and VandenBussche surveying 137 cytologists showed that the N/C
ratio assessed visually was more reproducible between observers when compared
with other cytological features such as hyperchromasia or coarse chromatin; how-
ever, there was a tendency to overestimate an N/C ratio that approximates 0.5 [47].
Of interest, one of the early studies, performed by Vaickus and Tambouret, after the
publication of the first edition of TPS demonstrated that morphologists are indeed
capable of rendering relatively accurate estimates of the N/C ratio, especially at
higher levels. They also found that the practice of specifying discrete decimal N/C
ratios for atypical cells would be valid [48]. Shortly thereafter, Hang et al. used digi-
tal image analysis validating TPS recommendations for an N/C ratio cutoff at 0.5
for the AUC category [49]. Choosing a discrete value was not without criticism.
Another study by Long et al. showed problems with inter-observer agreement; how-
ever, the article was published shortly after the release of TPS, and its features had
to be somewhat novel to the study group [50]. Subsequently, McIntyre et al., found
mean N/C ratios for the HGUC and SHGUC categories to be 0.57 and 0.53, sug-
gested reducing the N/C ratio below the current TPS threshold of 0.7 for these cat-
egories (see Chaps. 5 and 6); however, the authors recognized that HGUC cases also
contained individual cells with >0.7 ratio [51]. Thus the criteria for HGUC do func-
tion effectively. Overall, it appears that utilizing the N/C ratio criterion of ≥0.5 is
not only feasible, but when present, this criterion also provides a high predictive
power for HGUC in AUC specimens, justifying it as a required criterion for the
AUC category. (See Diagnostic Decision Tree in the Introduction) [47, 52].
PS Criteria Including Those for AUC Are Maintained for Upper

T
Tract Samples
As is well explained in Chap. 7, a number of studies of TPS demonstrated that the

diagnostic criteria function in samples derived from the lower urinary tract and
should be applied in a similar manner to the upper tract [16, 53, 54]. This includes
interpreting specimens exhibiting atypical urothelial cells. Instrumented specimens
from the upper tract are characteristically hypercellular and may contain tissue frag-
ments with reactive changes due to physical forces. Such changes do not constitute
a basis to render a diagnosis of AUC [52].
PS Criteria Including Those for AUC Are Currently Maintained

T
for All Preparation Types
According to a 2019 survey performed by the College of American Pathologists

(CAP), with participants from 20 countries including the USA and Canada, the
most common preparation methods used for urinary specimens were ThinPrep
(57.4%) (Hologic, Marlborough, MA) followed by Cytospin (45.5%) (ThermoFisher,
Fitchburg, WI) [33]. For a complete discussion of possible morphologic variations
that are technique dependent, see Chap. 10. There is some evidence supporting the
common use of TPS criteria among preparation types, but more studies are needed
(see Chap. 10).
Updates from The Paris System Survey (2020)
In preparation for the second edition of TPS, a survey querying utilization and func-
tionality of the system was compiled by TPS authors. The survey solicited recom-
mendations and suggestions for improvement from the participants. The survey was
sent in July 2020 to the American Society of Cytopathology and International
Academy of Cytology memberships via e-mail, website announcement, and social
media (Twitter and Facebook) for widespread distribution. The survey was open for
2 months following launch and gained responses from 523, of which, 451 passed
the initial screening question and were tabulated as participants. Answers were not
mandated; therefore, not all questions were answered by all participants. The vast
majority of the participants (344/370, 93%) reported processing urine specimens in
their own local cytology laboratories, and of those, a majority (214/261, 82%) were
using The Paris System to report urine cytology results in their institution. As seen
in the previous survey from the CAP, the most common preparation methods used
were ThinPrep (44.6%) followed by Cytospin (37%). The mean reported “atypia”
rates before and after implementation of TPS in the laboratories were 21.6% and
15.98% (p ≥ 0.01%), respectively. Regarding the question of how “atypical squa-
mous cells” should be categorized, 61% of the participants responded, “an atypical
category that is separate and distinct from the AUC category.” The majority of the
participants (156/215, 72.6%) agreed that the set of criteria for diagnosing voided
and instrumented urinary samples should be the same. Similarly, the majority of the
participants (167/215, 77.7%) agreed that the set of criteria for diagnosing lower
versus upper tract specimens should be the same. Again, the majority (nearly 70%)
of participants reported that the cytomorphologic criteria for the AUC category was
applied with relative ease, and was not restrictive [55].
Criteria
For the AUC category, a strict morphological definition and a description of the
characteristics of atypical urothelial cells are not feasible. AUC is multifactorial and
probabilistic as explained in the background paragraphs of this chapter, but the sav-
ing grace to the concept of AUC is that there are boundaries. Practitioners of the
cytologic method recognize that AUC represents a grey zone between reactive
changes and SHGUC, i.e., cytologic changes that fall short of SHGUC but are
beyond what is accepted for NHGUC. Degeneration in the cellular sample is always
a problem; the better the preservation, the more secure the interpretation. Diagnoses
of SHGUC and HGUC are best made on nondegenerated urothelial cells if at all
possible. Many samples interpreted as AUC may exhibit some degree of cellular
degeneration while eliciting worry for HGUC.
AUC is defined as cellular changes that fulfill the major (required) criterion in
addition to one minor criterion.
• Major criterion (required)

–– Urothelial cells with an increased nuclear cytoplasmic (N/C) ratio (≥0.5),
secondary to nuclear enlargement (Explanatory Note 1 and 2)
• Minor criteria (one required):
–– Nuclear hyperchromasia (Explanatory Note 3)
–– Irregular nuclear membranes (also known as chromatinic rim or nuclear con-
tour) (Explanatory Note 4)
–– Irregular, coarse, clumped chromatin
Based on the presence of the major criterion and one of the minor criteria noted
above, a diagnosis of AUC may be rendered. The morphology of normal basal uro-
thelial cells, typically observed in instrumented urine specimens, needs to be recog-
nized as “normal” or NHGUC despite the fact that they may have a high N/C ratio
and may appear mildly hyperchromatic (Fig. 4.4). These cells frequently occur in
groups and have uniform, round nuclei, and inconspicuous nucleoli with a finely
dispersed, smooth chromatin. Chapter 3 provides a detailed discussion of the cyto-
morphologic features of the NHGUC category. In cases where there are cells with
significant atypia, but due to extensive degeneration secondary to poor cellular pres-
ervation or obscuring elements a definitive diagnosis could not be rendered, the
diagnosis of AUC is a valid option.
Both the quality and quantity of atypical urothelial cells in a urine specimen are
important for the diagnosis. In a study reviewing subclassification of indeterminate
categories, cases with a benign/negative for HGUC outcome had an average of less
than 9 atypical urothelial cells, compared to cases with a “positive HGUC” outcome
in which >16 atypical urothelial cells were present [56]. Currently, there is no rec-
ommendation for counting the number of atypical urothelial cells for an AUC diag-
nosis; however, as the number of atypical cells with the described features increases,
so does the probability of malignancy.
Explanatory Notes
Explanatory Note 1: High N/C ratio HGUC cells typically show a high N/C ratio
approximating 0.7 [51]. For a diagnosis of AUC, the N/C ratio should be at least 0.5
[49]. The N/C ratio is increased due to nuclear enlargement as a result of increased
chromatin content relative to a smaller or no increase in cytoplasmic volume. If ele-
vated N/C ratio is the sole finding, i.e., there is no nuclear hyperchromasia, coarse
chromatin, or nuclear contour irregularity, then the case is unlikely to fit under the
AUC category but in NHGUC. A proviso is that any concern regarding HGUC is
grounds for investigation, discussion, and consultation. It is important to remember

that basal and intermediate urothelial cells, typically seen in instrumented specimens,
may have an increased N/C ratio; however, they lack nucleomegaly, hyperchromasia,
coarse chromatin pattern, and irregular nuclear contours. Figures 4.6, 4.7, 4.8, 4.9,
4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, and 4.19 demonstrate urothelial
cells with increased N/C ratios and various other abnormal morphologic findings.
a b
Fig. 4.18 Atypical urothelial cells (AUC). (a) A cohesive group of urothelial cells with high N/C
ratio, mild nuclear enlargement, and hyperchromasia on a Cytospin preparation. The chromatin
pattern is mostly uniform. (Bladder washing, Cytospin, high mag.). (b) Follow-up showed a low-
grade urothelial carcinoma on biopsy (urinary bladder biopsy, H&E, medium mag.)
a b
Fig. 4.19 (a–c) Atypical urothelial cells (AUC). (a) Single cells and groups of urothelial cells
with high N/C ratio and nuclear enlargement are depicted. (b–c) Compared with nearby unbrella
cells there are single urothelial cells with high N/C rtio and nuclear enlargement. However, the
chromatin is smooth and there is a lack of nuclear contour irregularity. The patient was post-BCG
therapy, and the follow-up urinary bladder biopsy showed reactive epithelial changes. (Catheterized
urine, Cytospin, high mag.)
Explanatory Note 2: Nuclear enlargement The nuclei of atypical urothelial cells

are usually larger than that of intermediate or basal urothelial cells. A study by
Vosoughi et al. showed that nuclear enlargement had a 95.8% positive predictive
value for HGUC [7]. This feature is not used as a major criterion, as HGUC cells
can be pleomorphic and different subtypes may show different morphologic fea-
tures. Notice in Fig. 4.6 both benign intermediate and basal urothelial cells and the
atypical urothelial cells have increased N/C ratios, but the increase in atypical cells
results from nuclear enlargement. Enlarged nuclei presumably have increased chro-
matin consistent with increased DNA synthesis and may be a sign of abnormal
ploidy associated with neoplastic growth.
Explanatory Note 3: Nuclear hyperchromasia An increase in the microscopic

optical density of the nuclear chromatin of urothelial cells, when compared with
that of normal superficial urothelial cells, is a result of increased light absorption.
Increases in chromatin concentration, increases in nuclear thickness, and increased
affinity for nuclear dyes are variably seen in neoplastic cells [29]. This relates to
Beer’s Law that relates the amount of light to the properties of the material through
which the light is passing. Of note, a small subset of HGUC cases have nuclear
hypochromasia, but if all the other criteria of HGUC are present, the pale chromatin
does not justify a diagnosis of AUC [57, 58]. Instead, a definitive HGUC diagnosis
should be rendered. See Chap. 6 for the discussion of hypochromatic HGUC. Nuclear
hyperchromasia is depicted in Figs. 4.11, 4.15, 4.16, 4.17, and 4.18.
Explanatory Note 3: Irregular nuclear contours Compared with the round shape
and smooth contours of the nuclei of normal urothelial cells, AUC usually have
irregular, variably thickened chromatin rims. The irregular contours and thickened
rims may relate to the variable amount of chromatin and lengths of DNA segments
attached to the nuclear envelope [44].
The “atypia” diagnosis, prior to TPS, was associated with a risk of subsequent
biopsy-proven HGUC that ranged from 8.3 to 37.5% [1, 3]. Based on results of the TPS
study group practice survey, we believe the rate of AUC category should not exceed
15%, and <10% should be an ultimate goal [55]. As further studies are performed uti-
lizing the criteria set forth in TPS, and as evidence-based data become available, there
will be further recommendations for the frequency and utility of the AUC interpreta-
tion. Post-TPS, the reported ROHM ranged from 24% to 53% (see Chap. 11).
Conclusion
Since the main aim of urine cytology according to TPS is to detect HGUC, a diag-
nosis of “atypia” is inappropriate for known benign cellular changes such as reac-
tive umbrella cells, polyomavirus or other viruses, granulomas, or changes due to
urolithiasis as discussed in Chap. 3. The target for AUC is HGUC, and only cells
worrisome for HGUC but lacking sufficient evidence for a more advanced
interpretation should be included in this category. With time and progressive bound-
ing of the AUC category, sensitivity and specificity for HGUC are expected to
improve, and thus increase the ROHM of patients in this diagnostic category while
simultaneously decreasing the rate of indeterminate cases. The exclusion of reac-
tive/reparative causes from AUC has resulted in a reduced atypia rate, sparing these
patients from an unnecessary neoplastic workup. Conversely, since TPS also
includes a “suspicious” category, SHGUC, we expect that some cases formerly
interpreted as AUC will show changes significant enough to be interpreted as
SHGUC, potentially resulting in a lower proportion of HGUC in the follow-up of
AUC. Thus, The Paris System intends to decrease the rate of “atypia” at both ends
of the diagnostic spectrum. For further discussion of SHGUC, see Chap. 5.
Cytologic reports that are unambiguous and issue a specific diagnostic category
mitigate confusion and anxiety among both clinical providers and patients and can
potentially help prevent unnecessary procedures. An article by Barkan, Wojcik, and
Pambuccian72 provides an overview and a roadmap to reduce atypia rates by outlin-
ing measures that can be taken by cytopathology practitioners [59].
Sample Reports
Example 1
Voided urine:
Interpretation: Atypical urothelial cells present
Example 2
Urine, bladder barbotage:
Interpretation: Atypical urothelial cells present. See note.
Note: The urine specimen contains rare urothelial cells with increased nuclear
cytoplasmic ratios, nuclear enlargement, and irregular nuclear membranes. The
paucity of these atypical urothelial cells limits further classification. According to
The Paris System for reporting urinary cytology, the “atypical urothelial cells” cat-
egory is associated with a 24–53% risk of high-grade malignancy. Follow-up is
recommended as clinically warranted.
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ogy: what are the helpful features? J Am Soc Cytopathol. 2015;4:183–9.
57. Renshaw AA, Gould EW. High-grade urothelial carcinoma with hypochromatic chromatin in
urine cytology. J Am Soc Cytopathol. 2021;10:25–8.
58. Pierconti F, et al. Hypochromatic large urothelial cells in urine cytology are indicative of high
grade urothelial carcinoma. APMIS. 2018;126:705–9.
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60. Rai S, et al. A quest for accuracy: evaluation of The Paris System in diagnosis of urothelial
carcinomas. J Cytol. 2019;36:169–73.
61. Vaheda Begam K, Vallamreddy SKR, Pratima J. Implementation of the Paris system versus
institutional diagnosis in the performance of urinary cytology: a 5 years correlative study of 74
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uous terminology to more objective criteria for reporting urine cytology. Cytopathol Off J Br
Soc Clin Cytol. 2017;28:509–15.
Suspicious for High-Grade Urothelial
Carcinoma (SHGUC) 5
Fadi Brimo, Manon Auger, Ivan Chebib, Tarik M. Elsheikh,
Mitsuru Kinjo, Eric Piaton, Marc Pusztaszeri,
and Tatsuro Shimokama
• Updated discussion on the risk of high-grade malignancy.

• Additional photos, including those of Cytospin, SurePath and ThinPrep
preparations.
• Additional and updated references.
Background
The diagnosis of “suspicious for HGUC” (SHGUC) is meant to reflect the presence
of urothelial cells with severe atypia that falls short for a diagnosis of high-grade
urothelial carcinoma (HGUC) but beyond atypia that is associated with the “atypi-
cal urothelial cells” (AUC) category. Since the publication of the first edition, many
studies have confirmed that a cytologic diagnosis of SHGUC confers a risk for
HGUC that falls in between that of AUC and HGUC cytological diagnoses, provid-
ing justification for keeping it as a separate diagnostic category [1–9].
Definition
This diagnosis is restrictively used in cases with abnormal urothelial cells that quan-
titatively fall short of a definitive diagnosis of HGUC.

https://doi.org/10.1007/978-3-030-88686-8_5
86 F. Brimo et al.
Criteria
A diagnosis of SHGUC (Figs. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, and 5.9) is defined
as non-superficial and nondegenerated urothelial cells showing increased nuclear
to cytoplasmic (N/C) ratio, at least 0.7, and in addition, at least two of the three
following features:
• Moderate to severe hyperchromasia.

• Irregular clumpy chromatin.
• Irregular nuclear membranes.
Until further data become available, the same qualitative morphological criteria
should be applied to different specimen types (voided and instrumented; lower tract
and upper tract). Using all the features listed above, the decision to assign a case
into the SHGUC or the HGUC cytological categories is mainly based on the number
of abnormal cells fulfilling the above criteria. Due to the lack of definitive data
derived from studies specifically addressing this quantitative issue, a strict cutoff
number of abnormal cells above which one can confidently assign a HGUC diagno-
sis cannot be determined [10–11].
Fig. 5.1 SHGUC. One

single abnormal well-
preserved intermediate
urothelial cell displays an
eccentric nucleus with
increased N/C ratio,
hyperchromasia, irregular
clumpy chromatin, and
mildly irregular nuclear
membrane. (Voided urine,
TP, high mag)
Fig. 5.2 SHGUC. Rare

but abnormal well-
urothelial cells show
increased N/C ratios,
hyperchromasia, and
irregular nuclear
membranes. (Catheterized
urine, CS, high mag)
5 Suspicious for High-Grade Urothelial Carcinoma (SHGUC) 87
Fig. 5.3 SHGUC. A few

abnormal intermediate
urothelial cells, many of
which are well-preserved
and feature an increased
N/C ratio, hyperchromasia,
irregular clumpy
chromatin, and severely
irregular nuclear
membranes. In this
example in which
pleomorphism is present, a
diagnosis of HGUC may
also be appropriate.
(Catheterized urine, CS,
med. mag)
Fig. 5.4 SHGUC. A cell cluster composed of six abnormal well-preserved intermediate urothelial
cells displays increased N/C ratios, hyperchromasia, clumpy chromatin, and irregular nuclear
membranes. Note that despite similar nuclear characteristics, there is a range of N/C ratios from
0.5 to 0.8. An HGUC diagnosis may be appropriate in this case, especially in the presence of a
previous history of HGUC. (Catheterized urine, CS, high mag)
88 F. Brimo et al.
Fig. 5.6 SHGUC. One

severely abnormal
urothelial cell (left) has an
increased N/C ratio and
coarse and clumpy
chromatin but regular
nuclear membranes. Also
present is a degenerated
atypical cell (top) that does
not fulfill the criteria for
SHGUC. (Cystoscopic
Fig. 5.7 SHGUC. One

single abnormal well-
urothelial cell has an
increased N/C ratio,
hyperchromasia, irregular
clumpy chromatin, and
smooth regular nuclear
membranes. Note the
severe hyperchromasia in
comparison to the normal
intermediate urothelial
cells (right). (Catheterized
Fig. 5.5 SHGUC. Few

cells exhibit an increased
N/C ratio, hyperchromasia,
and irregular nuclear
membranes in the absence
of clear chromatin details.
(Cystoscopic urine, CS,
high mag)
Fig. 5.8 SHGUC. One abnormal well-preserved intermediate urothelial cells has increased N/C
ratio, severe hyperchromasia, irregular nuclear membranes but no easily evaluable chromatin pat-
tern. The background contains smaller degenerated cells with hyperchromatic nuclei, red cell
membranes, and necrotic debris, suggesting an invasive lesion. The follow-up biopsy was invasive
high-grade urothelial carcinoma (voided urine, CS, high mag)
Fig. 5.9 SHGUC. Rare

but abnormal well-
urothelial cells display
increased N/C ratios,
moderate hyperchromasia,
irregular chromatin
patterns, and irregular
nuclear membranes.
(Voided urine, SurePath,
med mag)
Instead, a cutoff range of at least five to ten cells for lower tract samples and at least
ten cells for the upper tract is recommended based on the degree of abnormal nuclear
changes observed. This approach also reflects the results of the first TPS survey in
which 90% of participants reported preferring a range of five to ten cells rather than
setting up a rigid arbitrary cutoff [12]. Accordingly, a HGUC diagnosis should very
rarely be assigned in the presence of less than five abnormal well-preserved cells. In
comparison, in the presence of five to ten abnormal cells, the decision to assign a
“positive for HGUC” diagnosis should take into account the severity of atypia of all
the abnormal cells, the clinical context, as well as the specimen type. For example, in
the presence of a previous history of HGUC and/or in voided specimens which are
by nature less cellular than instrumented specimens and in which cellular degenera-
tion is frequent, as low as five well-preserved and severely abnormal cells with
the features listed above may be sufficient to render a definitive HGUC diagnosis.
90 F. Brimo et al.
On the other hand, at least ten abnormal cells are needed before labelling a case as
HGUC in instrumented specimens derived from the upper urinary tract (see Chaps. 6
and 7 for further discussion).
• The cells are usually seen as single cells although clusters of atypical cells may
also be present.
• Features that may be seen but do not necessarily need to be present are:
–– Eccentric nuclear location (Fig. 5.1).
–– Prominent nucleolus.
–– Necrotic background.
–– Pleomorphism.
–– Mitoses.
–– Apoptotic bodies.
Explanatory Notes
Explanatory Note 1 Increased N/C ratio generally refers to an enlarged nucleus

that occupies at least half of the surface of the cell provided the cell is not degener-
ated and the cytoplasm is complete. The N/C ratio of the abnormal cell should
exceed 0.7, although cases may have some abnormal cells having an N/C ratio
between 0.5 and 0.7.
Explanatory Note 2 Hyperchromasia refers to an increased density of the nuclear

chromatin of abnormal urothelial cells as compared with that of normal umbrella or
intermediate urothelial cells. The degree of hyperchromasia is moderate to severe; a
mild difference in the chromatin density between the questionable urothelial cell
assessed and the normal accompanying cells does not warrant a “suspicious” diag-
nosis (Fig. 5.10). However, since there is a variant of HGUC with hypochromatic
nuclei, all cellular criteria must be evaluated before the case category is chosen (see
Chap. 6).
Explanatory Note 3 In the absence of clear and evaluable chromatin details, the
irregular clumpy chromatin pattern is not required in the presence of the other fea-
tures (high N/C ratio, nucleomegaly, irregular nuclear membranes, hyperchromasia)
(Figs. 5.5, and 5.8). Similarly, the presence of nuclear membrane irregularity is not
required in the presence of the other features (increased N/C ratio, nucleomegaly,
hyperchromasia, irregular clumpy chromatin) (Figs. 5.6, and 5.7).
Explanatory Note 4 Intermediate urothelial cells with increased N/C ratio, nucleo-
megaly, and mild hyperchromasia in the absence of evaluable chromatin details and
irregular nuclear membranes should not be labelled as “suspicious” but as AUC
instead. Similarly, cells with increased N/C ratio and irregular nuclear membranes
in the absence of moderate or severe hyperchromasia should generally not be
labelled as “suspicious” (Figs. 5.11, and 5.12).
Explanatory Note 5 While the category of AUC includes a subset of cases showing
cellular degeneration, a SHGUC diagnosis should not be rendered solely on degen-
erated cells, although some of the severely atypical cells may be degenerated.
Cellular degeneration is often present in voided specimens and can take the form of
incomplete cytoplasm, poorly preserved chromatin details, or discontinuous nuclear
membranes. The resulting altered cellular morphology can be problematic from the
diagnostic standpoint for the following reasons:
• Nuclei may look “blown-up” resulting in a falsely increased N/C ratio (Figs. 5.13,
and 5.14).
• Cytoplasm may be incomplete which makes it difficult to assess the N/C ratio,
often making it appear increased (Fig. 5.14).
• Nuclear membranes may seem irregular from dehydration.
• Nuclei may look hyperchromatic as a feature of degeneration, and not as a result
of an abnormal chromatin (Figs. 5.13, and 5.14).
Explanatory Note 6 Although prominent nucleoli can be appreciated in HGUC

cells, this finding is not consistently present, and therefore this criterion is not
required for a diagnosis of SHGUC. In addition, visible to prominent nucleoli can
be seen in reactive urothelial cells, which may have an increased N/C ratio border-
ing on and sometimes exceeding 0.5; however, unlike cells in SHGUC or HGUC,
such reactive cells exhibit regular nuclear membranes and fine chromatin (Fig. 5.15).
Fig. 5.10 AUC. Cell clusters of well-preserved intermediate urothelial cells demonstrate variable
N/C ratios and hyperchromasia. The degree of hyperchromasia is mild in comparison to the normal
intermediate cell nucleus (upper right). In addition, the cells do not show clumpy chromatin pattern
or irregular nuclear membranes which preclude the assignment of a SHGUC diagnosis. (Voided
urine, TP, high mag)
92 F. Brimo et al.
Fig. 5.11 AUC. Abnormal

cells with increased N/C
ratios and irregular nuclear
membranes in the absence
of nuclear hyperchromasia
preclude a diagnosis of
SHGUC. The follow-up
diagnosis was
LGUC. (Voided urine, TP,
high mag)
Fig. 5.12 AUC. Interme-

diate urothelial cells with
increased N/C ratios and
irregular nuclear mem-
branes in the absence of
nuclear hyperchromasia
are insufficient criteria for
a diagnosis of
SHGUC. (Voided urine,
TP, high mag)
Fig. 5.13 AUC. One single and degenerated urothelial cell has an enlarged nucleus, with increased
N/C ratio, mild hyperchromasia, and irregularly distributed clumpy chromatin. Note the presence
of incomplete cytoplasm and poorly preserved chromatin details. A SHGUC diagnosis should not
be rendered on degenerated cells only. However, atypical degenerated hyperchromatic cells are
often seen in the background of cases diagnosed as SHGUC or HGUC. Consequently, the presence
of degenerated atypical hyperchromatic cells warrants careful evaluation in order to rule out the
presence of similar non-degenerated cells in which a SHGUC or a HGUC diagnosis can be ren-
dered. (Voided urine, TP, high mag)
a b
Fig. 5.14 (a) AUC. One single and degenerated urothelial cell displays an increased N/C ratio,
hyperchromasia, and irregularly distributed clumpy chromatin. Note the presence of incomplete
cytoplasm and discontinuous nuclear membranes. In the presence of cellular degeneration, a
SHGUC diagnosis should not be rendered. (Voided urine, TP, high mag.) (b) AUC. One single and
degenerated urothelial cell shows increased N/C ratio, hyperchromasia, and irregularly distributed
clumpy chromatin. Note the presence of incomplete cytoplasm and discontinuous nuclear mem-
branes (Voided urine, TP, high mag)
94 F. Brimo et al.
Fig. 5.15 NHGUC.
Reactive urothelial cells
often show increased N/C
ratios as seen in the current
example. However,
hyperchromasia is absent,
and the nuclei show fine
regularly distributed
chromatin with small
visible nucleoli. Nuclear
membranes are smooth and
regular. (Voided urine, TP,
high mag)
Prevalence of SHGUC
The reported prevalence of SHGUC among all cytologic diagnoses ranges from
0.2% to 6.6% depending on the study [13–15]. As such, suspicious specimens rep-
resent a small percentage of overall cases across studies, and this is likely due to the
strict criteria in TPS for the use of the SHGUC category, leaving this diagnosis only
for cases with a limited number of malignant cells.
Risk of Malignancy
Studies using TPS classification of urine cytology have shown that the risk of high-
grade malignancy (ROHM) that is associated with or subsequent to a diagnosis of
SHGUC ranges from 33.3% to 100%, which is intermediate between that of AUC
(range: 12.3–60.9%) and HGUC (range: 58.8–100%) diagnoses [1–9, 13–15] . In
the four studies with the largest number of patients with histologic follow-up,
ROHMs for the SHGUC category were between 80 and 89%, while they were
between 32.6% and 49% for AUC and all studies above 90% for HGUC [2, 5, 11,
13]. Taking into account all large published studies using TPS, the mean ROHM
weighted by sample size is 76.2% for the SHGUC category, in comparison to 38.5%
and 88.8% for the AUC and HGUC categories, respectively (see Chap. 11 for fur-
ther discussion).
Management
Patients with a SHGUC diagnosis should be clinically investigated in order to deter-

mine the presence of a HGUC. Management decision is made by the treating physi-
cian based on the patient history, clinical setting, as well as the cytologic findings
(see Chap. 12).
References
1. Stanzione N, Ahmed T, Fung PC, Cai D, Lu DY, Sumida LC, Moatamed NA. The con-
tinual impact of the Paris system on urine cytology, a 3-year experience. Cytopathology.
2020;31(1):35–40. PMID: 31596979.
2. de Paula R, Oliveira A, Nunes W, Bovolim G, Domingos T, De Brot L, Bezerra S, Cunha I,
Morini M, Saieg M. Two-year study on the application of the Paris system for urinary cytology
in a cancer centre. Cytopathology. 2020;31(1):41–6. PMID:
3. Wang Y, Auger M, Kanber Y, Caglar D, Brimo F. Implementing the Paris system for reporting
urinary cytology results in a decrease in the rate of the “atypical” category and an increase
in its prediction of subsequent high-grade urothelial carcinoma. Cancer Cytopathol. 2018;
PMID: 29278461.
4. Rohilla M, Singh P, Rajwanshi A, Gupta N, Srinivasan R, Dey P, Kakkar N. Cytohistological
correlation of urine cytology in a tertiary centre with application of the Paris system.
Cytopathology. 2018;29(5):436–43. PMID: 29920811.
5. Hassan M, Solanki S, Kassouf W, Kanber Y, Caglar D, Auger M, Brimo F. Impact of imple-
menting the Paris system for reporting urine cytology in the performance of urine cytology.
Am J Clin Pathol. 2016;146(3):384–90. PMID: 27543983.
6. Xing J, Monaco S, Pantanowitz L. The ability of the Paris system to stratify the risk of high-
grade urothelial carcinoma. J Am Soc Cytopathol [Internet]. Elsevier Inc; 2018;7(5):S43.
Aveailable from: https://doi.org/10.1016/j.jasc.2018.06.102.
7. Nguyen L, Nilforoushan N, Krane JF, Bose S, Bakkar R. Should "suspicious for high-grade
urothelial carcinoma" and "positive for high-grade urothelial carcinoma" remain separate cat-
egories?. Cancer Cytopathol. 2020. PMID: 33036060.
8. Zare S, Mirsadraei L, Reisian N, Liao X, Roma A, Shabaik A, Hasteh F. A single institutional
experience with the Paris system for reporting urinary cytology: correlation of cytology and
histology in 194 cases. Am J Clin Pathol. 2018;150(2):162–7. PMID: 29878037.
9. Granados R, Duarte JA, Corrales T, Camarmo E, Bajo P. Applying the Paris system for report-
ing urine cytology increases the rate of atypical urothelial cells in benign cases: a need for
patient management recommendations. Acta Cytol. 2017;61(1):71–6. PMID: 27838683.
10. Brimo F, Xu B, Kassouf W, Ahmadi-Kaliji B, Charbonneau M, Nahal A, Kanber Y, Caglar
D, Auger M. Urine cytology: does the number of atypical urothelial cells matter? A quali-
tative and quantitative study of 112 cases. J Am Soc Cytopathol. 2015;4(4):232–8. PMID:
31051759.
11. McCroskey Z, Bahar B, Hu Z, Wojcik E, Barkan G. Subclassifying atypia in urine cytology:
what are the helpful features? J Am Soc Cytopathol. 2015;4(4):183–9. PMID: 31051752.
12. Daniel F. I. Kurtycz, Fadi Brimo, Dorothy L. Rosenthal, Momin T. Siddiqui, Z. Laura Tabatabai,
Christopher J. VandenBussche, Eva M. Wojcik, Güliz A. Barkan, Perceptions of Paris: An
international survey in preparation for The Paris System for Reporting Urinary Cytology 2.0
(TPS 2.0), accepted, American Society of Cytopathology’s 66rd Annual Scientific Meeting, Las
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13. Meilleroux J, Daniel G, Aziza J, d’Aure DM, Quintyn-Ranty ML, Basset CML, Evrard SM,
Courtade-Saidi MM. One year of experience using the Paris system for reporting urinary cytol-
ogy. Cancer Cytopathol. 2018;126(6):430–6. PMID: 29663682.
14. Bakkar R, Mirocha J, Fan X, Frishberg DP, Peralta-Venturina M, de Zhai J, Bose S. Impact
of the Paris system for reporting urine cytopathology on predictive values of the equivocal
diagnostic categories and interobserver agreement. Cytojournal. 2019;16:21.
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Reporting Urinary Cytopathology: review of the published literature. J Am Soc Cytopathol.
2020:S2213–2945(20)30323–9.
High-Grade Urothelial Carcinoma
(HGUC) 6
Momin T. Siddiqui, Derek B. Allison, Guido Fadda,
Jee-Young Han, Patrick J. McIntire, Christopher L. Owens,
Z. Laura Tabatabai, Toyonori Tsuzuki, and Mingjuan Lisa Zhang
• Criteria for cytologic diagnosis of HGUC are internationally accepted and suc-
cessfully applied.
• Spectrum of N/C ratios is noted in malignant cells of HGUC.
• Hypochromasia is recognized as a rare occurrence and as a potential pitfall in
diagnosis of HGUC.
• Degenerative changes in HGUC are addressed.
• HGUC sub-types are described.
• Fibrovascular cores in HGUC are considered.
Background
In the first edition of TPS, the recommended criteria for diagnosing HGUC in urine
samples included a minimum of five to ten malignant cells. This range was estab-
lished to accommodate varying experience, patient populations and previous his-
tory, as well as type of specimen. Considering potential significant consequences of
HGUC diagnosis in the upper tract, higher number of malignant cells (>10) has
been recommended from upper tract specimens. The criteria for malignant cells
included an N/C ratio of 0.7 or greater, i.e., the cross-sectional area of the nucleus
occupying equal to or more than 70% of the cross-sectional area of the cell size,
nuclear hyperchromasia, irregular nuclear membranes, and coarse nuclear chroma-
tin [1]. Diagnosing HGUC is recognized as the gold standard for TPS, and hence the
above criteria have been widely applied by laboratories processing and reporting
urine cytology [2–4].

https://doi.org/10.1007/978-3-030-88686-8_6
98 M. T. Siddiqui et al.
Introduction
The TPS criteria for HGUC have been widely utilized for reporting urinary cytology
across the globe [2–7]. This fact has been affirmed by peer-reviewed publications
and a 2019 survey/poll conducted by the American Society of Cytopathology [5–7].
In these large cohort studies, with the largest number of patients with histologic
follow-up, the risk of high-grade malignancy for the HGUC category is all above
90%, confirming the high positive predictive value for HGUC by applying TPS
criteria [8–10]. This achievement is notable. And as such, the recommended cyto-
morphologic criteria remain unchanged in the second edition of TPS (Figs. 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 6.10, 6.11, and 6.12).
Fig. 6.1 HGUC
demonstrating a wide
range of cell sizes and
shapes. Nuclear
hyperchromasia is
pronounced (voided urine,
TP, low mag)
Fig. 6.2 HGUC with numerous malignant cells displaying heterogeneity. The tumor cells vary in
size and shape and are arranged in clusters and single cells. Necrosis is present in the background
(voided urine, SP, low mag)
6 High-Grade Urothelial Carcinoma (HGUC) 99
a b
Fig. 6.3 (a) Malignant cells are arranged in small groups and as single cells. Pleomorphism and
background necrosis are also present (voided urine, SP, medium mag.). (b) Malignant cells with
high N/C ratios and irregular nuclear membranes. The sample is stained lightly, so observers are
cautioned to use benign cells in the background as stain intensity controls. Also, note the presence
of lymphocytes, which can be used as a control for nuclear size (washing, TP, medium mag)
Fig. 6.4 Loosely cohesive

HGUC cells demonstrate
high N/C ratios which are
>0.7 (voided urine, TP,
high mag)
Fig. 6.5 An aggregate of

hyperchromatic HGUC
cells with high N/C ratios
which are >0.7 (voided
urine, TP, medium mag)
Fig. 6.6 A loosely

cohesive group of HGUC
cells with a wide range in
size (voided urine, TP,
high mag)
Fig. 6.7 Hyperchromatic
HGUC cells presenting as
a cohesive group. The
majority of tumor cells
have high N/C ratios
(bladder washing, TP,
high mag)
Fig. 6.8 Nuclear
membrane irregularity and
focal thickness of nuclear
membranes are evident in
this group of HGUC cells
high mag)
Fig. 6.9 HGUC cells with

coarsely clumped nuclear
chromatin, but with N/C
ratios <0.7. However, all
other features, including
enlarged cell size, are seen
in HGUC (voided urine,
TP, high mag)
Fig. 6.10 Coarse
chromatin and nuclear
membrane irregularity is
present in HGUC cells
high mag)
Fig. 6.11 Nuclear
membrane irregularity,
hyperchromasia, coarse
chromatin, and
cytoplasmic vacuolation
are present in HGUC. The
vacuoles probably reflect
degeneration but may also
be found in cells with
glandular differentiation.
The follow-up biopsy
confirmed the diagnosis of
HGUC with glandular
differentiation (bladder
washing, TP, high mag)
Fig. 6.12 HGUC cells

exhibiting nuclear
hyperchromasia, nuclear
membrane irregularity,
coarse chromatin, and
atypical mitoses. The N/C
ratios vary, from low to
very high (bladder
Reporting the TPS criteria for HGUC has resulted in a very high specificity, i.e.,
decreased false positives, for the diagnosis of HGUC [5]. These criteria may result
in a decrease in the diagnosis of HGUC, especially in degenerated specimens, in
which malignant cells can exhibit a reduction in the N/C ratio to less than 0.7 [5,
11]. Such scenarios lead to malignant cells being under-classified. Likewise, the
high specificity of the criteria may help shift a subset of cases from a suspicious to
a malignant diagnosis [5, 11]. Thus, the high specificity for HGUC ultimately
results in improved risk stratification for patients based on future risk of malignancy
[11]. Malignant cells with degenerative changes and a lower N/C ratio are addressed
in a later section as a variation from the recognized criteria for HGUC.
Hyperchromasia is one of the criteria for diagnosing HGUC [1]. However, after
the publication of TPS first edition, hypochromasia has been reported as a rare fea-
ture in HGUC cells in cytologic samples [12, 13]. Although, unusual, this is an
important variance to be recognized, particularly in instances where it is identified
in all of the malignant cells in a given specimen. Similarly, the first edition of TPS
did not address all of the known sub-types of HGUC, most of which are rarely
encountered. In this second edition, examples of the more aggressive sub-types,
such as plasmacytoid and micropapillary HGUC, are illustrated and discussed [14,
15]. TPS remains focused on a broader discussion of the morphologic criteria of
HGUC with a goal to be able to diagnose as many cases as possible and not be
detracted by potential outliers. Readers are encouraged to keep abreast of peer-
reviewed literature for infrequently encountered morphologic findings and incorpo-
rate them in the diagnosis, if clinically appropriate.
Required Cytomorphologic Criteria of Malignancy for HGUC
• Cellularity: At least five to ten malignant cells, for lower and upper tract speci-
mens, respectively.
• N/C ratio: 0.7 or greater (cross-sectional area of the nucleus occupying equal to
or more than 70% of the cross-sectional area of the cell size).
• Nucleus: Moderate to severe hyperchromasia.
• Nuclear membrane: Irregular in outline.
• Chromatin: Coarse/clumped.
Additional Cytomorphologic Features
• Cellular pleomorphism.
• Variation in size and shape of malignant cells, i.e., oval, round, elongated, and
spindle.
• Dense or vacuolated cytoplasm.
• Prominent nucleoli.
• Mitoses.
• Necrosis.
• Inflammation.
Variances Noted in the Cytomorphology of HGUC
N/C Ratio
HGUC can display a very wide range of N/C ratios in malignant cells (Figs. 6.13,
6.14) . However, an increased N/C ratio of at least 0.7 is a recommended benchmark
for diagnosing HGUC. Although this criterion is arguably the most restrictive, it
accomplishes the goal of TPS by producing a very high specificity for urinary
cytology [5, 11]. In most cases, the majority of HGUC cells will exhibit N/C ratios
greater than 0.7 but may also display a very wide range of N/C ratios, with a signifi-
cant proportion of malignant cells having a N/C ratio between 0.5 and 0.7.
Occasionally, malignant cells can be large with abundant cytoplasm, displaying N/C
ratios of <0.5, which even falls below the threshold for the AUC category (Figs. 6.15,
6.16, and 6.17) [14]. As a result, the use N/C ratio > 0.7 as a requirement for a
malignant diagnosis has been a topic of much debate and further brings up the issue
of a morphologist’s ability to assess N/C ratios accurately and reproducibly. In
almost all samples from HGUC, there will be enough cells meeting criteria with
N/C ratios equal to or greater than 0.7 to make the interpretation with confidence.
Fig. 6.13 Loosely
cohesive HGUC tumor
cells with a very wide
range of N/C ratios. The
nuclear features are helpful
in confirming the
diagnoses of malignant
cells with an N/C ratio
lower than 0.7 (voided
Fig. 6.14 A majority of

malignant cells with N/C
ratio lower than 0.7 but
classic features of
HGUC. An N/C ratio of at
least 0.7 is a benchmark
for diagnosing HGUC,
present in rare tumor cells
in this loosely cohesive
group (voided urine, TP,
high mag)
Fig. 6.15 Malignant cells

can be large with abundant
cytoplasm, creating a low
N/C ratio. All other
features of HGUC are
present. Note the tumor
necrosis clinging to the
cells (bladder washing, TP,
high mag)
Fig. 6.16 A multinucle-

ated malignant cell with
abundant cytoplasm,
possibly the result of
degeneration (voided urine,
SP, high mag)
Fig. 6.17 A single

malignant cell with very
low N/C ratio. The nuclear
features are consistent with
HGUC. If less than five
similar cells are present in
the specimen, then the
diagnosis of the sample
should be SHGUC (voided
The literature shows variable results. For example, Vaickus et al. found that mor-
phologists were relatively accurate in estimating N/C ratios without a measuring
device with greater accuracy achieved as the N/C ratio increased [16]. In contrast,
Layfield et al. concluded the opposite [17]. Additionally, Zhang et al. showed a
trend toward N/C ratio overestimation, especially for values approaching the cutoff
points of 0.5 and 0.7 [18]. As a result, digital image analysis (DIA) has the opportu-
nity to more accurately assess the N/C ratio, but studies are very limited on what the
actual cutoff values should be and how that would translate into traditional morpho-
logical assessment. McIntire et al. found that the HGUC and SHGUC categories
revealed cells with average N/C ratios of 0.57 and 0.53, respectively, suggesting that
the current cutoff value of 0.7 is too high [19]. Yet, this study also noted that malig-
nant cells with N/C ratios higher than 0.7 were also present in the samples. Even if
ideal N/C ratio cutoff values are determined by DIA, these values may not be mor-
phologically distinguishable to the unassisted eye and may not be meaningful, espe-
cially if morphologists tend to overestimate N/C ratios. When otherwise overtly
malignant features are present, where the cells of interest all have N/C ratios <0.7,
those cases can be placed in the “suspicious” category, thereby preserving high
specificity, but also indicating to the treating physician the likelihood of malignancy.
Hypochromasia
HGUC can present with hypochromatic nuclei (Figs. 6.18 and 6.19), usually as a
small proportion of malignant cells along with those exhibiting hyperchromatic
nuclei, which still adheres to the TPS criteria. It is rare to see hypochromatic malig-
nant cells as a solitary feature [12, 13]. Cells with hypochromatic nuclei can be
diagnostic for HGUC based on increased nuclear size, high N/C ratio (>0.7), irregu-
lar nuclear membranes, and coarse (frequently peripheral) chromatin in the absence
of hyperchromasia [12, 13].
Fig. 6.18 Hypochromatic
HGUC tumor cells are
rarely present as a solitary
finding. High N/C ratios
and nuclear membrane
irregularity are also present
to confirm the diagnosis
(voided urine, CS,
medium mag)
Fig. 6.19 Hypochromatic
HGUC tumor cells
predominate along with
rare hyperchromatic
HGUC cells (voided urine,
CS, medium mag)
Degenerative Changes
Degenerative changes are particularly identified in voided urine cytology samples,
where cells are free floating and separated from their nutrient supply for an unknown
period of time. TPS criteria recommend against the assessment of degenerated cells
for inclusion in any category since altered morphology may be misinterpreted, des-
ignating the sample incorrectly [1]. These degenerative changes may include a loss
of cytoplasm and nuclei that appear “blown-up” and enlarged, resulting in increased
N/C ratios. In contrast, some cells may actually pick up additional cytoplasmic vol-
ume in the form of bubbly vacuoles, resulting in a decrease in N/C ratios (Figs. 6.20
and 6.21). Furthermore, nuclear membranes may become irregular because of dehy-
dration or changes in osmotic forces, and nuclei may appear hyperchromatic due to
pyknosis rather than to abnormal chromatin. Therefore, cells with incomplete cyto-
plasm, discontinuous nuclear membranes, and poorly preserved chromatin should
be excluded from evaluation for HGUC and SHGUC categories. Conversely, HGUC
cells may also display degenerative features that can masquerade as benign changes,
such as mimicking BK viral cytopathic effect [5, 20]. Not uncommonly, cells with
overt malignant features may also include one or more degenerative changes; how-
ever, these cells should not count toward the minimum of five to ten nondegenerated
malignant cells to make a diagnosis of HGUC.
Fig. 6.20 Degenerated
tumor cells with
cytoplasmic vacuolation
and frayed cell membranes
medium mag)
Fig. 6.21 Degenerated
tumor cells with irregular
nuclear membranes and
pyknosis (bladder
washing, TP, medium mag)
Extremely Dark Chromatin

HGUC cells may have chromatin that is ink black and opaque, precluding any
assessment of whether the chromatin is coarse and clumpy. When this occurs, the
nuclei are referred to as India ink, coal-black, or jet black nuclei, which is a well-
recognized feature of HGUC, especially in degenerated HGUC cells. In fact, the
presence of such nuclei has been shown to be an independent predictor of malig-
nancy in urinary tract washing specimens and a common finding of HGUC in
Cytospin preparations [15, 21]. As a result, the presence of extremely dark and
opaque nuclei may equate with the combination of hyperchromatic nuclei and
coarse/clumped chromatin as criteria for malignancy. Cells displaying all the
“required” features of HGUC are usually abundant in classic HGUC specimens,
obviating reliance on unusual or unreliable features.
Important Histologic Variants of HGUC in Urine Cytology
There are 13 histologic variants of HGUC based on the 2016 WHO classification of
tumors of the urinary system [22]. These can pose a diagnostic challenge in urinary
cytology specimens because the classic features of HGUC, by definition in some
cases, may be absent. TPS has created an understandable diagnostic model, but
some variants of HGUC may not conform to the “required” features, potentially
resulting in a false-negative diagnosis. Fortunately, these variants are extremely rare
and are often associated with conventional components displaying cytomorphologic
features adherent to TPS criteria.
HGUC with squamous differentiation is defined by the presence of keratinization
and/or intercellular bridges as classic morphological features (Figs. 6.22 and 6.23).
Squamous cells are intermixed with malignant cells exhibiting classic features of
HGUC [1, 23]. The squamous cells may display hyperchromatic and spindle nuclei
with clumped chromatin. The cytoplasm is dense, keratinized, and orangeophilic.
Keratin flakes and necrosis are frequently observed in the background [1]. Similarly,
HGUC with glandular differentiation shows the presence of true glandular
Fig. 6.22 A few cells

show classic features of
HGUC adjacent to tumor
cells with squamous
differentiation (Bladder
Fig. 6.23 The squamous

cells have hyperchromatic
nuclei with clumped
chromatin. The cytoplasm
is dense, keratinized, and
orangeophilic. A
keratinized tad-pole cell
with a Herxheimer spiral is
also present. Keratin flakes
and necrosis are present in
the background (bladder
formation within groups of tumor cells [1, 23]. Mucin-filled goblet cells with hyper-
chromatic nuclei and coarse chromatin are present (Figs. 6.24 and 6.25). These can
be intermixed with malignant cells exhibiting classic features of HGUC [1, 23].
The nested variant of urothelial carcinoma is a rare malignancy characterized by
bland morphology and a deep and widely invasive pattern of growth. As a result,
even on biopsy, it is often difficult to distinguish superficially invasive nested vari-
ant urothelial carcinoma from von Brunn nests. In a study by Cardillo et al., the
authors were able to retrospectively identify cells in the urine that had a similar
morphology to the patient’s nested variant tumors on resection [24]. These features
included medium-sized, round-to-polygonal cells with abundant granular-to-dense
cytoplasm with distinct cell borders, slightly enlarged N/C ratios, irregular nuclear
contours, and occasional prominent nucleoli. However, given the subtle nature of
these findings, which may overlap with reactive changes, they recommended against
making this diagnosis on urinary cytology samples [24].
Micropapillary urothelial carcinoma is rare variant of HGUC. As a result, it is
important to recognize and sub-type this variant on histology, which, in addition to
Fig. 6.24 Mucin-filled
goblet cells with
hyperchromatic nuclei and
coarse chromatin are
present in HGUC with
glandular differentiation
high mag)
Fig. 6.25 Scattered single

malignant cells with
mucin-filled cytoplasm
(arrow) are representative
of glandular differentiation
medium mag)
high-grade cytologic features, is notable for forming tight infiltrating clusters that
lack fibrovascular cores and that are surrounded by retraction spaces on histologic
samples. Urinary cytology demonstrates tightly packed clusters in a three-
dimensional micropapillary configuration, which may show hyperchromasia, high
N/C ratio, and occasional vacuolated cells (Fig. 6.26) [23, 25, 26].
Plasmacytoid urothelial carcinoma is a poorly differentiated variant of HGUC
that is histologically characterized by infiltrating discohesive cells with eccentric
nuclei and abundant cytoplasm, resembling plasma cells. Rare reports have docu-
mented this variant in urinary cytology specimens, noting similar features to the
histologic correlate [25, 26]. The cytomorphology is characterized by large, disco-
hesive malignant cells with abundant and thick cytoplasm. Thus the tumor cells may
have an N/C ratio < 0.7. The nuclei are eccentrically placed and are hyperchromatic
with irregular nuclear membranes and coarse chromatin. These are clearly different
from benign cells or those seen in LGUN cases (Fig. 6.27) [25, 26].
Fig. 6.26 Tightly packed

three-dimensional
micropapillary clusters
with hyperchromasia, high
N/C ratio, and some
vacuolated cells in a case
of micropapillary HGUC
(voided urine, TP,
medium mag)
Fig. 6.27 Large,
discohesive hyperchromatic
malignant cells with
abundant and thick
cytoplasm and an
eccentrically placed
nucleus represent a
plasmacytoid HGUC
high mag)
Fibrovascular Cores
A papillary growth pattern is defined by the presence of three-dimensional cellular
clusters surrounding a central fibrovascular core. True papillary clusters are exceed-
ingly rare in urine cytology specimens, most likely due to the fact that intact papil-
lary tissue fragments do not routinely shed into the urine without some disrupting,
external force. Furthermore, papillary tissue fragments are not specific for a single
entity and may derive from a papilloma, papillary urothelial neoplasm of low malig-
nant potential (PUNLMP), low-grade papillary urothelial carcinoma, or high-grade
papillary urothelial carcinoma. On the other hand, papillary-like clusters, which
lack a central small capillary in the fibrovascular core, are frequently observed in
both voided and instrumented procedures, but their presence is much more common
in washing/barbotage procedures, due to disruptive forces. The diagnostic signifi-
cance of papillary or papillary-like clusters in urinary cytology is completely depen-
dent on the presence or absence of cytologic features for HGUC. Select cases that
contain true papillary fragments but that lack HGUC features may be placed in
NHGUC as a secondary diagnosis, i.e., low-grade urothelial neoplasm (LGUN); the
majority of cases with clusters of benign urothelial cells will also be placed in the
NHGUC category. Although HGUC typically presents as single cells and small
clusters, the presence of true papillary clusters containing cells with high-grade
cytologic features is consistent with a diagnosis of HGUC.
Effects of Cytopreparation on the Cytomorphology of HGUC

Various specimen preparation techniques can be used for processing urinary cytol-
ogy, including ThinPrep (TP), SurePath (SP), and Cytospin (CS), that are all
alcohol-fixed methods. The use of cell block material can be helpful in a subset of
cases, particularly those with large, three-dimensional clusters that are better visual-
ized on a tissue section [27]. In general, alcohol fixation shrinks cells, causing them
to round up as three-dimensional structures, leading to a relative increase in N/C
ratio compared to formalin fixation; but alcohol optimally preserves nuclear mate-
rial that can be well-visualized by Papanicolaou staining, allowing for easy and
accurate assessment of the chromatin. No single technique has been shown to be
superior to another, although head-to-head comparisons are relatively lacking in the
literature in the era of TPS. However, one study by Straccia et al. compared the
Cytospin method to the ThinPrep method and found that the method did not impact
the diagnosis of malignancy [28]. Ultimately, regardless of the specimen prepara-
tion technique, TPS criteria can be applied with confidence. The reader is referred
to Chap. 10, Cytopreparatory Techniques.
Sample Reports
Example 1
Satisfactory for evaluation.

Malignant.
High-grade urothelial carcinoma. See note.
Note: The specimen is cellular with numerous malignant cells. The malignant
cells have a high N/C ratio, hyperchromatic nuclei, irregular nuclear membranes,
and coarse chromatin.
Example 2

Malignant.
High-grade urothelial carcinoma. See note.
Note: Numerous urothelial cells with degenerative changes are present. Viable
malignant cells are identified. These are few, however, five malignant cells are pres-
ent in the specimen. The tumor cells have a high N/C ratio, hyperchromatic nuclei,
irregular nuclear membranes, and coarse chromatin. These findings support the
above diagnosis.
Example 3

Malignant.
High-grade urothelial carcinoma with squamous differentiation. See note.
Note: Numerous malignant cells consistent with high-grade urothelial carcinoma
are present. Additionally, malignant squamous cells with keratinization are also
present. These findings support the above diagnosis.
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Cytopathology of the Upper Urinary
Tract 7
Christopher J. VandenBussche, Jen-Fan Hang,
Patrick J. McIntire, Yurina Miki, Stephen Peyton,
Poonam Vohra, and Mingjuan Lisa Zhang
Background
Upper tract (renal and ureteral) urothelial carcinomas (UTUC) account for only
5–10% of all urothelial carcinomas [1–3]. UTUC tend to show higher grade and
stage and have a worse prognosis than urothelial carcinomas of the bladder (UCB)
[4, 5]. Cytological examination of voided urine (VU) or washings (barbotages)
obtained from the upper urinary tract (UUT) is often a component of the diagnostic
workup for UTUC. Tissue biopsies of UUT lesions can be challenging due to the
increased difficulty of visualization and access of these lesions during ureteroscopy.
Studies have shown that instrumented/selective UUT specimens (catheterized,
washings/barbotages, and brushings) are superior to VU for the detection of UTUC,
but the presence of instrumentation artifacts can impede cytological evaluation.
This chapter summarizes the utility of UUT cytology, the current understanding of
UTUC including its molecular biology, and reviews the relevant current literature.
Definition of Upper Tract Urothelial Carcinoma (UTUC)
Upper tract urothelial carcinoma (UTUC) refers to urothelial carcinomas arising in

the renal pelvis or ureter. Broadly speaking, urothelial carcinomas include carci-
noma in situ (CIS), high-grade urothelial carcinoma (HGUC), and low-grade uro-
thelial carcinoma (LGUC). When assessing upper tract lesions, the focus of The
Paris System (TPS) remains on the detection of CIS and HGUC, both referred to in
this chapter as upper tract high-grade urothelial carcinoma (UTHGUC).

https://doi.org/10.1007/978-3-030-88686-8_7
116 C. J. Vandenbussche et al.
Explanatory Note
In general, the assessment of upper tract lesions utilizes the same principles applied
by TPS to lower tract lesions. One key consideration is that upper tract lesions are
most frequently seen and evaluated on washing/barbotage specimens. However,
these specimens are not expected to differ greatly from those collected from the
bladder. A second key consideration is that UTHGUC cells may be seen in voided
urine (VU) specimens, as VU specimens contain a sampling of cells from through-
out the urinary tract. However, UTHGUC cells in VU specimens are more likely to
be degenerated due to the greater length of time these cells have spent between their
natural exfoliation and specimen collection. A third key consideration is that diag-
noses made on upper tract cytology specimens collected from specific anatomic
sites (“selective cytology”) can have significant clinical implications. As opposed to
VU specimens which cannot localize lesions within the urothelial tract, selective
cytology specimens can localize UTHGUC to a specific kidney or ureter and direct
definitive treatment, potentially without a concurrent or intervening tissue biopsy
specimen. That decision ultimately is made by the urologic surgeon.
Etiology of Upper Tract Urothelial Carcinoma
UTUC Prevalence
Synchronous UTUC and urothelial carcinomas of the bladder (UCB) are seen in
17% of cases [3]. Patients with primary UTUC have been reported to develop
recurrences in the bladder in 22–47% of cases and in the contralateral UUT in
2–6% of cases [3]. Molecular analysis on paired primary UTUC and recurrent
UCB tissues from the same patient demonstrated a shared clonal origin; both pri-
mary and recurrent tumors were usually high-grade [6]. This finding suggests a
common field cancerization effect or intravesicular tumor seeding. For UTUC,
alterations in FGFR3, KDM6A, and CCND1 were significantly associated with a
higher risk of developing a subsequent UCB, whereas TP53 mutations were asso-
ciated with a lower risk [6].
Molecular Biology of Upper Tract Urothelial Carcinoma
Molecular Biology of UTUC Vs. UCB
Both UTUC and UCB have high somatic mutation burdens and predominant apoli-
poprotein B mRNA editing enzyme catalytic polypeptide (APOBEC)-related muta-
tional signatures [6–10]. In UTUC, FGFR3 and HRAS are more frequently altered,
7 Cytopathology of the Upper Urinary Tract 117
whereas TP53, RB1, and ERBB2 are less frequently altered in comparison to UCB
[6, 7, 10]. These differences may be due to environmental factors that are more
specific to carcinogenesis in UTUC (such as phenacetin, Balkan endemic nephropa-
thy, Chinese herb nephropathy, and association with blackfoot disease) [11].
Aristolochic acid (AA), a compound contained in certain Balkan and Chinese herbal
products, is one of the most potent carcinogens associated with UTUC [12–14]. The
reader is referred to Chap. 1, Pathogenesis, for further discussion.
Molecular Biology of Upper Tract HGUC Vs. Upper Tract LGUC
Activating mutations in FGFR3 are significantly more common in upper tract

LGUCs (>70%) than in UTHGUC (~30%) [6, 7, 9, 10]. However, the frequency of
FGFR3 alterations is higher in UTHGUC than in muscle-invasive HGUC of the
bladder [6, 7]. In contrast, among UTUC, TP53 mutations are found in UTHGUC
but not in upper tract low-grade lesions. Mutations in FGFR3 and TP53 have been
found to be mutually exclusive, with the exception of a small subset of UTHGUC. This
finding may suggest that UTHGUC often arises de novo, while some upper tract
LGUC with FGFR3 alterations acquire a TP53 mutation over time and progress to
high-grade disease [6, 7, 9].
Criteria for Upper Tract Urothelial Carcinomas
General Criteria for HGUC
TPS cytomorphological criteria for HGUC is the same for upper and lower tract
specimens (Figs. 7.1, 7.2, 7.3, 7.4, and 7.5), and a detailed discussion can be
found in Chap. 6 [15–19]. A recent study using digital image analysis determined
that UTHGUCs have higher N/C ratios, smaller cell circumference, smaller
nuclei, and less cytoplasm compared to LTHUGC [20]. While these cells would
still qualify as malignant according to the TPS criteria, their smaller size may
cause them to be overlooked, underestimated, or confused with basal cells. Ideally,
a diagnosis of HGUC in a selective cytology specimen should be confirmed with
a tissue diagnosis prior to any definitive treatment. However, because a good-
quality tissue biopsy may be difficult to obtain from some upper tract lesions, a
diagnosis of HGUC should be made with extra caution in upper tract specimens.
See Explanatory Note 2.
a b
c d
Fig. 7.1 HGUC arising in the renal pelvis. (a) This field is cellular and contains cells of different
sizes and varied N/C ratios. A cluster of three particularly large cells can be seen in the top center.
Despite having minimal nuclear contour irregularities, the N/C ratios of these cells exceeds 0.7 and
the cells possess coarse chromatin, features concerning for HGUC. (Renal pelvis washing, SP,
medium mag.). (b) At higher magnification, the coarse chromatin pattern of these larger cells can
be better appreciated. Some of the smaller cells in the background also have high N/C ratios and
coarse chromatin and are also likely malignant. A mitotic figure (arrow) can be seen, a finding that
is not entirely specific for HGUC. (Renal pelvis washing, SP, high mag.). (c) Despite having a very
low N/C ratio, this huge cell has all the other features of malignancy: hyperchromasia, coarse
chromatin, and irregular nuclear contours. The abundant cytoplasm is likely a form of degenerative
change. (Renal pelvis washing, SP, high mag.). (d) The corresponding biopsy reveals papillary
HGUC. Note the full thickness of atypical urothelial cells on this papilla, demonstrating hyper-
chromasia, nuclear enlargement, anisonucleosis, and irregular nuclear contours. (Renal pelvis
biopsy, H&E, high mag)
a b
Fig. 7.2 HGUC with prominent cercariform cell formation. (a) The specimen is cellular and con-
tains numerous dispersed single atypical cells. At this magnification, great variation in nuclear size
can be seen. (Renal pelvis washing, SP, low mag.). (b) At higher magnification, the atypical cells
can be identified as malignant. They have coarse chromatin and variable N/C ratios. Some cells
have cytoplasmic tails, forming so-called cercariform cells. This feature is seen more often in
washing specimens containing papillary urothelial neoplasms of any grade and is not specific to
HGUC. (Renal pelvis washing, SP, medium mag)
a b
Fig. 7.3 Small fragments of HGUC. (a) This three-dimensional fragment contains cells with
hyperchromatic nuclei and minimal cytoplasm. While HGUC cells tend to be discohesive, frag-
ments of HGUC may be seen in instrumented specimens. Even here, the cells at the edges appear
discohesive. (Renal pelvis washing, TP, medium mag.) (b) A separate fragment of HGUC cells
from the same case demonstrates striking anisonucleosis. The cells have highly irregular nuclear
contours and the chromatin is too dark to assess for the coarse, clumpy chromatin often seen in
HGUC. However, this level of hyperchromasia is just as concerning as the presence of clumpy
chromatin. (Renal pelvis washing, TP, high mag)
a b
Fig. 7.4 HGUC in a pelvic washing with cell-in-cell forms. (a and b) The HGUC cells in these
two representative fields are present singly and in small fragments. Both fields show examples of
a HGUC cell’s cytoplasm enveloping a neighboring HGUC cell. This phenomenon has been
described as “clasping” (when the neighboring cell does not appear entirely enveloped) and “cell-
in-cell” (when the neighboring cell is completely enveloped). Other terms used include “cell can-
nibalism” and “hugging.” While this finding can be seen in HGUC, the finding is not specific and
can occasionally be seen with benign, reactive processes. (Renal pelvis washing, Cytospin,
medium mag)
a b
Fig. 7.5 HGUC. (a) In this ureteral washing specimen, the HGUC cells have features of malig-
nancy: high N/C ratios, coarse chromatin, and slightly irregular nuclear contours. The cells do not
demonstrate significant anisonucleosis, which is often seen in other specimens with HGUC but is
not required for a definitive diagnosis of malignancy. (Ureteral washing, SP, medium mag.) (b) The
presence of atypical keratinized cells can indicate squamous differentiation in a HGUC. In this
renal pelvic washing specimen, the keratinized cells have bland-appearing nuclei but brilliant
orange cytoplasm. Overtly malignant cells are seen in small- to medium-sized non-keratinized
fragments adjacent to the keratinized fragment. While their morphology is compatible with urothe-
lial carcinoma, squamous cell carcinoma can metastasize to the kidney from the lung. If uncer-
tainty exists as to whether this is a HGUC, the “Other” diagnostic category may be used. (Renal
pelvis washing, SP, high mag)
Explanatory Note 1
In a barbotage/washing specimen that adequately samples a suspicious lesion, many
abnormal cells should be seen in the specimen if the neoplasm is HGUC. In instances
where a distinct lesion cannot be seen on uroscopy and selective cytology is being
used to localize HGUC identified on a prior voided urine specimen, a flat CIS or
small papillary HGUC lesion could be present and yield only a smaller subpopula-
tion of malignant cells. Following treatment and resection, the identification of focal
areas of CIS may be challenging and perhaps not possible on gross and microscopic
examination of the resection specimen.
Explanatory Note 2
When diagnosing HGUC in upper tract specimens, circumspection is recommended,
favoring specificity over sensitivity. In other words, the diagnosis of HGUC in upper
tract specimens should be reserved for those cases with indisputable qualitative cri-
teria (Fig. 7.6 a). Due to the infrequency of diagnosing UTHGUC, review by a
second pathologist is prudent. While a diagnosis of suspicious for HGUC (SHGUC)
is acceptable for specimens that do not clearly contain HGUC cells, contacting the
clinician can allow discussion regarding the amount of suspicion one has for HGUC
as well as how the patient should be managed (Fig. 7.6 b).
a b
Fig. 7.6 Suspicious for HGUC. (a) There are five atypical urothelial cells in the center of the field.
While some of these cells meet the cytomorphologic criteria for HGUC, a higher threshold is rec-
ommended for making a diagnosis of HGUC in upper tract specimens as compared to lower tract
specimens. Based on the overall clinical picture, an upper tract specimen may be quantitatively or
qualitatively insufficient for HGUC. Direct communication with the clinical team in these circum-
stances to convey the level of concern for HGUC may be useful. (Renal pelvis washing, SP, high
mag.) (b) Several singly dispersed malignant cells are intermixed with fungal organisms, debris,
and acute inflammatory cells. While some of the cells meet the cytomorphologic criteria for
HGUC, other cells are obscured and degenerated. This specimen is at least suspicious for HGUC
and a diagnosis of HGUC may be rendered depending on what is seen in other fields and the clini-
cal scenario. (Nephrostomy tube, SP, high mag)
Upper Tract HGUC in Instrumented Specimens
Only a few pre-TPS studies have assessed specific cytomorphologic features predic-
tive of UTUC in UUT specimens [21]. Potts et al. [22] found that increased nuclear-
to-
cytoplasmic (N/C) ratio, anisonucleosis, and overlapping nuclei were
distinguishing features for identifying UTHGUC. Witte et al. [23] reviewed a cohort
of renal pelvis washing specimens and determined that high N/C ratio, isolated
cells, anisonucleosis, nuclear hyperchromasia, and coarse chromatin were predic-
tive of UTHGUC (Fig. 7.7).
Several studies have examined the cytomorphologic features of UTHGUC in the
context of TPS and thus provide insight into the usefulness of TPS criteria in this
setting [21]. Simon et al. found that the incidence of cytomorphologic alterations
among UTHGUC in UUT specimens was N/C ratios ≥0.7 (88%), hyperchromasia
(83%), coarse chromatin distribution (67%), and nuclear pleomorphism within cell
clusters (65%) [24]. The most consistent feature associated with a downgrade from
HGUC to SHGUC (60% of cases) was the lack of marked nuclear membrane irreg-
ularity, which was only seen in 23% of HGUC cases. The presence of coarse chro-
matin and bizarre single cells was more commonly seen in HGUC in comparison to
LGUC; these findings were statistically significant. Increased N/C ratios, nuclear
hyperchromasia, nuclear membrane irregularities, pleomorphism, and the presence
of mitoses and/or atypical degeneration were not statistically significant features in
differentiating between low-grade and HGUC of the UUT [43].
Fig. 7.7 Fragment of HGUC from the ureter. Upper tract specimens are obtained through instru-
mentation and thus fragments of HGUC, such as the one seen here, may be forcibly exfoliated
during collection. This contrasts to the HGUC cells seen in voided urine specimens, which natu-
rally exfoliate as predominantly single cells from the urothelial lining. Because the center of this
fragment contains overlapping cells, it can be difficult to discern the morphology of each individ-
ual cell. By looking at the edge of the fragment, the high N/C ratios and coarse chromatin can be
better appreciated. (Ureter washing, SP, medium mag)
During a retrospective review of 30 UUT cytology cases prepared using ThinPrep

with histologic follow-up, Xing et al. found a decrease in the number of cases called
SHGUC or positive for HGUC when TPS criteria were applied to specimens with
UTHGUC [25]. When these cases were reviewed, most failed to meet the TPS 1.0
requirement for hyperchromasia, as the neoplastic cells were predominantly hypo-
chromatic and not hyperchromatic [25].
Explanatory Note 1
While some studies have identified an association of nuclear hypochromasia with
UTHGUC, Renshaw and Gould reported a similar finding in HGUC cells in bladder
washing specimens (A. Renshaw, personal communication, April 15, 2021) [26]. It
is therefore possible that hypochromasia may be associated with instrumented spec-
imens rather than tumor location (Fig. 7.8).
Explanatory Note 2
Due to the discohesive nature of HGUC cells, they are often seen as single malig-
nant cells when they naturally exfoliate into urinary tract specimens. However,
HGUC may be forcibly exfoliated as large papillary fragments in instrumented uri-
nary tract specimens (Fig. 7.9). Therefore, one should not automatically assume that
the presence of urothelial tissue fragments with fibrovascular cores indicates a low-
grade lesion. If HGUC is present, the pathognomonic single cells meeting the crite-
ria of HGUC will confirm the diagnosis (Fig. 7.10).
a b
Fig. 7.8 Hypochromatic HGUC cells. (a) Occasionally HGUC cells may appear paradoxically
hypochromatic, a feature seen in many of the cells in this field. However, the malignant cells are
very large and have elevated N/C ratios, nuclear contour irregularities, and chromatin coarseness.
Because the chromatin stains faintly, these cells may be overlooked. (Renal pelvis washing, SP,
medium mag.) (b) A higher magnification shows the atypical nature of these hypochromatic cells,
with markedly atypical nuclear borders. The chromatin clumps are also small and difficult to dis-
cern, even at this high magnification. Note that some cells with dark chromatin can be seen in the
top right corner. (Renal pelvis washing, SP, high mag)
a b
c d
Fig. 7.9 Examples of HGUC in fragments. (a) Tissue fragments containing HGUC cells. While
the tissue fragments have a papillary configuration, they lack fibrovascular cores. Single HGUC
cells with variable amounts of atypia can be seen in the background. (Renal pelvis washing, SP,
medium mag.) (b) Separate field of (a) at higher magnification. The smaller fragment contains cells
with more severe atypia than the cells seen in the lower, larger fragment and demonstrates hyper-
chromasia, coarse chromatin, anisonucleosis, and, for some of the cells, high N/C ratios. (Renal
pelvis washing, SP, high mag.) (c) Numerous tissue fragments of variable sizes containing HGUC
cells. HGUC cells can also be seen dispersed in the background. Examination at higher magnifica-
tion is required to confirm the low power impression of HGUC. (Ureteral washing, SP, low mag.)
(d) Well-preserved HGUC cells in a fragment, with coarse chromatin, hyperchromasia, and high
N/C ratios. Instrumented specimens can obtain freshly exfoliated cells that have not been degener-
ating in the bladder. (Renal pelvis washing, SP, high mag)
Fig. 7.10 HGUC cells adjacent to an LGUC fragment. Patients may have both LGUC and HGUC
in the urinary tract, so it is most important to diagnose specimens based on features of HGUC
rather than the identification of low grade urothelial neoplasms. This patient had LGUC and CIS
in one ureter. The papillary lesion was identified by cystoscopy due to its papillary growth, but the
CIS lesion was not identified until subsequent biopsy. This field contains a combination rarely
seen – a papillary fragment of LGUC, identified by a fibrovascular stalk (arising from the right-
hand bottom corner) with a monotonous population of attached neoplastic cells – and a small
fragment of HGUC cells (left-hand bottom corner). (Ureteral washing, SP, medium mag)
Upper Tract HGUC in Voided Urine Specimens
The criteria for diagnosing UTHGUC cells in VU specimens are the same criteria
used to diagnose HGUC of the lower urinary tract, as the location of HGUC cannot
be determined in VU specimens. UTHGUC cells are more likely to be degenerated
in VU due to increased transit times and thus are more likely to be diagnosed as
atypical urothelial cells (AUC) or SHGUC rather than HGUC. Zhang et al. found
that among patients with biopsy-confirmed UTHGUC, 50% of preceding VU speci-
mens demonstrated high-grade cytomorphological features (SHGUC/HGUC) com-
pared to almost 90% of preceding UUT cytology specimens; 81% (26/32) of VU
samples were diagnosed as AUC or higher [27].
Upper Tract Low-Grade Urothelial Neoplasms (LGUN)
The instrumentation required to procure UUT cytology specimens can result in the
exfoliation of benign, reactive urothelial tissue fragments along with fragments of
neoplastic cells. This complicates the detection of LGUN, as LGUN tissue frag-
ments can have significant cytomorphologic overlap with tissue fragments contain-
ing benign and reactive urothelial cells (Figs. 7.11, 7.12, 7.13, 7.14, and 7.15). The
presence of urothelial tissue fragments with fibrovascular cores are considered con-
sistent with a clinical impression of LGUN, so long as the diagnostic features of
high-grade lesions are not seen in the specimen (Fig. 7.16). The cytomorphologic
features of LGUN are discussed in Chap. 3 (Figs. 7.17 and 7.18) and are applicable
to UT lesions. Small urothelial tissue fragments without fibrovascular cores and
with bland nuclei may arise from either a urothelial neoplasm or the normal urothe-
lial lining [25] [28–30].
a b
c d
Fig. 7.11 Fragments of low-grade urothelial neoplasm without fibrovascular cores. (a–d) This
patient was found to have a low-grade urothelial carcinoma on a concurrent biopsy. In this washing
specimen, there are numerous neoplastic cells both in variably sized fragments (a–c) as well as
dispersed single cells with cercariform features (d). The cells and their nuclei have an elongated,
spindly appearance, possibly due to cautery artefact, which causes some fragments to resemble a
spindle cell neoplasm (a–c). No features of HGUC can be identified. The cells have low N/C ratios
and bland chromatin; the nuclei are all similar in size. In this case, it is appropriate to make a diag-
nosis of NHGUC; the specimen provides a good example of how LGUN can be cellular and visu-
ally striking in washing specimens. (Renal pelvis washings, TP, medium mag)
Fig. 7.12 Benign urothelial tissue fragment. Instrumentation often forcibly exfoliates benign uro-
thelial tissue fragments that may have cytomorphologic overlap with LGUN fragments. Benign
urothelial tissue, however, does not form papillary fragments with fibrovascular cores. While some
anisonucleosis can be spotted in this fragment, other features are benign. The cells have pale chro-
matin and low N/C ratios. (Renal pelvis washing, Cytospin, medium mag)
a b
Fig. 7.13 Benign urothelial tissue fragments. (a) The nuclei of benign urothelial cells in frag-
ments may appear slightly darker and have more irregular contours when compared to singly dis-
persed urothelial cells. However, even at this magnification, one can see that these cells have
abundant cytoplasm, supporting a benign diagnosis. (Renal pelvis washing, SP, medium mag.) (b)
One benign feature in the small fragment at the top of the field is the presence of a “cytoplasmic
collar,” formed by centrally placed nuclei surrounded by a peripheral “collar” of granular cyto-
plasm. This configuration represents the asymmetric unit membrane, a pathognomonic feature of
superficial urothelium. Nuclei with prominent chromocenters and condensed rims of chromatin are
characteristic of superficial cells and should not be mistaken for the hyperchromasia and coarse
chromatin pattern seen in HGUC. (Renal pelvic washings, SP, medium mag)
a b
Fig. 7.14 Benign urothelial tissue fragments. (a) These are benign urothelial cells from a renal
pelvis washing. Note the presence of multinucleated umbrella cells with abundant cytoplasm,
single cells in the background with low N/C ratios and bland chromatin, and a three-dimensional
benign urothelial tissue fragment at the bottom left-hand side of the field. (Renal pelvis washing,
SP, medium mag.) (b) At higher magnification, the benign features of the tissue fragment seen in
(a) can be appreciated. The cells have nuclei of approximately the same size and abundant, foamy
cytoplasm that forms a peripheral cytoplasmic collar around the fragment. Most of the nuclei have
two to three prominent chromocenters, which could be mistaken for a coarse chromatin pattern.
However, the chromatin clumps are distributed similarly among the cells and do not vary in size.
By contrast, the chromatin clumps in HGUC tend to be nonuniform in size and unevenly distrib-
uted within the nucleus, with dissimilar chromatin patterns found among the carcinoma cells.
(Renal pelvis washing, SP, high mag)
a b
c d
Fig. 7.15 Benign urothelial tissue fragments demonstrating stone atypia. (a-d) These tissue frag-
ments originated in a renal pelvis washing from a patient with nephrolithiasis. The fragments are
of variable sizes; the larger three-dimensional fragments (d) make cytomorphology difficult to
assess. Stones in the urinary tract can cause reactive changes in benign urothelial cells and also
forcibly exfoliate urothelial tissue fragments as they pass through the ureter into the bladder. The
fragments contain cells with increased N/C ratios and dark, hyperchromatic nuclei. The nuclei may
be so hyperchromatic that the chromatin pattern cannot be assessed. In addition to increasing one’s
threshold for an atypical diagnosis in patients with urolithiasis, certain features can be reassuring.
The nuclei are usually small and do not vary greatly in size within the fragments. A cytoplasmic
collar may be present, and well-preserved single cells concerning for HGUC are absent in the
background. (a–b, renal pelvis washing, Cytospin, medium mag.; c, renal pelvis washing, SP,
medium mag)
a b
Fig. 7.16 True papillary tissue fragments arising from LGUC. (a) This patient had a LGUC in
their ureter. The washing procedure forcibly exfoliated this papillary fragment with a fibrovascular
stalk and additionally caused the detachment of numerous neoplastic cells (seen in the back-
ground). At this magnification, the neoplastic cells appear monotonous, whereas HGUC cells often
appear more pleomorphic. Because HGUC may coexist with LGUN and because LGUN lesions
may contain foci of HGUC, examination at a higher magnification is warranted to exclude the
presence of HGUC. (Renal pelvis washing, SP, low mag.) (b) Note the monotony of the cells
attached to this papillary fragment. The cells have round-to-oval nuclei with regular contours. The
nuclei are similar in size. Neoplastic cells can also be seen singly in the background and have
eccentrically placed nuclei with low N/C ratios (below 0.5). (Renal pelvis washing, SP, medium mag)
a b
Fig. 7.17 Cercariform cells derived from LGUN. (a) In washing specimens, urothelial cells
derived from papillary neoplasms may exfoliate as single cells. These cells may have cytoplasmic
tails, resulting in a “cercariform” appearance. Here, the nuclei are monomorphic and small, sug-
gesting that the neoplasm is low-grade. No cytomorphologic features of HGUC can be identified.
(Renal pelvis washing, TP, medium mag.) (b) Cells derived from a papillary LGUC appear both
epithelioid and spindled (cercariform). The epithelioid cells have eccentrically placed nuclei with
a bland chromatin pattern and regular nuclear contours. Some cells possess degenerative features,
such as small, pyknotic nuclei. Despite their hyperchromasia, these degenerating cells and their
nuclei are notably smaller than those seen in the well-preserved neoplastic cells. (Ureteral wash-
ing, SP, high mag)
a b
Fig. 7.18 LGUN cells with plasmacytoid morphology. (a and b) These are two representative
fields from a renal pelvis washing in a patient with renal LGUC. The neoplastic cells fill the fields
with rare benign umbrella cells seen in the background. The cells are monomorphic and have bland
chromatin. Their nuclei are mostly eccentrically placed, causing a plasmacytoid appearance. Some
cells appear to have irregular nuclear shapes, irregular contours, and nuclear grooves, and many
cells have N/C ratios above 0.5. While these findings technically meet the criteria for the AUC
category, the specimen could be diagnosed as NHGUC if the overall features are more suggestive
of LGUN. (Renal pelvis washing, Cytospin, high mag)
erformance of Urinary Cytology for Upper Tract

P
Urothelial Carcinoma
Historical Performance of Urinary Cytology for UTUC
Prior to the publication of TPS in 2016, numerous studies, dating back to the 1960s,
have examined the performance of urinary cytology for the diagnosis of UTUC. Most
studies have primarily focused on the utility of selective UUT specimens (i.e., renal
pelvis and/or ureteric washings/brushings) either in isolation or in comparison with
lower urinary tract cytology specimens, as well as other diagnostic modalities.
Furthermore, some studies have examined the prognostic value of UUT cytology in
predicting disease progression or recurrence in patients who have undergone surgical
treatment for UTUC. Unfortunately, many of these studies focus on the detection of
all urothelial carcinomas regardless of grade. Because of the difficulty in detecting
LGUN in urine cytology specimens, the performance of urine cytology for detecting
all urothelial carcinomas is typically worse than the performance for detecting only
high-grade lesions [31]. The practice of intermingling both low- and high-grade
lesions in these cohorts has resulted in urine cytology being historically associated
with low sensitivities and makes the results of these studies difficult to compare
directly with those obtained from studies in the TPS era. Combined analysis of pre-
TPS studies is further complicated by inconsistencies in the interpretation of an inde-
terminate cytological diagnoses since some studies considered an “atypical” urinary
cytological specimen as a “positive” diagnosis, while others considered it to be a
“negative” diagnosis, subsequently affecting statistical analysis, including sensitivity

calculations [21, 32].
A large-scale meta-analysis study by Potretzke et al. systematically reviewed
literature published between the years 2005 and 2016 on the performance of selec-
tive UUT cytology [33]. The study found that the overall sensitivity and specificity
of UUT cytology for diagnosing UTUC was 53% (95% CI = 42.3–63.6) and 90%
(95% CI = 85.4–93.2), respectively. UUT cytology has shown comparable diagnos-
tic performance to ureteroscopic tissue biopsies, but detection rates of UTUC were
improved when these two modalities were combined, with reported sensitivities of
up to 100% [34–41]. Tumor grade and stage can both impact the sensitivity of UUT
cytology in detecting UTUC, with many studies showing a higher accuracy rate for
detecting HGUC and/or muscle-invasive tumors (pT2–pT4) compared to LGUNs
and/or non-muscle-invasive tumors (pTa-pT1) in UUT cytology [23, 27, 34–37, 42,
43]. However, at least one study found that abnormalities on UUT cytology speci-
mens were not predictive of tumor grade or stage [44].
odern Performance of Urinary Cytology for Upper Tract

M
Urothelial Carcinoma
Modern Performance of Urinary Cytology for UTHGUC
The majority of post-TPS studies on UUT specimens were conducted using retro-
spective slide review and yielded widely variable diagnostic reporting rates of each
category. Some UUT studies showed a decrease in the rate of the AUC category,
whereas others showed no significant change [21, 24, 25, 27, 45–49]. These contra-
dictory trends differ from those seen in studies examining lower urinary tract cytol-
ogy specimens prospectively diagnosed following the implementation of TPS. In
these studies, the AUC rate usually declines following implementation of TPS [50].
The reported sensitivities of UUT cytology for HGUC after implementation of TPS
are not markedly different from those reported in pre-TPS literature (19–82%)
while maintaining excellent specificity (86–100%) and positive predictive value
(92–100%) [24, 25, 45–48]. As expected, the sensitivity of UUT cytology for pre-
dicting subsequent HGUC on tissue biopsy improves when the SHGUC and/or
AUC diagnostic categories are included in the positive cohort [24, 45, 46]. McIntire
et al. reported that application of TPS made UUT cytology nearly twice as likely
(63% vs. 34%) to be concordant with follow-up histology and increased the identi-
fication of HGUC as well as LGUN compared to using pre-TPS diagnoses [47].
The reported risk of malignancy (ROM) for UTUC based on UUT cytology for
each TPS category varies among different studies. These contradictory results may
be attributed to the sparse number of UUT cytology studies, the retrospective design
of most studies, and the limited cohort sizes within each diagnostic category avail-
able for inclusion in these studies [21, 25, 46–48]. Some studies reflected an overall
shift to downgrading diagnoses and highlighted how the strict application of TPS
criteria for UUT cytology can lead to a decrease in the frequency of positive diag-
noses and a concurrent increase in SHGUC and NHGUC diagnoses. Cited pre-
sumptive reasons included lack of marked nuclear membrane irregularities and
nuclear hypochromasia rather than irregular nuclear membranes and hyperchroma-
sia, both of which have been considered essential cytological criteria for the diagno-
sis of HGUC in the prior version of TPS [16, 19, 25, 27, 45, 51].
Modern Performance of Urinary Cytology for Upper Tract LGUN
Given that the primary goal of urine cytology is the detection of HGUC, the role of
TPS is limited for the identification of LGUN, which may demonstrate nonspecific
features [31]. The overall sensitivity for LGUN is much lower than that for HGUC,
with reported sensitivities of 46% for LGUN in UUT cytology prior to TPS vs.
0–34% (average 17%) after implementation of TPS [24, 25, 45–49].
Clinical Management of Upper Tract Urothelial Carcinoma
The accurate diagnosis and staging of UTUC can be challenging due to the limita-
tions of the endoscopic instrumentation that is utilized to obtain surgical biopsy
specimens. Thus, selective upper tract cytology can have significant impact on the
treatment decision. The decision to proceed with a nephroureterectomy based solely
on a cytology result (HGUC or SHGUC) without an endoscopically or radiographi-
cally visible lesion is generally not recommended; however, these patients should
be monitored closely to identify the source for the abnormal cells and be treated
accordingly in a timely fashion.
pecial Considerations for Collection, Preparation,

S
and Adequacy
Specimen Collection
VU specimens are limited by cellular degeneration and significantly decreased abil-

ity to localize lesions within the urinary tract. Suspicious or positive findings require
further investigation by uroscopy with tissue biopsy and/or UUT cytology. While
the cytological examination of VU has a clinically meaningful role in the diagnosis
and surveillance of UCB [52], its value in the diagnosis of UTHGUC is less clear
[32]. Zhang et al. found that among patients with biopsy-confirmed UTHGUC, 81%
of preceding VU samples were diagnosed as AUC or higher, suggesting that persis-
tent AUC diagnoses on serial VU specimens without presence of a bladder lesion
should prompt bilateral UUT specimens to exclude UTHGUC (Fig. 7.19) [27].
Fig. 7.19 Indeterminate cells in a voided urine, derived from an upper tract HGUC. Upper tract
HGUC cells naturally exfoliate into the urinary stream but may have degenerated before a voided
specimen is collected. These three urothelial cells are quite large compared to the background
inflammatory cells. The nuclei have irregular contours and dark chromatin, but the chromatin pat-
tern is obliterated, likely due to degeneration. However, these were the only atypical cells seen in
the specimen and the N/C ratios are lower that 0.5; a diagnosis of SHGUC rather than HGUC may
be appropriate depending on overall quantity and quality. (Voided urine, SP, high mag)
Numerous studies have shown that UUT cytology demonstrates significantly

improved performance over VU cytology for the detection of UTHGUC, with
reported sensitivities of 33%–100% for UUT specimens versus 11%–82.7% for VU
specimens, with the caveat that these studies used varying methodologies where
some considered the atypical and/or suspicious categories as “positive.” [27, 34, 35,
53–61] [62] This observation is unsurprising considering that UUT cytology offers
targeted sampling with a higher probability of obtaining better-preserved neoplas-
tic cells.
The Role of Cell Blocks
Cell block (CB) preparations help detect tissue fragments containing fibrovascular
cores and assess cytomorphologic features, thus aiding the diagnosis of both HGUC
(Fig. 7.20) as well as LGUN (Fig. 7.21). However, the performance of this practice
has not been rigorously studied in UUT cases. Xing et al. examined 12 CBs pre-
pared from UUT specimens and showed that they could provide additional informa-
tion for identifying LGUN, particularly when a biopsy of these UUT lesions was
not feasible [25]. Chan et al. reported that CBs represent a useful adjunctive method
a b
c d
Fig. 7.20 Cell block preparation containing HGUC. (a and b) Numerous HGUC cells are found
singly as well as in a tissue fragment. Note the anisonucleosis, elevated N/C ratios, and irregular
nuclear shapes. Many of these cells have a prominent nucleolus rather than clumped chromatin.
(Ureteral washing, TP, intermediate mag.) (c and d) The cell block preparation demonstrates con-
cordant morphology. However, the fibrovascular cores of true papillary fragments can be identified
in the cell block preparation (c). (Renal pelvis washing, H&E, low mag. [c] and high mag. [d])
for classification as “LGUN” within the “Negative for HGUC (NHGUC)” TPS
diagnostic category, but their study included primarily lower urinary tract cytology
specimens [63] They reported that a CB can increase the confidence of a LGUN
diagnosis. Dantey et al. showed that routinely preparing CBs for urine samples did
not improve their laboratory’s specimen adequacy rate [64]. Nonetheless, CBs of
urine samples did help lower the frequency of atypical diagnoses, from 33% to
16–19% when routinely or selectively prepared. Overall, these studies indicate that
CB preparations may be helpful in UUT specimens, as biopsy is not always possi-
ble, and washing/barbotage specimens are generally adequately cellular for CB cre-
ation. However, more data are needed on the performance of CBs in UUT specimens,
specifically. See Chap. 10, Cytopreparatory Techniques, for further discussion.
a b
Fig. 7.21 Cell block preparation containing LGUC. (a) Given the bland cytomorphology seen in
their cellular constituents, these benign-appearing tissue fragments could represent either frag-
ments of benign urothelial lining or fragments derived from an LGUN. No features of HGUC can
be identified. (Renal pelvis washing, TP, medium mag.) (b) The cell block preparation shows a
cross section of a papillary fragment containing a fibrovascular stalk. The cells have monomorphic
nuclei, corresponding to LGUN (and, in this case, LGUC). (Renal pelvis washing, H&E,
medium mag)
Adequacy
UUT cytology specimens are usually obtained through washing/barbotage, and thus
assessment for adequacy in these specimens differs from that of VU specimens.
Studies have shown that specimen volume and obscuring factors play an important
role in the sensitivity of voided urinary cytology specimens for HGUC, whereas the
number of urothelial cells per high-power field (HPF) is more important in washing/
barbotage specimens [65–67]. The minimum urothelial cell count predictably
affects adequacy depending on specimen preparation types, with one study showing
an increased false-negative rate in ThinPrep specimens containing less than 20 uro-
thelial cells per 10 HPFs, and another study showing an increased false-negative
rate in Cytospin specimens containing less than 10 urothelial cells per 10 HPFs [67,
68]. Refer to Chap. 2 on Adequacy for suggested guidelines.
Ancillary Testing in Upper Tract Urothelial Carcinoma
The use of fluorescence in situ hybridization (FISH) analysis as an adjunct to urine

cytology (in both VU and UUT specimens) has been shown to improve the detection
of UTUC [42, 43, 69–71]. While multiple studies found that FISH analysis
(UroVysion probe set) was at least equally sensitive and specific compared to cytol-
ogy alone, the combination of urine cytology (VU or UUT specimens) and FISH
offered a superior sensitivity (86–100%) and specificity (84–98%) compared to
either modality alone [42, 43, 69]. A recent study prospectively evaluated the utility
of UroVysion in UTUC and reported that combining UUT cytology and UroVysion
can improve the diagnostic accuracy of UTUC, especially for cases diagnosed as
AUC or SHGUC [48]. Further information on ancillary studies is presented in
Chap. 9.
Examples of Reports
Upper Tract HGUC in Instrumented Specimens (with Cell Block)
Final diagnosis:
High-grade urothelial carcinoma (HGUC); see comment.
Comment: The ThinPrep slide contains rare crowded clusters of hyperchromatic

cells with increased nuclear-to-cytoplasmic ratios. The corresponding cell block
with true papillary architecture and numerous single cells with nuclear enlargement,
nuclear membrane irregularities, and hyperchromasia confirm the diagnosis.
Increased mitosis and apoptosis are noted.
Upper Tract NHGUC in Voided Urine Specimens (with Cell Block)

(Upper Tract lesion by radiology)
Final diagnosis:
• Negative for high-grade urothelial carcinoma (NHGUC).

• Low-grade urothelial neoplasia (LGUN); see comment.
Comment: The ThinPrep slide is cellular, composed of numerous three-

dimensional clusters of cells with nuclear overlapping. Some fragments are three-
dimensional papillae. The cell block highlights true papillary architecture with
definite fibrovascular cores. The papillary clusters display minimal architectural
disarray and low nuclear-to-cytoplasmic ratios. No definite features of high-grade
urothelial carcinoma are noted in this sample. In the proper clinical context, the
findings correspond to a low-grade urothelial neoplasm (LGUN).
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Non-Urothelial Malignancies and Other
Miscellaneous Lesions 8
Tarik M. Elsheikh, Rana S. Hoda, Stefan E. Pambuccian,
Jae Y. Ro, and Sun Hee Sung
• Discussion of “atypical squamous cells” and their clinical significance

• More detailed discussions of primary and secondary malignancies
• Additional photos (45), including those of Cytospin, SurePath and ThinPrep
preparations, histologic correlation, and immunohistochemistry
• Sample reports
• Additional and updated references
Introduction
Non-urothelial malignancies (NUM) are infrequent and may present as primary or

secondary disease. They represent malignancies other than high-grade urothelial
carcinomas (HGUC) and can demonstrate epithelial (i.e., squamous, glandular, neu-
roendocrine), or non-epithelial (i.e., sarcomatoid, melanocytic, hematopoietic) dif-
ferentiation. NUM frequently pose diagnostic challenges for cytologists, due to
their morphologic overlap with HGUC and its variants.
Primary epithelial NUM, including squamous cell carcinoma, adenocarcinoma,
and small-cell carcinoma, have been shown to carry an aggressive biological behav-
ior and often present at an advanced stage. Secondary involvement of the urinary
bladder by epithelial NUM occurs through metastases from distant sites or by direct
extension from adjacent organs, such as the cervix, colorectum, and prostate. Non-
epithelial NUM, including sarcoma, melanoma, and lymphoma, can involve the
urinary tract as primary or secondary disease.
The presence of atypical squamous cells in urine cytology can be clinically sig-
nificant, as they may represent the only clue to the presence of a primary or second-
ary carcinoma. Their presence should be noted in the report, and clinicians made
aware of their significance. Other miscellaneous lesions such as paraganglioma and

https://doi.org/10.1007/978-3-030-88686-8_8
144 T. M. Elsheikh et al.
nephrogenic adenoma are extremely rare, and unlikely to be encountered in urine

cytology specimens. It is important, however, not to mistake them for NUM.
The clinical history in NUM is very important, and an accurate diagnosis may
require review of clinical and imaging data, previous pathology material, and utili-
zation of ancillary studies in a subset of cases. Urine cytology can be a helpful tool
in the initial diagnosis of primary or secondary NUM, which can be critical for
appropriate management and subsequent prognostication.
Non-urothelial Malignancies
Background
NUM of the bladder are uncommon, accounting for less than 5% of all bladder
tumors, and are seldom detected by urine cytology [1, 2]. They may present as pri-
mary or secondary disease and refer to malignancies other than HGUC. NUM may
demonstrate squamous, glandular, neuroendocrine, or sarcomatoid features among
other components. Approximately 90% of all NUM are epithelial in origin, and
these include carcinomas, such as squamous cell carcinoma (SqCC), adenocarci-
noma (AdCa) and small-cell carcinoma [2]. Non-epithelial malignancies include
sarcoma, melanoma, and lymphoma, with the latter two most often metastatic to the
bladder. The pathogenesis of these cancers is incompletely understood, but several
factors are believed to be contributing, such as metaplasia, chronic inflammation,
and development from multipotent stem cells [3].
Urine cytology findings of NUM have rarely been reported, and frequently pose
diagnostic challenges to the cytologist and pathologist, due to their morphological
overlap with HGUC and its variants. In a College of American Pathologists perfor-
mance comparison study that evaluated participant responses for the reference diag-
noses of HGUC, SqCC, AdCa, and benign diagnoses (including polyomavirus
infection and ileal loop urine) [4], less than half of the participants were able to
accurately identify SqCC or AdCa; and cases recognized as “malignant” were mis-
diagnosed as HGUC by 36–48% of participants. Moreover, the cytological distinc-
tion between HGUC with divergent differentiation from pure non-HGUC may not
be possible in cytological samples or in small biopsy specimens and frequently
requires surgical resection of the tumor with extensive histologic sampling.
Primary NUM usually pursue an aggressive clinical course and often present at
an advanced stage of disease. A multimodal approach using clinical details, imaging
results, and pathological diagnosis is vital for a prompt management decision and
early therapeutic intervention. The overall survival remains poor and, apart from
SqCC, shows similar outcomes to HGUC when controlled for gender, stage, and
grade of malignancy [5, 6]. A recent analysis of 222,435 bladder cancer cases from
the Surveillance, Epidemiology, and End Results registry (SEER, 2004–2016) con-
cluded that primary SqCC had worse survival across all stages, even after multivari-
ate adjustment and matching with urothelial bladder cancers. AdCa, neuroendocrine
carcinomas, and other non-urothelial carcinomas presented at higher stage than UC
8 Non-Urothelial Malignancies and Other Miscellaneous Lesions 145
and had worse outcomes than UC when they were organ-confined, but not when
they presented at an advanced stage [7]. It remains unclear, however, whether the
prognosis of NUM is worse than invasive HGUC, based on histologic type alone
[8]. A multi-institutional study of 1131 consecutive patients (1042 HGUC and 89
NUM) reported no differences in 5-year survival following radical cystectomy with
lymph node dissection [6]; multivariate analysis demonstrated that gender, clinical
stage, pathological stage, lymph node involvement, and lymph node dissection were
the independent predictive factors for survival, whereas histological type and grade
had no significant impact on survival.
Secondary tumors of the urinary bladder, upper urinary tract, and urethra repre-
sent rare events and are encountered more commonly in autopsies than in clinical
surgical pathology or cytopathology specimens. Secondary malignancies involving
the urinary tract may result in shedding of malignant cells in the urine by direct
invasion from malignancies arising in adjacent organs (anorectum; cervix and endo-
metrium in women; and prostate in men); transcoelomic spread through the perito-
neum in gastric and ovarian malignancies; lymphatic and hematogenous spread
from breast carcinomas, pulmonary carcinomas, and melanomas; and rarely from
various other malignancies. A multi-institutional study of 1042 UC and 89 NUM
reported no difference in 5-year survival following radical cystectomy [6].
Awareness of the patient’s prior history and comparison and correlation of pathol-
ogy material from prior malignancies is necessary to diagnose metastasis to the
urinary bladder and exclude the possibility of a new primary [9].
Liquid-based preparations (LBPs), such as ThinPrep (TP) (Hologic, Marlborough,
MA) and SurePath (SP) (BD-TriPath Imaging, Burlington, NC), have been increas-
ingly used for processing urinary cytology specimens. Several studies comparing
the older conventional sediment preparations, and the Cytospin (CS) centrifugal
method, to LBPs have shown no significant differences in the sensitivity of detect-
ing malignancies, especially AdCa and SqCC [10–13]. LBPs, however, are associ-
ated with certain subtle artifacts and cytomorphologic changes that are different
from CS, and need to be recognized by cytologists [14] (further discussion is pro-
vided in Chap. 10, Cytopreparatory Techniques). Generally, LBPs provide more
uniform distribution of cells with less cellular overlapping and a cleaner back-
ground, i.e., reduced blood and inflammatory debris, while CS tends to show more
clumped clusters with better preservation of architecture [13]. There is significant
shrinkage of cells associated with LBP, resulting in smaller sized cells compared to
CS. Malignant nuclear features are comparable, including nuclear irregularity and
high N/C ratio, but there appears to be a greater degree of nuclear hyperchromasia
associated with LBP.
Finally, ancillary studies such as immunohistochemistry (IHC) have a limited
role in urine cytology, as most cases are not of sufficient cellularity to allow for cell
block preparation. However, IHC performed on cell block or cytologic preparations
from specimens with adequate cellularity may be helpful in subclassifying or con-
firming NUM in a subset of cases.
Primary Epithelial Malignancies
Primary Squamous Cell Carcinoma
Background
Primary SqCC is the second most common malignant neoplasm of the urinary tract,
accounting for 2–5% of bladder cancers in the Western world [15]. Until recently, it
predominated in some sub-Saharan African countries (western, eastern, and south-
eastern) and Egypt, where Schistosoma haematobium infection was a major risk
factor [16]. SqCC associated with Schistosoma infection was known as bilharzial
SqCC (B-SqCC). The prevalence of B-SqCC appears to be decreasing in recent
years in Egypt and other countries, parallel with the continuing efforts to eradicate
Schistosoma infections [17]. Non-bilharzial SqCC (NB-SqCC), which is found in
areas without Schistosoma infection, has a different epidemiology, risk factor asso-
ciation, pathogenesis, and clinical behavior. In the USA, NB-SCC is more common
in the African American population and has more of a male to female preponder-
ance (4:1) than HGUC (3:1) [18]. In contrast to B-SqCC, which is a disease of
younger age, NB-SqCC occurs more commonly in older individuals in the seventh
decade of life (similar to HGUC of the bladder) [16]. NB-SqCC is associated with
keratinizing squamous metaplasia and leukoplakia secondary to recurrent chronic
infection, neurogenic bladder dysfunction, prolonged irritation from indwelling
catheters, foreign bodies, and stones. Other etiologic factors such as tobacco smok-
ing, cyclophosphamide therapy, and human papilloma virus infection have been
investigated but shown no definitive relationships [19].
Similar to HGUC, primary pure SqCC of the urinary tract most commonly pres-
ents with hematuria, dysuria and irritative symptoms. Most of these cancers are
moderately to poorly differentiated and present at an advanced stage with bladder
muscularis propria invasion. Although pathologic stage is highly correlated with
prognosis, B-SqCC appears to have a better prognosis than NB-SqCC [16].
Definition
Primary SqCC of the urinary tract is a malignant neoplasm that shows exclusively
squamous differentiation, without associated urothelial or glandular elements.
Therefore, the diagnosis of primary pure SqCC should be based on extensive biopsy
sampling or a resection specimen to exclude any squamous differentiation in asso-
ciation with invasive and/or in situ HGUC components (Fig. 8.1).
a b
Fig. 8.1 Primary pure squamous cell carcinoma (SqCC) of the urinary bladder. (a) Resection
specimen shows a muscle invasive carcinoma composed entirely of squamous components. (b)
Another case of SqCC (upper part of photo) is associated with calcified Schistosoma haematobium
eggs deposited in the stroma (lower part of photo). (H&E, medium mag.)
Cytologic Criteria
• Cellular specimen with numerous highly atypical squamous cells occurring as

isolated cells and/or in clusters (Fig. 8.2).
• Tumor cells are large, polygonal with keratinized/orangophilic cytoplasm, sharp
borders, and severely atypical hyperchromatic nuclei (Fig. 8.3). Fiber and tad-
pole cells, squamous pearls, and “cell in cell” arrangements may be present,
most often in tissue fragments.
• Groups or single non-keratinizing malignant squamous cells may be infrequent
or predominate in a specimen. Prominent nucleoli are often observed (Figs. 8.4
and 8.5).
• Background may show plaques and fragments of anucleated squamous cells
(“ghost cells”), small atypical parakeratotic cells, necrosis, red blood cells, and
neutrophils (Fig. 8.6).
a b
Fig. 8.2 SqCC of the urinary bladder. (a) Cellular specimen displays tightly cohesive clusters of
atypical keratinized squamous cells. (Voided urine, TP, medium mag.) (b) Loosely cohesive clus-
ters consist of highly atypical keratinized and non-keratinized squamous cells. (Instrumented
urine, TP, medium mag.)
a b
Fig. 8.3 SqCC of the urinary bladder. (a) Predominately single highly atypical keratinized squa-
mous cells are large, polygonal with high N/C ratio, sharp cytoplasmic borders, and hyperchro-
matic nuclei. (Voided urine, TP, high mag.) (b) Elongated keratinized “fiber” and “tadpole cells”
may also be present, as well as inflammatory cells and some debris in the background. (Bladder
Fig. 8.4 Primary SqCC of the urinary bladder. Tightly cohesive three-dimensional clusters of
non-keratinized squamous cells. The tumor cells show nuclear enlargement, high N/C ratio,
marked overlapping, and severe hyperchromasia. Occasional nucleoli are observed. In the absence
of a keratinizing component, it is difficult to distinguish this morphology from HGUC or AdCa.
(Bladder washing, CS, high mag.)
a b
Fig. 8.5 SqCC of the urinary bladder. Non-keratinized atypical squamous cells may be arranged
in loose clusters (a) and as single cells (b). The tumor cells have a metaplastic appearance with
rigid basophilic cytoplasm and nuclear elongation with marked hyperchromasia. In the absence of
keratinization, it is difficult to distinguish HGUC from non-keratinizing SqCC of the bladder.
(Instrumented urine, TP, high mag.)
Fig. 8.6 SqCC of urinary

bladder. A background of
necrotic debris and
inflammatory cells
surrounds rare atypical
keratinizing and non-
keratinizing squamous
cells. (Bladder washing,
TP, low mag.)
Explanatory Notes
Explanatory Note 1 Differential diagnosis of highly atypical or malignant keratin-

ized squamous cells includes primary or metastatic SqCC and HGUC with diver-
gent squamous differentiation (Fig. 8.7).
Explanatory Note 2 In absence of a known previous primary, HGUC with squa-

mous differentiation must be excluded first. The latter is the most common type of
divergent differentiation and is seen in up to 40% of invasive HGUC. Histologically,
the neoplasm is classified as “HGUC with squamous differentiation” in the pres-
ence of any HGUC component, invasive or in situ.
Explanatory Note 3 Non-keratinizing SqCC may be impossible to distinguish

cytologically from HGUC, especially if the malignant cells are mostly discohesive
(Fig. 8.5).
Explanatory Note 4 Any significant degree of atypia in squamous cells must be

interpreted with caution, as it may represent SqCC [17] (Fig. 8.8).
Explanatory Note 5 Mildly to markedly atypical keratinized squamous cells are

characterized by nuclear enlargement and hyperchromasia or prominent nuclear
membrane irregularity [18]. Differential diagnosis includes a variety of benign, dys-
plastic and malignant lesions, as well as direct extension or contamination from the
lower female genital tract or external male genitalia (see below discussion of atypi-
cal squamous cells).
Fig. 8.7 HGUC with

squamous differentiation.
Only highly atypical
keratinized squamous cells
are present in this urine.
Histologic follow-up (not
shown) revealed divergent
squamous differentiation
associated with invasive
HGUC. (Voided urine, TP,
high mag.)
Fig. 8.8 Atypical squamous cells (ASC). This specimen obtained from a 57-year-old female con-
tains rare clusters of keratinized squamous cells with significant atypia. A diagnosis of “ASC,
suspicious for squamous carcinoma” was rendered. Follow-up transurethral resection (TUR)
revealed Schistosoma-associated SqCC of the bladder. (Instrumented urine, TP, high mag.)
Atypical Squamous Cells
Background
The presence of atypical squamous cells (ASC) in urine cytology is a rare finding
and is seen in approximately 0.3–0.9% of urine specimens [20, 21]. There is a pau-
city of literature regarding the clinical significance of ASC, but what exists suggests
that any degree of squamous atypia should not be discounted. Bladder surface
mucosa frequently shows keratinizing squamous metaplasia with or without squa-
mous dysplasia. The majority of ASC are not associated with malignancy and rep-
resent reactive/inflammatory changes, most commonly due to vaginal contamination
or exfoliation from distal urethra. However, ASC may be associated with bladder or
cervical cancers in up to 20–30% of cases [20]. Association with carcinoma is even
higher in the presence of severe squamous atypia that is suspicious for carcinoma
[21]. In women, cystoscopy and biopsy and/or colposcopy may be needed to deter-
mine a bladder or lower female genital tract origin of the squamous atypia.
A diagnosis of ASC should be rendered in the presence of significant atypia that falls
short of a definitive diagnosis of malignancy, i.e., significant atypia with insufficient cel-
lularity or equivocal malignant cytologic features. There appears to be no substantial
association between the number of ASC and histological diagnosis, age, sex, or speci-
men type. However, the presence of highly suspicious cytology, i.e., extreme squamous
atypia and pleomorphism, can be associated with cancer in up to 60% of cases [18, 19].
Malignant conditions associated with ASC include primary bladder SqCC, cervical
SqCC, and HGUC with squamous differentiation. Occasionally isolated ASC may be
the only cytologic clue to the presence of an undetected or unsampled HGUC [22].
ASC with cytologic features of low-grade squamous intraepithelial lesion
(LSIL), including koilocytosis/HPV changes, most commonly originate from the
female genital tract and urethra in men [23]. There are no well-defined guidelines
regarding the management of these patients, but patients with persistent abnormali-
ties and/or immunosuppression should be closely followed. In women, LSIL in
urine cytology is highly associated with HPV infection in the genital tract; there-
fore, these findings should be correlated with a corresponding Pap test. Men, with
LSIL in their urine, should be considered for genitourinary examination to evaluate
for genital warts [23]. Benign conditions associated with ASC include squamous
metaplasia of bladder mucosa, radiation therapy effect, and contamination from
distal urethral or genital tract mucosa. Atypical parakeratosis (dyskeratosis) may be
associated with condyloma of bladder or urethra, squamous papilloma, dysplasia,
and bladder irritation or may represent a genital contaminant [24].
More recently, HPV testing using noninvasive techniques such as self-collected
urine and cervicovaginal samplings has been investigated as a possible alternative to
clinician-collected cervicovaginal samples and as means of increasing participation
in screening programs. In countries without well-developed cervical cancer screen-
ing programs, participation rate is low and up to 50–80% of women are not screened
[25]. Noninvasive urine-based high-risk HPV (hrHPV) testing can be an attractive
option for women who are reluctant to undergo gynecologic examination, as self-
collected samples seem to be preferred over clinician-collected ones [26].
Furthermore, urine samples could potentially be collected at home and sent by mail
to the laboratory for hrHPV testing, removing the need for an initial clinic-based
pelvic exam. Whereas most studies have shown that self-collected vaginal samples
appear to have similar sensitivity to clinician-collected cervical samples, results of
urine HPV testing have been variable and inconsistent [27]. Reported sensitivities
of urine hrHPV testing for detection of HGSIL+ ranged from 45% to 80–100%, and
specificities ranged from 25 to 53% among patients referred for colposcopy, which
are significantly lower than self-collected and provider-collected cervicovaginal
samples (94% sensitivity) [28]. Although, based on available literature, urine HPV
testing appears to be a promising secondary option to cervicovaginal sampling, fur-
ther studies are needed to evaluate and improve the accuracy of urine hrHPV testing
for detection of HGSIL+, especially in a primary screening population and/or in
women with limited access to health care.
Definition
Keratinized squamous cells with large and hyperchromatic nuclei, high N/C ratio,
abnormal nuclear or cytoplasmic shapes, and densely orangophilic cytoplasm. The
atypical cells may occur as groups or isolated cells but lack qualitative and/or quan-
titative unequivocal criteria of malignancy.
Explanatory Notes
Explanatory Note 1 ASC are defined as atypical keratinized cells, since cytologic
distinction between non-keratinized SqCC and HGUC is often difficult and some-
times impossible (Figs. 8.8 and 8.9).
Explanatory Note 2 The presence of ASC should be noted in the report and clini-
cians made aware of their significance. They are particularly clinically significant if
they persist in subsequent specimens.
Explanatory Note 3 The presence of mild to moderate keratinizing squamous

atypia should be reported as ASC (Fig. 8.10). However, marked atypia that is highly
suspicious for carcinoma should be diagnosed as “ASC, suspicious for SqCC or
squamous differentiation in HGUC,” because of the high association with malig-
nancy (Figs. 8.7–8.9).
Explanatory Note 4 Atypia consistent with LSIL, including koilocytosis/HPV

changes, most commonly originate from the female genital tract and urethra in men,
including cervical LSIL and urethral condyloma, respectively (Fig. 8.11).
Fig. 8.9 Atypical squamous cells (ASC), suspicious for squamous cell carcinoma (SqCC). This
patient was recently diagnosed with SqCC of the penile meatal opening. The specimen shows
singly scattered atypical non-keratinized squamous cells with high N/C ratio and marked hyper-
chromasia. Absent the history, it would be impossible to distinguish these atypical non-keratinized
squamous cells from HGUC. (Voided urine, TP, high mag.)
a b
Fig. 8.10 Atypical squamous cells (ASC). (a) Atypical keratinized and non-keratinized cells have
moderate atypia, and increased N/C ratios. The nuclei are enlarged and hyperchromatic, but have
a smudgy, degenerated appearance. (Bladder washing, TP, medium mag.) (b) Follow-up resection
revealed a noninvasive papillary HGUC with squamous differentiation. Inset shows surface kera-
tinized squamous cells, identical to those observed in the cytology specimen. (H&E, low mag.;
inset, high mag)
a b
Fig. 8.11 Atypical squamous cells (ASC). (a) Rare keratinized ASC with mild to moderate atypia
is present in this specimen from a 50-year-old female. (Voided urine, TP, high mag.). (b) Follow-up
Pap test reveals low-grade squamous intraepithelial lesion and positivity for high-risk HPV. (Pap
test, TP, medium mag.). ASC in this urine specimen represented contamination from the geni-
tal tract
Primary Adenocarcinoma
Background
AdCa may be primary or metastatic. Primary AdCa is rare, accounting for 0.5–2%
of all bladder malignancies, whereas HGUC with glandular differentiation is more
prevalent [29]. Histologically, primary AdCa has a pure glandular phenotype and
includes bladder AdCa and urachal AdCa, which are indistinguishable cytohisto-
logically. Urachal AdCa, however, tends to be located in the bladder dome and/or
anterior wall with an epicenter in the bladder wall, no association with surface intes-
tinal metaplasia, and typically affects a younger patient population [15, 30]. Primary
AdCa is similar to HGUC in age and gender distribution, with peak incidence in
sixth decade, and is more common in males [31]. Clinical features at presentation
include hematuria, dysuria, urinary frequency, and rarely mucosuria. Risk factors
for primary AdCa include intestinal metaplasia, particularly in patients with bladder
exstrophy, schistosomiasis, diverticula, and chronic irritation and obstruction [15,
30, 32]. Prognosis is best predicted by pathological stage but overall tends to
be poor.
Primary AdCa is histologically subclassified as enteric, mucinous, mixed enteric-
mucinous, and AdCa, not otherwise specified (AdCa-NOS); mixed enteric-mucinous
is the most common subtype. Enteric AdCa is morphologically identical to its intes-
tinal counterpart, while mucinous AdCa shows nests of tumor cells associated with
abundant mucin and may show variable signet ring cell morphology. AdCa-NOS
terminology is used when morphology does not resemble a specific subtype [31,
33]. Clear cell carcinoma is a rare distinctive variant of AdCa that is seen more com-
monly in females and in urethra more than bladder [24]. Divergent glandular dif-
ferentiation has been reported in up to 18% of invasive HGUC, so it should be
excluded before establishing a diagnosis of pure AdCa [34]. Secondary involvement
of the bladder and upper urinary tract by AdCa is more common than primary uri-
nary tract adenocarcinomas and may occur by direct extension from adjacent organs
or metastasis from distant sites (see later discussion of secondary epithelial malig-
nancies). Cytologic examination alone cannot distinguish primary from secondary
AdCa; therefore, clinical and pathologic correlation is warranted.
Definition
Primary AdCa of the urinary bladder is characterized by pure glandular

differentiation.
Criteria
• Variable cellularity.
• Enteric AdCa shows similar morphology to colonic AdCa (Fig. 8.12), including
columnar cell clusters and single degenerated cells in a background of necrosis
and mucin. Nuclei are large and elongated, vesicular or hyperchromatic, with
irregular shapes, and visible or prominent nucleoli. The cytoplasm may be vacu-
olated (Fig. 8.13a).
• Mucinous AdCa shows rounded three-dimensional clusters of crowded cells,
with variable atypia, small to moderate amount of delicate cytoplasm, occasional
cytoplasmic vacuoles, and medium-sized nuclei with distinct nucleoli. Mucin is
present in the background. Signet ring cells may be present, displaying large
cytoplasmic mucin-containing vacuoles that displace and push crescent-shaped
hyperchromatic nuclei to the periphery of the cells. Signet ring cells may also be
associated with enteric AdCa or AdCa-NOS (Fig. 8.13b).
a b
Fig. 8.12 Primary enteric adenocarcinoma (AdCa) of the urinary bladder. (a) Cytology demon-
strates a cluster of cells with enlarged elongated palisading nuclei, hyperchromasia, irregular
nuclear membranes, and prominent nucleoli. (Voided urine, TP, high mag.). (b) Resected bladder
shows AdCa exhibiting similar morphology to its colorectal counterpart. (H&E, medium mag.)
a b
Fig. 8.13 Primary enteric AdCa of the urinary bladder. In this case, the malignant cells have more
voluminous cytoplasm with prominent vacuolization, but characteristic palisading morphology is
not demonstrated. (a) There is elongation of the nuclei with significant pleomorphism. (b) Some
of the cells display signet ring appearance, created by a large cytoplasmic vacuole pushing the
crescent-shaped hyperchromatic nucleus to the periphery of the cell. (a, b: Catheterized urine, CS,
high mag.). Although histologic resection demonstrated enteric AdCa, cytologic features in this
urinary cytology do not allow for determination of subtype of AdCa
• Clear cell AdCa presents in clusters and has large cells with abundant hypervacu-
olated cytoplasm, centrally located nuclei, prominent nucleoli, and occasional
hobnail configurations (Fig. 8.14).
• In most instances, AdCa does not show distinctive cytomorphologic features to
allow for determination of the subtype. These cases are best reported as AdCa-
NOS, with appropriate differential diagnosis listed in an accompanying com-
ment (Figs. 8.13 and 8.15).
a b
Fig. 8.14 Clear cell AdCa of the urethra. (a) Specimens are usually of high cellularity with cohe-
sive clusters of malignant cells. The tumor cells have abundant vacuolated and/or eosinophilic
cytoplasm, centrally placed nuclei, and prominent nucleoli. (Bladder washing, TP, high mag.). (b)
Resection revealed clear cell AdCa of the urethra invading the bladder. The tumor had a tubulo-
papillary architecture, lined by highly pleomorphic cells with abundant eosinophilic or clear cyto-
plasm, and frequent hobnailing. (H&E, high mag.)
Fig. 8.15 Primary AdCa,

not otherwise specified
(AdCa-NOS) displays a
cluster of cells with
eccentrically placed
irregular nuclei, prominent
nucleoli, and finely
vacuolated cytoplasm.
Although the cytology of
the malignant cells is
consistent with AdCa,
HGUC with glandular
differentiation and
secondary AdCa should
also be considered in the
differential diagnosis.
(Voided urine, TP, high
mag.)
Explanatory Notes
• Explanatory Note 1: The diagnosis of AdCa in urine cytology can be challeng-

ing, and such a diagnosis should only be rendered in the presence of adequate
cellularity and significant atypia. In addition, the cytomorphology of AdCa may
differ according to the subtype of AdCa, as discussed above [23, 25, 26].
• Explanatory Note 2: UC with glandular differentiation is more common than
pure AdCa and, therefore, should be included in the differential diagnosis of
AdCa. Such a distinction, however, may not be possible by cytologic examina-
tion alone and often requires histologic confirmation.
• Explanatory Note 3: Differential diagnosis of low-grade AdCa includes glandu-
lar cells from a vaginal or intestinal tract fistula, cystitis glandularis, intestinal
metaplasia, nephrogenic metaplasia/adenoma, Mullerian lesions (endometriosis
and endocervicosis), and reactive/degenerative tubular cells [15, 24, 35]. In the
absence of unequivocal malignant cytologic features or limited cellularity, it is
best to report those findings as “atypical glandular cells” or “suspicious for ade-
nocarcinoma,” depending on the degree of atypia (Fig. 8.16). Histologic sam-
pling can be recommended in such instances to help arrive at a more definitive
diagnosis, recognizing that cytology has alerted clinicians to the possibility of a
more significant lesion.
• Explanatory Note 4: In some instances it may be difficult to distinguish AdCa
from HGUC [24, 36]. The malignant cells may lack columnar appearance,
nuclear palisading, and cytoplasmic vacuolization characteristic of AdCa
(Fig. 8.17).
• Explanatory Note 5: There is significant overlap in cytomorphologic and immu-
nohistochemistry (IHC) profiles of primary and secondary AdCa, especially
those of colorectal origin. Clinical and imaging correlations are necessary to
exclude secondary/metastatic AdCa. IHC has a limited role in this distinction but
can be of value in occasional cases (see below).
• Explanatory Note 6: Cytology of enteric AdCa is similar to that of colorectal
carcinoma. Also, IHC profile of enteric AdCa overlaps with colorectal AdCa, as
both cancers express CK20, CDX2, and SATB2. However, positive nuclear beta-
catenin staining favors colorectal carcinoma (80% of cases), as opposed to mem-
branous staining in primary bladder AdCa [29–31].
• Explanatory Note 7: Plasmacytoid carcinoma is a rare variant of UC, character-
ized by cells that resemble plasma cells. The malignant cells invade the bladder
wall in a discohesive pattern and have clear or eosinophilic cytoplasm and eccen-
trically placed, enlarged hyperchromatic nuclei [37]. Some of the malignant cells
may contain cytoplasmic vacuoles imparting a signet ring-like appearance and
hence can be confused with AdCa containing signet ring cells. As a matter of
fact, most cases previously reported in the literature as signet ring cell AdCa of
bladder would now be classified as plasmacytoid UC [34]. Therefore, the pres-
ence of signet ring cells in urine cytology should raise the differential diagnosis
of primary carcinomas such as mucinous AdCa with signet ring features, HGUC
with divergent glandular differentiation, plasmacytoid UC, and metastatic gas-
trointestinal AdCa or breast lobular adenocarcinoma [24]. CK7, CK20, GATA3

and uroplakin II, and, to a lesser degree, p63 are expressed in most plasmacytoid
UCs, which may help to distinguish them from metastatic carcinomas [38, 39].
Other non-epithelial malignancies that may have a plasmacytoid morphology
include melanoma, lymphoma, and plasmacytoma.
• Explanatory Note 8: Clear cell carcinoma is a rare variant of primary AdCa but
may also be admixed with HGUC or AdCa-NOS (Fig. 8.14). Major differential
diagnosis is renal cell carcinomas and nephrogenic adenoma [30, 32, 35].
a b
Fig. 8.16 Atypical glandular cells (AGC). (a) This urine specimen from a 69-year-old male con-
tains rare clusters of glandular cells with mild atypia, including slightly enlarged palisaded nuclei,
and nuclear hyperchromasia. Inflammatory cells are present in the background. A diagnosis of
AGC was rendered in this case, with a comment/note stating that the differential diagnosis included
benign nonneoplastic conditions versus low-grade AdCa. Follow-up (not shown) revealed florid
cystitis glandularis. (Instrumented urine, CS, high mag.). (b) This specimen, from a 73-year-old
female, contains a single cluster of highly atypical glandular cells. No history was available at the
time of evaluation of the cytology specimen. A diagnosis of “AGC, suspicious for AdCa” was
rendered, with appropriate differential diagnosis listed. Follow-up revealed that the patient had
stage 4 ovarian serous carcinoma; therefore, this most likely represented secondary involvement of
the urinary tract. (Voided urine, TP, high mag.)
a b
Fig. 8.17 AdCa-NOS. (a) This urine from a 67-year-old female contains single and loosely cohe-
sive atypical cells with enlarged hyperchromatic nuclei, high N/C ratio, and marked pleomor-
phism. The case was signed out as “HGUC.” (Voided urine, CS, high mag.). (b) Follow-up
cystectomy revealed primary AdCa of the urinary bladder, enteric type. (H&E, medium mag.). In
some instances, the AdCa cells tend to round up in cytologic specimens, and it may be extremely
difficult to distinguish them from HGUC. Therefore, rendering HGUC diagnosis was not unrea-
sonable in this case, since it resulted in further workup of the patient
Neuroendocrine Tumors
Background
Neuroendocrine tumors are a diverse group of bladder tumors that include well-
differentiated neuroendocrine tumors (WDNET), neuroendocrine carcinomas
(NEC), and paraganglioma. WDNET has been previously referred to as carcinoid,
but that terminology is currently not recommended [40]. NECs are rare and consist
of small-cell carcinoma (SmCC) and large-cell NEC (LCNEC). SmCC is the most
common of these cancers and accounts for less than 1% of malignant bladder
tumors. In addition to bladder, NEC can involve the kidney and prostate and are
morphologically, immunohistochemically, and ultrastructurally similar to their pul-
monary counterparts. NEC can also involve the bladder secondarily. Only a few
cases of LCNEC and WDNET of bladder have been reported [40].
SmCC is often associated with HGUC and other divergent histologic subtypes,
suggesting a urothelial origin. In over half of the cases, there is association with
other carcinomatous components, such as in situ or invasive HGUC, SqCC, AdCa,
sarcomatoid carcinoma, or mixtures of these components [40, 41]. This supports the
most widely accepted view that NEC of the bladder originates from a pluripotential
stem cell that can differentiate into various cell types, suggesting a common clonal
origin of the neuroendocrine, urothelial, squamous, and AdCa components. Like
pulmonary SmCC, it is closely associated with tobacco smoking and tends to pursue
an aggressive course and present at an advanced stage. Differential diagnosis
includes metastatic SmCC, which is very rare, lymphoma, and HGUC. IHC profile
commonly includes the expression of chromogranin, synaptophysin, and CD56,
while TTF1 is expressed in about 50% of cases. As opposed to high-grade UC,
SmCC shows variable GATA3 expression and is negative for p63 and CK20 [40].
LCNEC and WDNET are extremely rare and unlikely to be encountered in urine
cytology. LCNEC of the bladder is a high-grade malignancy that exhibits neuroen-
docrine features by light microscopy and increased mitotic activity and expresses
neuroendocrine markers. Only a few cases have been reported in the surgical pathol-
ogy literature, and some of these were associated with HGUC [42]. Most cases have
followed an aggressive course. Differential diagnosis includes HGUC, SqCC, and
AdCa-NOS. WDNETs of the urinary bladder are exceedingly rare with few docu-
mented cases reported in the surgical pathology literature. Age range is similar to
HGUC, and the most common presenting symptom is hematuria. Morphologically
it is similar to its counterparts in other sites and consists of uniform cells with round
nuclei that may be eccentrically placed, salt and pepper chromatin, and minimal to
moderate amount of cytoplasm. WDNET can be confused with well-differentiated
AdCa or nonneoplastic lesions. Other differential diagnostic considerations include
paraganglioma, HGUC, and lymphoma [43]. The tumor cells of WDNET show
positive staining for neuroendocrine markers and occasionally PSA but no other
prostatic markers [40]. Paraganglioma is discussed later with “other miscellaneous
lesions.”
Definition of SmCC
SmCC is a high-grade malignant neoplasm with neuroendocrine differentiation. It

is positive for neuroendocrine markers such as synaptophysin and chromogranin
and may be positive for TTF1. SmCC may be mixed with variable components of
HGUC or other divergent histologies.
Criteria
• Cellularity is usually moderate to high.

• Cells may be arranged singly, or in loosely or tightly cohesive clusters (Fig. 8.18).
Occasionally, there may be an appreciation of a linear pattern or rosette formation.
a b
Fig. 8.18 Primary small-cell carcinoma (SmCC) of the urinary bladder. (a) Urine cytology speci-
men is highly cellular and shows tightly cohesive clusters of small, individual cells. The tumor
cells have minimal amount of cytoplasm, round to oval nuclei, and salt and pepper chromatin pat-
tern. Nucleoli are absent or inconspicuous. Individual cell necrosis and apoptosis are also noticed.
Background is bloody with necrotic debris. (Voided urine, CS, medium mag.). (b) Resection speci-
men revealed SmCC with histologic appearance similar to its pulmonary counterpart. The tumor
cells were positive for neuroendocrine markers (not shown) and had no associated divergent his-
tologies. (H&E, high mag.)
• Tumor cells are small to medium in size (two to three times the size of lympho-
cytes or red blood cells), with scanty cytoplasm and high N/C ratio (Fig. 8.19).
• Nuclei are round to oval, hyperchromatic with finely granular evenly distributed
or smudged chromatin, ill-defined membranes, and focal to prominent nuclear
molding. Nucleoli are absent or inconspicuous (Fig. 8.19).
• In LBPs, compared to conventional preparations, tumor cells are often smaller
and rounder, and crush artifact or molding may be less apparent [34] (Fig. 8.20).
• Single-cell necrosis (apoptosis) is present and may be extensive.
• Background may be hemorrhagic and necrotic (Fig. 8.18).
a b
Fig. 8.19 SmCC of the bladder. (a) Cytology displays cells arranged in a loosely cohesive cluster.
Tumor cells are small, show high N/C ratio, round to oval hyperchromatic nuclei with evenly dis-
tributed fine to coarse chromatin, and absent or inconspicuous nucleoli. Nuclear overlapping and
spindling are more pronounced than molding, and the background is clean, compared to Cytospin
preparations (see Fig. 8.18a). (Voided urine, TP, high mag.). (b) In this sample, the cytologic fea-
tures are similar to “a,” but nuclear molding and linear arrangement of the malignant cells are more
pronounced. (Bladder washing, SurePath, high mag.)
Explanatory Notes
Explanatory Note 1 Urine cytology has a low sensitivity and specificity for detect-
ing SmCC, particularly when cellularity is sparse or in cases intermixed with
HGUC. The diagnosis of SmCC, however, can be suggested or made when classic
cytomorphology is appreciated, which is similar to the exfoliative cytology of
SmCC from other sites [10, 41, 44, 45].
Explanatory Note 2 IHC can be confirmatory in a subset of SmCC cases with

adequate cellularity. Tumor cells are usually positive for chromogranin and synap-
tophysin, and TTF1 may be positive (Fig. 8.20).
Explanatory Note 3 The cytological differential diagnosis includes metastatic

SmCC, SqCC with basaloid features, LCNEC, HGUC, lymphoma, melanoma, and
other small-cell malignancies (Fig. 8.21). It is also important to consider benign
conditions in the differential diagnosis, such as inflammatory infiltrate, follicular
cystitis, and BCG-associated changes [42, 46].
Explanatory Note 4 Timely and accurate cytological diagnosis of SmCC is impor-

tant to ensure a prompt clinical workup for these highly aggressive tumors [47].
a b
Fig. 8.20 Primary SmCC. (a) Urine cytology from a 76-year-old man shows a predominantly
discohesive cell pattern. The tumor cells are small (approximately two to three times the size of
RBCs or neutrophils) and have scant cytoplasm. The nuclei are round and have finely to coarsely
granular chromatin. Single-cell necrosis is present. No nuclear molding or spindling is appreci-
ated. This morphology also raises the differential diagnosis of lymphoma, melanoma, and
HGUC. (Voided urine, TP, high mag.). (b) Immunohistochemistry performed on TP slides demon-
strates positive staining with synaptophysin and chromogranin. (Synaptophysin, high mag.)
Fig. 8.21 Non-keratinizing SqCC. Voided urine from a 48-year-old man with locally advanced
anal squamous cell carcinoma. The cytology consists of tightly cohesive clusters of basaloid tumor
cells with overlapping round to oval, hyperchromatic and irregular nuclei, and ill-defined cyto-
plasm. This cytologic appearance can be confused with SmCC, especially in the absence of clinical
history or previous pathology for comparison. Features favoring SqCC over SmCC are the open
chromatin pattern of nuclei and presence of conspicuous nucleoli. (Voided urine, CS, high mag.)
Secondary Epithelial Malignancies
Background
Secondary malignancies rarely involve the urinary tract, often presenting as a soli-
tary mass and accounting for approximately 2% of surgically resected bladder
tumors. Most of those malignancies (approximately 70%) represent direct extension
from adjacent organs, such as colorectum, prostate, and female genital tract [48]. A
smaller proportion of secondary tumors (<30%) represent metastases from distant
sites, via lympho-hematogenous spread [49]. The site of the tumor within the bladder
may suggest its origin, for example, cancers of prostate and cervix tend to invade the
bladder neck and trigone, while colorectal cancers more commonly involve the fun-
dus [50]. Distant metastases to the bladder are typically a late manifestation of dis-
seminated disease and more commonly occur with cancers of the breast, stomach,
and lung. Lymphoma and melanoma can also involve the bladder secondarily and are
discussed later with “non-epithelial malignancies.” Other reported primary sites of
origin include the kidney, pancreas, and ovary [49]. The urothelium is usually spared
in metastatic disease, which provides an important clue to the histologic diagnosis,
but may also explain the rarity of exfoliated malignant cells in urine cytology [51].
Whereas the cytohistologic appearance of secondary malignancies may be distinc-
tive, many extravesical cancers can mimic primary urothelial or non-urothelial malig-
nancies. This is especially problematic with AdCa, which represents over 50% of all
secondary bladder malignancies [48]. The rarity of secondary carcinomas and overlap
of their cytomorphologic features with those of primary carcinomas make an accurate
diagnosis of metastasis challenging. In a 10-year retrospective review of all urine
cytologies from biopsy-proven NUM patients, 25 urine samples from 14 patients
showed metastases, accounting for <1% of all urine specimens. Most cases were rec-
ognized as being atypical or malignant but were frequently mistaken for a urothelial
origin [36]. Although the morphologic overlap in some cases may not allow for distin-
guishing NUM from HGUC, especially if there is no previous history of malignancy,
malignant cells from NUM in urine may signify the initial presentation of metastasis
[49]. Therefore, familiarity with characteristic cytologic features that may serve as
clues, review of pertinent clinical information and histologic samples, and application
of ancillary studies can lead to a more specific diagnosis in a subset of these cases.
Colorectal AdCa
Colorectal AdCa is the most frequent of all secondary bladder tumors and tends to
involve the bladder by means of direct extension. Approximately 21% and 12% of
secondary malignancies are from the colon and rectum, respectively [48]. Typical
cytologic features include pleomorphic cells with elongated nuclei, irregular nuclear
borders, coarse chromatin, and often inconspicuous nucleoli. The cytoplasm is usu-
ally vacuolated (Fig. 8.22). Often there is background necrosis, but this is best
observed in conventional preparations, as this tends to be difficult to appreciate and
minimal to absent in LBP [36]. Cytomorphologic and IHC features of colorectal
a b
Fig. 8.22 Colorectal AdCa. (a) Clusters of pleomorphic columnar cells display elongated irregu-
lar nuclei and coarse chromatin. The cytoplasm is vacuolated, with occasional signet ring cells.
The background shows necrosis and inflammatory cells. Follow-up revealed rectal AdCa directly
invading the bladder. (Bladder washing, SurePath, medium mag.). (b) This patient had metastatic
colon AdCa to the bladder diagnosed via bladder biopsy, collected at the same time with urine
cytology. The malignant cells are columnar with elongated hyperchromatic nuclei and vacuolated
cytoplasm. Notice that the background in TP tends to be cleaner than that of SurePath or Cytospins.
(Bladder wash, TP, high mag.). Cytologic features of colorectal AdCa overlap with those of pri-
mary bladder AdCa, enteric type
AdCa overlap with primary bladder AdCa, enteric and mucinous types [49]. In the
absence of clinical history, high-grade colorectal AdCa is not uncommonly misdi-
agnosed as HGUC. The combination of atypical columnar cells and necrosis are the
best clues to suspecting colorectal carcinoma. IHC markers such as CK20, beta-
catenin, CDX2, and SATB2, all of which are usually positive, may help clarify the
differential diagnosis (see previous discussion of primary AdCa) [9, 52].
Prostate AdCa
Prostate cancer is the second most common non-primary malignancy of the urinary
tract and accounts for approximately 20% of secondary malignancies [48]. It tends
to involve the bladder and urethra by either direct extension or lymphatic spread
[36]. In urine cytology, prostatic AdCa is almost always associated with clinically
advanced stage, high Gleason score, and a known history [53]. Hematuria and outlet
obstruction are often presenting symptoms [54].
Characteristic cytologic features of lower-grade prostatic AdCa include large
cells with uniform appearance, arranged in cohesive groups and syncytia. The
a b
Fig. 8.23 Prostate AdCa. (a) Characteristic cytologic features of prostatic AdCa are best appreci-
ated in lower Gleason score carcinomas. The tumor cells are large, arranged in cohesive groups,
and have a uniform appearance. The cells have abundant delicate cytoplasm, round to oval nuclei,
fine chromatin, and prominent nucleoli. (Instrumented urine, TP, high mag.). (b) In higher Gleason
score AdCa, the tumor cells tend to have less voluminous cytoplasm but retain the uniform appear-
ance and prominence of nucleoli. There is a suggestion of acinar formation at the periphery of the
cluster. Follow-up biopsy (not shown) revealed prostatic AdCa, Gleason score 8. (Bladder wash-
ing, SurePath, high mag.)
malignant cells have delicate vacuolated cytoplasm, round to oval nuclei that may
be eccentrically placed, fine chromatin, and prominent nucleoli (Fig. 8.23a).
Occasionally, acinar formation is appreciated (Fig. 8.23b). In some cases, however,
cytomorphology may be difficult to distinguish from HGUC (Fig. 8.24), especially
when the prostatic carcinoma is of high Gleason score, of ductal type, or has hyper-
chromatic nuclei without nucleolar prominence [36, 48, 53]. In contrast to HGUC,
however, high-grade prostate AdCa tends to still retain relatively uniform nuclei and
prominent nucleoli. IHC can be of value, as prostate AdCa expresses NKX3.1 and
PSA (Fig. 8.24), while HGUC expresses p63 and GATA3, but the latter two markers
are often lost in poorly differentiated HGUC [49, 50, 54].
Gynecologic Malignancies
Involvement of the urinary bladder by gynecologic cancers is uncommon and may

occur via direct extension and transperitoneal or lymphatic-hematogenous spread.
Direct extension by cervical SqCC is most common and accounts for approximately
10% of secondary malignancies [48], whereas ovarian and endometrial AdCas
account for approximately 6% and 4%, respectively. By cytologic evaluation alone,
it is impossible to distinguish secondary SqCC from primary SqCC or from
a b
c d
Fig. 8.24 Prostate AdCa. This specimen is from an 80-year-old male, who presented with urinary
retention and elevated serum PSA levels. (a) Urine cytology is hypercellular and contains a disco-
hesive population of intermediate sized cells with minimal dense cytoplasm. The nuclei are round
with conspicuous nucleoli and relative uniformity. (Voided urine, TP, high mag.). (b) The cell block
reveals similar cytologic features, including hyperchromatic nuclei and occasional prominent
nucleoli. Differential diagnosis includes HGUC, prostatic AdCa, poorly differentiated carcinoma-
NOS, and melanoma. (Cell block of voided urine, H&E, high mag.). (c) There is strong positive
staining of the tumor cells with NKX3.1 and PSA and negative staining with P63 and GATA3.
(NKX3.1 immunohistochemistry, high mag.). (d) Follow-up prostate biopsies confirm AdCa,
Gleason score 10-grade group 5. (H&E, high mag.)
squamous differentiation in HGUC. Clinical findings and histologic confirmation

are often needed for a definitive diagnosis. Clinical clues are the younger median
age of cervical carcinoma patients (49 years) compared to HGUC patients (73 years)
and that most patients have a known history of cervical cancer [55] (Fig. 8.25).
Although p16 is diffusely expressed immunohistochemically in most cervical
SqCCs, it is of limited diagnostic value because it is also expressed in 25–37% of
primary SqCC of bladder and up to 80% of HGUC [56]. HPV in situ hybridization,
however, is a good discriminator between metastatic cervical carcinoma and
HGUC [57].
Metastatic ovarian AdCa, particularly high-grade serous carcinoma, is character-
ized by clusters of large pleomorphic cells with high-grade nuclei and may show
papillary configuration. This morphology, however, can be quite deceptive, and can
also be easily mistaken for HGUC. If ovarian carcinoma is suspected, IHC positiv-
ity for PAX8 and WT1 and negativity for p63 and GATA3 could help confirm the
diagnosis [55]. Endometrial AdCa can also be difficult to distinguish from primary
bladder AdCa or HGUC [29], emphasizing the importance of correlation with clini-
cal and imaging findings in those cases (Fig. 8.26).
Fig. 8.25 Cervical
SqCC. This 51-year-old
female presented with
malignant squamous cells
in the urine cytology.
There is focal suggestion
of keratinization. It is not
possible to distinguish
between primary and
secondary SqCC or HGUC
with squamous
differentiation, based on
cytologic exam alone.
Clinical correlation is
needed in such instances.
Clinical follow-up revealed
locally advanced SqCC of
the cervix. (Instrumented
urine, TP, high mag.)
a b
Fig. 8.26 Endometrial serous AdCa. This urine cytology is from a 61-year-old female. (a) There
are many tightly cohesive clusters of large pleomorphic cells, with marked nuclear irregularity and
high N/C ratio. A papillary configuration is suggested. (Voided urine, TP, high mag). (b) An endo-
metrial biopsy, performed 1 week prior, demonstrated serous papillary AdCa, FIGO grade 3.
(H&E, high mag.) Therefore, the cytologic features are consistent with metastatic high-grade
serous carcinoma
Breast AdCa
Breast AdCa is one of the more common primary sites responsible for distant metas-
tases to the bladder and may be of lobular or ductal type. It accounts for approxi-
mately 2.5% of secondary malignancies involving the urinary bladder [48] and is
seldom reported in urine cytology [32, 58]. In some instances, the bladder may be
the sole organ involved by metastatic breast carcinoma. Lobular carcinoma tumor
cells may be arranged in a linear (single file) configuration but more often present
as single cells or small groups. Tumor cells are small with high N/C ratio, eccentri-
cally placed nuclei, and small or inconspicuous nucleoli (Fig. 8.27). Occasionally,
the center of the cytoplasmic vacuole shows mucin condensation (“bull’s eye” or
“targetoid” body). Lobular carcinoma of breast can resemble primary signet ring
cell AdCa, plasmacytoid UC, melanoma, neuroendocrine tumors, and plasmacy-
toma. Ductal carcinoma is rarely encountered in the urine and presents as tightly
cohesive clusters of atypical cells with somewhat uniform appearance (Fig.8.28).
a b
Fig. 8.27 Breast lobular carcinoma. This patient is a 48-year-old woman with a remote history of
lobular carcinoma of the breast. (a) Urine cytology reveals discohesive small to intermediate sized
cells, with high N/C ratios, hyperchromatic nuclei, and small nucleoli. Some cells have eccentri-
cally placed nuclei with a plasmacytoid appearance. Immunohistochemistry performed on the cell
block (not shown) revealed positive staining of the tumor cells for gross cystic disease fluid pro-
tein-15 (GCDFP-15). (Voided urine, TP, high mag.). (b) Follow-up transurethral resection revealed
metastatic lobular carcinoma diffusely invading the urinary bladder and undermining and penetrat-
ing the surface urothelium. (H&E, medium mag.)
a b
Fig. 8.28 Metastatic breast ductal AdCa. Both of these patients had a previous history of invasive
breast ducal carcinoma. (a) In this first patient, the tumor cells are forming a cellular sphere (“mor-
ula”), which is commonly seen in metastatic breast ductal carcinomas involving exfoliative fluid
cytologies. The cluster has a sharp community border, and tumor cells are relatively uniform.
(Voided urine, TP, high mag.). (b) In this second case, the tumor cells are more loosely cohesive
with suggestion of acinar formation but are not forming spheres. The neoplastic cells have a mod-
erate amount of cytoplasm, round to oval nuclei, and prominent nucleoli. (Voided urine, CS,
high mag.)
Clinical history in combination with IHC markers, including mammaglobin and

GCDFP-15, which are positive in most lobular carcinomas and a large percentage
of ductal carcinoma, can help confirm the diagnosis. GATA3 shows positive stain-
ing in both metastatic breast carcinoma and HGUC and is therefore not helpful in
excluding plasmacytoid UC [59].
Gastric AdCa
Metastatic gastric AdCa to the urinary bladder and upper urinary tract is rare,
accounting for less than 5% of secondary malignancies of the bladder. The bladder
tends to be involved in advanced stages of disease, and most cases are associated
with peritoneal dissemination. Sporadic case reports describe urine cytologic
Fig. 8.29 Metastatic
gastric signet ring cell
AdCa from a 78-year-old
male with advanced stage
disease, including
peritoneal dissemination.
The tumor cells are
small- to medium-sized,
are discohesive, and have
cytoplasmic vacuoles that
indent and displace the
nuclei to the periphery.
(Catheterized urine, TP,
high mag.)
findings in metastatic signet ring cell carcinoma, which is an aggressive variant of

gastric AdCa [60]. Urine cytology shows tumor cells that are small in size and
arranged as single cells and small clusters. The presence of signet ring cells raises
the differential diagnosis of primary mucinous AdCa, mucinous colorectal AdCa,
and plasmacytoid UC (Fig. 8.29). Metastatic intestinal-type gastric carcinoma has
overlapping cytologic features with primary AdCa, HGUC with glandular differen-
tiation, and colorectal carcinoma. Currently, IHC plays a limited role in the differ-
ential diagnosis, but clinical history and histologic correlation with the primary
gastric cancer are paramount in arriving at an accurate diagnosis.
Lung Carcinoma
While lung carcinoma is a common form of cancer, metastases to the bladder are
relatively uncommon, accounting for <3% of secondary malignancies. AdCa is the
most common histologic type of pulmonary carcinoma metastatic to the bladder and
upper urinary tract, followed by SqCC [61]. The efficacy of urine cytology in assess-
ment of metastatic lung carcinoma is unclear, as it may be associated with signifi-
cant false negative results [62]. Cytomorphologic features alone cannot distinguish
metastatic lung AdCa or SqCC from primary bladder or other metastatic carcino-
mas; therefore, careful evaluation of the clinical history is essential. IHC may be
helpful in a subset of cases, as TTF1 and napsin A positivity are consistent with lung
AdCa, whereas primary bladder AdCa is not expected to stain for these markers
(Fig. 8.30).
a b
Fig. 8.30 Lung AdCa. This specimen is from a 91-year-old man with stage 4 lung AdCa. (a) Urine
cytology reveals a few clusters of large pleomorphic malignant cells with abundant delicate cyto-
plasm. The nuclei are round to oval shaped, have open chromatin, and have prominent nucleoli.
Based on cytologic evaluation alone, it is not possible to determine the primary origin of AdCa.
(Voided urine, TP, high mag.). (b) TTF1 IHC, performed on cell block, shows positive staining of
the malignant cells, consistent with clinical history of lung AdCa. (TTF1, high mag.)
Renal Cell Carcinoma
Metastatic renal cell carcinoma (RCC) to the urinary bladder is unusual and it is
exceedingly uncommon for the malignant cells to be detected in urine cytology.
This is in large part due to the fact that most RCCs are currently detected by imag-
ing studies before they erode and shed cells into the urine [53]. Therefore, urine
cytology plays no significant role in the diagnosis of RCC [63]. As expected, most
reported metastatic RCCs to the bladder are of clear cell type [9]. Cytomorphologic
findings include large tumor cells with abundant hypervacuolated clear cytoplasm,
round to oval nuclei, and occasionally prominent nucleoli (Fig. 8.31). Granular
eosinophilic cells with pyknotic nuclei and indistinct cytoplasmic borders are
believed to be the results of degenerative changes caused by the urinary environ-
ment. Differential diagnosis includes HGUC with clear cell features and clear cell
variant of primary AdCa. In many instances the malignant cells are very degener-
ated and difficult to separate from benign renal tubular cells or reactive urothelial
cells [63] (Fig. 8.32).
a b
Fig. 8.31 Renal cell carcinoma (RCC). This urine specimen is from a patient with an established
history of advanced RCC. (a) On lower power exam, there are cohesive clusters of large tumor
cells with abundant cytoplasm that has a hypervacuolated and/or granular appearance. (b) High
power illustrates the cytoplasmic quality and enlarged round nuclei with large prominent nucleoli.
(a, b: Catheterized urines, CS, medium and high mag.)
a b
Fig. 8.32 Atypical cells suspicious for RCC. The specimen is from a 79-year-old man without
clinical history at the time of evaluation of urine cytology. (a) Rare discohesive large cells with
abundant granular cytoplasm are seen. The nuclei have small, distinct nucleoli, but no significant
pleomorphism. The background is bloody. This case was initially reported out as reactive/repara-
tive changes. (Voided urine, CS, high mag.). (b) Subsequently provided clinical history and review
of surgical resection revealed an advanced RCC that eroded into the renal pelvis. (H&E, high
mag.). In retrospect, these atypical cells most likely represented RCC
Non-epithelial Malignancies
Background
Non-epithelial malignancies account for less than 0.5% of all bladder tumors and
may primarily or secondarily involve the urinary tract. They include sarcoma, mela-
noma, and lymphoma [8]. Due to their rarity, inexperience and/or lack of awareness
of cytologists with their occurrence in this setting, and tendency for these tumors to
involve the bladder wall rather than the mucosal surface, they are seldomly diag-
nosed in urine cytology specimens. Pathogenesis of primary non-epithelial malig-
nancies is incompletely understood, but several factors are believed to be contributing
agents, such as metaplasia, chronic infection, and development from multipotent
stem cells [64].
Sarcoma
Overall, primary sarcomas of the urinary tract are exceptionally rare, except for
embryonal rhabdomyosarcoma in the pediatric age group.
Leiomyosarcoma is the most common urinary bladder sarcoma in adults,
accounting for 1% of all bladder malignancies, and most often has high-grade mor-
phology [64]. Patients usually present with hematuria and a palpable pelvic mass,
and mean age at presentation is 65 years. Leiomyosarcoma has been reported to be
often associated with prior cyclophosphamide therapy for neoplastic and nonneo-
plastic conditions.
In adults, rhabdomyosarcomatous differentiation occurs more commonly as a
component of sarcomatoid HGUC; only rare cases of urinary tract pure rhabdomyo-
sarcoma or angiosarcoma have been reported in the literature [65]. Adult sarcomas
generally share an extremely aggressive biologic behavior, regardless of the histo-
logic subtype.
Diagnosis of sarcoma by urine cytology is seldom made, taking into account the
rarity of those lesions in the urinary tract, infrequent exfoliation, and the lack of
experience of cytologists with their appearance. Only a few cases have been reported
in the English literature [66, 67]. High-grade sarcoma is easier to detect since the
tumor cells tend to have a spindle or epithelioid appearance and show significant
cytologic atypia and pleomorphism. In the absence of significant atypia, differential
diagnosis of spindle cell lesions includes low-grade sarcomas and benign nonneo-
plastic and neoplastic processes such as postoperative spindle cell proliferation and
inflammatory myofibroblastic tumor (also known as inflammatory pseudotumor),
which cannot be distinguished by urine cytologic evaluation.
Definition
Malignant mesenchymal tumor showing smooth muscle, skeletal muscle, endothe-

lial, or no specific differentiation.
Criteria
• Moderate to high cellularity.

• Sheets and/or scattered spindle cells with moderate to severe nuclear
pleomorphism.
• Moderate amount of ill-defined cytoplasm.
• In leiomyosarcoma, there are large hyperchromatic, oval to spindle-shaped
nuclei with blunt ends, coarse chromatin, and occasional nucleoli (Fig. 8.33).
• In high-grade or pleomorphic sarcoma, any pleomorphic malignancy should
enter into the differential diagnosis (Fig. 8.34). Diagnostic considerations include
HGUC with sarcomatoid differentiation, sarcoma, secondary sarcomatoid carci-
noma from other sites (such as the kidney), melanoma, and lymphoma.
a b
Fig. 8.33 Leiomyosarcoma of the urinary bladder. (a) Urinary cytology shows fragments of atypi-
cal spindle cells amid stroma containing inflammatory cells. The cells contain large elongated
(“cigar”-shaped) mildly hyperchromatic nuclei with a moderate amount of ill-defined cytoplasm.
(Bladder washing, CS, medium mag.). (b) Histology of the resected leiomyosarcoma demonstrates
bundles of spindle-shaped cells in profile and cross section with elongated and pleomorphic nuclei.
(Biopsy, H&E, medium mag.)
a b
Fig. 8.34 Pleomorphic rhabdomyosarcoma of the bladder. (a) Cytology demonstrates numerous
highly pleomorphic and bizarre malignant cells with spindle and epithelioid features. Individual
cell necrosis is evident in the background. Differential diagnosis based on this morphology
includes any pleomorphic malignancy, including sarcoma, sarcomatoid carcinoma, and melanoma.
(Bladder washing, SP, medium mag.). (b) Histology shows pleomorphic rhabdomyosarcoma.
(H&E, high mag.)
Explanatory Notes
Explanatory Note 1 Before considering a diagnosis of sarcoma in limited samples,

sarcomatoid carcinoma should first be excluded (Fig. 8.35).
Explanatory Note 2 Differential diagnosis of malignant spindle cells should

include primary or secondary sarcomatoid carcinoma, melanoma, and lymphoma.
IHC may play a role in confirming a new diagnosis or recurrent disease in a subset
of cases with highly cellular specimens, where a cell block or additional cytology
preparations can be prepared for ancillary studies.
Explanatory Note 3 In general terms, the presence of atypical spindle cells in urine
indicates the need for a more invasive diagnostic procedure to arrive at a precise
histologic diagnosis or subclassification, as a comprehensive panel of IHC stains
may not be feasible on a limited cytological sample [68].
Fig. 8.35 Sarcomatoid
HGUC. This 63-year-old
male presented with gross
hematuria. Urine cytology
contains atypical spindle
cells and individual cell
necrosis. This case was
reported as “high grade
spindle cell malignancy,
can’t exclude HGUC.”
(Voided urine, TP, high
mag.). Follow-up
cystoscopy and TURP
showed a muscle invasive
sarcomatoid HGUC
Malignant Melanoma
Background
Malignant melanoma of the urinary tract is very rare, accounting for less than 1% of
all melanomas. Most melanomas are metastatic from another site and tend to involve
the kidney and bladder [36]. Some cutaneous melanomas of the glans penis or vulva
extend into the urethra and urinary bladder. Primary melanoma is postulated to arise
from neural crest cells and most commonly arises in the urethra, whereas only few
cases have been reported in the urinary bladder [69]. Metastatic melanoma accounts
for approximately 4% of all secondary malignancies of the bladder and generally
has a poor prognosis [48]. Hematuria is the most common presenting symptom.
Definition
Malignant cells of melanocytic origin.
Criteria (Fig. 8.36)
• Individually scattered large atypical cells with round to oval nuclei and abundant
cytoplasm.
• The malignant cells may have epithelioid or spindle appearance.
• Nuclei are large, pleomorphic, and may be eccentrically placed.
• Prominent nucleoli with occasional nuclear pseudoinclusions.
• The cytoplasm may contain dark finely dusty brown to black melanin pig-
ment [70].
• Pigment granules may be present in the background or in macrophages in cases
of widely metastatic melanoma with melanuria [71].
a b
Fig. 8.36 Malignant melanoma. (a) Metastatic cutaneous melanoma displays scattered large atyp-
ical cells with round to oval nuclei and variable amount of cytoplasm. The cytoplasm contains dark
dusty brown melanin pigment. The nuclei are enlarged and have prominent nucleoli. (Voided urine,
TP, high mag). (b) In this primary urethral melanoma, the malignant cells are huge and show
extremely pleomorphic nuclei that are eccentrically placed and have macronucleoli. In contrast to
specimen “a,” the cytoplasm is dense and amelanotic. (Urethrovaginal fluid, TP, high mag.)
Explanatory Notes
Explanatory Note 1 Metastatic melanoma is more common than primary

melanoma.
Explanatory Note 2 Melanoma may be suspected if classic cytologic features are

present, such as cytoplasmic melanin pigment and intranuclear inclusions.
Explanatory Note 3 In the absence of classic cytologic features, it may be difficult

to distinguish melanoma from HGUC or other malignancies such as poorly differ-
entiated carcinoma, lymphoma, plasmacytoma, and sarcoma.
Explanatory Note 4 In the presence of a known clinical history of melanoma,

abnormal cells in urine should be carefully evaluated to exclude metastatic
melanoma.
Explanatory Note 5 IHC melanocytic markers are needed for confirmation, includ-
ing positive staining with S100, HMB45, Melan A, and SOX10; and generally
n egative staining with cytokeratins and lymphoid markers (although rare aberrant
expression of cytokeratin staining has been reported in melanoma) [72].
Hematopoietic Malignancies
Background
Hematopoietic malignancies, such as lymphoma and plasmacytoma, are unusual in

the urinary tract and may involve it as a primary tumor or more commonly as a
component of systemic disease. Clinically, they present most often as a solitary
mass and less frequently as multiple nodules [73]. These tumors typically involve
the bladder wall with overlying normal urothelium, so they may not shed many cells
into the urine. Patients often display nonspecific symptoms, such as hematuria, dys-
uria, back/abdominal pain, and urinary frequency.
Lymphoma
Primary lymphoma accounts for 0.2% of all extranodal lymphomas and <1% of all
bladder tumors. Secondary involvement of the urinary tract occurs more often [74].
The majority of primary bladder lymphomas (80%) are low-grade, extranodal mar-
ginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) origin.
High-grade lymphomas are rarer, with diffuse large B-cell lymphoma (DLBCL)
being most common. Other types, such as anaplastic large-cell lymphoma (ALCL)
and Burkitt’s and mantle cell lymphomas, have been seldom reported. Primary
MALT lymphoma is commonly associated with chronic cystitis and affects adults
older than 60 years of age. It is usually localized at time of diagnosis and often has
excellent prognosis following local resection [73, 74]. On the other hand, primary
high-grade types and secondary lymphomas tend to have a poor prognosis and may
be treated with various therapeutic options, including chemotherapy, radiotherapy,
and surgery, alone or in combination.
The cytologic features of lymphoma in the urine are similar to those seen in
other sites. DLBCL is characterized by large cells with increased N/C ratio, round
to oval nuclei, dense chromatin, and prominent nucleoli (Fig. 8.37). The cells are
most commonly arranged singly, but they may form cohesive clusters, especially in
ALCL. Abundant necrotic debris and numerous apoptotic bodies are common. The
atypical lymphoid cells resemble centroblasts with multiple smaller peripherally
located nucleoli, or immunoblasts with central large nucleoli. Multinucleated
forms and plasmacytoid cells may be seen in ALCL. MALT lymphoma shows
medium-sized cells with round nuclei, dispersed chromatin, and inconspicuous
nucleoli [75]. They may resemble small lymphocytes or have a plasmacytic appear-
ance (Fig. 8.38).
Cytologic diagnosis of lymphoma in urine specimens is very challenging, espe-
cially if there are no clinical symptoms or known clinical history. Also, because of
a b
Fig. 8.37 Diffuse large B-cell lymphoma (DLBCL). (a) This 59-year-old woman presented with
gross hematuria, urinary frequency and lower abdominal pain. Urine cytology demonstrates a dis-
persed bi-morphic population of small and large lymphoid cells. The larger cells predominate and
show clumped chromatin, prominent single or multiple nucleoli, and variably stripped cytoplasm.
Follow-up biopsy (not shown) revealed a primary DLBCL in the bladder dome. (Voided urine, CS,
medium mag.). (b) This is a case of secondary DLBCL involving the bladder, showing a monomor-
phic population of large cells with round to oval nuclei, vesicular chromatin, and prominent nucle-
oli. Some of the cells have a plasmacytoid appearance. Differential diagnosis includes high-grade
carcinoma and melanoma. (Voided urine, TP, high mag.)
its rare occurrence, lymphoma may not be entertained in the differential diagnosis
of abnormal cytology. High-grade lymphoma is easily identified as abnormal or
malignant but can be misdiagnosed as HGUC because of the high N/C ratio and
hyperchromasia of the nuclei (Fig. 8.37a). The initial clinical impression may be
misleading and suggestive of HGUC [74], since most lymphomas present as a soli-
tary mass. Background tumor diathesis and numerous apoptotic bodies should
raise the possibility of lymphoma. Differential diagnosis of high-grade lymphoma
includes HGUC, SmCC, melanoma, and sarcoma. IHC positivity for lymphoid
markers such as CD20, CD79a, and bcl-2 help support the diagnosis, but ulti-
mately histologic confirmation is needed. MALT lymphoma is more challenging to
diagnose than high-grade lymphoma, as it may be difficult or impossible to distin-
guish it from reactive lymphoid cells (Fig. 8.38). The diagnosis of MALT lym-
phoma should be considered in a lymphoid-rich urine specimen, if the patient has
a previous history of lymphoma, or there is a clinical presentation of a blad-
der mass.
Fig. 8.38 Marginal zone

(MALT) lymphoma from a
78-year-old woman with a
history of chronic cystitis.
Urine contains
monomorphic small- to
medium-sized atypical
centrocyte-like
lymphocytes. Follow-up
biopsy (not shown)
revealed MALT
lymphoma, confirmed by
IHC and flow cytometry.
(Voided urine, Cytospins,
high mag.)
Plasmacytoma
Plasmacytoma of the urinary bladder is very rare, accounting for <5% of all plasma
cell neoplasms. For a diagnosis of primary plasmacytoma, there must be no evi-
dence of plasmacytoma elsewhere, including a negative bone marrow biopsy [73].
An occasional case report documenting plasmacytoma in urine can be found in the
literature [76]. Cytologic features are similar to plasmacytoma at other sites. The
neoplastic cells are large, singly dispersed, with dense basophilic cytoplasm and
eccentrically placed nuclei. The nuclear chromatin has peripheral clumping in a
clockface distribution and may show prominent nucleoli. Differential diagnosis
includes HGUC, plasmacytoid UC, lymphoma, melanoma, NET, and sarcoma. IHC
or surgical biopsy is needed to establish a definitive diagnosis. Prognosis of primary
plasmacytoma is improved with early detection and combination radiation therapy
and surgery. Chemotherapy is utilized in high-risk patients and radiation-resistant
tumors [73].
Other Miscellaneous Lesions
Two lesions that are extremely rare to encounter in urine cytology are briefly dis-
cussed in this section. They include paraganglioma and nephrogenic adenoma.
Paraganglioma
Paraganglioma is the preferred term for extra-adrenal pheochromocytoma.

Approximately 10% of paragangliomas occur in extra-adrenal sites, and of these,
less than 10% are localized in the bladder wall (<0.05% of all bladder tumors) [40].
They are thought to arise from embryonic nests of chromaffin cells in the sympa-
thetic plexus of detrusor muscle. Approximately 15% have a malignant behavior
[8]. They tend to be functional and occur mostly in young adult Caucasians. Patients
commonly present with hypertension, headache, and hematuria. They are usually
treated by partial cystectomy. Prognosis after surgery of stage T1 or T2 is excellent,

but T3 and T4 stage tumors have a risk of recurrence or metastases [40]. Based on
the histology or cytology alone, determination of benign vs. malignant tumor behav-
ior is difficult. It has been recently reported that large tumor size, a higher number
of mitoses, and the presence of lymphovascular tumor invasion and succinate dehy-
drogenase B (SDHB) mutations suggest malignant behavior of paraganglioma
[40, 77].
Although FNA cytologic features of paragangliomas from different sites are well
documented, there exists only a single case report describing urine cytology fea-
tures [78]. This is not surprising, since most bladder paragangliomas arise deep in
the bladder wall and are unlikely to exfoliate. Urine cytology shows large epitheli-
oid cells arranged in loose clusters and singly scattered, with moderate amount of
cytoplasm, round to oval nuclei with fine chromatin, and inconspicuous nucleoli
(Fig. 8.39). There is moderate anisonucleosis, and few naked nuclei in the back-
ground, but no significant nuclear pleomorphism [78]. The major differential diag-
nosis is HGUC, but the latter is characterized by higher N/C ratio and marked
nuclear hyperchromasia and/or nuclear irregularities. IHC profile of paraganglioma
includes positive staining for neuroendocrine markers, such as chromogranin and
a b
Fig. 8.39 Paraganglioma. (a) Urine cytology shows a loose cluster of large epithelioid cells with
abundant delicate cytoplasm, anisonucleosis, round to oval nuclei with fine chromatin, and incon-
spicuous nucleoli. (Bladder washing, SurePath, high mag.). (b) Bladder tumor resection displays
cells arranged in a nested (zellballen)/trabecular pattern, separated by delicate vasculature. The
cells are large and have acidophilic cytoplasm, round to oval nuclei, and prominent nucleoli.
(H&E, high mag.)
synaptophysin. However, similar to HGUC, paraganglioma commonly expresses

GATA3, so caution is needed in utilizing this marker to distinguish it from HGUC.
Nephrogenic Adenoma
Nephrogenic adenoma (NA) is an uncommon benign lesion of the urothelial mucosa

that most commonly involves the urinary bladder. It is postulated to develop as a
result of traumatic injury, and implantation of exfoliated renal tubular epithelium.
Histologically, they display various growth patterns, including tubular, cystic, tubu-
locystic, papillary, and flat [79], and are lined by low cuboidal to low columnar cells
with eosinophilic cytoplasm. Clear cell change, degenerative atypia, and hobnail
appearance can occur. NA shows diffuse positive IHC staining with PAX8. Urinary
cytologic features have been described in limited retrospective studies and include
the presence of vacuolated polygonal cells, arranged in small groups, papillae, or as
isolated cells [80]. Columnar cells may be present singly or in groups. There is
generally mild or absent nuclear atypia, but occasionally there are slightly increased
N/C ratio, nuclear hyperchromasia, and prominent nucleoli. Differential diagnostic
considerations include reactive changes, instrumentation effect, HGUC, clear cell
AdCa, and RCC, but the latter two usually demonstrate significant cytologic atypia.
More recently, those previously described urinary cytologic features of NA have
been challenged, suggesting that they are nonspecific and are not characteristic of
NA [81]. In that study, all urine cases that had NA biopsies and corresponding urine
cytologic features fulfilling the previously described criteria showed negative PAX8
staining of the suspected lesional cells, which confirmed the absence of NA cells in
these cytologic specimens. This is not surprising, since most NAs are small or
located beneath the surface urothelium, and therefore do not shed cells into the
urine. When NA involves the surface urothelium and/or sheds cells, they are often
indistinguishable from benign urothelial cells with reactive or degenerative changes,
or instrumentation effect (Fig. 8.40). This has also been our experience, as most
urine specimens corresponding to NA biopsies at our institutions are reported as
negative, with occasional cases reported as atypical. A similar opinion was voiced
by Dr. Leopold Koss in his monograph, where he stated that he has “personally not
observed any cell changes that could be considered characteristic of this lesion”
[82]. In summary, it is not possible to prospectively diagnose NA on urine cytology
with certainty, but fortunately other major diagnostic considerations include benign
conditions that have no clinical impact [73].
a b
c d
Fig. 8.40 Nephrogenic adenoma (NA), from a 64-year-old man. (a) Histology shows characteris-
tic tubulocystic and papillary architecture of NA, which involves the urothelial surface and lamina
propria. The tubules are closely packed and associated with background inflammation. A small
uninvolved portion of the urothelium is present at left top corner of photo (*). (H&E, low mag.).
(b) NA shows PAX8 positivity but spares the portion of uninvolved urothelium (*). (c) Urine cytol-
ogy demonstrates rare groups of large cells with abundant dense cytoplasm, round nuclei, and
small nucleoli. There is no significant atypia. (Instrumented urine, TP, high mag.). (d) Lesional
cells of NA have abundant eosinophilic dense cytoplasm with round nuclei and small nucleoli
(right part of photo); these cells have similar cytologic appearance to those observed in corre-
sponding urine cytology. However, it would be very difficult, in isolation, to distinguish between
the lesional cells and adjacent uninvolved urothelial cells (left part of photo). (H&E, high mag.)
Sample Reports
If urine cytology is interpreted as positive for “NUM,” it is implied that the sample
is adequate for evaluation and diagnostic of malignancy. “Suspicious for NUM”
implies that the cytologic findings are highly suspicious but not diagnostic of malig-
nancy. “Atypical cells present” implies that the atypia is of uncertain nature or sig-
nificance, and it would be best to categorize the cell type, if possible, i.e., squamous,
glandular, spindle, lymphoid, etc. Narrative notes or comments should be used to
explain or subclassify the malignant, suspicious, or atypical findings and offer a
potential differential diagnosis. A microscopic description is optional.
Example 1
Malignant
Keratinizing squamous cell carcinoma
Note: A pure population of malignant keratinizing squamous cells is present.

This may represent divergent squamous differentiation associated with urothelial
carcinoma or a primary or secondary pure squamous cell carcinoma. Clinical cor-
relation and histologic sampling may be needed for definitive subclassification of
the carcinoma.
Example 2
Malignant
High-grade carcinoma with squamous features
Note: There is admixture of malignant keratinizing squamous cells and malig-
nant non-keratinizing cells. The latter may represent high-grade urothelial carci-
noma versus non-keratinizing squamous carcinoma component. Differential
diagnosis includes urothelial carcinoma with squamous differentiation, primary
pure squamous carcinoma, and secondary squamous cell carcinoma extending from
the genital tract. Clinical correlation is recommended.
Example 3
Suspicious for malignancy

Atypical squamous cells, suspicious for squamous cell carcinoma
Note: Highly atypical keratinized squamous cells are present. These findings can
be associated with urinary tract or genital tract malignancies in a significant propor-
tion of cases. Clinical correlation and additional sampling are recommended, as
clinically indicated.
Example 4
Atypical squamous cells with mild atypia

Note: Although mild squamous atypia is most commonly associated with reac-
tive/inflammatory changes, a significant minority of cases may be associated with
bladder or genital tract carcinoma, condyloma, and dysplasia. This atypia is particu-
larly significant if it persists in subsequent specimens.
Example 5
Malignant
Adenocarcinoma
Note: The malignant cells have columnar appearance with elongated hyperchro-
matic nuclei, and necrosis in the background. Major diagnostic considerations
include primary adenocarcinoma, urothelial carcinoma with divergent glandular
differentiation, and metastatic adenocarcinoma from other sites such as colorectum.
Clinical correlation is recommended.
Example 6
Malignant
Adenocarcinoma with signet ring cell features
Note: The malignant cells show signet ring cell features. Signet ring cells may be
associated with several carcinomas, including plasmacytoid urothelial carcinoma,
primary adenocarcinoma, and metastatic adenocarcinoma from other sites such as
gastrointestinal tract and breast. Clinical correlation is recommended.
Example 7
Malignant
Adenocarcinoma
Note: There are cohesive clusters of highly atypical glandular cells, consistent
with adenocarcinoma. Although high-grade urothelial carcinoma cells are not seen,
urothelial carcinoma with glandular differentiation cannot be excluded. Differential
diagnosis also includes primary and secondary adenocarcinoma.
Example 8
Suspicious for malignancy

Atypical glandular cells, suspicious for adenocarcinoma
Note: There is a single cohesive cluster of atypical glandular cells with marked
atypia. Due to the limited cellularity of the atypical cells, a definitive diagnosis is
not rendered. Additional sampling is recommended for a more definitive diagnosis.
Example 9
Malignant
Small-cell (high-grade neuroendocrine) carcinoma
Note: The specimen contains many clusters and single highly atypical small cells
with prominent nuclear molding/spindling and apoptosis. Immunohistochemistry
(IHC) performed on the cell block demonstrated positive staining with synaptophy-
sin and chromogranin and negative staining with CD45, p63, and SOX10. The com-
bined cytologic and IHC findings are consistent with small-cell carcinoma.
Example 10
Malignant
Adenocarcinoma, consistent with prostate primary
Note: Previous history of high-grade prostatic adenocarcinoma is noted. Rare
groups of atypical glandular cells with relatively uniform enlarged nuclei and prom-
inent nucleoli are present. Immunohistochemistry performed on cytologic prepara-
tions showed positive staining with NKX3.1 and PSA and negative staining with
GATA3 and p63, consistent with prostatic adenocarcinoma.
Example 11
Atypical spindle cells, suspicious for malignancy

Note: Rare highly atypical spindle cells are present. There is no definite epithe-
lial differentiation identified in this limited specimen. These findings are suspicious,
but not diagnostic of malignancy. Diagnostic considerations include reactive pro-
cess, inflammatory myofibroblastic tumor, sarcomatoid carcinoma, sarcoma, and
melanoma. Histologic confirmation is recommended, as clinically indicated.
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Ancillary Studies in Urinary Cytology
9
Lukas Bubendorf, Nancy P. Caraway, Andrew H. Fischer,
Ruth L. Katz, Fernando Schmitt, Margareta Strojan Fležar,
Theodorus H. Van der Kwast, and Philippe Vielh
Summary of Changes in the Second Edition.
• Updated literature review on ancillary testing including new markers and assays.
• Recent data on ancillary testing in the era of TPS.
• New section on the promise of next-generation sequencing in urinary cytology.
Background
Urine cytology and cystoscopy with biopsy still remain the only means of definitive
diagnosis of urothelial carcinoma (UC). Though cytology and cystoscopy are com-
plementary, together they fail to detect a significant number of patients with UC. The
difficulties and challenges of urinary cytology have been driving the search for bio-
markers and development of commercial diagnostic assays to improve its sensitivity
for detecting urothelial carcinoma. In the past two decades, many different assays,
or ancillary tests, have been developed to overcome the limitations of urinary cytol-
ogy and improve the timely detection of UC. In parallel, the number of review
articles on urinary markers for detection of bladder cancer (UC) has steeply risen.
Despite the countless markers that have been proposed, only a few have reached the
stage of US Food and Drug Administration (FDA)-approved assays for diagnostic
application. The ancillary tests that have recognized utility include UroVysion®
Fluorescence in situ Hybridization (U-FISH; Abbott Laboratories, Abbott Park, IL,
USA); BTA™ (Polymedco Inc., Cortlandt Manor, NY, USA); and NMP22™
(Bladder Check) test (Alere Inc., Waltham, MA, USA). Several other tests for
tumor-associated antigens and PCR-based tests for mutations have been proposed,
and within the past several years, next-generation sequencing (NGS) has begun to
be applied to detect mutations or epigenetic changes of UC.

https://doi.org/10.1007/978-3-030-88686-8_9
194 L. Bubendorf et al.
Evaluation of individual markers has been hindered by the complexity of the disease
diagnosis and behavior, variable clinical endpoints, and the different types of treatment
dependent upon tumor grade and stage. Importantly, many studies have been done with-
out reference to cytology results classified according to TPS, making interpretation dif-
ficult because cytological findings and results of ancillary tests often cannot be linked
back to particular stages or grades of UC. These factors make it at least challenging if
not impossible to define an optimal role for ancillary studies through review of the litera-
ture. Concerns about cost-effectiveness of ancillary testing are another source of ongo-
ing discussions. For these reasons, no single ancillary test is clearly being recommended
as part of the routine evaluation in the guidelines of the American Urological Association
and the European Association of Urology at this time [1, 2].
There are basically two types of ancillary tests: those that are based on cytologi-
cal preparations (cell-based tests) and those that rely on a non-morphologic analysis
of urinary fluids (liquid-based tests). Liquid-based tests, such as NMP22, are meant
to be applied in the office of the urologist to identify patients with high risk of pri-
mary urothelial carcinoma or recurrence. Cell-based tests dovetail with the activi-
ties of the Cytopathology Laboratory and generally benefit from input from the
cytologist about whether a particular sample should be tested. Cell-based tests also
offer the potential to provide feedback to cytologists on a per cell basis about the
significance of atypical cytologic findings, potentially leading to future improve-
ment in criteria and the performance of cytologic diagnosis.
Many of the controversies on the value of ancillary testing in urinary cytology are
caused by a lack of clear indications for testing. Major unresolved questions are the
threshold risk of UC to invoke ancillary testing, whether ancillary studies should be
routinely performed on patients with an atypical cytology, whether cytologists or urol-
ogists are in the best position to order ancillary cell-based testing, and whether ancil-
lary tests have value as a quality improvement measure for the cytology laboratory.
TPS provides an opportunity to define and narrow down the diagnostic catego-
ries in which ancillary testing of urinary specimens is most rewarding and to avoid
unnecessary testing in areas with minimal or no added value. TPS has laid the
ground for prospective studies in search of cost-effective combinations of urine
cytology with ancillary testing to improve diagnosis and clinical outcome of
UC. According to a survey by the College of American Pathologists (CAP) covering
the time period from 2014 to 2016, ancillary testing of urinary tract cytology was
performed by 128 of 596 laboratories (21.4%), and UroVysion® fluorescence in
situ hybridization (U-FISH; Abbott Laboratories, Abbott Park, IL, USA) was the
most commonly used ancillary test (83.6%) followed by FISH tests from other man-
ufacturers (14.8%) [3]. While we will summarize several proposed diagnostic mark-
ers, we will therefore place a special emphasis on U-FISH that has been approved
by the FDA for diagnosis of UC in patients with hematuria and/or monitoring for
tumor recurrence in patients previously diagnosed with bladder cancer. To date,
U-FISH is considered the most promising ancillary test in urinary cytology and
therefore described in a separate section of this chapter [1, 4, 5]. This test is based
on the detection of numerical and structural chromosomal aberrations, which are a
hallmark of cancer, but only rarely seen in benign cells. ImmunoCyt/UCyt+®
(uCyt; DiagnoCure Inc., Quebec, Canada), another cell-based test, relies on the
9 Ancillary Studies in Urinary Cytology 195
detection of three proteins that are preferentially expressed by UC cells as opposed

to benign cells [6]. Although approved by the FDA, this test is no longer commer-
cially available and is not discussed further in this chapter.
re-Analytical Procedures, Technique, and Evaluation

P
of UroVysion® FISH
The commercially available multitarget multicolor U-FISH assay contains four sin-
gle-stranded DNA probes. Three probes are chromosome enumeration probes (CEP)
targeting pericentromeric regions of chromosomes 3, 7, and 17. Another probe is a
locus-specific identifier (LSI) probe targeting 9p21 locus on gene p16. All probes are
directly labeled with fluorescent dyes, specifically CEP 3 SpectrumRed, CEP 7
SpectrumGreen, CEP 17 SpectrumAqua, and LSI 9p21 SpectrumGold. There is a
similar FISH test that is labeled as an in vitro diagnostic (IVD) product (ZytoLight®
SPEC CDKN2A/CEN 3/7/17 Quadruple Color Probe; ZytoVision, Bremerhaven,
Germany). The same technical principles apply as for U-FISH. However, there are
no published data on its performance in urinary tract cytology.
Specimen Preparation
The U-FISH test was originally intended for use on voided urine specimens.
However, later studies have proved its use on other fluid specimens from the urinary
tract, e.g., bladder washings and upper urinary tract washings. Fresh urinary speci-
mens without pre-fixation must be processed to cell preparations on glass slides as
soon as possible after collection (within 3 hours). If necessary, fluid specimens may
be prefixed with 2% polyethylene glycol in 50% ethanol (Carbowax), PreservCyt®
(Hologic, Marlborough, MA), or other preservatives (e.g., an equal volume of 50%
ethanol) and preferably processed within 72 hours. Different techniques of slide
preparation can be used for U-FISH test. See Chap. 10 for appropriate specimen
preparation methods. There should be 100 to 200 well-preserved cells in the target
area for hybridization, spread in a moderately cellular monolayer, and fixed by
alcohol-based fixatives.
Unstained slide preparations are often used for the assay. However, U-FISH is
also applicable to original slides that have been stained according to the Papanicolaou
method to enable cytomorphological evaluation before the FISH assay. A disadvan-
tage of performing FISH on a slide that has been pre-stained is that the Eosin Azure
(EA) dye can cause fluorescence: if the stain is not completely washed away during
FISH, high background can be seen. Decreasing the EA concentration for samples
destined for FISH can be helpful to keep background fluorescence low. Areas with
abnormal urothelial cells on the Papanicolaou preparation can be relocated after
FISH, as described in more detail in the section on automation.
The basic principles of the U-FISH assay are illustrated in Fig. 9.1. In addition,
the technical procedures including slide pretreatment and hybridization are
described in the Appendix.
Fig. 9.1 Basic principles of U-FISH assay. The assay contains directly fluorescent dye-labeled
single-stranded DNA probes. Pre-analytical procedures include slide preparation (including pre-
treatment of pre-stained slides). UroVysion assay starts with probe preparation and specimen –
target DNA denaturation followed by DNA hybridization of probes to target DNA sequences.
Analysis of FISH signals is performed under epi-fluorescent microscope. CEP centromere enu-
meration probe, LSI locus-specific identifier probe, chr chromosome, loc locus
Analysis of FISH Signals
An epi-fluorescence microscope equipped with a 100-watt mercury lamp or LED

light sources and appropriate filters is recommended to detect multicolor fluorescent
signals. Specimens should be scanned at 400x magnification to locate cells with
nuclear signals and then analyzed under 600x to 1000x magnification. Systematic
analysis of signals following a standardized and repeatable approach is essential
(e.g., starting analysis in the upper left quadrant of the target area and continue scan-
ning from left to right and top to bottom). Signals should be counted in abnormal
cells as defined by their nuclear features, namely, enlarged nuclear size, irregular
nuclear borders, and “patchy” 4,6-diamidine, 2-phenylindole dihydrochloride
(DAPI) staining. Besides the single cells, cell clusters may be evaluated, but only if
overlapping nuclei can be individually distinguished. The number of signals for all
4 probes should be counted and recorded as abnormal when there is a gain (≥2 sig-
nals) for 2 or more chromosomes 3 (red), 7 (green), and 17 (aqua) or there is a loss
of both copies of 9p21 (gold) as suggested by the manufacturer. The test is consid-
ered positive when ≥4 of the 25 analyzed cells show gains for 2 or more chromo-
somes or ≥ 12 of the 25 cells have zero 9p21 signals. If this criterion for test
positivity is not met, analysis should be continued until the entire sample is ana-
lyzed. Some authors have recommended excluding rare tetraploid or octoploid cells
(cells with exactly four or exactly eight copies of each probe) unless they are numer-
ous (e.g., ≥10) [7–9], since tetraploid or even octoploid cells can occur in benign
a b c
Fig. 9.2 (a–c) Schematic illustration of U-FISH findings. (a) Nuclei of a FISH-negative benign
cell with 2 signals for the chromosomes 3 (red), 7 (green), and 17 (aqua) and for 9p21 (gold). (b)
U-FISH-positive cell with increased and unbalanced number of chromosomes 3 (red) and 17
(aqua) and heterozygous loss of 9p21 (gold). (c) Tetraploid FISH pattern with four signals of each
FISH probe. This pattern is not specific for urothelial carcinoma but commonly seen in reactive
urothelial cells
reactive cells (e.g., umbrella cells) and may give rise to false-positive FISH results.
Along this line, it is specifically mentioned in the package insert that results at or
near the cut-off point should be interpreted with caution. A schematic illustration of
typical U-FISH findings is shown in Fig. 9.2.
There are several conditions that may prevent accurate FISH examination,
including the presence of lubricant jelly, corpora amylacea, degenerated cells, crys-
tals, sperm, and bacteria.
In the case of performing FISH on conduit urines, interference by inflammation
and cell debris may occur. Squamous contamination, especially in urines from
female subjects, may obscure urothelial cells. If there are only a few atypical cells
present, then a targeted FISH approach, using the same Papanicolaou stained prepa-
ration as the cytology slide, is advisable.
Imaging and Automation of UroVysion® FISH Analysis
Advantages of an Automated Imaging System
Because manual FISH analysis has a relatively slow throughput and the fluorescent
signals fade over time [10], automated imaging systems are increasingly being uti-
lized, especially in institutions with high volumes of FISH tests. Reviewing repre-
sentative digitized images instead of a FISH slide at the microscope can lead to
dramatic savings in pathologist time [11]. Imaging systems may also be imple-
mented for improved productivity, quality control, archiving of images, and
increased accuracy. We estimate that almost 50% of the laboratories performing
U-FISH analysis in the United States use automated systems. In some labs there
was a combination of both manual and automated use. In non-US countries, auto-
mated imaging systems for U-FISH analysis are less commonly used.
Studies have shown a high concordance (>98%) between the manual method and
the automated method of reviewing cells. In several cases deemed negative by the
manual method, machine-assisted interpretation showed a positive result for abnor-
mal cells which was subsequently verified within a short interval by cystoscopy
[11]. In addition, the ability to enlarge cells with questionable signals and enhance
weak signals is advantageous in reducing false positives or false negatives.
Disadvantages of automation may include a higher number of unsatisfactory
specimens due to scant cellularity or clumping, in which case the manual system of
review is employed. Factors to consider before purchasing an imaging system
include financial cost, space requirements, information technology system needs or
modifications, validation of system, training programs for personnel, continuing
education, maintenance of system, workflow and patterns of reimbursement [11].
Details of Automated Imaging Systems
Imaging systems consist of an automated scanning microscope coupled with soft-

ware for image analysis. Some are approved or validated by the FDA for the detec-
tion, classification, and enumeration of urine specimen cells from individuals with
a history of urothelial carcinoma probed by the U-FISH Kit. Imaging systems that
have been approved by the FDA include the Duet TM System™ (Bio View, LTD,
Billerica, MA) (Figs. 9.3 and 9.4) and the Ikoniscope oncoFISH Bladder Test
Fig. 9.3 Automated scanning instrument showing workstation with concurrent acquisition of
DAPI images and FISH images illustrated on screen
Fig. 9.4 Urine from a patient with a history of high-grade urothelial carcinoma. Histogram of
U-FISH abnormal cells showing 14 out of 25 cells with polysomy for at least 2 chromosomes per
cell. This is an abnormal result and consistent with high-grade urothelial carcinoma. Although a
total of 99 cells were scored, reporting is based on 25 of the most morphologically abnormal
cells scored
System (Ikonsys, Inc., New Haven, CT). Technical details of these imaging systems
are described in the Appendix. Automated systems allow for interactive rejection or
acceptance of cells, so that overlapping cells or degenerated cells can be rejected
[12]. The imaged cells are then formatted in a gallery on a screen for review. Normal
and abnormal cells can be segregated and then can be reclassified as needed [11].
Extraneous cells such as neutrophils and lymphocytes are automatically excluded
from analysis.
Target FISH
FISH may also be performed on Papanicolaou-stained monolayer preparations that

have been pre-scanned by the imaging system with atypical urothelial cells selected
by the operator (Fig. 9.5). The slide is subsequently destained with acid alcohol and
hybridized with the four-color probe set. The hybridized cells are then matched in
perfect registration with the pre-selected cells so that these same cells can be ana-
lyzed for abnormal FISH signals. Such “Target FISH” has been shown to be more
accurate than conventional cytology [13]. In laboratories that do not have an auto-
mated imaging system in place, targeted U-FISH analysis of atypical urothelial cells
can also be achieved by usage of a standard fluorescence microscope equipped with
an automated stage and a camera allowing for interactive automated relocation and
imaging of representative cells for review and documentation [14, 15]. Target FISH
Fig. 9.5 (a–d) Examples of U-FISH and corresponding urinary cytology (Papanicolaou stain).
Chromosomes 3 in red, 7 in green, 17 in aqua, and 9p21 in gold. (a) Benign bi- or multi-nucleate
umbrella cell with pronounced reactive changes in a patient with irritative bladder (bladder wash-
ing). Highly increased number of all FISH signals due to repeated doubling of the genome (i.e.,
endoreplication), but no unbalanced copy number changes and no 9p21 deletion. No evidence of
urothelial carcinoma after 8-year follow-up. (b) Sheets of mildly atypical urothelial cells in a
washing of the renal pelvis. Normal FISH result (two copies per chromosome), consistent with
reactive changes. No histological follow-up. (c) Atypical urothelial cells suspicious of high-grade
urothelial carcinoma (SHGUC) in a washing of the renal pelvis. Positive U-FISH result with
increased copy numbers of all three chromosomes and a homozygous deletion of 9p21. Note the
normal cell nucleus with retained 9p21 signals as internal normal control (right lower corner).
Nephrectomy revealed noninvasive high-grade papillary urothelial carcinoma of the renal pelvis.
(d) U-FISH-positive atypical urothelial cells (SHGUC) with increased copy number of all five
chromosomes but no 9p21 deletion. Note the normal cell nucleus with normal copy numbers (right
lower corner). Histology revealed invasive high-grade papillary urothelial carcinoma (pT1) with
focal CIS
enables the reviewer to associate aneuploidy with cytologic features on a per cell
basis. This also provides an opportunity to the cytopathologists to improve their
skills in differentiating morphological features of benign/reactive and neoplastic
cells using the chromosomal status as a reference.
Reporting of Results
One or several pathologists can sign out cases by using remote computers situated
away from the main automated scanning microscope equipped with appropriate
software [11, 16]. In manual screening a minimum of 25 abnormal cells is evalu-
ated. In contrast, the automated systems require a minimum of 100 urothelial cells
without any chromosomal aberrations to be classified as normal [11]. Using either
method, only 25 of the most abnormal cells are reported.
Factors affecting the scanning performance include the cellularity, hybridization

efficiency, and cleanliness of the slide and coverslip. In addition, slides with marked
bacterial contamination [10] or obscuring inflammation are difficult to analyze. The
CAP requires photographic or digitized images for all FISH assays that should
include at least one cell image for assays with normal results and two cell images
for assays with abnormal results (images of at least two cells are required to docu-
ment all abnormalities). Images on all FISH assays testing for neoplastic conditions
must be retained for documentation for a minimum of 10 years (CAP requirement
CyG.43300). In addition, CAP requires pathologist and technologist participation in
proficiency testing [11].
Performance of UroVysion® FISH Testing
U-FISH testing of voided urine has been approved by the FDA as an aid for initial
diagnosis of bladder cancer in patients with hematuria and/or subsequent monitor-
ing for tumor recurrence in patients previously diagnosed with bladder cancer. In
routine practice, there are several additional situations in which U-FISH is fre-
quently used [14]. In a meta-analysis, the pooled sensitivity and specificity of FISH
were 72% and 83%, respectively, as compared with 42% and 96%, respectively, for
cytology [17]. However, there is a range of U-FISH results in the literature [9, 18–
21]. Like urine cytology, performance of FISH is better in patients with high-grade
UC. Analyzing these published results is challenging due to great variability of
critical parameters including type of material (voided urine versus bladder wash-
ing), clinical situation (history or no history of bladder cancer), concurrent cystos-
copy findings, length and type of follow-up, proportion of low-grade and high-grade
tumors, local experience in FISH analysis, and the definition of a positive result,
among others. This emphasizes the need for well-controlled and independently
funded studies to clarify the performance of FISH in clearly defined situations and
to analyze the cost-benefit ratio for both the laboratory and patient management.
UroVysion® FISH in Atypical Urinary Cytology
Atypical urinary cytology has emerged as the most rewarding application of U-FISH
analysis [7, 15, 21–30]. U-FISH is currently regarded as the most useful marker in
the setting of a negative cystoscopy and atypical cytology, according to the
International Consultations on Urological Diseases, the European Society of
Urology, and the Guidelines of the American Urological Association [1, 4]. In an
early pivotal study, U-FISH in 120 urine specimens revealed a sensitivity of 100%,
89%, and 60% in patients with suspicious, atypical, and negative cytology, respec-
tively, while the overall specificity was 97% [31]. This is in line with another study
showing that the sensitivity of U-FISH to detect UC in patients with atypical cytol-
ogy and equivocal or negative cystoscopy was 100% with a specificity ranging from
60% to 100% depending on whether or not there was a history of UC or a lesion at
cystoscopy [28]. The low positive predictive value in patients with a history of UC
and a negative cystoscopy in this study was explained by early (“anticipatory”)
detection of neoplastic cells that preceded the appearance of established cancer. In
fact, other studies have shown that such anticipatory positive U-FISH results predict
recurrence in patients under surveillance with atypical or suspicious cytology and a
negative cystoscopy [23, 29, 32]. Previous data on U-FISH results in conjunction
with atypical cytology samples are hampered by the inconsistent definitions and
variable prevalence of “atypia” in the pre-TPS era. In a recent study with prospec-
tively determined TPS categories, 40/90 (44%) of AUC cases were U-FISH posi-
tive, and 25/28 (89%) of these U-FISH-positive patients with follow-up were
diagnosed with UC [15].
Despite these promising data, performing U-FISH on a urine sample with atypi-
cal cytology at the time of a negative surveillance cystoscopy is not yet generally
recommended since the U-FISH result is still unlikely to change the currently estab-
lished surveillance strategies in non-muscle-invasive UC.
U-FISH is particularly helpful in the notoriously difficult construct of atypical
cytology after intravesical bacillus Calmette-Guerin (BCG) treatment of high-grade
non-muscle-invasive UC [1, 27, 33–35]. Patients with a positive post-BCG cytology
(HGUC) or FISH result have a substantially higher risk of recurrence when com-
pared to the patients with negative results (NHGUC) [27, 36, 37]. In a recent meta-
analysis, the preferred timing of post-BCG U-FISH analysis was proposed at
3 months after initial transurethral resection of non-muscle-invasive bladder cancer
[37]. Washing cytologies of the upper urinary tract (UUT) can also be challenging
due to instrumentation-related changes that might lead to false-positive cytology
results. On the other hand, accurate diagnosis of UUT UC is critical to avoid delay
of diagnosis. Published reports suggest that U-FISH also appears as a helpful tool to
increase the sensitivity for the detection of UC of the UUT [14, 38–41]. In our expe-
rience, FISH is very helpful in atypical UUT washing cytology. However, one needs
to be familiar with tetraploid U-FISH patterns that may occur in reactive conditions,
e.g., renal calculi, in order to avoid false-positive FISH diagnoses (see Pitfalls of
UroVysion® FISH Analysis). Examples of U-FISH in atypical urinary cytology are
shown in Fig. 9.5.
Change of TPS Category after U-FISH Analysis
Clinically useful management guidance can be achieved by harmonizing the

U-FISH and cytology results into one unified TPS report, as shown in examples at
the end of this chapter. For instance, an initial AUC can be switched to NHGUC in
case of a negative U-FISH result (especially if target FISH specifically interrogates
the AUC in question), since HGUC virtually always has abnormal numbers of chro-
mosomes. Conversely, a U-FISH-positive result justifies changing SHGUC to
HGUC. Harmonizing AUC with a positive U-FISH result is controversial. Although
an unequivocally positive U-FISH result in case of AUC is virtually diagnostic of
urothelial neoplasia, there is no appropriate TPS category covering this situation.
Therefore, a new category “urothelial neoplasia diagnosed by U-FISH, not other-

wise specified (UNF-NOS)” has recently been proposed [15]. Depending on the
degree of cytological atypia and the patient’s disease history, a change from AUC to
SHGUC is another option. Sufficient expertise in targeted U-FISH analysis for pre-
cise correlation with the atypical cells is a precondition for reassigning AUC based
on U-FISH.
Cost-Effectiveness of UroVysion® FISH
There have been concerns about the cost-effectiveness of the relatively expensive
U-FISH testing, mainly because of the low positive predictive value. If applied to
unselected patients with hematuria (in whom the prevalence of UC is about 13%)
[42] and recognizing the limited clinical value of enhanced detection of low-grade
UC, FISH results cost the patient a high price [14]. However, recent data suggest
cost-effectiveness of U-FISH in patients with atypical cytology and equivocal or
negative cystoscopy by avoiding unnecessary biopsies in patients with a negative
FISH result [22]. Decreasing anxiety for the patient and clinician is also beneficial.
Pitfalls in UroVysion® FISH Analysis
Despite the evident utility of FISH in atypical urinary cytology, there is a possibility
of false-positive results. Besides “anticipatory positive” FISH results, low specific-
ity and low PPV in some studies might also be due to an increased number of tetra-
ploid or dividing cells under reactive conditions. Although tetraploid cells are not
specifically mentioned in the scoring guidelines of the manufacturer, it has now
been recognized that rare cells with a tetraploid signal pattern (i.e., four signals of
each probe) should not be taken as an unequivocally FISH-positive result but inter-
preted with caution [8, 43–47]. In contrast, unbalanced numerical changes of one or
more chromosomes (e.g., 2, 3, 5) or loss of 9p21 is virtually specific for neoplasia
in bladder cytology. An exception is that pelvic irradiation (e.g., of the prostate or
the uterus) often results in permanent chromosomal aberrations carrying a risk of a
false-positive diagnosis by U-FISH. This is particularly the case for polysomies,
whereas homozygous or heterozygous deletion of 9p21 remains highly specific for
neoplasia even after irradiation. This emphasizes the need of appropriate clinical
information and consideration of the typical post-irradiation cytological changes.
Decoy cells, i.e., polyoma-infected urothelial cells, can easily mislead to a false-
positive cytological diagnosis of UC if one is not familiar with the morphological
spectrum of decoy cells. Although there have been reports on rare FISH-positive
decoy cells, most data suggest that polyomavirus infection does not interfere with
U-FISH results [48]. While rare, tumors other than UC involving the urinary tract
such as adenocarcinoma, small cell carcinoma, and renal cell carcinoma have been
found to have positive U-FISH results in urinary specimens [32].
Target FISH, in which there is cytological pre-screening of slides and cells in

question prior to FISH, has been proposed as another way of making the FISH
analysis more precise and specific. Pre-screening avoids the analysis of obviously
reactive bystander cells or the analysis of remaining material that might be devoid
of the atypical cells and therefore not representative of the original slide [12, 15, 43,
44]. This approach requires hybridization and scoring of the original stained slides
and automated relocalization of the cells in question, ideally coupled with image-
aided interpretation.
FDA-Approved Liquid-Based Tests
Liquid- or non-cell-based tests can be applied to voided urine samples either with-
out the need for further preparation or on sediments of centrifuged urine samples.
The two most commonly applied liquid-based urine tests on pure voided urine sam-
ples are the BTA™ (Bladder Tumor Antigen) (Polymedco Inc., Cortlandt Manor,
NY, USA) and the NMP22™ (Nuclear Matrix Protein) BladderChek test (Alere
Inc., Waltham, MA, USA), both approved by the FDA, both for detection of UC in
symptomatic patients and for monitoring of patients with a history of UC. For both
tests, a qualitative point-of-care test (BTA stat™ and NMP22-BladderChek™) and
a quantitative version (NMP22™ and BTA TRAK ™) are available. The latter tests
require a dedicated laboratory with specialized personnel. The advantage of a point-
of-care test is that it gives an immediate result just prior to cystoscopy. A positive
urine test may lead to a diagnostic bias, i.e., with the knowledge of a positive urine
test result, the urologist may be more likely to detect a bladder neoplasm at cystos-
copy [49]. These tests have been developed for both voided urine samples (not first-
morning urine) and catheter-collected urines but not for barbotage fluids.
BTA Test
The bladder tumor antigen (BTA) is a complement factor H-related protein secreted
by malignant cells, which was shown to be elevated in urine samples of patients
with bladder cancer [50]. There are two approved versions of this test for bladder
cancer follow-up in concurrent use with cystoscopy: the quantitative ELISA test
BTA TRAK and the qualitative point-of-care test BTA stat (Polymedco Inc.,
Cortlandt Manor, New York, USA) [51]. For the BTA™ test, the urine should be
collected without preservatives or fixatives in a clean urine cup and labeled
appropriately.
In different reviews and meta-analysis, the BTA stat has an overall sensitivity
and specificity of 64% (range 58–69%) and 77% (range 73–81%), respectively [51].
The specificity of the BTA™ tests may be overrated by some studies by their exclu-
sion of patients with urinary bladder infection and renal or bladder stones. The BTA
TRAK™ test has an overall sensitivity of 65% (range 54–75%) and specificity of
74% (range 64–82%) respectively [51].
A systematic review [52] demonstrated how bladder cancer patient selection

may influence in particular the sensitivity of studied biomarkers. In one review, the
BTA stat™ test had a 12% lower sensitivity when studies included in the review
were restricted to only those with patients under surveillance (sensitivity 58%), as
compared to reviews lacking this selection criterion (70%). For the BTA TRAK™
test, the sensitivity was minimally influenced by this selection (sensitivity 71% and
69%, respectively). The specificity of the BTA stat™ and BTA TRAK™ test was
similar in patients under surveillance (73% for BTA stat™ and 66% for BTA
TRAK™) compared with unselected populations (75% for BTA stat™ and 65% for
BTA TRAK™). In healthy persons without genitourinary signs or symptoms, the
specificity of BTA stat is 97%, but in patients with benign genitourinary conditions,
the specificity is only 46% [53].
In conclusion, the BTA stat™ and BTA TRAK™ tests are approved by the FDA
to detect bladder cancer in symptomatic patients and for surveillance of patients
with UC. These tests have a higher sensitivity than cytology, but like NMP22, the
BTA assays suffer from a higher false-positive rate in patients with benign urinary
tract diseases which limits their use in clinical practice [54].
The NMP22 Test
Nuclear matrix proteins (NMPs) are a family of nuclear structural proteins [55].
Several of them are overexpressed in UC, like NMP22, and released into the urine
upon apoptosis of tumor cells. However, elevated NMP22 levels may correspond to
the cellularity and cell turnover in the bladder rather than representing a tumor-
specific protein [56]. Both quantitative ELISA NMP22 Test Kit for bladder cancer
and qualitative NMP22 BladderChek point-of-care (POC) tests (Matritech, Newton,
Massachusetts) have been approved by the US FDA, the first one in 1996 and the
second one in 2002 [57]. They are used in the context of diagnosis and monitoring
for bladder cancer recurrence. The test should not be performed on voided urine
samples obtained within 5 days after instrumentation of the urinary bladder as this
may evoke wound repair potentially resulting in a false-positive result. Variation has
been reported among studies with some very low sensitivity values [54]. Moreover,
false-positive results of the NMP22 tests are more common in patients with benign
bladder conditions such as infection, stones, inflammation, and hematuria that need
to be distinguished clinically from bladder cancer [54].
Other Liquid-Based Biomarkers
A wide variety of other potential ancillary tests have emerged based on protein
detection, microRNAs, gene expression profiling, epigenetic changes, and PCR-
based detection. A few of these tests are described in this section, and Table 9.1
summarizes their performance [58–62].
The ADXBladder test (Arquer Diagnostics, Ltd., Sutherland, United Kingdom)

is a recently developed ELISA test based on the detection of the proliferation-
associated marker minichromosome maintenance protein 5 (MCM5). Its sensitivity
and negative predictive value to detect UC recurrences in voided urines during fol-
low-up of non-muscle-invasive bladder cancer exceed that of cytology in two stud-
ies [63, 64], but its specificity is lower (Table 9.1). Nevertheless, its performance
characteristics in this setting are somewhat better than the FDA-approved BTA and
NMP22 tests [54]. A drawback is that inflammatory and reactive conditions also
lower its specificity.
Table 9.1 Performance characteristics of selected ancillary tests in comparison with cytology for
bladder cancer monitoring. Methodological differences in the studies make it difficult to compare
sensitivity, specificity, and negative predictive value (NPV)
Test Target molecule Sensitivity Specificity NPV Comment
Cytology [54, NA 34–42 96–99 68–99 Performance depends
89–91] on grade of
UC. Interlaboratory
variation
UroVysion Chromosome 3, 72 83 – Performance is lower
FISHa [17] 7, 17 in low-grade UC
enumeration
and 9p21 locus
BTA stat and Human 65 74 – Performance is lower
TRAKa [51, 92] complement in low-grade
factor H-related UC. Lower specificity
protein in benign non-
neoplastic urologic
disorders
NMP22a [47, 54] Nuclear matrix 52–59 87–89 – Variation in
protein performance between
studies. False positives
in patients with
non-neoplastic
urologic diseases
ADXBLADDER Single protein 45–88 70–73 74–100 ELISA test, adversely
[54] (MCM5) affected by
inflammation or
instrumentation
CxBladder mRNA (5 gene 93–95 96–97 Not influenced by
Monitor [59] expression previous BCG therapy
[93] markers
combined with
2 clinical
features)
(continued)
Table 9.1 (continued)
Test Target molecule Sensitivity Specificity NPV Comment
Bladder Methylated 62–95 82–88 79–99 Not influenced by
EpiCheck [54, DNA (15 inflammation or
68, 94] markers) hematuria. Lower
sensitivity for
low-grade UC
compared to
high-grade UC
Uromonitor-V2a PCR DNA (3 73–93 86 95 Not influenced by
[75, 76] markers; TERT, inflammation.
FGFR3 and Roughly equivalent
KRAS) performance for
low-grade and
high-grade UC
uCAPP-Seq [87] NGS-based test 84 96 – Somewhat better
assessing 20 sensitivity for
mutations high-grade UC
compared to
low-grade UC
AssureMDx [95] NGS-based 93 86 – Better test
test, assessing performance for
mutations in high-grade than
TERT, FGFR3, low-grade UC.
HRAS, and
DNA
methylation of
OTX1,
ONECUT2, and
TWIST1
UroSeek [81, 96] NGS-based 83 99 – Complementary to
test, mutations cytology. Equivalent
in 11 genes, performance for
and detection of low-grade and
aneuploidy high-grade UC. Lower
sensitivity in a cohort
followed for
recurrence
FDA-approved tests
a
CxBladder Monitor (Pacific Edge Diagnostics, Dunedin, New Zealand) is a test

based on the expression profile of five genes assessed by quantitative reverse-
transcription PCR in combination with two simple clinical parameters: [1] whether
the previous tumor was primary or recurrent and [2] the time since the previous
tumor was resected. The test derives sensitivity and specificity by including mRNAs
that are specific for urothelial carcinoma cells as well as mRNAs specific to inflam-
matory cells. Earlier studies determined the proprietary algorithm for an optimal
cut-off, and subsequent validation studies showed a considerably higher sensitivity

and somewhat improved negative predictive value as compared to cytology, NMP22,
BladderChek, and ELISA for detection of bladder cancer recurrences [59]. BCG
treatment effects do not cause false-positive results, a major advantage of CxBladder
Monitor.
At least 98.5% of UC have detectable alterations in the genome as an underlying
abnormality [65]. There are two types of heritable genomic changes in UC: changes
in the DNA sequence itself (i.e., mutations) and “epigenetic” changes that are heri-
table between cell divisions. A common epigenetic change in UC is alteration in the
methylation of the cytosine base of DNA that heritably alters expression of a gene.
Hypermethylation of promoter islands of tumor suppressor genes in particular
occurs early in bladder carcinogenesis [66] and shows specificity for bladder cancer
progression [67]. EpiCheck (Nucleix, Rehovot, Israel) is a commercially available
test assessing the methylation of 15 genes. Its detection kit makes use of a methyl-
ation-sensitive restriction enzyme treatment (which cleaves and eliminates non-
methylated sequences from normal cells) followed by real-time quantitative
PCR. Based on a proprietary algorithm, a score is calculated ranging from 0 to 100,
and a score ≥ 60 is considered positive. Its performance (Table 9.1) may exceed that
of cytology, BTA stat, NMP 22, and U-FISH [68]. A drawback to its routine employ-
ment in UC patient follow-up is the need for a dedicated molecular laboratory to
perform real-time PCR. It also has lower sensitivity for low-grade UC compared to
high-grade UC [69]. One study, comparing cytology using TPS terminology and
EpiCheck testing in patients followed for their UC, reported that EpiCheck would
have value only within diagnostic categories of AUC and SHGUC. For urine cytol-
ogy cases reported as NHGUC or positive for HGUC, the EpiCheck test was not
helpful in identifying patients with recurrence of high-grade UC [70].
Mutations in the promoter region of the TERT gene are an early event in bladder
carcinogenesis, occurring in up to 80% of bladder cancers, irrespective of grade or
stage [71, 72]. In addition, FGFR3 gain-of-function point mutations occur in about
70% of low-grade UCs [73], and mutations in KRAS genes (mutually exclusive for
mutations in FGFR3) occur in about 13% of bladder cancers [74]. The commer-
cially available Uromonitor-V2® test, which has recently been approved by the
FDA, is a highly sensitive real-time quantitative multiplex PCR test designed to
identify hotspots in the TERT, FGFR3, and KRAS genes; presence of any mutation
is considered a positive test result. The test has a reported analytical sensitivity of
0.7%, or the theoretical ability to detect about 1% of malignant cells in a sample. In
two studies its performance exceeded that of cytology [75, 76]. Larger preferably
prospective studies are needed to further validate the potential of this urine test in
the context of UC monitoring. Other tests, combining FGFR3 mutation analysis
with methylation markers [77], e.g., the Urodiag® PCR kit [78], might further
improve the performance characteristics of molecular tests on voided urine.
Ancillary Tests Based on Next-Generation Sequencing Technology
Next-generation sequencing (NGS) is a technique for acquiring a DNA sequence

from each starting molecule of DNA in a sample. Many different mutations can be
present in different cases of UC, and by permitting the identification of multiple
mutations simultaneously, NGS offers the potential for a sensitive assay detecting
all the genetic varieties of UC.
NGS also helps to solve an important technical challenge of ancillary testing: the
ability to detect rare tumor cells among an excess of normal cells. Cytologists are
well-aware that urine samples may contain an overwhelming number of inflamma-
tory cells and benign urothelial or squamous cells. PCR-based tests can be designed
to specifically amplify mutant alleles (e.g., by restriction enzyme digestion of non-
mutant alleles), but development of PCR-based assays based on simultaneous detec-
tion of multiple different potential targets is much more challenging.
The ability to detect rare mutations is dependent on the proportion of tumor cells
and the number of independent sequences that are obtained. To detect a mutated
gene when the tumor cells comprise only 1% of the sample, at least 200 sequences
(when each cell has two alleles of the gene) are necessary. In fact, many more times
this number of sequences (or “reads”) are needed to account for the probability that
a sample of DNA molecules will contain the mutated gene and to be able to get at
least a few independent reads of the mutation to verify that it is not an artifact.
The reagent costs for NGS have dramatically decreased, though the full cost of
NGS includes DNA isolation, library production, technician time, computational
costs, and the NGS machine itself. The lower costs have recently made it economi-
cally feasible to directly assay for many mutations simultaneously with an analyti-
cal sensitivity of about 1% tumor cells. A number of potential NGS-based ancillary
tests for bladder cancer detection have emerged, though there are not yet sufficient
prospective studies for any to be FDA-approved or accepted by the major interna-
tional urologic societies [79].
Mutations, epigenetic alterations, and even copy number changes can be detected
by NGS. Epigenetic changes (DNA methylation) are detected by using a chemical
treatment (bisulfite) to convert the methylated cytosines to a nucleotide that is ulti-
mately sequenced as a thymidine. The locations of methylated cytosines can then be
deduced computationally. Copy number changes (which can represent aneuploidy)
are deduced based on the number of individual reads.
Different genetic changes are found in low-grade and high-grade UC, but overall
both low-grade and high-grade UC have a comparable high (>95%) prevalence of
genetic changes [80–82]. Thus, NGS-based assays have the potential to have high
sensitivity across the entire spectrum of UC. In contrast, FISH testing has lower
sensitivity for low-grade UC [14].
Another value of NGS ancillary tests is that particular mutations in various UCs
correlate usefully with clinical or therapeutic features [83]. For example, there are
mutations that correlate with grade and stage at presentation, risk of recurrence, and
response to therapy. In case of carcinoma in situ (CIS), urine samples may contain
a higher number and proportion of malignant cells than matched histologic biopsies
[84], which are often denuded due to exfoliation of CIS cells. Thus, NGS may be an
important ancillary test in patients with a positive urine cytology when searching for
genetic alterations that might predict the response to intravesical BCG treatment.
There are many approaches for detecting tumor cell mutations when the propor-
tion of tumor cells is relatively low. One approach is using cell-free DNA (DNA in
suspension or not associated with cells) within a urine sample. The appeal of cell-
free DNA is the expectation that tumor cells have a relatively higher turnover than
benign cells, such that fragments of their genome will be enriched in the fluid com-
pared to the genomes of normal cells. In fact, the performance of cellular DNA- and
cell-free DNA-based NGS assays has been comparable [82, 85].
For cell-based assays, microfluidic techniques have been developed attempting
to enrich tumor cells based on physical properties or surface antigens. Initial posi-
tive results using microfluidics to enrich tumor cell DNA have not been validated
prospectively [86]. Cytologists are aware that UC cells can have a wide variety of
physical shapes and sizes, making microfluidic approaches seem difficult.
Enrichment of tumor DNA can also be accomplished through genetic techniques.
For example, uCAPP-Seq enriches mutant alleles by pre-hybridizing to immobi-
lized probes that recognize the mutation [87]. When applied to cell-free urine sam-
ples, uCAPP-Seq had a sensitivity of 84% when blinded to tumor mutation status
[87]. Laser capture microdissection of individual cells, as guided by cytologic
assessment, has been proposed as another means of enriching tumor cells or exclud-
ing obviously normal cells [83]. This “cytologically targeted NGS” would ulti-
mately allow morphologic features to be correlated on a per cell basis—analogous
to target FISH.
At least 25 publications of ancillary NGS using a wide variety of platforms have
been published [79]. The performances of the various studies are difficult to com-
pare since prevalence of disease in different studies varies widely, and some studies
only assessed urine samples in which there were known mutations in a bladder
tumor sample from the same patient. Table 9.1 lists selected ancillary tests in rela-
tion to conventional cytology.
Conclusions
The existing evidence strongly points towards a utility of U-FISH in the setting of
atypical urinary cytology. Since the definition of “atypia” and its boundaries with
“suspicious” have been variable in the past, further studies are needed to better
determine the performance of UroVysion® FISH and other ancillary tests in the
more stringently and precisely defined AUC and SHGUC categories of TPS. Other
urine-based biomarkers for diagnosis and follow-up of urothelial neoplasia have
limited value for the time being, and many require further validation. Liquid-based
tests done outside of the cytology laboratory such as BTA and NMP22 tests have a
sensitivity comparable to that of cytology for high-grade tumors and are superior for
the clinically less important low-grade tumors. Although urine marker-guided fol-
low-up of patients with bladder cancer appears feasible, studies proving the efficacy
of this concept and demonstrating an added value for patients or the health system
are lacking. Therefore, based on current levels of evidence, systematic marker-
guided follow-up is still not recommended [1, 2, 4]. However, this might change in
the near future given the promising results of new molecular tests. According to the
current EAU guidelines, diagnostic markers might be most useful in patients with
negative cystoscopy and UUT work-up to identify patients at increased risk of dis-
ease recurrence and possibly progression [88]. Until there are new international
consensus recommendations on ancillary testing in urinary cytology, U-FISH
should best be used judiciously on the basis of cytological findings in an individual
case after prior consultation with the treating physicians or as a reflex test. For the
future, assessing multiple biomarkers simultaneously, for example, by using next-
generation sequencing, is likely to be most sensitive, therefore having a sufficiently
high negative predictive value to help avoid unnecessary cystoscopies. There are
several opportunities for future studies to specify the role of ancillary testing in
urinary cytology. Well-controlled and independently funded studies are needed to
clarify the performance of U-FISH and other tests in clearly defined situations and
to analyze the cost-benefit ratio for both the laboratory and patient management. For
example, there is still insufficient clinical evidence to change the currently estab-
lished surveillance strategies in non-muscle-invasive UC based on U-FISH or other
tests in urines with atypical cytology at the time of a negative surveillance cystos-
copy. It also remains to be studied to what degree targeted U-FISH of atypical or
suspicious cytology after automated relocation can help the cytopathologists to
improve their skills in differentiating morphological features of benign/reactive and
neoplastic cells using the chromosomal status as a reference. Whether U-FISH or
other molecular test results should be integrated in new TPS categories will be a
subject of future discussions.
Sample Reports
he Influence of UroVysion FISH on the Interpretation

T
of Cytological Findings in Urinary Samples
Example 1 (AUC, FISH Negative)
Renal pelvis (washing): Several clusters of atypical urothelial cells of unknown

significance.
TPS category: AUC.
Results of UroVysion FISH Testing (Chromosomes 3, 7, 17, and 9p21): Negative.
Final diagnosis (cytology & FISH): NHGUC.
Note: the absence of chromosomal aberrations is in favor of reactive urothelial
changes.
However, a low-grade urothelial lesion cannot be excluded with certainty, since
20–30% of these are negative by this FISH test.
Example 2 (SHGUC after BCG, FISH Positive)
Bladder (washing): Numerous atypical urothelial cells, suspicious of high-grade

urothelial carcinoma.
TPS category: SHGUC.
Results of UroVysion FISH Testing (Chromosomes 3, 7, 17, and 9p21): Positive
(22/25 cells).
Final diagnosis (cytology & FISH): HGUC.
Note: Almost all atypical cells that were scored revealed pronounced chromo-
somal aberrations including unbalanced polysomy of the chromosomes 3, 7, and 17
and a relative loss of 9p21. This positive result confirms a diagnosis of high-grade
urothelial carcinoma (at least carcinoma in situ).
xample 3 (AUC in Bladder Washing from Negative Surveillance

E
Cystoscopy, FISH Positive)
Bladder (washing): Monotonous population of mildly atypical cells, cannot exclude

urothelial low-grade lesion.
TPS category: NHGUC.
(18/25 cells).
Final diagnosis (cytology & FISH): Consistent with low-grade urothelial neo-
plasm (LGUN).
Note: The majority of atypical cells that were scored revealed chromosomal
aberrations including polysomy of the chromosomes 3, 7, and 17 (3–4 signals
instead of 2 signals per chromosome) and a loss of 9p21 (1–2 signals). This positive
test result is consistent with a low-grade urothelial lesion.
Example 4 (AUC in Voided Urine, FISH Positive)
Voided urine: Several degenerated urothelial cells with nuclear atypia of uncertain
significance.
TPS category: AUC.
(10/25 cells).
Final diagnosis (cytology & FISH): Consistent with urothelial neoplasia
(see note).
Note: Ten of the 25 atypical cells that were scored revealed unequivocal chromo-
somal aberrations including polysomy of chromosomes 3 and 17 and homozygous
deletion of 9p21. This positive test result is in favor of urothelial neoplasia. A dis-
tinction between low-grade and high-grade lesion is not possible. We advise further
investigations.
Example 5 (AUC in Bladder Washing, Tetraploid FISH Pattern)
Bladder washing: Several urothelial cells with nuclear abnormalities of unknown

significance favor reactive changes.
TPS category: AUC.
Results of UroVysion FISH Testing (Chromosomes 3, 7, 17, and 9p21):
Several urothelial cells with a tetraploid chromosomal pattern (6/25).
Final diagnosis (cytology & FISH): NHGUC (see note).
Note: FISH analysis revealed several cells with a tetraploid pattern (four instead
of two signals of each chromosome) but no other abnormalities. Rare tetraploid
cells are often found in reactive conditions and are unlikely to point towards urothe-
lial neoplasia. This test result is consistent with reactive urothelial changes.
Appendix
UroVysion® Assay
The slide pretreatment with protease uncovers target DNA and is recommended in
Pap-stained specimens. Decolorization is not mandatory since the stain is removed
during further phases of FISH procedure. If using archival slides, remove the cover-
slip and mounting medium in xylene. Place the slides in 1% acid alcohol (HCL and
70% alcohol) overnight or until decolorized. The U-FISH assay can subsequently
be conducted either manually or automatically. The first step is denaturation of
specimen DNA to expose single-stranded target DNA. U-FISH probes should be
prepared accordingly and applied to the selected area of the slide. The area should
be coverslipped and sealed immediately to ensure optimal conditions. Hybridization
of probes to target DNA sequences follows under appropriate conditions. The pro-
cedure is finished with post-hybridization washes to remove excessive probes.
Slides should be dried in a dark area. The exact procedure of the FISH assay is
described in the UroVysion kit datasheet. The procedure should be validated in each
individual laboratory, together with positive and negative controls, to ensure opti-
mal hybridization. Afterwards the specimen chosen for analysis is stained by DAPI
solution. Slides are coverslipped and stored at −20 °C in the dark until analysis.
Automated Imaging Systems for UroVysion® FISH Analysis
The Duet TM System™ workstation integrates a microscope, CCD camera, motor-

ized stage or slide-loader, computer, keyboard, mouse, joystick, monitor, and a
dedicated software program. Up to 200 slides that have undergone the FISH proce-
dure can be loaded and run overnight for inspection the following day. This latter
feature may be suitable for diagnostic laboratories that receive high volumes of
abnormal or atypical urines. Similarly, the Ikoniscope oncoFISH Bladder Test
System has an automated scanning microscope system coupled with an image
analysis work station. It features automated slide loading and handling, low and
high magnification scanning to identify cells of interest, and digital image acquisi-
tion [10]. The MetaSystems uses an automated fluorescent scanning microscope
and analysis software with “tile-sampling” method [16].
The BioView Duet System™ scans cells that are imaged at high resolution
(under oil immersion) both in bright light illumination and in fluorescent illumina-
tion. Cells are classified by the system according to their morphological features,
according to their staining on bright field (Giemsa or Papanicolaou stains, if target
FISH is used), and according to the pattern of fluorescent signals. The automated
microscope has micrometer-level precision in the X, Y, and Z axes which allows it
to focus on cells and retain coordinate information for target cells. There are two
modes of operation: [1] automatic scanning, which provides a gallery of all fields of
view, and [2] manual scanning, which provides interactive control allowing the user
to select the fields of view using either bright-field or fluorescent illumination.
Similar to manual scoring, the automated system scans the FISH slides by locat-
ing and scoring the nuclei exhibiting abnormalities such as enlargement, irregular
borders, and patchy DAPI staining. As identification of abnormal or malignant cells
based solely on aberrant morphology may be misleading, the BioView System™
classifies cells both by morphology on the DAPI fluorescence and by superimposed
FISH signals. Cells are ranked based on a combination of nuclear features, includ-
ing size, shape, DAPI intensity, and DAPI standard deviation inside the nucleus.
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Cytopreparatory Techniques
10
Donna K. Russell, Willam N. Crabtree, and Gary W. Gill
• Detailed preparation techniques for urine cytology

• Additional images/graphics of cytologic preparation types
• Cell block preparation techniques
• Standard Operation Procedures (SOPS) for urine preparation
• Additional/updated references
Background
Regardless of the source of cells or reporting system, the role of cytopreparation in

reliable diagnostic results must be recognized and explicitly detailed because speci-
men processing can affect the cytomorphological findings at least as much as the
underlying biology of the cells or the expertise of the cytologist. Cytopreparation is
a combination of methods for optimizing and standardizing the collection, prepara-
tion, and analysis of cytologic samples in ways that promote the detection of cells
of interest and accurate interpretation of nuclear morphology.
For these reasons, laboratories are wise to invest in the valuable technical skillset
known as cytopreparation. This practice involves the analysis of each step, device,
method, and reagents that the cells encounter between removal from the body and
morphological analysis at the microscope. Collectively, cytopreparatory techniques
that a lab uses for processing a specimen constitute a standard operating procedure
(SOP), each one with its own applicable terms of cellular preservation and flatten-
ing, chromatin distribution and particle size, biochemical makeup, stainability,
interaction with light, and cost. Laboratorians must often balance the trade-offs of
all the steps required by the device, preparation methods, and reagents in order to
arrive at a SOP that best matches the laboratory’s clinical needs and financial
constraints.

https://doi.org/10.1007/978-3-030-88686-8_10
222 D. K. Russell et al.
Specimen Collection
In terms of cellularity and preservation, there is a trade-off between the invasive-

ness of the clinical procedure and the cellular yield: bladder washings are best,
catheterized urines next, and voided specimens are third. The collection method
should not impact the processing method unless the volume of instrumented spec-
imens is artificially high. Chapter 2 discusses the impact of collection techniques
on adequacy.
Assessment
Gross examination of the fluid is noted upon receipt in the cytopathology laboratory.
Physical features such as volume of sample, color, clarity, opalescence, and viscos-
ity are then documented in the cytopathology report [1]. If the cell sample is low in
volume, is clear in appearance, and contains only a few cells, the method of prepara-
tion should include a concentrating cytocentrifugation or a liquid-based preparation
method. The method of cytopreparation may be selected on the basis of the physical
properties of the specimen and the patient’s clinical history. For example, highly
viscous specimens may require a smear technique or mucolytic agents, and bloody
specimens will benefit from methods that segregate epithelial cells from red blood
cells. Highly cellular specimens are ideal for smeared preparations, whereas sparsely
cellular specimens will require multiple centrifugation steps and special cell con-
solidation processing.
Processing
A collection method should harvest well-preserved cells that reliably represent any
urinary tract lesion that might be present. As urine itself may degrade cells that have
been sitting in the bladder for extended periods of time, cells expelled during the
first void of the morning are less than optimal; it is best to discard the initial voided
urine and use the next voided fresh urine after the patient drinks 8–16 oz. of water.
Clinically the laboratory usually receives sterile, pyrogen-free balanced salt solu-
tions from specimens associated with bladder instrumentation. Normal saline (i.e.,
0.9% NaCl w/v) is discouraged [2] as it may ruin cell morphology [3]. Saline may
be isotonic, but it is not physiologic.
Ideally, freshly collected urine specimens should be processed promptly after
collection. Cells in urine specimens may be degenerated at the time of collection,
and no amount of preservative can save them and may even increase cellular degen-
eration [4, 5]. Process urine specimens within about 4 hours, and if this is not fea-
sible, specimens may be refrigerated for up to 12 hours without noticeable effects
on specimen quality. However, refrigeration accelerates the precipitation of salts
in urines.
10 Cytopreparatory Techniques 223
Preparation Methods
Accurate diagnostic interpretation is not possible without optimal specimen prepa-

ration. Preparation types include liquid-based preparations, cytocentrifugation
preparations, conventional direct smears, and cell blocks. In a recent survey per-
formed by the College of American Pathologists, of the 738 laboratories that
responded to the question regarding the urinary tract specimen types processed in
their laboratories, 735 (99.6%) reported processing voided urine specimens, 653
(88.5%) reported processing catheterized urine, and 639 (86.6%) reported process-
ing bladder washing specimens [6].
The most commonly used processing methods were ThinPrep (424/739; 57.4%)
and Cytospin preparations (336/739; 45.5%). SurePath was performed in only 49 of
739 laboratories (6.6%). In addition, 202 of 739 laboratories (27.3%) reported using
cell block preparations; of these, 197 gave the percentage of the urinary cytology
cases for which a cell block was prepared. Cell block preparations were performed
for all cases by 15.7% (31/197) of laboratories; 44.7% (88/197) performed cell
blocks for fewer than 10% of the cases; and the remaining 40% (78 of 197) per-
formed cell blocks as deemed appropriate [6].
Following good principles and practices of cytopreparation, all processing meth-
ods will produce satisfactory results [4]. An international survey conducted in antic-
ipation of TPS 2.0, supported by the American Society of Cytopathology (ASC) and
the International Academy of Cytology (IAC) involving 55 countries, revealed prac-
tice patterns in urinary cytology. The most common types of urine fluid preparation
were specimens processed by Hologic ThinPrep® (44.4%), Thermo Scientific
Cytospin® (37.3%), BD SurePath® (8.8%), Direct smear (8.1%), and Millipore®
filtration (0.7%) [7] (Fig. 10.1). If the sample is cloudy and has a cell pellet after
centrifugation, preparation methods may include a cell block method.
8.14%
44.41%
37.29%
8.81%
Direct Smear (conventional) CytoSpin® Millipore® SurePath® ThinPrep®
Other
Fig. 10.1 Paris 2.0 International survey of urine preparation types

If a physician requests fluorescence in situ hybridization (FISH), whether reflex-

ive, i.e., if atypical urothelial cells are present, or regardless of interpretation, the
specimen is prepared using liquid-based processing techniques and is sent to a cyto-
genetics laboratory for FISH testing. All fixed urine specimens are suitable for FISH
analysis for one week at room temperature. Refer to Chap. 9 for ancillary testing
procedures for FISH.
Liquid Based Preparation
ThinPrep® Liquid-Based Preparation

(Hologic, Inc., Marlborough, MA, USA)
The specimen is collected fresh or with added preservative and concentrated by
conventional centrifugation. A SOP of the centrifugation process is found in the
laboratory procedure manual (Appendix A). Centrifugation of the urine sample is
performed after gross description and electronic accessioning is performed.
Samples are poured into one or two 50 mL conical tubes and capped. Exact
amounts are poured to balance the centrifuge. Conical tubes are placed in the
centrifuge and spun for 2500 rpm for 5 minutes. If the specimen is bloody, 30 mL
of CytoLyt™ solution can be added after initial spinning to lyse the blood. After
mixing, the sample can be re-centrifuged at 2500 rpm for 5 additional minutes.
Tubes are removed from the centrifuge, and the supernatant is decanted. A pipette
is used to resuspend the sediment. Three to five drops are added to the PreservCyt™
manufacturer’s non-gynecologic vial (Fig. 10.2). The non-gynecologic
PreservCyt™vial is labeled with the specimen number and patient name. The cap
is removed from the vial which is placed on the ThinPrep® 2000 processor for
specimen processing, producing a liquid-based preparation ready for Papanicolaou
staining (Fig. 10.3). A gentle dispersion step breaks up blood, mucus, and nondi-
agnostic debris and thoroughly mixes the cell sample. The cells are then collected
on a ThinPrep Filter specifically designed to collect diagnostic cells using the
ThinPrep 2000 Processor. The device constantly monitors the rate of flow through
the ThinPrep Filter during the collection process in order to prevent the cellular
presentation from being too scant or too dense. A thin layer of cells is then roboti-
cally transferred to a glass slide in a 20-mm-diameter circle, and the slide is auto-
matically deposited into a fixative solution.
The ThinPrep® 5000 processor (Fig. 10.4) delivers a fully automated slide prep-
aration option for non-gynecologic urine samples. It is a continuous hands-free pro-
cessor and prepares up to 20 samples per batch, which allows 45 minutes of free
walk-away time for the cytopreparatory technologist. SOP for ThinPrep preparation
is documented in the laboratory procedure manual (Appendix B). (For exact details,
refer to manufacturer’s procedure manual.) [8, 9].
Fig. 10.2 ThinPrep®
liquid-based preparation
technique
1. Combine into
50 mL tube
2. Concentrate
by Centrifugation
3. Pour Off
Supernatant
and Resuspend
Cell Pellet
4. Add an Appropriate
Amount of Specimen
to the PreservCyt
Solution Vial
5. Run on ThinPrepTM
2000 Processor Using
Sequence 2 or
ThinPrepTM 5000
Processor Using
sequence Non-Gyn
Fig. 10.3 High-grade
urothelial carcinoma.
(ThinPrep® preparation,
voided urine, Papanicolaou
stain, high magnification)
Fig. 10.4 Hologic
ThinPrep® 5000®
Processor
Cytocentrifugation
Thermo Scientific™ Cytospin™ Cytocentrifuge

(Thermo Fisher Scientific, Waltham, MA, USA)
The specimen is collected fresh or with added preservative. Centrifugation of the
urine sample is performed after gross description and electronic accessioning is
performed. Samples are poured into 1–2 50 mL conical tubes and capped. Exact
amounts are poured to balance the centrifuge. Conical tubes are placed in the cen-
trifuge and spun at 2500 rpm for 5 minutes. Tubes are removed from the centrifuge,
and the supernatant is decanted. A pipette is used to resuspend the sediment.
Cytocentrifugation with the Cytospin deposits the cells onto a 6 mm circle on a
vertical slide while the fluid is removed by the surrounding absorbent paper. A slide
(label at the top) is placed in the clip with an absorbent paper on top and the plastic
funnel chamber on top of both. The Cytospin clip is closed by swinging the wire
toward the clip back and hooking it closed. The circle of the funnel and the slide
must match up exactly. Slide clips are placed in the centrifuge opposite one another
for balancing, and specimen is added to the chamber. Five to eight drops are placed
in the chamber for clear fluid and one to two drops for cloudy fluids (Fig. 10.5). The
sample chambers are capped, and the cytocentrifuge unit cover is closed (Fig. 10.6).
Specimens are spun at 800 rpm for 5 minutes.
Fig. 10.5 Thermo
Scientific™ Cytospin™
preparation technique
1. Combine into
a 50 mL tube
2. Concentrate
by Centrifugation
3. Pour Off
Supernatant
and Resuspend
Cell Pellet
4. Run on Shandon
Cytocentrifuge
Fig. 10.6 Thermo Scientific™ Cytospin™ instrument
Fig. 10.7 Negative for

high-grade urothelial
carcinoma. (Cytospin™
preparation, bladder
washing, Papanicolaou
The program will begin when “Start” is selected, and the unit will chime when
complete. Once complete, the top of the cytocentrifuge is removed, the slide clip
unit opened, and the paper and slide are removed. The slide is placed in 95% alcohol
and is stained with the Papanicolaou stain (Fig. 10.7). (For exact details, refer to
manufacturer’s procedure manual.) [10].
Direct Smear Preparation
Centrifugation of the urine sample is performed following gross description and

electronic accessioning. Sample types differ; they may be dilute or concentrated. If
the sample is dilute, consider a cytocentrifugation or liquid-based procedure.
Specimens that are concentrated and display a pellet upon centrifugation can be
prepared by the direct smear technique. Samples that are bloody can be centrifuged
with a lysing agent. A unique specimen number is attached to each urine sample.
Every specimen is prepared depending upon the quantity and physical characteris-
tics of fluid received. Samples are poured into 50 mL conical tubes and capped. To
balance the centrifuge, exact amounts of water and urine are poured into oppo-
site tubes.
Conical tubes are placed in the centrifuge and spun at 2500 rpm for 5 minutes.
The tube is removed from the centrifuge, and the supernatant is decanted. Two
slides are labeled with the patient’s last name, accession number, and type of urine
submitted. A pipette is used to take sediment remaining in the conical tube. A few
drops are placed on a labeled slide, and the second slide is used to smear the prepa-
ration. The slide is placed immediately in 95% ethanol, or a spray fixation may be
used prior to Papanicolaou staining (Fig. 10.8). Direct smear preparations often
exhibit considerable cell loss and should be avoided unless more optimal slide prep-
aration methods are unavailable. Spray fixative can mitigate cell loss by adhering
the fixed cells onto the slide.
Liquid Based Preparation
BD Totalys™ MultiProcessor and BD Totalys™ SlidePrep (SurePath®)

(Becton Dickinson and Company, Sparks, MD, USA)
BD Totalys™ SlidePrep prepares and stains non-gynecologic samples. It is a
fully automated slide preparation and staining system when used with the BD

carcinoma. (Direct smear,
bladder washing,
Papanicolaou stain, high
magnification)
Totalys™ MultiProcessor (Fig. 10.9). The multiprocessor is an automated sample

transfer, centrifugation, aspiration, and decanting system for cytology. It minimizes
the hands-on time for preparation. The sample is collected fresh or with added pre-
servative and is concentrated by centrifugation. The Laboratory Information System
Fig. 10.9 BD Totalys™

1. Combine into
50 mL tube
2. Automated sample transfer,

centrifugation, aspiration and
decanting for cytology preparation.
3. Run on the
BD TotalysTM
SlidePrep
(LIS) provides sample barcode identification to prepare for diagnostic systems. It

features an optional aliquot system that transfers specimen to secondary vials for
ancillary testing. The tubes are loaded onto the BD Totalys™ SlidePrep.
The system provides a full chain of custody by automatically scanning each slide
and matching it to the associated specimen. The system has flexibility; it allows for
both liquid-based Pap tests and non-gynecologic samples on the same system. A
graphical interface with touch screen takes users through each step and offers ran-
dom loading to reduce manual steps. It offers consistent results and stains individual
chambers using fresh reagents for each specimen and reduces the risk of contamina-
tion while ensuring sample match and chain of custody through automatic slide
barcode scanning (Fig. 10.10). A single run of 48 slides can be processed in 68 min-
utes and is completely automatic. It reduces cytopreparatory staff time to allow
performance of additional tasks in the laboratory. The SOP for BD Totalys™
SlidePrep preparation is available in the laboratory procedure manual (Appendix
C). A Papanicolaou-stained liquid-based SurePath slide is produced (Fig. 10.11).
The cells are presented within a 13 mm-diameter circle. (For exact details, refer to
manufacturer’s procedure manual.) [11, 12].
Fig. 10.10 BD Totalys™ SlidePrep instrument

urothelial carcinoma. (BD
Totalys™ SurePath®
preparation, bladder
washing, Papanicolaou
Millipore Filtration (EMD Millipore, Billerica, MA)
The specimen is collected fresh or with added preservative and concentrated by

conventional centrifugation. The supernatant is discarded, and the cell button is
resuspended by being vortexed in a few mL of balanced electrolyte solution. The
resuspended cells are processed by membrane filtration (i.e., filtered at 100 mmHg
negative pressure on a 5 μm pore size, 47 mm-diameter Millipore filter) that was
rinsed with balanced electrolyte solution and fixed in situ by adding 95% ethanol.
The cells are kept wet (i.e., not allowed to air-dry), and the filter is transferred to a
Petri dish of alcohol. Subsequently, the filter preparation is Papanicolaou stained
and coverslipped (Fig. 10.12). Membrane filtration is labor-intensive and is rarely
used these days.

carcinoma. (Millipore®
filter preparation, bladder
diversion, Papanicolaou
Cell Block Preparations
Most anatomic pathology laboratories prefer to perform ancillary tests on cell block
material, since cell block processing for ancillary studies is similar to standard his-
tology. Laboratories should have alternative validated protocols for pure cytology
samples. The standard staining methods used are hematoxylin and eosin (H&E) for
formalin-fixed, paraffin-embedded (FFPE) specimens. Cell blocks (CBs) have
many advantages and play a pivotal role in cytology [13, 14]. Currently, the utility
of CBs is recognized due to their role in ancillary testing, particularly in immuno-
chemistry, as well as capturing tissue fragments akin to biopsy material.
CB can be made from aggregation of exfoliated cells from fluid cytology. Cells
are resuspended in fluids by agitation immediately prior to processing the fluid to
ensure homogeneous distribution of the cell types throughout the specimen. The
architecture present in CB is more reminiscent of histology and is therefore familiar
to surgical pathologists (Fig. 10.13). Multiple sections for tests and controls can be
obtained from a single block. CB preparation methods vary among laboratories.
Evaluation in urine cytology can contribute to a final diagnosis in a range of cases
including benign, atypical, high-grade urothelial carcinoma and metastatic disease
[15]. Diagnostic architectural features can be observed in specimens that have a
large sediment when centrifuged. CB can demonstrate LGUC with fibrovascular
cores (Fig. 10.14).
In a study by Dantey et al., data revealed preparing CB for urine samples rou-
tinely did not improve the laboratory’s specimen adequacy rate. However, they can
assist to decrease the frequency of indeterminate diagnoses. Routine preparation of
CB, however, is expensive and provides minimal added clinical benefits, the authors
concluded [16].
Wilson et al. prepared CB cytology when a visible pellet was observed after
centrifugation [17]. Despite low urine volume in a number of cases in the study,
sufficient cellularity was available to produce diagnostic material. This recent study
urothelial carcinoma. (Cell
block preparation, bladder
washing, hematoxylin &
eosin stain, high
magnification)
a b
Fig. 10.14 Low-grade urothelial carcinoma with fibrovascular cores. (a) Millipore® filter prepa-
ration, bladder washing, Papanicolaou stain, low magnification. (b) Cell block preparation, bladder
washing, hematoxylin & eosin stain, low magnification
suggests CB preparation is useful on cases demonstrating atypical urothelial cells

(AUC) or suspicious for high-grade urothelial carcinoma (SHGUC) [17] for histo-
logic examination and IHC, which is valuable when a non-urothelial malignancy is
considered.
Cell block preparation methods may include traditional sediment, agar/gelatin,
plasma-thrombin, collodion bag, Cellient®, or Thermo Scientific™ Shandon™
Cytoblock™ cell block. If the cell sample is bloody, the sample can be centrifuged
first with a lysing fixative, prior to preparation. A common lysing agent used is gla-
cial acetic acid. Other commercial products are available. Additional washings with
a lysing fixative may be required in extremely bloody samples [1].
Centrifugation Sediment Cell Block
Urine samples are poured into two 50 mL conical centrifuge tubes labeled with
patient identification. The tubes are placed in the centrifuge and spun at 2400 rpm
for 5 minutes to achieve a concentrated cell pellet. The supernatant is poured off. If
the cell pellet is not cohesive, formalin can be added, and the sample is spun at
2400 rpm for an additional 5 minutes. Allow the tube to sit for an additional
30–60 minutes to harden. The hardened button is removed with a metal spatula by
loosening the edges so it breaks free of the tube. The cell pellet is placed on histol-
ogy tissue paper moistened with neutral buffered formalin. Tissue paper is folded
around the cell pellet and placed in a histology cassette labeled with patient identi-
fication (Fig. 10.15). Once all steps are complete, the tissue cassette is placed in a
Fig. 10.15 Centrifugation
sediment cell block
CENTRIFUGE
Urine Sample Sediment
Decant Supernatant Remove Excess

Supernatant
10% FORMALIN
Clot Formation Clot to Paraffin

Block
a b
Fig. 10.16 Metastatic adenocarcinoma. (a) Cell block preparation, bladder washing, hematoxylin
& eosin stain, high magnification. (b) Cell block preparation, bladder washing, Kreyberg mucin
histochemical stain, high magnification
formalin filled cup for additional time in formalin. The time is noted on the
requisition.
Time in formalin should be at least 6 hours of fixation for specific ancillary test-
ing IHC protocols [18, 19]. Quality control procedures documenting correct patient
identification and accession number, as well as any additional instructions, e.g.,
immunochemical protocol, may be incorporated in the cell block SOP. The speci-
men is then processed routinely in the histopathology laboratory and stained with
H&E stain according to the laboratory procedure for cell block preparation
(Appendix D). Immunostains can be ordered on the cell block material (Fig. 10.16).
If the cell pellet is extremely small after spinning the sample, HistoGel™/agar, col-
lodion, or Cellient® automated cell block preparation methods will conserve the
cellular material [20].
Shandon Cytoblock™ Cell Block
Thermo Scientific™ Cytoblock™ Cell Block Preparation System

(Thermo Fisher Scientific, Waltham, MA, USA)
This system is designed to facilitate the preparation of paraffin-embedded cell
suspensions, cell aggregates, and tissue fragments [21]. The Cytoblock™ system
simplifies the preparation of paraffin blocks from a kit and can be used to process
cell blocks from Fine Needle Aspiration (FNA), urine and serous fluids, and resid-
ual sediment from other cytologic samples. The Cytoblock™ kit consists of 50
Cytoblock™ cassettes with backing papers and board inserts, Reagent-1 (clear)
and Reagent-2 (colored). Patient information is recorded on Cytoblock™. The

specimen should be fixed in 10% formalin for approximately 30 minutes prior to
beginning Cytoblock™ preparation in the Cytospin. The fixed cells are concen-
trated by centrifugation, and excess fluid is decanted. If the total amount of speci-
men is two drops or less, add four drops of Reagent-1. A pipette is used to vortex
the sample. The Cytoblock™ cassette is then assembled in the Shandon Cytoclip™.
One to three drops of Reagent-1 are placed in the center of the well. A Cytofunnel™
disposable chamber is placed over the prepared Cytoblock™, and the metal clip
is attached. Spin for 5 minutes at 1500 rpm on low acceleration. Once complete,
carefully remove the Cytoblock™ from the metal clips. The Cytoblock™ is
placed in formalin for tissue processing.
Cellient® Cell Block
Cellient® Automated Cell Block System

(Hologic, Inc., Marlborough, MA, USA)
This technique is commonly used when the specimen consists of small tissue
fragments and is made directly from a liquid-based preparation vial. It is a fully
automated CB system. The specimen is centrifuged in a 50 mL conical tube for
5 minutes at 2400 rpm. Once the specimen is spun, the supernatant is poured off.
Only three drops of cell sediment are added to the PreservCyt™ vial to be fixed. The
vial is placed in the Cellient® automated CB system for processing (Fig. 10.17).
The average preparation time for each CB is 45 minutes. The specimen is then rou-
tinely processed in the histology laboratory and stained with H&E. (For exact
details, refer to manufacturer’s procedure manual.) [20] Validation of immuno-
chemistry using this technique must be performed in the laboratory because it
involves alcohol fixation prior to formalin fixation.
Fig. 10.17 Cellient®
automated cell block
1. Combine into
a 50 mL tube
10 min
2. Concentrate
by Centrifugation
3. Pour Off
Supernatant
and Resuspend
Cell Pellet
4. Add an Appropriate
Amount of Specimen
to the PreservCyt
Solution Vial
5. Run on Cellient®
Automated Cell
Block System
Discussion
A cursory review of the scientific literature reveals no generally accepted best mate-
rials and methods of collecting and processing urine to detect urothelial malignan-
cies. Various citations come to different conclusions about the pros and cons of
urine that is voided spontaneously versus catheterized versus bladder washings
[22]. The number of urine specimens [23], whether urine should be collected fresh
or preserved [5], and whether to keep urine at room temperature or refrigerated are
debated pre-analytical steps [5, 22–24]. Additionally pondered is the question of
hours at these temperatures, the number of urine specimens needed to improve sen-
sitivity [24], which processing method is best [25–27], and the number of cells to
define adequacy [28, 29]. Some basic principles appear to have broad consensus and
are practiced by laboratories worldwide.
Conclusions and Recommendations
Cytology terminology reporting systems are essential to good patient management,

and good cytopreparatory techniques are essential to producing cells suitable for
being described by standardized terminology reporting systems. The word “tech-
nique” is deliberately chosen, as it means a particular way of doing something—
compared to another similar-appearing way—that makes a useful difference.
The urine collection and processing guidance in this chapter must be followed by
consistently good staining and mounting techniques for best results. Readers are
referred elsewhere for that guidance [1, 4, 15, 30, 31].
The Johns Hopkins Hospital Cytopreparatory Laboratory is named in honor and
memory of Gary Gill, cytotechnologist extraordinaire. Inscribed on the bronze
plaque at the entrance are words chosen by Gary: “Quality begins here!”
ppendix A: Centrifugation Process Standard

A
Operating Procedure
Title
Centrifugation Process
Purpose
Centrifugation is used to concentrate cellular material from a fluid-based sample for

cytologic evaluation. Fluid-based samples include effusions, washes, urines, lavage
specimens, and other body discharges.
Responsibilities
Approval and signatories for this document are determined in accordance with the
cytopathology laboratory.
Responsibilities specific to the process outlined in this document are listed below.
Role(s) Responsibilities
Cytopreparatory Technicians Follow procedure.
Cytotechnologists Follow procedure
Equipment
( a) Sorvall ST16 120 V Centrifuge

(i) Serial #
(ii) The centrifuge is calibrated annually at 4600RPM at 2 minutes (timed with
a tachometer)
(iii) For service and maintenance, call Clinical Engineering at 5–5501
Procedure
1. After gently dispersing the specimen by shaking, it is poured into 50 mL centri-
fuge tubes. If there is not enough of the sample to fill two like centrifuge tubes
with equal volumes of fluid, a balance tube filled with water is used to balance
the centrifuge.
2. There are four separate holding chambers for the centrifuge tubes. Each chamber
is self-contained and covered by a plastic cover that is snapped into place. Each
chamber holds four 50 mL conical tubes.
3. Open the centrifuge lid, remove the plastic domes, and place specimens in to the
chamber being careful to properly balance the centrifuge
(a) To balance the centrifuge, place an identical volume tube into the directly
opposite chamber in the spot.
4. Close the lid and press the start button. The setting is preset at 2400RPM for
5 minutes.
5. When complete the centrifuge will alert that the process is done. The lid is
opened and the specimens are removed and can proceed with processing
Training
List role(s) of applicable staff Note which of the following are applicable: Read/review, Quiz (Knowledge Check),
and/or Skills Assessment.
Cytopreparatory Technicians Read/review
Cytotechnologists Read/review
Revision History
Previous Version Revised Document
Reason for Revision
Number Version Date
Appendix B: ThinPrep™ Process Standard Operating Procedure
Title
ThinPrep™ NonGynecologic Processing
Purpose
ThinPrep® technology for the preparation of non-gynecologic cytology specimens

is used on almost all cytology specimens in the laboratory. Specimens can be col-
lected directly into CytoLyt® solution or can be submitted fresh, in saline, or in
formalin for ThinPrep® processing.
Scope
Non-GYN cytological specimens
Responsibilities
cytopathology laboratory.
Responsibilities specific to the process outlined in this document are listed below.
Cytopreparatory Technicians Follow procedure.
Cytotechnologists Follow procedure
Procedure
Body Cavity Fluids, Urine, CSF, Cyst Fluid

1. All specimens are received fresh.
2. Gently shake the gross sample to redistribute the material which may have set-
tled since time of procurement.
3. Pour sample into 50 mL centrifuge tube(s) and centrifuge for 5 minutes at
2400RPM.
4. Decant supernatant. Add 30 mL of CytoLyt® solution. Gently redistribute the

material and centrifuge for 5 minutes at 2400RPM.
5. Decant supernatant. If sample is still grossly bloody, repeat step 4.
6. Resuspend the sample; there should be a total of 0.5–1.0 cc, and add 8–10 drops
of the sample to the vial of PreservCyt® solution.
7. Place the PreservCyt® vial onto the ThinPrep® Processor. Close door and select
non-gyn sequence (2) on key pad.
Fine Needle Aspiration

1. Specimen is collected in saline, in formalin, or in CytoLyt® solution during fine
needle aspiration procedures.
2. Centrifuge for 5 minutes at 2400 RPM.
3. Decant supernatant. Resuspend button and add 8–10 drops to PreservCyt® vial.
4. Place the PreservCyt® vial onto the ThinPrep® Processor. Close door and select
Mucoid Samples (Sputum, Bronchial Wash, etc.)

1. Place sample into 30 mL CytoLyt® solution.
2. Use vortex instrument to disperse mucus.
3. Centrifuge for 5 minutes at 2400 RPM.
4. Decant supernatant. Resuspend button and add 8–10 drops to PreservCyt® vial.
5. Place the PreservCyt vial onto the ThinPrep® Processor. Close door and select
The ThinPrep® Processor has a separate procedure manual covering specimen

preparation, maintenance, and trouble shooting. This manual is available in the
cytopreparation area.
Training
List role(s) of applicable staff Note which of the following are applicable: Read/review, Quiz
(Knowledge Check), and/or Skills Assessment.
Cytopreparatory Technicians Read/review
Cytotechnologists Read/review
Revision History
Reason for Revision
Number Version Date
ppendix C: SurePath™ Preparation Using BD Totalys™

A
MultiProcessor for Non-gynecologic Processing
Standard Operating Procedure
Title
SurePath™ Preparation Using Totalys™ Multiprocessor for Non-Gynecologic Processing
Purpose
A SurePath preparation is a monolayer technology that uses sedimentation and sam-
ple enrichment techniques to produce a specimen for cytologic examination.
Totalys MultiProcessor can be used to automate the front-end processing with
chain of custody and error reconciliation prior to slide processing.
Scope
All cytology staff
Definitions
The specimens are from a variety of body sites including major body cavity fluids,
bronchoscopic specimens, urines, brushings and FNA rinses, etc.
Reagents
1. Density reagent
2. GYN preservative
3. Tris buffered saline working solution
4. Alcohol blend rinse
5. SurePath prep Stain
6. Cleaning alcohol (95% ETOH)
7. Deionized water
8. Xylene
9. 50/50 xylene/absolute ETOH mix
10. 100% ETOH
Equipment/instrumentation/consumables
1. SurePath vials
2. SlidePrep instrument
3. Multi-mix vortex
4. SurePath pre-coat microscopic slides
5. Slide and tube racks
6. Centrifuge tubes
7. Centrifuge
8. Settling chambers
9. Disposable transfer/aspirator tips
Procedure
Note: All procedures involving the specimen should be performed underneath
the biohazard fume hood.
1. Access the specimen into the laboratory computer system to obtain a cytology
specimen number (see procedure on accessioning). Label the specimen con-
tainer with a printed 2-D Sato label.
2. It is important to ensure that the SurePath vial BDVA barcode is overla-
beled with the appropriate CoPath 2-D accession label.
3. Thoroughly mix fluid submitted by vigorously agitating bottle or specimen
container.
4. Any brush accompanying a specimen should be removed from the submit-
ted container and placed directly in the large opening of the SurePath vial.
The original container should be centrifuged with any pellet formed also
added to the same vial to ensure complete transfer.
5. Label a centrifuge tube with unique identifiers for the specimen using 1 inch
Sato labels.
6. Attempt to aliquot at least 50 ml of specimen into a 50 ml centrifuge tube when-
ever possible. For smaller volumes the entire specimen may be used.
7. In a balanced centrifuge, centrifuge the specimens on program 3 in the Jouan
centrifuge.
8. When finished. Remove specimens and decant supernatant into formalin discard
container.
9. Add 5 drops of centrifuged pellet (or in scant specimens all available) directly to
previously labeled SurePath vial using a transfer pipette.
ppendix D: Cytocentrifuge Cell Block Process Standard

A
Operating Procedure
Title
Cytocentrifuge Cell Block Standard Process
Purpose
The following procedure for preparation of cell blocks can be applied to any non-
gynecologic or gynecologic specimen received by the Cytopathology Department.
This procedure will be used most commonly on serous effusions, pelvic/abdominal
washes from the female genital tract, and fine needle aspirates. A cell block may be
performed on virtually any sediment that exists in a centrifuge tube after a specimen
has been spun down. Cell blocks are the preparation of choice for any specimen that
requires special stains or immunohistochemical stains. Cell blocks should be con-

sidered on any specimen that reveals visible sediment after centrifugation.
Responsibilities
cytopathology laboratory. Responsibilities specific to the process outlined in this
document are listed below.
Cytotechnologist Follow policy.
Cytopreparatory technician Follow policy.
General Guidelines/Policy
1. Spin down specimen to achieve a concentrated cell button. (Centrifuge

5 min/2400 rpm.)
2. Pour off supernatant.
3. Add 5 cc buffered formalin, if necessary and if needed, spin to fix specimen.
(Centrifuge 5 min/2400 rpm.)
4. Label cell block cassette with patient name on the side and accession number
on the front. ‘8 mm’ designation is placed on side of the cassette if requested.
5. After sitting in formalin, remove hardened cell button from centrifuge tube with
metal spatula. Keep the cell block intact if possible.
6. Loosen the edges of the cell button until it breaks free of the centrifuge tube.
7. Place on blue histology tissue paper moistened with buffered formalin.
8. Fold tissue paper around cell block section(s) and place in histology cassette.
Folded edges of the blue tissue paper should face UP. Snap cassette shut and
place in white specimen cup containing buffered formalin. Use enough forma-
lin to cover cassette.
9. When “8 mm” is requested, immunohistochemistry and/or special stains will
already have the menu of stains to be performed, via computer or written request
by the pathologist.
10. Cell blocks are submitted on the Cytology cell block submission form to the
Histology laboratory every day.
Patient name and accession number should be verified and documented by two
people on the cell block submission form before submitting to Histology. An
accessioning sticker must be used to identify patient and Cytology accession num-
ber for each cassette.
All cell block material is fixed in 10% formalin (unless lymphoma protocol
is requested) for at least 6–72 hours.
Training
List role(s) of applicable staff Note which of the following are applicable: Read/review, Quiz
(Knowledge Check), and/or Skills Assessment.
Cytotechnologist Read/review
Cytopreparatory technician Read/review
Revision History
Reason for Revision
Number Version Date
References
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totalys-system/totalys-multiprocessor.
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totalys-slideprep.
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2018;29(6):505–24.
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add value to the diagnosis? A pilot study. J Am Soc Cytopathol. 2021;10:47–55.
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Cytoblok%20IFU.pdf/
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p. 148–61.
Risk of High-Grade Malignancy (ROHM)
11
Mauro Saieg, Güliz A. Barkan, Fadi Brimo,
Ashish Chandra, Tarik M. Elsheikh, Ricardo G. Pastorello,
Marcus L. Quek, Jianyu Rao, Momin T Siddiqui,
Z. Laura Tabatabai, Christopher J. VandenBussche,
and Philippe Vielh
Background
Since its first edition in 2016, The Paris System for Reporting Urinary Cytology
(TPS) [1] has been adopted worldwide in hospitals and laboratories for the cytologi-
cal diagnosis of urothelial malignancies. This has resulted in a large number of
publications that reported varied experiences. Besides providing clarity of commu-
nication, one of the major goals of a standardized reporting system is to yield an
estimated risk of malignancy (ROM) for each diagnostic category in order to prop-
erly guide patient management. Since the main goal of urine cytology is the detec-
tion of clinically significant cancers, particularly high-grade urothelial carcinoma
(HGUC), it seems more appropriate to utilize the term “risk of high-grade malig-
nancy (ROHM)” rather than ROM. Low-grade urothelial neoplasms (LGUN), on
the other hand, by definition have minimal cytologic atypia, and accurate diagnosis
of these lesions by cytomorphology alone is practically impossible. Thus, this sec-
ond TPS edition incorporates the term “ROHM,” rather than “ROM.”
Current Evidence for ROHM
A literature search yielded 11 studies with more than 100 samples each and avail-
able results on subsequent biopsy and/or resection for patients with a previous cyto-
logical diagnosis using TPS. These data were included to estimate ROHM for each
category of TPS (Table 11.1). Overall, a total of 2292 cases were available. The
ROHM ranged from 8.7% to 36.8% for NHGUC, 12.3% to 60.9%% for AUC,
33.3% to 100% for SHGUC, and 58.8% to 100% for HGUC. Mean ROHM weighted
by sample size (and standard deviations) from all studies was calculated at 15.7%
(± 7.8%), 38.5% (± 14.3%), 76.2% (± 17.2%), and 88.8% (± 12.7%) for NHGUC,
AUC, SHGUC, and HGUC, respectively. It is important to note that the majority of
these studies retrospectively applied TPS classification to cases that were signed out
prior to the introduction of TPS (Table 11.1, “reclassified pre-TPS”). Hence, these

https://doi.org/10.1007/978-3-030-88686-8_11
250 M. Saieg et al.
Table 11.1 Distribution of risks of high-grade malignancy within each diagnostic category after
the implementation of TPS across included 11 studies
Study Number of cases with ROHM (%)
Author, year cohort available histology NHGUC AUC SHGUC HGUC
Stanzione et al., P 294 14.4 44.3a 88.9 97.4
2020 [3]
de Paula et al., P 499 11 32.6 80 92.3
2019 [4]
Bakkar et al., 2019 R 100 29.7 60.9 100 100
[6]
Wang et al., 2018 P 167 17.7 50 76.4 89.1
[7]
Meilleroux et al., P 229 8.7 49 87 91
2018 [2]
Xing et al., 2018 P 150 27 59 93 100
[8]
Rohilla et al., 2018 R 244 11.6 12.3 33.3 58.8
[9]
Zare et al., 2018 R 194 9.9 17.4 72 96.3
[10]
Suh et al., 2017 R 142 13.5 38.1 59.1 68
[11]
Granados et al., R 149 36.8 40 75 87.1
2017 [12]
Hassan et al., 2016 R 124 17.8 53.1 83.3 100
[13]
TPS The Paris System for Reporting Urine Cytology, ROHM risk of high-grade malignancy,
NHGUC negative for high-grade urothelial carcinoma, AUC atypical urothelial cells, SHGUC sus-
picious for high-grade urothelial carcinoma, HGUC high-grade urothelial carcinoma, R reclassi-
fied pre-TPS, P post-TPS
a
AUC”, “AUC, NOS/reactive,” and “AUC, favor LG” grouped together
publications might suffer from review bias, as pathologists may be more inclined to
upgrade indeterminate diagnoses in a research setting when there is no associated
clinical implication. Accordingly, when ROHM was calculated separately for pre-
and post-TPS studies, mean ROHM was significantly lower for the AUC and
SHGUC categories in the retrospective (pre-TPS) studies (32% and 65%, respec-
tively) versus prospective (post-TPS) studies (43 and 84%, respectively). Another
potential bias in ROHM may be attributed to differences in age and gender. However,
data regarding these possible differences is not extractable from the available stud-
ies, and different numbers by age and gender should be incorporated in future
reports for further refinement of the ROHM of the distinct categories in the third
version of TPS. In addition, all of these studies used histological follow-up to esti-
mate ROHM, which may overestimate ROHM due to selection bias. Therefore, the
actual ROHMs should be expected to be in the mid-range of what has been pub-
lished in the literature. Nonetheless, most studies have shown progressively higher
ROHM across the diagnostic categories. Within the three studies with the largest
number of patients that had histological follow-up, ROHMs for the HGUC were
11 Risk of High-Grade Malignancy (ROHM) 251
above 90%, emphasizing the high positive predictive value of TPS [2–4]. Indeed, it
would be important for future studies to have a standardized way to determine
ROHM, making them easier to compare and reflecting a final mean number closer
to actual clinical practice. Of interest, a recent study proposed the use of likelihood
ratios (LR) and pre-test probability (based on clinical and epidemiological param-
eters) to assess a more personalized post-test probability of having malignancy. This
would minimize the bias in number when comparing multiple studies, but is depen-
dent upon the clinical parameters pre-test, not always available [5].
Data on ROHM associated with a nondiagnostic/unsatisfactory (ND/U) urine
cytology is scant. As these cases represent a minority of specimens, their estimated
risks may be particularly prone to overestimation in limited cohorts [8]. In addition,
several authors have opted to exclude them from their analyses. In these few efforts
that reported ND/U cases with histological follow-up, the associated ROHM ranged
from 0 to 15.6% [2, 4, 14]. Of particular interest, a recent, larger study detailed
outcomes of 116 ND/U specimens from 106 patients, including histological and
cytological follow-up, with a NPV of 84.4% [14].
Among cases classified as atypical (AUC), a decrease in the use of this category
was noted in the majority of reports following implementation of TPS [2, 3, 6, 7,
15–18]. This was likely due to the clearly defined cytomorphological features of
atypia by TPS and inclusion of reactive conditions such as previous radiation, BCG
therapy, lithiasis, or manipulation within the NHGUC category [1, 19]. The major-
ity of the studies have also shown an increased ROHM for AUC category post-TPS,
reflecting a more meticulous use of this term, based on the clear recommendations
by the first edition of TPS [2, 3, 6–8, 13].
ROHM in Low-Grade Neoplasms
Urine cytology performs poorly in detecting low-grade neoplasms, as mentioned

before. However, the LGUN category included in the first edition of TPS was
reserved for the exceedingly rare cases in which distinct fibrovascular cores can be
appreciated on cytology, and the absence of high-grade features. Due to the rarity of
this scenario, only three series in the literature reported more than ten LGUN cytol-
ogy cases, with histological follow-up within their cohorts, corresponding from 1%
to 13% of the study samples. In these series, the risk of low-grade neoplasm (RLGN)
associated with this cytological diagnosis ranged from 45.5% to 100% (weighted
mean = 78.1% ± 22.3%) [2, 17, 20]. In these same studies, the ROHM among those
specimens ranged from 0% to 54.1% (weighted mean = 21.9% ± 22.3%). Again,
these numbers should be examined with caution, given the limited sample sizes. On
the other hand, when looking at all tumors reported ultimately as LGUN on histol-
ogy, the majority of corresponding cytologies were diagnosed as NHGUC. This is
an expected finding, considering the known morphological overlap of LGUN and
normal/reactive urothelium in cytology samples [3]. Of note, in four studies [3, 17,
20, 21], cytology cases with a corresponding histologic diagnosis of LGUN were
primarily assigned to the AUC category (Table 11.2).
252 M. Saieg et al.
Table 11.2 Distribution of TPS categories initially assigned to cases ultimately reported as LGUN
on histology
Number of TPS categories % (n)
cases with
histologic
Study diagnosis of
Author, year cohort LGUN NHGUC AUC SHGUC HGUC LGUN
Stanzione et al., P 58 41.4 (24) 56.9 (33)a 1.7 (1) 0 (0) a
2020 [3]
Anbardar et al., R 22 4.5 (1) 18.2 (4) 31.8 (7) 40.9 (9) 4.5 (1)
2020 [21]
de Paula et al., P 88 93.2 (82) 4.5 (4) 0 (0) 2.3 (2) 0 (0)
2019 [4]
Vallamreddy R 32 18.8 (6) 21.9 (7) 6.3 (2) 0 (0) 53.1 (17)
et al., 2019 [17]
Rai et al., 2019 R 16 37.5 (6) 12.5 (2) 25 (4) 0 (0) 25 (4)
[22]
Wang et al., P 46 69.6 (32) 23.9 (11) 4.3 (2) 2.2 (1) b
2018 [23]
Meilleroux P 62 72.6 (45) 16.1 (10) 1.6 (1) 0 (0) 8.1 (5)
et al., 2018 [2]
Xing et al., P 27 85.2 (23) 14.8 (4) 0 (0) 0 (0) 0 (0)
2018 [8]
Zare et al., 2018 R 52 80.8 (42) 9.6 (5) 3.8 (2) 1.9 (1) 3.8 (2)
[10]
Roy et al., 2017 R 34 14.7 (5) 23.5 (8) 5.9 (2) 11.8 (4) 29.4 (10)
[20]
Granados et al., R 40 60 (24) 25 (10) 7.5 (3) 7.5 (3) 0 (0)
2017 [12]
Malviya et al., R 5 40 (2) 20 (1) 20 (1) 20 (1) 0 (0)
2017 [24]
Suh et al., 2017 R 27 33.3 (9) 11.1 (3) 37 (10) 18.5 (5) 0 (0)
[11]
Hassan et al., R 25 72 (18) 28 (7) 0 (0) 0 (0) 0 (0)
2016 [13]
TPS The Paris System for Reporting Urine Cytology, LGUN low-grade urothelial neoplasm, ND/U
nondiagnostic/unsatisfactory, NHGUC negative for high-grade urothelial carcinoma, AUC atypical
urothelial cells, SHGUC suspicious for high-grade urothelial carcinoma, HGUC high-grade uro-
thelial carcinoma, R reclassified pre-TPS, P post-TPS
a
“AUC”, “AUC, NOS/reactive,” and “AUC, favor LG” grouped together
b
The LGUN category was excluded from this study
Overall Performance of TPS
Several studies have reported diagnostic test measures for urinary cytology after
implementation of TPS. Overall sensitivity ranged from 40% to 84.7%, specificity
from 73% to 100%, PPV from 68% to 100%, and NPV from 46% to 88.2% [2, 4, 6,
9–12]. It is noteworthy that these studies can be heterogeneous in their methods. The
calculation of accuracy measures assumes that a test has only two possible results:
benign or malignant. However, urine cytology is significantly more complex, as it
includes additional diagnostic categories that are not simply “benign or malignant,”
such as ND, AUC, SHGUC, and LGUN. Consequently, different authors have used
distinct approaches on converting multiple reporting categories into a dichotomous
system for these analyses. Despite this limitation, most studies that report their expe-
rience before and after implementation of TPS exhibit an increased diagnostic sensi-
tivity for HGUC after putting TPS into practice [2, 6, 10–12]. This is a great advantage
as urinary cytology is primarily used for symptomatic screening and urothelial can-
cer surveillance. In addition, calculation of ROHM mostly relies on surgical pathol-
ogy interpretation, which in itself is not immune to bias, depending on multiple
factors. Also, it is important to remember that in any study reporting a cytology-his-
tology correlation, a verification bias exists; that is, not all cytology cases are fol-
lowed up by surgical procedures and there are other clinical factors that influence
management strategy.
Conclusion
The reported experience on the implementation of TPS classification worldwide has

shown an increased sensitivity in urinary cytology, along with progressively higher
ROHM across the diagnostic categories. Hence, according to this experience and
available published data, Table 11.3 shows the estimated ROHM for each assigned
category (weighted by sample size and incorporated standard deviations) and pres-
ents updated numbers to the original values proposed in the first edition of TPS.
Table 11.3 Estimated risk of high-grade malignancy (ROHM) for each category of The Paris
System for Reporting Urinary Cytology
TPS category Risk of high-grade malignancy
ND 0–16%
NHGUC 8–24%
LGUN 0–44%
AUC 24–53%
SHGUC 59–94%
HGUC/malignant 76–100%
254 M. Saieg et al.
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Courtade-Saidi MM. One year of experience using the Paris System for Reporting Urinary
Cytology. Cancer Cytopathol. 2018;126(6):430–6. PMID: 29663682.
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in a cancer centre. Cytopathology. 2020;31(1):41–6. PMID: 31654587.
5. Myles N, Auger M, Kanber Y, Caglar D, Kassouf W, Brimo F. Evidence-based diagnos-
tic accuracy measurement in urine cytology using likelihhod ratios. J Am Soc Cytopathol.
2021;10:71–8.
6. Bakkar R, Mirocha J, Fan X, Frishberg DP, de Peralta-Venturina M, Zhai J, Bose S. Impact
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diagnostic categories and interobserver agreement. Cytojournal. 2019;16(21)
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8. Xing J, Monaco S, Pantanowitz L. The ability of the Paris system to stratify the risk of high-
grade urothelial carcinoma. J Am Soc Cytopathol [Internet]. Elsevier Inc; 2018;7(5):S43.
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9. Rohilla M, Singh P, Rajwanshi A, Gupta N, Srinivasan R, Dey P, Kakkar N. Cytohistological
correlation of urine cytology in a tertiary centre with application of the Paris system.
Cytopathology. 2018;29(5):436–43. PMID: 29920811.
10. Zare S, Mirsadraei L, Reisian N, Liao X, Roma A, Shabaik A, Hasteh F. A single institutional
experience with the Paris system for reporting urinary cytology: correlation of cytology and
histology in 194 cases. Am J Clin Pathol. 2018;150(2):162–7. PMID: 29878037.
11. Suh J, Go H, Sung C, Baek S, Hwang H, Jeong S, Cho Y. Modification of The Paris System
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12. Granados R, Duarte JA, Corrales T, Camarmo E, Bajo P. Applying the Paris system for report-
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Clinical Management
12
Marcus L. Quek, Trinity J. Bivalacqua, Ashish M. Kamat,
Mauro Saieg, Alexander I. Sankin, Yuji Tokuda,
and Bas WG van Rhijn
• Guideline changes for evaluation of microscopic hematuria

• Guideline changes for surveillance of non-muscle-invasive bladder cancer
• Use of reflex biomarker assays (UroVysion FISH, ImmunoCyt, Cxbladder) for
adjudication of atypical urine cytology
• Role for enhanced cystoscopic techniques (fluorescence “blue light” cystoscopy,
narrowband imaging)
Background
Urine cytology remains an important adjunctive test in the diagnostic armamentar-

ium of the urologist. The utility of cytology is dependent on several factors includ-
ing the disease process, the adequacy of the specimen collection, and the experience
of the cytopathologist. As such, the clinician must understand and appreciate the
inherent limitations of cytology. The implementation of The Paris System (TPS) has
standardized the diagnostic criteria, thus limiting interobserver variability in cyto-
logic interpretation and creating a more reproducible diagnostic tool [1].
For the patient undergoing the initial evaluation for a suspected urothelial malig-
nancy, urine cytology may play an important role in risk stratification. The finding
of a negative cytology result in the face of an obvious papillary bladder tumor
should not come as a surprise given the lack of cytologic atypia in most low-grade
noninvasive tumors. In this setting, the urologist can be reassured that an underlying
high-grade invasive lesion or CIS is less likely to be present. A positive cytology
without a tumor should raise suspicion of a missed lesion and/or missed CIS, for
which adjuncts to regular cystoscopy (such as fluorescence cystoscopy, narrowband
imaging, and directed or random biopsies of the bladder) and evaluation of the pros-
tatic urethra and upper tracts should be considered [2].

https://doi.org/10.1007/978-3-030-88686-8_12
258 M. L. Quek et al.
The clinical scenarios in which urine cytology is performed include the evalua-
tion of hematuria and irritative voiding symptoms, initial work-up for a suspected
urothelial malignancy, and surveillance following treatment for urothelial cancer.
The decision to obtain a voided or instrumented (barbotage/washing/brushing)
cytology depends on the clinical setting. Since the introduction of TPS in 2016,
several publications have reported the risk of high-grade malignancy associated
with the various diagnostic categories (see Chap. 11). Herein, we review the clinical
management considerations for each of these diagnoses.
anagement of Negative for High-Grade Urothelial

M
Carcinoma (NHGUC)
Urine cytology has always demonstrated accuracy in detecting high-grade tumors

and carcinoma in situ (CIS) [3, 4]. Mortality from bladder cancer is overwhelmingly
due to these lesions. The designation of NHGUC as a distinct diagnostic category
acknowledges the inherent limitations of cytology in detecting low-grade noninva-
sive cancers which represent the majority of bladder cancer diagnoses. It is there-
fore not surprising that historically the overall sensitivity for cytology to detect
bladder cancer was low [3]. TPS recognized that lesions without cytologic atypia,
specifically LGUN, are virtually impossible to diagnose, unless the cellular sample
contains tissue fragments with fibrovascular cores. Furthermore, whether these low-
grade neoplasms are actually cancers is now undergoing debate (see Chap. 1). The
acceptance of standardized criteria for this category has had the resulting beneficial
effect of decreasing the number of specimens allocated to the “wastebasket” diag-
nostic category of “atypical urothelial cells” (see Chap. 4).
The primary concern facing the clinician is whether there is a “clinically signifi-
cant” lesion somewhere in the urinary tract. From the standpoint of the urologist and
especially the patient, the first question is whether there is a malignancy and, sec-
ondly, whether that neoplasm is malignant and potentially lethal. While cytology
may not perform well in answering the first question (due to the poor sensitivity to
detect low-grade noninvasive tumors), its ability to provide insight into the second
question can be very useful depending on the clinical setting.
The role of urine cytology in the initial evaluation for hematuria or irritative
voiding symptoms is controversial. In fact, the American Urological Association
(AUA)/Society of Urodynamics, Female Pelvic Medicine, and Urogenital
Reconstruction (SUFU) Guidelines on Microhematuria do not recommend the use
of cytology in the initial evaluation of microscopic hematuria [5]. The Guidelines
leave open the option for urine cytology for those with persistent microhematuria
who have irritative voiding symptoms following a negative evaluation or risk factors
for carcinoma in situ (CIS). The Guidelines recommend a patient-centered approach
to diagnostic testing for microhematuria based on the risk for urothelial malignancy.
Patients are classified as low, intermediate, or high risk depending on several risk
factors including age, gender, degree of hematuria, irritative urinary symptoms,
smoking history, chemical exposure, history of cyclophosphamide use or pelvic
12 Clinical Management 259
radiotherapy, chronic indwelling foreign body in the urinary tract, or family history
of urothelial carcinoma or Lynch syndrome. Depending on the risk category, the
diagnostic work-up might include upper tract imaging (renal ultrasonography, CT
or MR urography) and cystoscopy.
Urine cytology plays a critical role in the ongoing surveillance for urothelial
recurrences following therapy. While most bladder recurrences will often be
detected by routine surveillance cystoscopy, upper urinary tract and prostatic or
urethral recurrences can be more difficult to diagnose in a timely manner. Anterior
bladder wall and bladder neck lesions can occasionally be missed, while carcinoma
in situ may not always be readily distinguishable cystoscopically from other benign
inflammatory conditions. Thoughtful utilization of contrast-enhanced imaging
along with urine cytology represents the primary initial diagnostic modalities when
direct endoscopic visualization can be difficult or impractical.
The current AUA/Society of Urologic Oncology (SUO) Guidelines recommend
a risk-stratified approach in surveillance of non-muscle-invasive bladder cancer [2].
The risk categories are based on factors such as tumor grade, stage, multifocality,
size, and recurrence. Urine cytology or other urinary biomarkers are not recom-
mended for patients with a history of low-risk cancer and a normal cystoscopy.
Surveillance for intermediate-risk disease should include both cystoscopy and
cytology every 3–6 months for 2 years, then 6–12 months for 2 years, and then
annually thereafter. High-risk patients who have a negative first surveillance cystos-
copy should be followed with cystoscopy and cytology every 3–4 months for
2 years, then 6 months for 2 years, and then annually thereafter.
Urine cytology is also an essential component in the surveillance of patients who
undergo radical cystectomy and urinary diversion, as these patients remain at risk
for recurrences in the remnant urothelium (upper tracts and urethra) [6, 7]. Prior to
TPS, the diagnostic yield of cytology in the setting of a urinary diversion was vari-
able due to the interpretive challenges related to glandular and inflammatory cells
and necrotic debris or mucous from the bowel segment. While some diagnostic
challenges remain [8], the rates of “atypical” diagnoses in urinary diversion patients
should approximate the rates in those without an interposed bowel segment [6].
Voided specimens for those with orthotopic neobladders, catheterized specimens
from incontinent and continent cutaneous diversions, and urethral washings provide
important diagnostic information some times before lesions become radiographi-
cally or symptomatically evident. Risk factors for urethral and upper tract recur-
rences following radical cystectomy have previously been described [9–12]. A
NHGUC diagnosis is reassuring, and the patient may continue routine surveillance
at intervals commensurate with the risk of recurrence.
Management of Atypical Urothelial Cells (AUC)
The management of “atypical urothelial cells (AUC)” has always presented a diag-
nostic dilemma for the urologist. Traditionally, this category has included a wide
spectrum of benign and malignant conditions lacking definitive cytologic criteria.
The use of TPS has decreased the rate of AUC diagnoses, as known benign condi-
tions such as reactive/inflammatory changes and cellular changes associated with
polyomavirus and urolithiasis are now categorized as NHGUC. As a result, there
may be an increased risk of malignancy associated with the AUC diagnosis due to
the defined criteria for this category (see Chap. 11). The importance of communica-
tion between the cytopathologist and clinician cannot be emphasized enough in
relaying the degree of suspicion for an underlying malignancy.
From the standpoint of the urologist, the work-up for AUC should be individual-
ized based on the risk assessment of the patient. Those with hematuria or persistent
irritative voiding symptoms still require a thorough evaluation with upper tract
imaging and cystoscopy. A patient with known risk factors for urothelial carcinoma
and an atypical cytology should prompt consideration of performing further testing
to rule out malignancy. For patients with a prior history of urothelial malignancy,
the extent of the work-up is dependent on the clinical suspicion for recurrent dis-
ease. Evaluation of the upper urinary tract and urethra may be considered if not
recently performed. The utility of random bladder biopsies of “normal”-appearing
urothelium is controversial, but may identify unsuspected carcinoma in situ [13, 14].
The role of additional molecular testing, such as UroVysion FISH and other
urinary biomarkers, remains to be determined (see Chap. 9) [3, 4, 15]. Several cen-
ters have instituted reflex UroVysion FISH testing for adjudication of AUC diagno-
ses, whereby a positive FISH assay is managed similarly to a suspicious diagnosis
and a negative FISH test is followed expectantly [16–19]. Other urinary biomarker
assays (such as ImmunoCyt, Cxbladder) have also been studied as adjunctive tests
when an AUC diagnosis is encountered [20, 21]. Further study is needed to confirm
the utility of these and other biomarker tests in this setting.
anagement of Suspicious (SHGUC) and Positive

M
for High-Grade Urothelial Carcinoma (HGUC)
From a practical standpoint, the clinical management of “suspicious for HGUC”

(SHGUC) is identical to a HGUC diagnosis. These patients require active investiga-
tion in order to identify the source of the suspicious or positive cells. When a “suspi-
cious” or “positive” for HGUC cytology result is obtained in the setting of a
low-grade noninvasive bladder cancer (LGUC), consideration should strongly be
given to further evaluation for other high-grade lesions or CIS utilizing adjuncts to
regular cystoscopy (fluorescence cystoscopy, narrowband imaging, directed/ran-
dom bladder biopsies) and evaluation of the prostatic urethra and upper tracts.
Upper tract evaluation with multiphase contrast-enhanced cross-sectional imaging
with computerized tomography (CT urography) is considered the current standard
imaging modality. For those with contraindications to contrast administration, alter-
natives may include magnetic resonance urography (MRU) and renal ultrasound
with retrograde pyelography. Any upper tract abnormalities should be further evalu-
ated with direct endoscopic (ureteroscopic) visualization with biopsies of any suspi-
cious areas if feasible. Cystourethroscopy remains the mainstay of evaluation of the
lower urinary tract. Biopsies and formal transurethral resection of mucosal abnor-
malities are indicated. For those with negative upper tract imaging and cystoscopy,
selective cytologic sampling from both upper tracts may help further localize lesions
such as CIS which may be difficult to visualize radiographically. Enhanced direct
endoscopic visualization techniques, such as fluorescence (“Blue Light”) cystos-
copy with hexaminolevulinate or narrowband imaging, have demonstrated improved
ability to detect more subtle lesions compared with conventional white light cystos-
copy [22–28].
For the subset of patients being followed post-cystectomy and urinary diversion,
a finding of suspicious or positive for HGUC also warrants a thorough investigation.
Recurrences in the intestinal segment itself are extremely unlikely [29] .
Investigations should focus on the upper tracts as well as the urethra. Prostatic stro-
mal invasion and anterior vaginal wall involvement with urothelial carcinoma por-
tend a high risk for urethral recurrences following cystectomy, especially in those
diverted by means of a cutaneous diversion (ileal conduit or continence cutaneous
reservoir) [9, 10, 12]. Urethral wash cytologies are sometimes employed in the rou-
tine follow-up of these patients (if a prophylactic urethrectomy is not performed)
based on the specific risk factors (multifocal CIS, tumors at the bladder neck or
prostatic urethra). For those with orthotopic neobladders, a positive/suspicious
voided cytology may be further investigated with cystoscopy and biopsies as indi-
cated. Risk factors for upper tract recurrences have previously been reported [7, 11].
Patients with suspected upper tract recurrences following cystectomy and diversion
often require direct percutaneous access to the upper tract for antegrade ureterore-
noscopy since retrograde access through the ureteroenteric anastomosis may be
challenging. Management of lesions identified in the remnant urothelium can be
treated via endoscopic resection (antegrade/retrograde), instillation of topical intra-
cavitary (pyelocaliceal) immuno- or chemotherapeutics, and surgical resection
(nephroureterectomy, urethrectomy) depending on the clinical scenario [9, 30].
Histologic evidence of divergent differentiation (such as squamous, glandular,
micropapillary, plasmacytoid, and sarcomatoid) in urothelial carcinoma is often
associated with a more aggressive phenotype. Several studies have shown a lower
success rate of intravesical therapy for these tumors, and hence the option of “early”
radical cystectomy for non-muscle-invasive disease should be discussed with the
patient [31].
Management of Low-Grade Urothelial Neoplasms (LGUN)
As mentioned previously the ability to diagnose LGUN based on cytology can be

challenging. The majority of bladder cancers present as low-grade noninvasive pap-
illary tumors. While these typically demonstrate a low likelihood of metastasis,
recurrences are common. Transurethral resection establishes the histologic diagno-
sis and is therapeutic for most solitary low-grade tumors. A single postoperative
instillation of intravesical chemotherapy (such as mitomycin C or gemcitabine) has
shown some benefit in decreasing the frequency of recurrences [32]. Routine
surveillance cystoscopies may be performed in the office at regular intervals. Both

the AUA and European Association of Urology (EAU) recommend a risk-adapted
surveillance protocol for non-muscle-invasive bladder cancer [2, 33]. The decision
to give adjuvant intravesical therapy (chemotherapy or immunotherapy) is based on
the risk of recurrence and progression [34]. The indications include large tumors,
tumor multifocality, presence of CIS, any high-grade component, invasion into the
lamina propria, and prior tumor recurrences [2]. Various agents have been utilized
including cytotoxic chemotherapy (mitomycin, doxorubicin, gemcitabine) as well
as immunomodulatory agents (BCG, interferon). Standard induction courses
(weekly intravesical treatments) may be followed by maintenance treatments
assuming a favorable response.
Management of Non-urothelial Tumors
Non-urothelial carcinomas account for approximately 10% of bladder cancers (see

Chap. 8). The distinction between primary non-urothelial malignancy and urothelial
carcinoma with divergent histologic differentiation can be difficult, if not impossi-
ble, to make by urinary cytology alone. These tumors tend to have a more aggres-
sive phenotype, typically presenting with invasive or locally advanced disease [31].
The histologic diagnosis is made by transurethral resection. Complete resection of
all visible tumors, when appropriate, is still recommended in order to distinguish
between a pure and mixed histology (which, even then, is not always possible
depending on the sampling). Due to their rarity, prospective randomized trials com-
paring treatments are lacking. Consideration should strongly be given to a planned
multidisciplinary approach, employing surgery, systemic chemotherapy, and radia-
tion on an individualized basis.
Squamous cell carcinoma is the second most common type of bladder cancer and
is often associated with chronic inflammation from schistosomal infection, recur-
rent urinary tract infections, chronic indwelling catheterization, and bladder calculi.
Radical cystectomy remains the standard treatment for primary squamous cell car-
cinoma of the bladder. Radiotherapy with concurrent chemotherapy may be given
for unresectable or residual disease.
Primary adenocarcinomas are associated with bladder exstrophy, urachal anoma-
lies, and chronic inflammation of the bladder. Malignancies from other sites
(colorectal, prostatic, breast) must be ruled out. Again, due to the rarity of the dis-
ease, comparative trials are lacking and the default standard treatment remains radi-
cal cystectomy. For urachal carcinomas, wide local excision of the umbilicus and
urachal remnant with cystectomy and pelvic lymphadenectomy is indicated. Unlike
urothelial carcinomas which tend to be multifocal due to the field effect nature of
the urothelium, primary adenocarcinomas tend to be solitary. As such, partial cys-
tectomy of the dome (for urachal-associated tumors) or those located within a diver-
ticulum may be considered in carefully selected cases.
Neuroendocrine (NE) tumors of the urinary tract have a propensity for systemic
involvement. Both pure NE carcinomas and urothelial carcinomas with NE
differentiation should be managed with a planned multimodality treatment approach

[35]. Neoadjuvant systemic chemotherapy followed by radical cystectomy has dem-
onstrated superior outcomes compared with radical cystectomy alone or with adju-
vant postoperative chemotherapy [36]. Combination chemotherapy with definitive
radiotherapy (analogous to the management of small cell lung cancer) may also be
considered in select cases [37].
Management of the Unsatisfactory Specimen
“Unsatisfactory” specimens may arise for a number of reasons. Water-based lubri-

cants commonly utilized for urethral catheterization and transurethral procedures
may impair specimen processing and can lead to an unsatisfactory diagnosis. The
management of this should be left at the discretion of the clinician. Depending on
the risk for a significant lesion, a repeat specimen may be obtained, if practical.
Quality control assessments by communication between the cytopathologists and
clinical staff will determine the reason for the “unsatisfactory” collection so that
repeat sampling under optimal conditions will be more likely to yield diagnostic
information.
Conclusions
Since 2016, The Paris System has been widely adopted and now represents the
standard nomenclature for urine cytology reporting. As the end-users of the urine
cytology report, we applaud the international cytopathology community in stan-
dardizing the criteria and methodology and bringing uniformity to the diagnostic
reporting. The evaluation and management described above represent only general
recommendations to provide insight for the cytopathologist to understand how a
given cytologic diagnosis may impact clinical decision-making. The ultimate care
of the patient should be individualized based on all available clinical information.
Communication between the clinician and cytopathologist is essential in order to
optimally utilize this powerful diagnostic tool.
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The History of Urinary Cytology:
The Long and Winding Road to Paris 2.0 13
Stefan E. Pambuccian
History has a good way to tell us, not only where we have been but also where we
are going, as William Osler [1] liked to quote the English churchman and historian
Thomas Fuller (1608–1661) [2]:
History maketh a young man to be old, without either wrinkles or gray hair; privileging him
with the experience of age, without the infirmities or inconveniences thereof. Yea, it not
only maketh things past, present, but enableth one to make rational conjecture of things to
come …. Old actions return again, furbished over with some new and different
circumstances.
Ancient History
Urine has had many uses in history. In Ancient Rome, urine was used to launder the
emblematic Roman togas, to fertilize fields and grow juicier pomegranates, and
even to whiten the teeth. Other nonmedical uses encountered through history
included the preparation of cosmetics, leather softening, textile dyeing, and the
preparation of gunpowder.1 Medical uses of human and animal urine as an ingredi-
ent of various remedies are found in all ancient cultures, starting with the Hindu
culture, to the Greco-Roman world, and even persisted into the late Middle Ages
and early Enlightenment, when urine was still recommended by some of the most
famous medical personalities. They included the French surgeon Ambroise Paré
(1510–1590), the English philosopher and chemist Robert Boyle (1627–1691), and
English physician Thomas Willis (1621–1675), who recommended urine to disin-
fect wounds and to treat cuts and burns and various external and internal ailments
1
Gunpowder to Teeth Whitener: The Science behind Historic Uses of Urine M Kumar - Smithsonian
Magazine (smithsonian. com), July–August. 2013 accession 3.1.2021
https://www.smithsonianmag.com/search/?q=Gunpowder%20to%20Teeth%20Whitener:%20
The%20Science%20behind%20Historic%20Uses%20of%20Urine

https://doi.org/10.1007/978-3-030-88686-8_13
268 S. E. Pambuccian
[3, 4]. While we may be surprised, shocked, repulsed, or disgusted by these and
other historical scatological therapeutic practices [5], we cannot be but fascinated
by the diagnostic use of urine through the ages. In many areas it is still a common
but not medically recommended practice to urinate on a sea urchin sting, jellyfish
sting, or a stingray wound.2 Through five millennia, witnessing the rise and fall of
empires, the examination of urine was considered one of the most informative diag-
nostic tools. Urine, after all, is an easily accessible fluid. The examination of urine
reached its apogee in the middle ages, when uroscopy was essentially the diagnosis.
The practice of uroscopy in the Middle Ages has inspired numerous manuscript
illuminations, paintings, and other works of art [6] and was considered the symbol
of the physician and its patron saint, Saint Cosmas [7] (Figs. 13.1, 13.2, 13.3,
and 13.4).
All major cultures left written and archeological evidence of the diagnostic value
of the examination of urine. Sickness and death have been plaguing humankind
since its earliest days, and humans have always struggled to understand and cure or
alleviate disease. Specialized “healers” have probably existed since the dawn of
civilization. The writings from Mesopotamia, Egypt, China, India, and Ancient
Greece all attest to the presence of healers. Depending on the culture, such healers
had different concepts of disease and employed a combination of magical/religious
and empiric/rational approaches, the relative importance of which varied in cultures
and different times. The oversimplified concept of a magical/religious healer, priest,
or exorcist of the Mesopotamian and Egyptian cultures changes abruptly in Ancient
Greece to a rational physician practicing Hippocratic medicine. In all these cultures,
just like today, sick people have sought help in multiple ways, starting from self-
help, prayer, and seeking the advice of friends and family and ultimately moving on
a b c
Fig. 13.1 Stained glass showing uroscopy: (a) Chartres Cathedral, thirteenth-century France.
Emperor Constantine, who has leprosy, consults a physician. (b, c) The monkey as a doctor exam-
ining a vial of urine. Detail from the Pilgrimage window. York Minster, fourteenth century
https://www.scientificamerican.com/article/fact-or-fiction-urinating/
2
13 The History of Urinary Cytology: The Long and Winding Road to Paris 2.0 269
Fig. 13.2 Left: A surgeon letting blood from a woman’s arm and a physician examining a urine
flask. Oil painting by a Flemish painter, 18th (?) century. Credit: Wellcome Collection. Attribution
4.0 International (Creative Commons (CC) BY 4.0). Right: doctor inspecting a urine flask in his
“home office.” Notice the wicker basket in which the urine was received. Evert Oudendijck, Dutch
painter (1648–1695)
Fig. 13.3 St. Cosmas and St. Damian (the patron saints of surgeons, physicians, and pharmacists).
St Cosmas (left) is frequently depicted holding a matula (urine flask). Woodcut attributed to Hans
Wechtlin from the 1517 book Feldbuch der Wundarzney of Hans von Gersdorff (1455–1529). The
framed image depicts Saint Cosmas and Saint Damian in an oil painting. Credit: Wellcome
Collection, London, UK. Attribution 4.0 International (Creative Commons (CC) BY 4.0)
Fig. 13.4 Illuminations from medieval manuscripts showing physicians performing uroscopy. (a)
Mystère de la Vengeance by Eustache Marcadé, Bruges, c. 1465 (London, British Library). (b) De
proprietatibus rerum by Bartholomaeus Anglicus, fifteenth century, France (Biblioteque Nationale
Francaise)
to seeking professional help. Based on a patient’s religion, understanding of the

cause of disease, personal experience, wealth, and the local availability of healers,
the professional help sought by an individual may have been religious or magical or
empirical and rational or various combinations thereof.
The cause of disease was sometimes obvious, as in traumatisms, wounds, and
insect and animal bites, due to the immediate witnessing of cause and effect; how-
ever, the etiology of most diseases was inscrutable. It was only in 1761 that Morgagni
(1682–1771) published De sedibus et causis morborum (i.e., of the Seats and Causes
of Diseases) that tied gross morphologic autopsy changes to clinical findings.
Certainly, it was not until the nineteenth century that modern knowledge about cell
theory, microbiology, and pathology began to evolve [8].
Since it would have been impossible for individuals of the time to understand the
real genetic or environmental causes of diseases, many of which still remain
unknown, different cultures blamed disease on supernatural forces, such as ghosts,
demons, and sins against gods. Alternatively, they may have held liable more natural
environmental causes, like exposure to cold and humid conditions, or excesses in
work, food, drink, or sex. The arrows of Apollo or crimes against the gods by mem-
bers of the family or city were enough reason for plague and disease.
Despite differences in their concepts of pathogenesis, healers from all these cul-
tures had to establish a diagnosis in order to fight the specific supernatural or natural
causes of disease by magical rituals, prayers and incantations, exorcisms, amulets,

fumigations, herbal remedies, or surgery. Diagnosis has relied, since the oldest
times, on objective, mostly visual, clues to complement the subjective clues gath-
ered from the patient’s account of the disease. These clues have included, like in
current medical practice, any changes in the sick person’s general appearance, pos-
ture, movement, color of the skin, and the appearance of their excreta. The appear-
ance, consistency, smell, and sometimes even the taste of blood, urine, feces, sweat,
sputum, and vomit could give the ancient medical practitioner a glimpse into the
inner workings of the body [9, 10].
Before the systematic performance of autopsies, the physiological processes that
gave rise to bodily fluids and excreta were a complete mystery to the ancient
Mesopotamian, Egyptian, Indian, Chinese, and Greco-Roman physicians, who had
to rely on alternative explanations for physiological and pathological processes.
Despite all the bloodletting, anatomic dissection was forbidden by Roman law.
Most ancient cultures had a holistic concept of disease, based on the person’s rela-
tionship with the universe, a universe that included both natural and supernatural
forces with which humans had to be in harmony. Rational natural philosophies that
developed with pre-Socratic philosophers in Greece in the sixth to fifth centuries
BCE and most likely independently in the Far East postulated the presence of ele-
mental matter in constant movement and balance as the basis for all things in the
universe. In Greek philosophy, these were the four essential elements: earth, fire,
water, and air, while in Chinese thought, they were wood, fire, earth, metal, and
water. These elements corresponded in the human body to fluids (humors) or ener-
gies that circulated within the body and were in tune with the surrounding universe
and corresponding to qualities of warmth and cold, dryness and moisture, to fullness
or emptiness, to colors, to the seasons, and even to the position of the stars. The
humors or energies were in balance and circulated freely within the body of a
healthy person, while sickness was caused by an imbalance between the fluids or
energies, the blockage of their circulation, or their contamination with poisons or
malefic energies.
Based on these theoretical considerations, Mesopotamian [11], Egyptian [12,
13], Indian, and Greco-Roman medical practitioners have used the appearance of
urine, its consistency, sometimes its smell, and even its taste to diagnose disease.
Studying the history of urine-based diagnosis, it is fascinating to realize that obser-
vations made on the urine of sick people were transmitted from civilization to civi-
lization. They were adapted to different worldviews and religions, some surviving
to our days in folk medicine and traditional or alternative medicine systems. The
history of uroscopy can, like the mythical Ariadne’s thread, guide the modern physi-
cian through the labyrinth of medical history, to discover the interconnectedness of
healing practices in major civilizations. One can retrace observations on the appear-
ance of the urine made in Mesopotamia 3000 years BCE to the Greek [14], Roman
and Byzantine, Islamic, and Medieval European cultures, demonstrating a remark-
able continuity of thought across time, geography, and religions [11]. For instance,
turbid white urine was compared to the urine of a donkey in the 40 tablets making
up the “Diagnostic Handbook” of the chief scholar of the Babylonian king
Adad-apla-iddina (1067–1046 BCE), but representing the transcription of an older

text from about 1600 BC. The same comparison with the urine of a donkey is found
in the works of the father of medicine, the Ancient Greek physician Hippocrates of
Kos (460–370 BCE), the sixth-century Byzantine Greek physician Aëtius of Amida,
the great eleventh-century Persian physician and philosopher Avicenna (Ibn Sina,
980–1037), the twelfth-century Sephardic Jewish physician Maimonides (Moses
ben Maimon, 1135–1204), and the thirteenth-century Byzantine writers Johannes
Zacharias Actuarius (1275–1328) and Nicephorus Blemmydes (1197–1272).
In the course of two millennia, uroscopy observations circled around the
Mediterranean. They can be traced westwards from Ancient Mesopotamia to
Ancient Greece and Rome and then eastwards to Asia Minor, back to Mesopotamia
and Persia, and then again westwards to North Africa and the Iberian peninsula in
the centuries after the fall of Rome. From Iberian Spain, teachings traveled to the
Italian peninsula and finally to France, England, the German-speaking countries,
and Scandinavia [15]. Some of the Mesopotamian “omens” reached Knidos and
Kos in Ancient Greece and were incorporated into the “sentences,” “prognosis,” and
“aphorisms” of the Hippocratic corpus in the fifth to fourth century BCE. In the
second century AD, Galen of Pergamon [16] incorporated these observations into
complex humoral physiologic interpretations, which remained the dominant theo-
retical background of medical practice for more than a millennium [17]. It took 14
centuries to challenge Galen’s infallibility through the demonstration of some of his
anatomical errors by Andreas Vesalius (1514–1564). Of interest, Galen postulated
there are three stages of “digestion,” each producing a waste product. The waste
products from the first digestion, occurring in the stomach, are the feces. The waste
products from the second digestion, occurring in the liver, were bile, black bile, and
urine. The third digestion, occurring in the other members (parts of the body), leaves
a variety of residues, including the urinary sediments, sputum, sweat, and so on.
According to this theory, urine was a fluid that reflected the inner workings of the
body and was therefore considered to be an easily accessible window into the
unseen digestive processes [18]. Based on Galen’s theory, the seventh-century
Byzantine physician Theophilus Protospatharios of Constantinople systematized
the examination of the urine, which he defined as a filtrate of blood. He described
ten colors ranging from white to black, thick or thin consistency, and the appearance
of the sediment in his book De urinis, which would constitute the basis for future
uroscopy texts [19, 20]. Viewed through intellectual lenses, some of these theories
are not entirely wrong and are significant scholarly achievements that did eventually
lead us to where we are today.
After the Arab conquests of the seventh century, the uroscopy observations of
Hippocrates and Galen were translated in Baghdad into Syriac and Arabic by the
Nestorian Christian physician Iohannitius (Hunayn ibn Ishaq, 809–873). They were
then incorporated into the works of the Persian Islamic Golden Age physicians Haly
Abbas (‘Ali ibn al-'Abbas al-Majusi (930–994), Rhazes (Abū Bakr Muhammad ibn
Zakariyyā al-Rāzī, 850–923), and Avicenna (Ibn Sina, 980–1037) [21]. Uroscopy
then travelled back westwards to North Africa, influencing the Arab-Jewish physi-
cian Isaac Judaeus (Isaac Israeli ben Solomon, 832–932) [22, 23] who worked in
Fig. 13.5 Map of the countries surrounding the Mediterranean Sea which played a role in the
spread of the knowledge about urine diagnosis
Egypt and Kairouan (currently Tunisia), and further westwards to the Iberian
Peninsula, to Cordoba (Al-Andalus), where they were included in the works of
Maimonides [24, 25] (Fig. 13.5).
In the following centuries, the Arabic texts on uroscopy authored by these physi-
cians were translated into Greek and Latin in Constantinople [26, 27] and Salerno
[28–31] in Southern Italy, from where uroscopy diffused to the major European
medical schools of Montpellier and Paris. Throughout this journey, the “judgement
of urines” became more and more complex, the number of colors of urine described
became more numerous, and the relative importance of uroscopy in the diagnostic
process increased. As the number of urine colors increased, naming them became
insufficient, and hand-painted urine wheels, depicting the various nuances of urine
color, were included in uroscopy manuscripts and the first printed uroscopy manu-
als. These urine wheels were considered so important that they were included in
foldable vade mecums (Latin, “go with me”) or pocket references that were placed
in bags or pouches hanging from the belts of the physicians of the time [32]. The
ownership of a urine wheel and the ability to interpret it constituted the closest
approximation to a medical license at the time (Fig. 13.6).
In the Middle Ages, when monastic medicine, societal norms, and concepts of
“modesty” made physical examination of patients, and in particular of women, dif-
ficult or impossible, the objective diagnostic methods were reduced to taking the
pulse and looking at the urine. Doctors educated at one of the rare universities of the
Middle Ages were few, were sometimes related to monasteries, and ordinarily did
not make house calls. The habit therefore ensued to send off urine in a rounded-
bottom flask that mimicked the shape of the bladder, called matula, which was
transported in a specially made basket. This practice, probably the first example of
Fig. 13.6 A portable physician’s folding almanac from the fifteenth century containing tables use-
ful for field practice, including a table of urine colors, next to zodiacal tables with moon and sun
phases and zodiacal-humoral correspondences, and bloodletting points (British Library, Harley
MS 5311). Hand-colored urine wheel from Fasciculo de medicina, attributed to Johannes de
Ketham, a professor of medicine in Vienna about 1460, and published in Venice in 1493 by Zuane
& Gregorio di Gregorii. This is the first illustrated medical book, a compendium of several differ-
ent, independently authored medical treatises covering a wide spectrum of the medical knowledge
of the time, including uroscopy, bloodletting, wound treatment, anatomical dissection, and wom-
en’s health
telemedicine, resulted in the physician being frequently depicted as looking into an

upheld urine glass, an image that morphed into the depiction of alchemists and
chemists [33] (Fig. 13.7).
The practice of basing a diagnosis solely on the examination of the patient’s
urine (uromancy), frequently with no or only incomplete history obtained from the
person delivering the urine-containing matula, probably led to results that were poor
even for the low expectations of the people of that time. The use of uroscopy as the
sole diagnostic tool, coupled with the popular belief that uroscopy can diagnose
pregnancy, and even the sex of the baby, led to excesses in the use of uroscopy,
which was taken up by laymen and charlatans, its fall into derision and disgrace
[35–37], and finally its abandonment in the seventeenth century [38]. This was not
before Paracelsus (Philippus Aureolus Theophrastus Bombastus von Hohenheim,
1493–1541) and his followers added alchemical methods to uroscopy. He remains a
very controversial figure to this day. Paracelsus was the first to openly break with
Fig. 13.7 Depiction of the matula after Joannes Zacharias Actuarius (1275–1328) [34]. Note that
the regions of the matula are corresponding to the parts of the human body. Regions 1–4, numbered
from the fundus of the matula, correspond to the belly and the urinary tract and regions 3–8 to the
thorax and 10–11 to the head. Human-shaped vessel to distill the urine (“Vase Distillatorio ad
Urinam”) made of “white venetian blown glass, substantially transparent,” known as anatomical
alembic or anatomic furnace (“Furnace Anatomica”), from the “Aurora Thesaurusque
Philosophorum, Theophrasti Paracelsi,” published in Basel in 1577, a treatise attributed to the
Belgian alchemist Gerard Dorn (1530–1584)
Galenism. Teaching in his native German rather than the customary Latin of his
times, he told his students that his shoe buckles contained more wisdom than Galen
and Avicenna [39]. Paracelsus refuted the diagnosis by uroscopy and pulse taking
practiced by the physicians of his times as impostures, commenting “their ignorance
cannot justify their fantastic theories. All they can do is gaze at piss.” In fact, prac-
titioners of uromancy acquired the appellation “Pisse Prophets.” Paracelsus none-
theless continued examining the urine of patients, through what he called chemical
“dissection” of the urine. He used alchemical extraction, coagulation, and distilla-
tion of the urine as diagnostic methods. Using such methods, he and his followers
would determine if the patient had bladder or kidney stones and what kind of stones
they were. They demonstrated albuminuria by precipitation of proteins when acid
(vinegar) was added to the urine [40, 41] and determined the weight (specific grav-
ity) of urine. They also introduced the quantification of substances it contained [42],
the fractionated distillation of urine, and the observation of the colors of the flames
after burning of different distillation residues.
One of his followers, the Swiss Leonard Thurneysser (1531–1595), used the
“anatomical furnace” to have an even more detailed analysis of the urine by frac-
tionated distillation at different temperatures to determine the presence and quantity
of elements of sulfur, salt, and mercury in urine. For a time, he had a very successful
practice and established the first “telemedicine” center in Berlin, where he was
examining patients’ urines sent from all over Europe and sent them back the pre-
scribed medications (Fig. 13.8).
Another alchemist, Hennig Brand (1630–1692) of Hamburg, studying urine,
accidentally discovered a chemical element, which he named phosphorus (Greek
for “light-bearer”) because the substance that he isolated from the heated resi-
dues from boiled-down urine emitted a pale-green glow. Despite the arcane,
strange, and complex theoretical underpinnings, the alchemical understanding of
disease was a step forward and laid the foundations of iatrochemistry, which
evolved into clinical chemistry. By the middle of the nineteenth century, the pres-
ence and quantity of most of the normal chemical constituents of urine were
known, and chemical methods of examining urine started to be routinely used in
the clinical diagnosis.
Fig. 13.8 Stained-glass window panel made by Christoph Murer in1579, showing Leonhard
Thurneysser zum Thurn (1531–1596) testing urine for a potentate in the Middle East (Kunstmuseum
Basel, Isobel Leybold-Johnson in Basel) http://www.swissinfo.ch/eng/culture/basel-s-16th-century-
superstar/28863378
The excesses of uroscopy and the fact that it was based on the Galenic theory of
humors can lead to the impression that the Late Antiquity, Byzantine, Islamic, and
Medieval Latin Western civilizations were a period of stagnation of medicine. However,
this period should not be perceived only as a relay station for the transmission of the
wisdom of Ancient Greek medicine. The authors of this period, while striving to main-
tain tradition, added new empirical data and practical therapeutic measures that lead to
improvements in medical and surgical care. Even while they claimed to follow Galen’s
theory, in their comments and explanations, they made subtle changes in its interpreta-
tion [28] and established a rational, almost algorithmic system of diagnostic decision-
making. This elaborate diagnostic system could be conveyed by formal instruction,
through textbooks and taught in the classroom [18], and could even be written in verse,
for ease of memorization [43]. Uroscopy also arguably constituted the first laboratory
test and highlighted the importance of observation. In today’s extensively technolo-
gized laboratory, it reminds us that sometimes simple observations, such as the change
in the color of urine, may have very important diagnostic connotations. The color of
urine, its viscosity and specific gravity, its clarity, and the presence of precipitates and
floating particles, all features that were assessed by uroscopy, are still diagnostically
useful. Modern-day urine wheels can be devised to depict the significance of urine
color for diagnosis [44], and observing the color of urine in the bag of catheterized
patients can still give important diagnostic clues [45] (Fig. 13.9).
Fig. 13.9 A physician

examining a urine flask.
1780 oil painting by the
German-born Dutch
painter Willem Joseph
Laquy after a 1677
painting by the Dutch
Golden Age painter Jacob
Toorenvliet (1635–1719)
Credit: Wellcome
Collection
The sixteenth century saw not only the birth of medical alchemy, which morphed
into iatrochemistry (or chemiatry), which in turn gave rise to clinical chemistry and
biochemistry, but also the invention of a device that was destined to change the
practice of medicine completely, the microscope. Towards the end of the sixteenth
century, simple compound microscopes were made by combining lenses and put-
ting them at the ends of a telescoping tube, by the Dutch spectacle makers, Hans
Janssen and his son Zaccharias. Such very primitive devices had 9x magnification,
and the blurry images were mostly used for the entertainment of nobility. In time,
bigger and better instruments were built, and in the early seventeenth century, lay-
men and scientists used them to extend the reach of visual observation of all kind of
objects: plants, insects, and human tissues and body fluids. Despite the difficulties
that must have been encountered in examining a transparent liquid under the primi-
tive microscopes of the time, urine was one of the first body fluids to be subjected
to microscopic analysis. Initially the microscopic examination of urine was proba-
bly done mostly out of curiosity, and outside the clinical context, it likely had little
impact on the medical field. It is worth mentioning that the microscopic examina-
tion of urine was not mentioned by some of the most prominent microscopists of the
time, who were interested in the urinary tract, including Marcello Malpighi
(1628–1694) and Lorenzo Bellini (1643–1704).
The first to describe the microscopic appearance of crystals (most likely uric acid
or oxalate crystals) in the urinary sediment as “a heap of rhomboidal bricks” was the
Provençal polymath Nicolas-Claude Fabri de Peiresc (1580–1637), according to his
biographer [46]. In 1630 he examined “sandy urine” and thought that he found the
cause of urinary calculi. A few decades later, in 1665, the English physicist Robert
Hooke (1635–1703) collated his microscopic observations in a beautifully illus-
trated tome “Micrographia.” In addition to coining the name “cells” to the spaces he
saw looking at a sliver of cork through his brass microscope, he also illustrated
crystals seen in the urine sediment and crystals formed by freezing urine [47]
(Fig. 13.10).
Starting around 1673, the Dutch draper (cloth retailer) Anton van Leeuwenhoek
(1632–1723) started reporting to the scientific community of the Royal Society of
London his observations made with his simple single-lens, yet powerful micro-
scopes capable of magnifications of up to 200–370 times. The metal construction of
his microscopes made the examination of fluids difficult, but he did report observa-
tions on urine crystals and other body fluids [48] (Fig. 13.11).
The first physician to perform microscopic studies on urine was probably the
Danish physician and member of the Leopoldina Academy Jørgen Hermansen Hahn
of Copenhagen (1647–1699). Better known under his Latinized name Georgius
Hannaeus, he published in 1687 an account of the microscopic appearance of “sand”
from urinary calculi, obtained after drying the urine sediment on paper and obtain-
ing a “farinaceous” powder. Also interested in urinary stones, the Leyden physician
and pioneer of bedside teaching, Hermann Boerhaave (1668–1738), studied the
urine of normal people to determine if they were predisposed to urolithiasis [49].
Primitive microscopes were expensive, were technically difficult to use, and pro-
duced poor images that could easily lead to misinterpretations. Their use was not
Fig. 13.10 Hooke’s 1665 depiction of urinary sediment crystals and crystals formed by freezing
urine. From “Micrographia: or some physiological descriptions of minute bodies made by magni-
fying glasses with observations and Inquiries thereupon” by Robert Hooke, Fellow of the Royal
Society, 1665. (Credit: Engraving from Micrographia, 1665, by Robert Hooke. Credit: Project
Gutenberg License)
Fig. 13.11 Hannaeus’ 1687 report on urinary sand

accepted by the medical community until more and more sophisticated and power-
ful microscopes were built, assuring physicians that the objects seen under the
microscope were “real” and not artifacts.
In the meantime, the eighteenth century, which was relatively quiet in regard to
microscopy, saw a dramatic change in the way disease was conceived. Humoral
medicine still conceived all diseases as involving the whole body as imbalances
between humors, the visible manifestations reflecting the local impact of this imbal-
ance. As indicated above, these concepts changed due to the publication in 1761 of
the results of 640 autopsies with clinicopathologic correlations (historiae anatomo-
medicae) by the 79-year-old Giovanni Battista Morgagni (1682–1771) [8, 50]. In
his influential work, The Seats and Causes of Diseases, the professor of anatomy at
the University of Padua proposed that most diseases do not diffusely involve the
whole body, but originate and reside locally in its organs. Despite the fact that
Morgagni was a pupil of Antonio Maria Valsalva (1666–1723), who had been men-
tored by the great microscopy pioneer Marcello Malpighi (1628–1694), Morgagni
only rarely used the microscope.
he Beginnings of the Study of Cells in the Urine

T
(Urine Cytology)
It is frequently assumed that the study of cells in the urine, or urinary tract cytology,
started with the works of Papanicolaou. This is hardly surprising, since Papanicolaou
himself was unaware of the rich history of cytology and entitled his first clinical
cytology contribution “New Cancer Diagnosis.”
The history of urine cytology started in the nineteenth century, but was “discon-
tinuous” [51] and followed a tortuous path. It had many promising starts that were
cut short by the researchers’ career changes, personal tragedies, historical events
like revolutions and wars, or merely lack of interest on the part of the medical estab-
lishment. During the 1830s, urine microscopy was “rediscovered” [52] as an impor-
tant clinical tool in Paris and then quickly disseminated to German-speaking
countries and Great Britain.
In order to understand the background against which this rediscovery has
occurred, we need to review the changes in medical thinking brought about by the
Paris clinical school in the first decades of the nineteenth century, which refined the
concept of disease and pioneered the nosology and classification of diseases. The
concept of disease endured another major change through the work of Marie-
Francois Xavier Bichat (1771–1802), chief physician at the Hotel-Dieu, Paris. A
product of the widespread changes in medical education and practice brought about
by the French revolution, Bichat, a member of the Paris clinical school, introduced
the notion of tissues as distinct entities. In his 1800 “Treatise on Membranes” [53]
Bichat distinguished 21 types of tissues, different combinations of which formed
the organs of the body, and maintained that diseases attacked tissues rather than
whole organs (“…we must consider disease not from the standpoint of the com-
pound organs but from the standpoint of their different textures, which are almost
always attacked separately”). Remarkably, Bichat had these insights without the use
of a microscope through performing numerous autopsies, which may have contrib-
uted to his untimely death at age of 31. Half a century later, the German pathologist
Rudolf Ludwig Carl Virchow (1821–1902) further narrowed down the site of dis-
eases to the cell [54]. Although Virchow advocated this cellular view and promoted
it to the scientific medical community, both the aphorism “omnis cellula e cellula”
usually attributed to him and the concept of the cell as the site of disease had been
previously stated by the French scientist and revolutionary François-Vincent Raspail
(1794–1878) in 1825 [55] and 1843 [56].
In the aftermath of the reforms brought by the French Revolution, medicine was
taught at the bedside in hospitals, rather than in auditoria and lecture halls. The clinical
experience was fostered in contrast to the sterile memorization of “classic” textbooks.
This practice encouraged the observation of the patient throughout the entire course of
the disease, and, if the disease proved fatal, correlation with autopsy findings. This led
to the birth of the clinique (clinic or teaching hospital) in Paris, a medical teaching
model still used to this day. The success of this observation-based clinical teaching
model, with the motto “peu lire et beaucoup voir” (“read little but see a lot”), was made
possible by the very large number of patients (up to 5000 per year) seen by doctors and
students at that time. The observation of patients was coupled with the examination of
patients aided by new or improved methods of physical examination (auscultation and
percussion), statistical thinking, and the increasing importance of pathologic correla-
tion [57]. Based on radical empiricism and opposition to any abstract theory, system, or
philosophical thinking, it was so successful in patient care that it made Paris the mecca
of the medical world, attracting students and physicians from other European countries
and North America. This led to the dissemination of clinicopathologic or anatomo-
clinical, as it was called at the time, thinking to other countries.
Gross pathologic examination was the cornerstone of the scientific method of the
time, but as soon as microscopes became more reliable, microscopic examination
was seen by some as an extension of macroscopic examination and was rapidly
embraced. This occurred in the 1820s when achromatic lenses, small enough to be
used in a microscope, were created through the synchronous achievements of com-
peting and collaborating artisans and physicists across Europe, including Giovanni
Battista Amici (1786–1863) in Italy, Joseph Jackson Lister (1786–1869) in England,
Charles Louis Chevalier (1804–1859) in France, and Joseph Von Fraunhofer
(1787–1826) in Bavaria.
Achromatic lenses reduced the distortion factor from 20% to 3%; controlled iri-
descence, removing the fringes of color around objects; and eliminated the reticular
image, a historical artifact that plagued early microscopists [58]. Therefore, despite
their initial significant cost, which may have reached a person’s yearly salary, such
microscopes were quickly embraced in Paris, Berlin, Vienna, and London and revo-
lutionized biologic and medical thinking through the development of the cell theory
around 1840, which states that the cell is the quantum minimum of all plant and
animal life.
Before 1837, when Gabriel Valentin (1810–1883), a Swiss-German professor at
the University of Bern, developed a double-bladed knife for this purpose, obtaining
tissue slices thin enough to be examined by transmission optical microscopy

required extraordinary skill and luck. Most early microscopic observations were
made on squashed bits of tissue, on touch imprints, on cells “teased out” of tissues
with the help of needles, or on body fluids, secretions, and excretions. These were
usually examined unstained, apart from the occasional use of iodine, because bio-
logical stains were only developed half a century later [59]. Although it might cur-
rently appear incomprehensible how detailed cytologic and histologic observations
can be made without fixation and staining, the authors of the microscopic revolution
that occurred in the 1830s and 1840s demonstrate that “with patience and careful
observation there is little which cannot be seen” even without the use of stains [60].
The advances in clinical chemistry made in the second part of the eighteenth
century and first part of the nineteenth century established chemical analysis of the
urine as the scientific method to study urine; consequently, the initial work of
microscopists was disparaged. It was perceived inferior to that of chemists, because
like clerks, microscopists just recorded their observations [61]. The first to advocate
the clinical use of the microscope for the study of urine, secretions, and excretions
was Alfred Marie François Donné (1801–1870). After completing his medical stud-
ies at the Paris medical school, Donné became “chef-de-clinique” (roughly equiva-
lent to today’s chief resident) at the medical clinic of the Hôpital de la Charité. He
was quickly attracted to pathology and microscopy. His mentor, the famous cardi-
ologist and neurologist Jean-Baptiste Bouillaud (1796–1881), encouraged his
endeavors. After graduating in 1831 with the doctoral thesis “Physiologic and
chemical-microscopic studies of the cells of the blood, pus, mucus and those of the
eye humors,” Donné continued his research on the microscopic findings of urine,
blood, pus, mucus, genital secretions, tears, and milk. After failing the “agrégation”
examination, which would have allowed him to be a candidate for a faculty position
at the medical school in 1835, Donné obtained a position of under-librarian at the
Paris Medical School. He was also given the apparently unprecedented privilege to
use a large lecture hall at the Medical School’s Hôpital des Cliniques. After equip-
ping this lecture hall with 20 modern microscopes acquired from his own funds, he
started to give private lectures on the clinical use of the microscope to an audience
of about 60 French and international students in 1837. The lectures were followed
by practical microscope sessions conducted with the help of his assistant, Jean-
Bernard-Léon Foucault (1819–1868), who first started a medical career, but later,
under Donné’s guidance, became a famous physicist, still remembered for the pen-
dulum he designed in 1851 to give direct evidence of the earth’s rotation. Foucault’s
inventivity proved useful, since he devised a carbon-based electric arc lamp as a
light source for his evening microscope sessions, replacing the flammable (and
explosive) mix of oxygen and hydrogen used at that time. He later used a similar
electric arc lamp to provide the first urban lighting solution, replacing the much less
powerful gaslights and making Paris the “city of lights.” Another one of Foucault’s
inventions was the adaptation of the new photographic technique invented around
the same time by Louis-Jacques-Mandé Daguerre (1787–1851) to the microscope.
In 1839, Donne published a monograph describing the components of the urinary
sediment, including epithelial cells, mucus, pus, blood cells, fat, sperm, prostatic
concretions, and various crystals [62]. He had previously described Trichomonas

(1836) and subsequently described platelets (1842) and leukemia (1844). He pub-
lished an atlas to accompany his lectures (Cours de microscopie) [63] containing the
first microphotographs (“daguerreotypes”) of various cells and structures, including
urinary crystals (1845) [64]. In his “Cours de microscopie,“ [63] Donné mentions,
without going into details, that urine can contain numerous foreign bodies and
pathologic products like tubercular or cancerous matter, among other materials. The
many discoveries made in examining urine under the microscope made Alfred
Donné declare in 1844 that “the study of urine is, I may say, the triumph of the
microscope” [65] (Fig. 13.12).
Despite these major achievements, his most important contribution may have
been his immensely popular and influential course on the use of the microscope as
a clinical diagnostic tool. His passion and conviction in the future of the microscope
inspired many French and foreign physicians, including Hermann Lebert
(1813–1878) and John Hughes Bennett (1812–1875). American physicians also
took his enthusiasm for clinical diagnostic microscopy back to their home countries.
Donne’s emphasis on the practical use of the microscope in clinical diagnosis con-
trasted with the use of the microscope to provide an abstract understanding of dis-
ease championed by the German microscopists. This approach secured Donné a
place in the history of pathology and him to be considered among the “Great
Physicians of the 19th Century” [66].
Fig. 13.12 Example of daguerreotype of urinary sediment included in Donne and Foucault’s atlas
showing “white filaments” (left) and red blood cells, some crenated (right) in the urine
Around the same time, Eugène-Napoléon Vigla (1813–1872) (Fig. 13.13), the
assistant of the founder of French nephrology, Pierre François Olive Rayer
(1793–1867), published his findings of the microscopic appearance of the crystals,
red cells, white cells, and epithelial cells most often seen of the urinary sediment in
various forms of kidney diseases [67, 68]. Vigla, who had taken Donné’s micros-
copy course [69], described the normal epithelial elements that “desquamate con-
tinuously” (“éprouve une desquamation continuelle dans l’état naturel”) and do not
represent a sign of a disease of the kidney or bladder. In women, when in doubt
about the urinary or genital tract origin of the epithelial cells, he recommended the
use of catheterized urine.
In the following years, urine microscopy was used more frequently, and a series
of new findings were described in rapid succession. Dysmorphic red blood cells of
Bright’s disease were described in 1841 by the French physician Louis Alfred
Becquerel (1814–1862) [70]. Urinary casts were described in 1844 concomitantly
by Julius Vogel (1814–1880), Johann Joseph Scherer (1814–1869), and Johann
Franz Simon (1807–1843) [71]. Simon’s drawings of casts were included by the
Guy’s Hospital physician Golding Bird (1814–1854) in his influential book on
Fig. 13.13 Eugène-Napoléon
Vigla, Lithograph by
Bornemann after Charles
Reutlinger Wellcome
Library, London
urinary sediment examination [52]. Urine microscopy became accepted as a stan-

dard clinical practice in the second half of the nineteenth century, at least in part due
to the books authored by Golding Bird and by another British pioneer of clinical
pathology, Lionel Beale (1829–1906) [72], who had opened a private reference
laboratory in London in 1852.
Until the middle of the nineteenth century, tumors were, in general, not a major
topic of pathologic study, both due to the preoccupation of physicians and scientists
with the much more common infectious diseases and due to the fact that relatively
short life expectancy of the population made tumors relatively infrequent. This was
particularly true regarding bladder tumors, which were much less common at that
time than bladder stones, the main concern of surgeons and lithotomists of the time.
As time progressed bladder stones became relatively rare, and bladder tumors much
more common, due to a combination of mutagenic factors, including smoking,
exposure to chemical carcinogens, and increased longevity.
The first depiction of a bladder tumor is that by the Dutch physician and praelec-
tor of the Amsterdam surgeon’s guild Frederik Ruysch (1638–1731) [73]. Rare scat-
tered descriptions of bladder tumors operated upon, most likely because they were
confused with bladder stones, can be found in the literature, but were unusual before
the availability of an instrument to visualize the inside of the urinary bladder, the
cystoscope. For instance, in the 1875 statistics of the Charité-Hospital in Berlin,
where Virchow was professor, of the 15,294 cases treated, most were diagnosed as
infectious or venereal diseases, only 157 as tumors, of which 111 were carcinomas.
Of the 49 carcinomas treated surgically, only 1 was a bladder cancer [74].
The introduction of the microscope into the pathologist’s armamentarium stimu-
lated a sudden interest in tumor pathology, for which the Berlin pathologist, physi-
ologist, and embryologist Johannes Petrus Müller (1801–1858) was particularly
responsible. In 1838, one year before his pupil Schwann formulated the cell theory,
Müller demonstrated that cancer was an abnormal growth made of abnormal cells
[75]. Pathologists in Edinburgh, Paris, Berlin, and Vienna became interested in the
pathology of tumors, and in the middle of the nineteenth century, the pathology of
bladder tumors and especially of papillary tumors of the bladder was described by
John Hughes Bennett [76], Joseph von Gerlach [77], and Carl Freiherr von
Rokitansky (1804–1878) [78].
The first microscopic descriptions of cancer cells in the urine were from cases of
advanced cancer in which fragments of large tumors broke off and were “expecto-
rated,” “vomited,” or eliminated in the urine, a phenomenon that was referred to as
“spontaneous biopsy” [51]. Although until now the first description of cancer cells
in the urine was attributed to Lambl (1856), or Sanders (1864) and Dickinson
(1869), the first to describe tumor cells in the urine appears to be the British surgeon
Charles Hewitt Moore (1821–1870) [79]. After obtaining a medical degree from the
St. Bartholomew’s Hospital in 1842, Moore became assistant to the anatomist and
surgeon Frederic Carpenter Skey (1798–1872) and Clinical Clerk to the physician
Sir George Burrows, to both of whom he attributed his precision of thought and
accuracy of observation. Moore then pursued 3 years of postgraduate studies in
Vienna and Berlin, where he studied pathology and microscopy. In Vienna he
studied with Rokitansky, whose “Manual of Pathological Anatomy” he translated

into English. On return to England, he became lecturer on anatomy and surgeon at
the Middlesex Hospital. For many years, starting in 1848, he was in charge of the
hospital’s Special Cancer Wards, probably the world’s first cancer center. His obitu-
ary describes him as “Sedate, cautious, and self-possessed, never speaking unless he
had something of value to say, or without careful previous consideration, and put-
ting conscience into every work he undertook” [80]. He was the first to advocate in
1867 breast cancer operations consisting of mastectomy and axillary dissection, as
anything less was “a mistaken kindness to the patient” [81].
In a case read before the Royal Society on June 22, 1852, Moore described the
case of a 53-year-old man who had a large pulsating groin tumor that was operated
upon under the impression it represented an aneurysm. The patient unfortunately
succumbed soon after the operation. The autopsy found that the pulsating tumor
was a massive metastatic adenopathy encasing the bifurcation of the common iliac
artery. The primary tumor was a prostate cancer, which the author and his contem-
poraries considered rare. Although the bladder appeared normal, the urine “pressed
out through the urethra after death was abundantly pervaded with cancer-cells.”
The cancer cells were “large irregular aggregations of spherical cells, caudate cells,
and circular cells with one, six, or more sub-cells. Besides these, there were epithe-
lium scales, and single round globules or cells.” During life, the patient had had
some urine examined grossly after it was obtained through a catheter; but since “it
was quite clear,” it was not further examined microscopically. He concludes his
communication by stating that: “It is a matter of little practical importance whether
these elements of the malignant disease were shed by the kidney or the prostate. It
is, however, worthy of remark that microscopic examination of an excretion,
though an unusual practice, might have the effect of determining the nature of a
doubtful case, and preventing a [needless] operation” [79] (Fig. 13.14).
The first to describe bladder tumor cells in the urine was Wilhelm (Vilém Dušan)
Lambl (1824–1895), a Czech physician and revolutionary working in Prague, then
Fig. 13.14 Charles Moore and the title page of his 1852 paper. (From Robinson [81])
part of the Austro-Hungarian Empire. In his study, first presented at the meeting of
the College of Physicians of Prague in November 1854 and published in 1856,
Lambl describes the microscopic findings of ten patients with bladder cancer,
mostly from the surgical clinic of Franz (Jiří František) Pitha (1810–1875). Some
tumors had been studied at autopsy, but some were diagnosed intra vitam in urinary
specimens, usually obtained through catheterization. The article contains extensive
descriptions of the microscopic findings and four lithographs beautifully depicting
the cytology of the tumors. In his paper, Lambl, who is remembered for his descrip-
tion of Giardia and of aortic valve excrescences that bear his name, not only states
that a diagnosis of bladder cancer is possible through the microscopic examination
of the urine but also that such a diagnosis can be performed at the patient’s bedside.
Contrary to the general thinking of the time, the diagnosis of tumors was usually
made based on their gross appearance. Even for pathologists with experience with
the use of the microscope, the diagnosis of cancer was made based on the demon-
stration of invasion. In addition, the question whether papillary bladder tumors
without demonstrable invasion are benign or malignant had not been solved and
would haunt pathologists to this day. One of the greatest pathologists of that time,
Rokitansky in Vienna considered all papillary (villous) tumors of the bladder as
cancer (“Zottenkrebs”) [78], while Virchow thought that there are both benign and
malignant papillary bladder tumors, but papillary tumors are mostly benign [82,
83]. Lambl’s paper elicited a negative reaction, both because he was recommending
the diagnosis of bladder cancer by a cytologic method that cannot demonstrate inva-
sion and the fact that he considered, like Rokitansky, that all papillary bladder
tumors are malignant (“Harnblasenkrebs”). Ernst Leberecht Wagner (1829–1888),
professor of pathology in Leipzig, wrote a detailed and thoughtful review of Lambl’s
paper for the “1856 Year Book in Medicine” [84]. He argues that neither cell
enlargement and rounding, nor nuclear enlargement or multinucleation, which
could be seen in the urine sediment, can answer the question if we are dealing with
a cancerous or non-cancerous papillary bladder tumor. In his view, the diagnosis of
cancer could only be made in the presence of fragments of the flat bladder mucosa
showing cancer, in addition to the papillary structures. He adds that, based on these
criteria, he had diagnosed two cases of cancer in the urine, a primary cancer of the
bladder and a metastatic gastric cancer. A similar position was held by Gustaf
Vilhelm Johan von Düben (1822–1892), the chair of Pathological Anatomy at the
Karolinska Institute [85], and despite Lambl’s insistence on the possibility of diag-
nosing bladder cancer microscopically in the urine in a follow-up paper in Virchow’s
Archiv [86], this idea was not generally accepted by the pathology community
(Fig. 13.15).
Nonetheless, some of Lambl’s ideas and drawings were taken up by the German
pathologist Karl Julius Vogel (1814–1880), who included them in his very success-
ful book on the chemical and microscopic analysis of urine written together with the
German chemist Carl Theodor Ludwig Neubauer (1830–1879). In the 1830s, Vogel
had travelled to Paris during his medical studies, where he became familiar with the
use of the microscope, probably by attending Donné’s clinical microscopy course.
Vogel and Neubauer’s book became a standard reference manual on the laboratory
Fig. 13.15 Wilhelm Lambl and the depiction of papillary tumor cells and fragments in the urine
in his 1856 paper
examination of urine for 50 years and was translated into English, French, and
Russian. The third edition of this book, published in 1858 [87], contains drawings
of cancer cells in the urinary sediment made by Vogel “in part from the valuable
work of Dr. Lambl,…, and partly from my own observation.” He describes the blad-
der cancer cells as “isolated cells, mostly irregular in form, partly caudate, and
partly branched, rather large, containing a large nucleus.” (Fig. 13.16).
A few years later, in 1864, the Edinburgh pathologist William Rutherford Sanders
(1828–1881), who had learned microscopy in Heidelberg with Friedrich Gustav
Jacob Henle (1809–1885), reports the case of a 43-year-old man with an extensive
“dendritic” (papillary) bladder cancer that discharged fragments into the urine, but
had no accompanying hematuria [88]. The tumor fragments formed plugs that inter-
mittently obstructed the urethra and under the microscope showed “peculiar con-
centric globular bodies,” which most likely represented squamous pearls.
The next case of bladder cancer diagnosed in the urine was reported by William
Howship Dickinson (1832–1913), an “all-around physician” [89], pathologist, and
specialist in childhood disease and kidney diseases, who would become president of
both the Pathological Society of London (1889–1990) and the Royal Medical and
Chirurgical Society (1897). In 1869, he reported an unusual case, in which tumor
fragments as large as a bean were passed in the urine without accompanying symp-
toms [90]. Microscopic examination of the small quantity of bloody urine in which
Fig. 13.16 Julius Vogel and depiction of bladder cancer fragments and single cells, partially
drawn after Lambl, in his book
the masses lay “abounded with large cells, presenting a great variety of shapes;
some were spheroidal, some spindle-shaped, others irregularly angular, and curved.
There could be no doubt that the masses described formed parts of an organised
cellular growth, such as would belong to a malignant tumour, apparently of the
encephaloid kind.” He confesses that he had never previously found anything diag-
nostic in cases of cervical or bladder cancer that he had examined microscopically.
After he acknowledges Sanders’ prior contribution, Dickinson states that, while
passage of cancer tissue fragments is rare, the passage of fragments of “villous
growth” (which he considers benign) is not, as he has observed it a number of times.
He states that: “When, however, the bladder has become the seat of a villous growth,
it not unfrequently happens that loops and fragments of blood vessels become
detached and pass out with the urine, and give conclusive evidence of the nature of
the disease,” since they are “easily recognised under the microscope.” Dickinson
also cautions that the microscopic suspicion of malignant disease of the bladder or
kidney is often a source of erroneous diagnosis of cancer. He writes: “Under cir-
cumstances of irritation and disturbance large cells, presenting quite the ideal of
cancer cells, of all shapes and characters, regular and irregular, rounded, angular,
and elongated, are occasionally thrown off in abundance from various parts of the
urinary membrane. No isolated or detached cells, therefore, found in the urine, can
be regarded as proof of cancer in the urinary tract, while the absence of such cells
presents no argument in favour of the freedom of the bladder and kidney from
malignant growths.” (Fig. 13.17).
In the following years, bladder tumor cells were occasionally described and
depicted in the urine by authors from France, Austria, and Germany. In Vienna,
which had become a center of medical progress, the surgeon/urologist Robert
Ultzmann (1842–1889) and the chemist Karl Berthold Hofmann (1842–1922) pub-
lished a beautifully illustrated atlas on urinary sediments in 1871 [91]. The book
includes drawings depicting papillae and single cells found in the urine in a case of
Vincenz Czerny (1842–1916) and states that this is a very rare finding. It also
advises that when only single cells (frequently unusually large, caudate cells, with
multiple nuclei) are present, a probable diagnosis should only be made when they
are seen in great quantity (Fig. 13.18).
In his 1874 book, the Paris pathologist Charles Philippe Robin (1821–1885) and
former student of Donne writes that: “The papillary epithelia, or fungating vegeta-
tions of the bladder neck, may be recognized by the character of this epithelium,
when these vegetations detach themselves from their surface, and form sediments in
the urine. The cells are sometimes isolated, sometimes united in tatters or papillary
sheaths. Sometimes papillae or clusters of papillae containing a vascular loop, and
covered with their epithelium, are also seen.”
In 1877, the German pathologist Max Perls (1843–1881), the inventor of the iron
stain that bears his name, published a beautifully illustrated textbook of pathology.
This included drawings made by his former assistant, Ziesing, depicting a urinary
bladder villous tumor and cancer cells (Fig. 13.17). Perls died at the age of 38 of
typhus acquired through the practice of pathology, and the second edition of his
book was published under the care of Friedrich Neelsen (1854–1898), the co-
developer of the Ziehl-Neelsen stain. The drawings of the cytology of bladder
Fig. 13.17 Drawing from Dickinson’s paper depicting bladder cancer in the urine. Portrait of
William Howship Dickinson from his obituary in Br Med J Jan 18, 1913
Fig. 13.18 Bladder cancer in the urinary sediment depicted in Ultzmann and Hofmann’s atlas
a b c
Fig. 13.19 Drawings by Dr. Ziesing from Perls’ book showing a fresh papillary tumor of the blad-
der, magnified 50× (a) and 250× (b), and cells from a papillary carcinoma of the bladder (c)
tumors were reproduced in other authors’ pathology textbooks, including a book on

the pathology of tumors published by the Chicago surgeon Nicholas Senn
(1844–1908) in 1895 [92] (Fig. 13.19).
In the United States, the Philadelphia clinician and pathologist James Tyson
(1841–1919) illustrated urinary sediment epithelial cells in his 1878 book on urine
examination [93] and comments that elements of “morbid growths” are seldom
found in the urine and may occur as single cells and fragments. Cells “may be sus-
pected to me of morbid origin by their large size, multinuclear character, large size
of the nuclei, and diversity of cell-forms. Fragments of cancerous growths which get
into the urine are generally from the villous kind, and may show the capillary vessels
which make up the villus, with or without the epithelial covering. Fragments,
suitable. for examination, are sometimes withdrawn with the catheter” [93]
(Fig. 13.20).
Towards the end of the nineteenth century, the Danish surgeon Niels Thorkild
Rovsing (1862–1927) published a paper on the diagnosis of kidney cancer [94], in
which he emphasized the diagnostic value of finding abnormal large round or
spindle-shaped cells sometimes with fatty degeneration in the urine.
The last decades of the nineteenth century witnessed extraordinary progress in
the study of bladder tumors, especially due to the introduction of cystoscopy into
routine urologic practice [95–97] and progress in surgical technique, anesthesia,
and antisepsis, which allowed the performance of cystectomies and other bladder
operations. During the same period, microscopes were significantly improved
through the development of focusable condensers, homogeneous immersion, and
apochromatic lenses at Carl Zeiss’s [98] workshop, based on Ernst Abbe’s research.
The last two decades of the nineteenth century were also the time in which most of
the histopathology techniques that are still in use today were perfected. Edwin
Klebs (1834–1913) introduced paraffin embedding and fixation with Zenker’s solu-
tion in 1869; aniline dyes (methylene blue and others, including eosin) were first
used as cytologic stains by Paul Ehrlich (1854–1915) in 1878. This was around the
same time that multiple researchers used the combinations of hematoxylin and
eosin [99]. Formalin fixation was introduced by Isaac Blum (1833–1903) in 1893,
and before the end of the nineteenth century, Paul Meyer (1848–1923) formulated
the currently used hematoxylin-eosin. Dmitri Leonidovich Romanowsky
(1861–1921) formulated a stain consisting of mixture of eosin and aged methylene
blue that was modified multiple times and proved to be useful in hematology, cytol-
ogy, and bacteriology [100–102]. By the turn of the century, most histologic
Fig. 13.20 James Tyson and the depiction of urinary sediment cells in his 1878 book
techniques that we use today, including formalin fixation, paraffin embedding, and
cutting thin sections with a rotary microtome, were already in widespread use in
laboratories on both sides of the Atlantic and histopathology began to dominate
pathology. At the same time, the cytologic methods, which had been used until then,
were largely abandoned by pathologists.
In these conditions, the microscopic diagnosis of urinary sediments was prac-
ticed predominantly by clinicians with an interest in nephrology or urology or by
“hybrid” clinician-pathologists. Towards the end of the nineteenth century, two
American clinician/pathologists practicing in New York City published an interest-
ing work on the urinary cytologic diagnosis of bladder tumors. The first of these was
the New York University Hospital Professor of Pathology and Clinical Medicine,
Farquhar “Frank” Ferguson (1851–1907). In 1892, he presented the results of his
new method of microscopic diagnosis of bladder tumors at the New York
Pathological Society. This was the first reported use of cellblock preparations,
4 years before Bahrenberg’s use of cellblock preparation obtained through paraffin
embedding clotted ascitic fluid [103]. However, his method, which he considered
superior to that of direct smears from the urinary sediment, was very tedious and
consisted of filtering the urine collected over 24 hours through a cheesecloth, hard-
ening the resulting sediment with alcohol, embedding it in celloidin, and cutting
100–200 sections from this. The sections were then screened at low power; if “any-
thing of importance” was found at screening, the sections were stained and exam-
ined at high power. Despite the fact that Dr. Ferguson stated that he has made the
diagnosis of bladder tumors on several occasions by this method and that “a greater
adoption of this method would soon enlarge our knowledge of tumors of the blad-
der,” this method was too cumbersome for clinical use and was to our knowledge
not adopted by others. Ferguson also acknowledged that one of his New York col-
leagues, Dr. Louis Heitzmann, was also engaged in the cytologic diagnosis of blad-
der cancer for several years. Louis Heitzmann (1864–1939) had been taught
microscopy by his father, the Austrian pathologist Carl Heitzmann (1836–1896)
who immigrated to the United States after failing in his attempt to succeed
Rokitansky as chair of pathology in Vienna. He was a consulting physician at Lenox
Hill Hospital, New York, and Professor of Pathology at the New York Medical
College. Louis Heitzmann published the results of his experience with urinary
cytology in the diagnosis of bladder tumors in a successful book first printed in
1899 [104], successive editions of which followed until 1936. The criteria Heitzmann
uses for the diagnosis of bladder papilloma (the equivalent of low-grade urothelial
neoplasms in our current classification) are very similar to those used in The Paris
System, i.e., the presence of fibrovascular cores: “The characteristic features of a
papilloma are peculiar connective tissue shreds that are, as a rule, abundant. They
are long, or extremely irregular, frequently branching in different directions and
often assume the shape of coils or knobs… in which capillary blood vessels filled
with blood corpuscles are seen coursing in various directions.” “The connective tis-
sue shreds found in villous cancer may be even larger and more irregular than those
seen in papilloma…. A number of these shreds contain large irregular cancer epithe-
lia, sometimes even small nests, a feature never found in papilloma.” In 1911, the
American surgeon/urologist George Austin Wyeth, Sr. (1877–1964) used

Heitzmann’s criteria to diagnose an upper urinary tract carcinoma preoperatively by
urine cytology while training in Berlin with the pioneer in modern urologic surgery
and discoverer of Actinomyces, James Israel (1848–1926) [105] (Fig. 13.21).
At the beginning of the twentieth century, George Leslie Eastes (1870–1944)
who was the director of the Clinical Pathology Laboratory of the Clinical Research
Association in London published his experience with over 6000 “microscopical
examinations of urine … all of which were conducted on deposits obtained by cen-
trifugalisation” (sic) [106]. He states “the diseases of the bladder that cannot be
diagnosed by an examination of the deposit are very few. Cystitis can be distin-
guished from localised ulceration, papillomatous and epitheliomatous neoplasms
recognized… this can be done by a consideration of the character of the epithelial
elements present.” “In epithelioma the cells are highly pleomorphic … they tend to
come away in groups, are granular, and contain a large and evident nucleus. But the
most characteristic cells are found in cases of villous growth: papillomata…. The
cells in these cases are of exceptional length, of a remarkable thinness, the greater
thickness, being that of the nucleus, with other cells, which are shorter and more
pleomorphic.” (Fig. 13.22).
The value of urinary cytology in the diagnosis of bladder cancer was also recog-
nized across the Atlantic, in the United States, as highlighted by the Boston surgeon
Arthur Tracy Cabot (1852–1912). He writes in his 1909 paper on the treatment of
bladder tumors that “microscopical examination of the urine follows or should fol-
low immediately upon the appearance of symptoms, because in fortunate cases bits
of villi…..in characteristic shapes …make the diagnosis clear.” He cautions that
“villi of the imperfect type are sometimes found in connection with inflammatory
conditions of the bladder” [107].
a b c
Fig. 13.21 (a, b) Urine sediment findings in papilloma (left) and papillary (“villous”) carcinoma
(right) depicted in Heitzmann’s book. (c) Urine sediment findings in a carcinoma of the renal pel-
vis diagnosed preoperatively by Wyeth
Fig. 13.22 Drawings of renal cell carcinoma and papillary bladder tumor from Eastes’ article
In his 1914 paper, the Philadelphia physician Augustus Theodore Gaillard

(1871–1924) also stressed the role of urinary cytology in the diagnosis of bladder
tumors stating that “Papilloma of the bladder should especially be mentioned,
because of its comparative frequency, and the striking microscopical evidences in
the urine when this benign tumor is present.” “…. rarely absent are the peculiarly-
shaped connective-tissue shreds, once seen never forgotten, and of themselves
almost pathognomonic. These shreds are very long, very irregular, having a ten-
dency to coil or knob-like formations, and frequently contain fat globules or inflam-
matory corpuscles” [108, 109].
Although microscopic studies on urinary sediments were usually made on
unstained smears, stains for the microscopic examination of cytologic specimens
had started to be used during the last decades of the nineteenth century. Paul Ehrlich
had introduced smearing (“Ausstrich”) of blood and bodily fluids, heat-drying, and
staining with more and more complex stains starting in 1879.
Starting in 1894, Siegfried Grosz (1869–1922) and R. Knapp working in Vienna,
in the chemical pathology laboratory led by Ernst Freund (1863–1946), reported on
the results of using sodium alizarin sulfonate (Alizarin Red S) stain on fresh wet
urinary sediments. Based on the observation that a 1% aqueous alizarin solution
stains acids yellow, weak bases red, and strong bases violet, these researchers
attempted to determine the upper urinary tract versus bladder origin of the cells and
mucus present in the urinary sediment, which would have been useful to differenti-
ate between pyelonephritis and cystitis. The German physician Adolf Bauer later
used the same staining technique to diagnose papillary bladder tumors in the urine
sediment [110, 111].
At the turn of the twentieth century, the famous French physician and bacteriolo-
gist George Fernand Isidore Widal (1862–1929) together with Paul Ravaut
(1872–1934) coined the term “cytodiagnosis” and used fixation with alcohol-ether
of dried smears of body fluids [112, 113]. In his 1903 book on cytodiagnosis, the
Paris physician Marcel Labbé (1870–1939) gives a detailed description of the
methodology [114], consisting of centrifugation of the fluid, smearing the centrifu-

gate onto slides, air-drying them, and post-fixing them with alcohol-ether. This was
followed by staining the slides with thionin-eosin-hematein, Unna’s blue (poly-
chrome methylene blue), or Ehrlich’s triacid (a mixture of orange G, acid fuchsin,
and methyl green). However, Labbé mentions that for urine specimens, in which the
cells are already degenerated, he and his colleague Leon Bernard (1872–1934) had
better results with fresh (unfixed) centrifugates stained between the slide and cover-
slip with a drop of a 1% aqueous solution of methylene blue or Unna’s blue and
examined immediately (Fig. 13.23).
The Swedish pathologist Johan Ulrik Theodor Quensel (1863–1934) success-
fully used a similar staining technique applied to fresh urine centrifugates, the
supravital staining with cadmium-methylene blue-Sudan III. In 1918, Quensel
published an extensive 340-page report documenting very promising results
obtained with this method. He was able to diagnose 7 of 12 papillomas and 8 of
13 carcinomas, including a metastatic breast cancer [115]. Quensel stresses the
difficulty of differentiating benign from malignant papillary neoplasms and the
value of enlarged (3–4 μ) and elongated nucleoli for the diagnosis of malignancy
(Fig. 13.24).
In 1925, the University of Helsinki surgeon Fjalar Jaakko Stenius (1891–1926)
published a study supporting the usefulness of cytology for bladder tumor diagnosis
[116]. He fixed the smears made from bladder washing sediments with formalin and
stained them with Weigert’s hematoxylin. Like Quensel, he performed detailed
measurements and stressed the value of nuclear and nucleolar enlargement and of
the nucleocytoplasmic ratio for the diagnosis of bladder tumors. Stenius, who died
at the age of 35, had also proposed an interesting classification of bladder tumors,
into papillomas, malignant papillomas, and papillary and solid carcinomas [117],
roughly corresponding to low-grade urothelial carcinoma, noninvasive high-grade
papillary urothelial carcinoma, and invasive urothelial carcinoma. The conclusions
Fig. 13.23 Medals of Dr. Widal and Dr. Ravaut

Fig. 13.24 Ulrik Quensel and the urinary sediment finding in bladder cancer depicted in his paper.
J Ulrik T Quensel, https://sok.riksarkivet.se/sbl/artikel/7457, Swedish biographical lexicon (art by
Margareta Åman). (Retrieved 2020-07-02)
to his cytology study were that the cystoscopically obtained bladder washings are
useful in the diagnosis of malignant (but not benign) papillomas and invasive papil-
lary and solid carcinomas. He considers the presence of epithelial cells with large
nuclei (over 13×7 μ) and nucleoli over 2–3 μ as suspicious for malignancy and cells
with nucleoli over 2–3 μ as malignant.
In the late 1920s, the London pathologist Leonard Stanley Dudgeon (1876–1938),
working at the St. Thomas Hospital, introduced a form of intraoperative diagnosis
based on smears made from scraping the surface of fresh operative specimens [118].
The smears were wet-stained with Schaudinn’s solution (a mix of 2 volumes of
mercuric chloride, one volume of alcohol, and glacial acetic acid) and then stained
with Mayer’s hemalum and counterstained with a weak eosin solution. A paper co-
authored by the young Australian-born surgery trainee, Norman Rupert Barrett
(1903–1979) [119], who would attain eponymic fame for his description of the
columnar-lined esophagus, summarized over 1000 cases examined by this tech-
nique. The paper provides a detailed description and depiction of the cytologic cri-
teria of malignancy that can hardly be improved upon even today. The paper finally
laid to rest the misconception started by Virchow and repeated almost verbatim by
surgeons and pathologists that cancer cannot be diagnosed from the appearance of
its cells. This was still the prevailing opinion during Dudgeon’s times, as stated by
the influential surgeon and cancer specialist Sir John Bland-Sutton (1855–1936):
“in the appearance of a cell from a cancer there is nothing characteristic of the dis-
ease, nothing that would lead a pathologist to identify it as a malignant cell. Cancer
can only be identified in sections showing the relation of cells to each other in a
group.” [120]
In addition, Dudgeon and Barrett’s paper also stresses the value of the method in
the diagnosis of bladder papillomas, in which early diagnosis of malignant change
can be made (Fig. 13.25).
Fig. 13.25 Professor Leonard S. Dudgeon and Norman Barrett, the latter as a surgical resident, at
St. Thomas Hospital, London. (Bamforth [167]; Lord [168]). Below, images from Dudgeon and
Barrett’s paper with the following captions. Left: Bladder papilloma showing the regular size of
the individual cells, placards of cells, and the abundance of isolated cells, all with a large amount
of cytoplasm. Right: On the right side of the circle, bladder carcinoma showing cells which have a
large nucleus and very little cytoplasm
The 1920s was a decade in which different researchers working independently of

one another were able to consistently diagnose bladder tumors using different stains
and wet fixation techniques applied to urine sediments or cytologic scrape prepara-
tions from fresh tissues. Another study published during this decade addresses the
value of cell blocks (“cell buttons”) in the diagnosis of bladder tumors. Frederick
S. Mandelbaum (1867–1926), the first Mount Sinai, New York pathologist, who had
trained with Paul Ehrlich and Rudolf Virchow in Berlin, and Richard Paltauf
(1858–1924) in Vienna developed around 1900 a cell block technique and applied it
to pus, body fluids, and urine. In 1917, he published the details of the technique,
which was very similar to that used today [121]. He gave the task to review the
accumulated experience with these cases to the young surgeon Abraham Philip
Zemansky, Jr. (1900–1928). In 1928, Zemansky reported the results of 914 of these
cases that had been reported since 1912, which included 46 cases of urine or bladder
washings [122]. He concluded that the cell block method is valuable in the study of
pleural and peritoneal fluids but of doubtful value in the diagnosis of urines/bladder
wash. His conclusion is based on the fact that only two of 46 cases showed tumor
cells in the cell blocks from urinary tract specimens, a conclusion the validity of
which is difficult to assess without knowing the prevalence of urinary tract malig-
nancy in the population from which these samples were obtained. In addition, cyto-
histologic correlation showed that, of the cases with surgical or autopsy follow-up,
only one of five cases of carcinoma of the bladder and none of six kidney tumors
showed tumor cells in the urine cell blocks. To explain the lack of sensitivity of this
method, he mentions the small quantity of urine received in the laboratory
(30–50 ml), which is a small fraction of the 24 hour urine output and is not sufficient
for the detection of tumor cells. He also mentions the technical difficulties encoun-
tered in embedding and cutting the minute amount of centrifugate obtained from
these specimens and the cellular degeneration caused by the hypotonic urine.
Unfortunately, Zemansky died the same year from septicemia after a mastoid opera-
tion at the age of 28.
In his study published in 1925, the University of Buffalo urologic surgeon
Frederick J. Parmenter (1880–1962) concluded that “the cytological examination of
the urine is a valuable aid to diagnosis and should be practiced as a routine proce-
dure,” that “its greatest value is for suspected tumors of the upper part of the
tract”….., and finally that “only positive results are of value” [123]. These conclu-
sions are largely valid to this day.
Despite this evidence and prior studies documenting the usefulness of urine cytol-
ogy for the diagnosis of bladder tumors, no widespread use of urinary cytology is
documented until 1945. While it is very important to ask the question why it took
urinary cytopathology so long to thrive [124], it was not for lack of studies docu-
menting its value, as can be surmised by the preceding examples. Also in doubt: “the
interest was there; the findings were striking; but the impetus of a devoted, painstak-
ing scientist was lacking to elevate the subject to consistent scientific duplicability”
[51]. At least in the case of urine cytology, “painstaking scientists” had been per-
forming study after study reaching the same conclusion that cytology was a valid and
valuable method for bladder cancer diagnosis. The problem was most likely that they
were ahead of their times. In his insightful analysis, Dr. James R. Wright, Jr. con-
cluded that the most important factor was the need for paradigm shifts in the under-
standing of cancer [124], from gross to microscopic and from the level of tissues to
the level of cells. According to Thomas Kuhn, such paradigm shifts are fundamental
changes in the basic concepts, experimental practices, techniques, and values shared
by members of a scientific community (in this case the medical community). Such
shifts do not occur suddenly, but gradually. Although the new paradigm, in this case
the diagnosis of cancer by microscopic examination of tissues and then of cells, may
gain followers, its adoption may take time. As the German physicist Max Planck
(1858–1947) puts it: “a new scientific truth does not triumph by convincing its oppo-
nents and making them see the light, but rather because its opponents eventually die,
and a new generation grows up that is familiar with it.”
For Virchow, the father of “cellular pathology,” “cancer” was defined by four major
characteristics: local progression, recurrence after resection, lymphadenopathy, and
distant metastases, all of which are clinical or gross features [125]. The only micro-
scopic feature that Virchow accepted was the presence of invasion. He did not accept
cellular malignancy criteria. He wrote “Indeed, there are no cells or nuclei that would
be characteristic of cancer” and “although I do not deny that [...] the size of the nuclei
and nucleoli could give meaningful moments for the diagnosis, it was impossible for
me to find anything specific in them124”. The enormous influence that Rudolf Virchow
had on the pathology of the nineteenth century, amplified by his network of former
trainees who occupied the chairs of almost all pathology departments in German-
speaking universities, drowned out the opposing views voiced in the middle of the
nineteenth century by the Danish pathologist Adolph Hannover (1814–1894) and the
former trainees of Alfred Donné, Hermann Lebert, and John Hughes Bennett, who
believed that the cellular changes in cancer are specific enough to allow a diagnosis.
Therefore, the paradigm shift could only occur with a new generation of pathologists,
who were not trained in Virchow’s dogma of nonspecificity of cancer cells and open
to the idea that a diagnosis of cancer is possible through microscopic examination of
the cells. This occurred in the late 1920s and 1930s, when cytologic diagnosis of can-
cer was brought back into the scientific discourse by pathologists from different coun-
tries, focusing on different areas of pathology. Leonard Dudgeon in London used
cytology to perform intraoperative diagnoses, Aurel A. Babeş (1886–1961) in
Bucharest, Romania, pioneered the cytologic diagnosis of cervical cancer [126], while
Fred Stewart in New York diagnosed thousands of cases of various cancers in needle
aspiration biopsies [127]. The explanation for why it took another two decades until
cytologic diagnosis was finally embraced is complex. For one thing, the proponents of
the cytologic method were not self-promoting, were also involved in other activities,
and did not exclusively focus on cytologic diagnosis. They failed to seek or gain the
support of medical societies interested in fighting cancer, which would prove to be
crucial in the future success of the method.
Finally, there was no perceived need for this method, which may have required
technical and interpretive skills that few pathologists of the time possessed, espe-
cially in areas that were relatively easily amenable to surgical biopsy. In the case of
urine cytology, with the widespread use of cystoscopy, which allowed for an easy
tissue diagnosis, many doubted the value of the cytologic diagnosis for clinical
decision-making. The fact that cytology was of little use in the diagnosis of well-
differentiated papillary tumors classified by some as papillomas and by others as
carcinomas was another argument against the use of this method, especially since
these tumors were easily diagnosed by cystoscopy. An additional obstacle in the
acceptance of urine cytology was the fact, alluded to by some of the early
proponents of cytology, that the lack of pathologists’ cytology training and experi-
ence had resulted in false-positive diagnoses, which made clinicians distrust
the method.
The Modern Era
In 1944–1945, George Nicholas Papanicolaou (1883–1962) started a collaboration

with the urologist Victor Fray Marshall (1913–2001) to study the value of urinary
sediment cytology for the diagnosis of bladder tumors [128]. This came on the heels
of the success of his paper on the value of the vaginal smear for the diagnosis of
uterine cancer, co-authored with the gynecologist Herbert Frederick Traut
(1894–1963) [129]. For the diagnosis of urinary sediments, Papanicolaou used the
same staining methodology as for vaginal smears. Before staining, the fresh urine
specimen was mixed with half its volume of 95% alcohol to avoid cellular degenera-
tion; centrifugation concentrated the cellularity of the specimen. He fixed the fresh
smears of the centrifugate by immersing them into a 50/50 mix of 95% alcohol and
ether and stained them with an early version of the stain that bears his name, a com-
bination of five stains (hematoxylin and alcoholic solutions of light green SF,
Bismarck brown, eosin, and orange G). He settled on this combination of stains
through trial and error, after having first used hematoxylin and an aqueous solution
of eosin to stain unfixed smears, then hematoxylin, and aqueous solutions of eosin
and water blue to stain alcohol/ether fixed smears [130, 131]. For the first 83
patients, Marshall and Papanicolaou reported that the diagnostic results were 88.8%
correct “positive” and 60% correct “negative.” As Koss later writes, these results
were “hardly worthy of note, were it not for the illustrious authors and the fact that
the article appeared in the exclusive scientific journal Science” [132]. Nonetheless,
Papanicolaou persisted and published a larger study, including 240 cases [133]
2 years after the original report of 83 cases. He applied his now famous five-tiered
class system to these cases, noting that classes I and II may be considered as nega-
tive, III as suspicious, and IV and V as positive:
• Class I. Absence of abnormal or atypical cells.

• Class II. Atypical cells present but without abnormal features.
• Class III. Cells with abnormal features but not sufficiently pathognomonic.
• Class IV. Fair number of pathognomonic cells and cell clusters.
• Class V. Large number of conclusive cells and cell clusters.
The criteria of malignancy that he used in urine sediments were the same, namely,
“modifications and abnormalities of the nucleus (enlargement, anisonucleosis, frag-
mentation, hyperchromatosis [sic], granular arrangement of the chromatin, promi-
nence of nucleoli, etc.), changes affecting the cytoplasm (basophilia, vacuolation,
leucocytic infiltration, etc.), and significant deviation of cells from their normal size
and form.” Papanicolaou also notes that catheterized urine specimens are generally
preferable to voided urine specimens. He also clearly states that “the smear
technique should not be considered as a substitute for biopsy, curettage or any other
standard method of diagnosis, but as an adjunct to them.” (Fig. 13.26).
It is important to note that these results were taking advantage of the fact that
the Memorial Hospital (currently the Memorial Sloan Kettering Cancer Center)
and the New York Hospital had the practice of interpreting low-grade papillary
Fig. 13.26 George Papanicolaou, with his chief cytotechnologist, Charlotte Street, and Dr. John
Seybolt, his collaborator on urine cytology studies. (From the George Papanicolaou, MD
Collection, Cornell University Medical Center)
lesions as “papilloma,” in the tradition of Dr. James Stephen Ewing (1866–1944)

and his successor Fred Waldorf Stewart (1894–1991) [134]. Other institutions
using the same nomenclature for the diagnosis of papillary bladder lesions
reported similar results [135, 136]; however, other institutions were diagnosing
such “papillomas” as Grade I papillary carcinoma, and their results in diagnosing
bladder “cancer” were significantly inferior [137]. For example, in a 1954 review
of the use of cytology in genitourinary and pulmonary tumors [138], the Mayo
Clinic pathologist, John R. McDonald (1910–1977), remarked that the “examina-
tion of urinary sediment for malignant cells in suspected lesions of the bladder is,
in my opinion, of extremely questionable value.” This is based on a sensitivity of
less than 50% for bladder “cancers” (many of which would have been diagnosed
as “papillomas” at the Memorial Hospital) and an 8% false-positive rate, which
McDonald considered “a percentage of error that is too great to make this a rea-
sonable procedure.” McDonald also noted: “it is exceedingly difficult to distin-
guish benign papillomas unless fronds of tissue are exfoliated.” He concedes that
the “examination of urine for malignant cells might prove of some value as a
follow-up procedure in vesical tumors.” In addition, McDonald opined that tech-
nical preparation details, i.e., whether cell block preparations or smears are pref-
erable and which stain is used, “matter little in the ultimate diagnosis.” Finally,
referring to the reporting of the cytology results, he states “it is easier on every-
one, including one’s own conscience, to classify results of smears in exfoliative
cytologic diagnosis as either positive or negative” [138].
Other studies performed at large institutions were also less than enthusiastic
about the value of urine cytology in the diagnosis of bladder cancer [139]. However,
the success of the smear method for uterine cancer diagnosis, Papanicolaou’s per-
sistence on reporting the results of large studies [140], and the reputation of Dr.
Marshall [141], who popularized and promoted the method within the urology com-
munity [142], led to the gradual adoption of this method.
Given the success of the dyestuff workers’ urine screening campaigns in the United
Kingdom [143], and that of vaginal smear screening in the early diagnosis of cervical
cancer, the Stanford urologists, Joseph C. Presti, Sr. and Henry M. Weyrauch, assessed
the value of urine cytology as a mass-screening method. The study failed to discover
even a single tumor of the urinary tract among 1575 individuals whose urines were
examined cytologically. The authors concluded “it seems doubtful that the Papanicolaou
examination of the urine is applicable to mass screening in an effort to diagnose malig-
nancy early in this system” [144]. The same study also found an unacceptably high
percentage (22%) of unsatisfactory smears, defined as “less than six cells, to permit
intelligent interpretation.” Of the 39 (2.5%) cases that had “doubtful” (i.e., atypical/
suspicious) results, half were retested with urine cytology, all with negative results.
Therefore, at the end of the 1950s, many of the problems that would plague urinary
cytology in the following years were already reported. Firstly, urine cytology has a
very low sensitivity for low-grade papillary transitional cell (urothelial) neoplasms,
and if such neoplasms are considered “cancer,” the sensitivity of urinary cytology will
appear very low. After the adoption of the 1973 World Health Organization typing of
urinary bladder tumors [145], which classified almost all papillary tumors as papillary
carcinomas, the performance characteristics of urine cytology versus histology

decreased significantly [146, 147]. Secondly, voided urine specimens may have very
low cellularity, and, in the absence of agreed-upon criteria for adequacy, a very large
proportion of urine specimens may be reported as unsatisfactory. Thirdly, urinary tract
cytology is usually not reported dichotomously (positive/negative), and a significant
proportion of urine specimens, even from individuals without symptoms referable to
the urinary tract, may be reported as atypical/suspicious.
The following decades have witnessed attempts by cytopathologists to overcome
some of these problems and improve the performance of urine cytology. The first
important problem was to increase the sensitivity of urine cytology, especially for
low-grade urothelial neoplasms. According to the 1973, 2000, and 2016 World
Health Organization classification schemes, most low-grade papillary urothelial
neoplasms are diagnosed as “carcinoma,” despite the absence of invasion and the
presence of no or only minimal cytologic atypia. This situation, which is not encoun-
tered in any other organ system, makes it highly unlikely or impossible to diagnose
low-grade papillary “carcinomas” in urine cytology, unless their defining feature,
the papilla with vascular core, is present in the urine sediment.
With the introduction of increasingly powerful computers in the 1970s, research-
ers have tried to identify subtle cellular changes that are not recognized by routine
cytologic examination, to increase the sensitivity of urine cytology for low-grade
urothelial neoplasm. Detailed computerized morphometric and karyometric mea-
surements of the nuclear size, nucleocytoplasmic ratios, nuclear chromatin, and tex-
ture were made by Leopold Koss (1920–2012) and his collaborators in an attempt
to refine our subjective criteria for benign, atypical, malignant, and degenerated
cells in urine sediments [148–153]. One of his associates, Myron Melamed
(1927–2013), investigated flow cytometry and its application to urine cytology
[154, 155]. Because of the physical properties of large variably shaped polygonal
urothelial cells in a liquid medium, the technology was considered unsuitable for
urine. The detection of aneuploidy was refined with the use of nuclear stains and
image cytometry performed on standard microscopic slide preparations, which
allows for digital analysis of individual cells [156]. While all these technological
advances contributed to our better understanding of urothelial carcinoma and its
potential diagnosis by urine cytology, they could only marginally improve the sen-
sitivity and specificity of conventional urinary cytology and, after being popular in
the 1980s, were eventually abandoned (Fig. 13.27).
The fate of other urine-based tests that were promoted in next decades to aid or
replace urine cytology was similar. Two biochemical urine tests were introduced in
the 1990s: one for nuclear matrix protein 22 (NMP22), which is involved in the distri-
bution of chromatin during mitosis, and one for bladder tumor antigen (BTA), the
human complement factor-H related protein. These were followed in the 2000s by
two additional ancillary tests. The first was an immunocytochemical test (ImmunoCyt/
uCyt+), designed to detect mucins/glycoproteins on surfaces of exfoliated malignant
cells with a cocktail of three fluorescein-labeled antibodies (LDQ10, M344, and CEA
19A211). The second was an interphase cytogenetic test, fluorescent in situ hybridiza-
tion (FISH) to detect polysomy of chromosomes 3, 7, and 17 and/or deletion of 9p21
Fig. 13.27 Dr. Leopold Koss. (From Steven I. Hajdu and Hormoz Ehya. Foundation of Diagnostic
Cytology Annals of Clinical & Laboratory Science, vol. 38, no. 3, 2008). Myron Melamed, from
M. B. Zaman. Myron R. Melamed, MD, 1927–2013 Journal of the American Society of
Cytopathology 2014, 3 (1), 2–4)
(UroVysion® test), the latter deletion relates to the loss of the p16 (CDKN2A) sup-
pressor gene. These tests typically have higher sensitivity but lower specificity than
urine cytology and frequently have problems similar to urine cytology, with lower
sensitivity in the detection of low-grade papillary neoplasms (see TPS 2.0 Chap. 10).
The initially high enthusiasm in using these tests in combination with, or instead of,
urine cytology has been dampened by their lower analytical sensitivity and specificity
in real-world use than was reported in the initial studies. In addition, their use has been
stymied by relatively high costs and lack of a clear place in the management of patients
for the diagnosis or surveillance of bladder cancer.
In recent years, an ever-growing number of multiplex molecular assays using
reverse transcription quantitative polymerase chain reaction or massive parallel
sequencing have been proposed or introduced in practice. These assays assess for
various genetic or epigenetic abnormalities that are found in urothelial carcinomas,
including mutant FGFR3 which characterizes low-grade papillary urothelial neo-
plasms (CertNDx). On the other hand, TERT promoter and TP53 usually characterize
high-grade urothelial carcinoma (UroSEEK). Despite promising initial results, the
value of these tests, cost-effectiveness, and their place in the management of the
patient suspected of, or under surveillance for, urothelial carcinoma is still unclear.
In addition to the difficulty diagnosing low-grade neoplasms was the large propor-
tion of unsatisfactory specimens, especially for voided urines. This was addressed
with a number of technical advances meant to increase the concentration of the cells
present in the sample. These methods include filter techniques (Millipore, Nuclepore),
cytocentrifugation, and liquid-based cytology. Even in the face of improved method-
ology that readily increased the cellularity of the samples prepared by these tech-
niques, it was still not entirely clear what constituted an adequate urine sample.
A nagging and persistent problem, which seemed to get worse rather than better
with time, was the large proportion of indeterminate diagnoses rendered in urine
cytology. In cytopathology, the rates of atypical and suspicious diagnoses are in gen-
eral associated with factors that are related to the patient (prior instrumentation or
therapy), the specimen (poor preparation, poor staining), and those attributable to the
pathologist (inadequate training and ability to make a diagnosis) [157]. In urinary
cytopathology, additional factors that can explain the high rate of atypical and suspi-
cious diagnoses are the low comfort level of pathologists with urine specimens, the
inherent difficulty and subjectivity of the diagnosis, as attested by relatively high
interobserver variability in urinary cytology interpretations, and, most importantly,
the lack of clear agreed-upon diagnostic categories and definitions. It is difficult to
communicate findings between facilities when local diagnostic opinions differ. It is
difficult to train cohorts of residents without clear consistent landmarks.
A number of classifications have been proposed starting in the 1980s [158–
161], all representing an improvement over Papanicolaou’s numeric class system,
but none of them was widely adopted or solved the problem of indeterminate
diagnoses. In 2013, two proposals, one representing a radical rethinking of uri-
nary cytology reporting, by the Johns Hopkins Hospital group led by Dr. Dorothy
L. Rosenthal [162, 163], and the other proposing a subclassification of the atypi-
cal group, made by a French group led by Dr. Eric Piaton [164], renewed the
interest in a new, reproducible, and practical classification of urine cytology diag-
noses. In the immediate aftermath of these proposals, Drs. Dorothy L. Rosenthal
and Eva M. Wojcik convened a meeting at the 18th International Congress of
Cytology held on May 26–30, 2013, in Paris, to discuss the possibility of a new
classification of urine cytology. On May 28, 2013, in a small room of the Palais
Des Congrès, in which the congress was held, the speakers of the two sessions on
urinary cytology met to discuss the potential new classification. They included, in
addition to Eva Wojcik and Dorothy Rosenthal, Güliz Barkan, Lukas Bubendorf,
Margareta Strojan Fležar, Drs. Rana Hoda, Daniel Kurtycz, Stefan Pambuccian,
Eric Piaton, Marcus Quek, and Momin T. Siddiqui. The president of the American
Society of Cytopathology (ASC), Ritu Nayar, and the president of the International
Academy of Cytology (IAC), Philippe Vielh, were also present at this inaugural
Paris meeting. The group agreed that a new, standardized terminology for report-
ing urinary cytology based upon histopathology and clinical outcomes was
needed. The agreed-upon guiding principle for the new classification would be
that urine cytology is meant to diagnose the clinically important high-grade uro-
thelial carcinoma capable of local invasion and distant spread. Cellular changes
that did not indicate a significant risk for high-grade urothelial carcinoma would
be classified under and relegated to the negative diagnostic category (“Negative
for high grade urothelial carcinoma”), including changes possibly attributable to
a low-grade neoplasm. An important principle of this system was that all
diagnostic categories should be clearly defined and have strict diagnostic criteria,
restricting the use of “atypical” to cases in which the cytologic features cannot be
attributed to a known etiology [165]. It was also agreed that the new classification
would be designated The Paris System (TPS), analogous to The Bethesda System,
to reflect the location of the first meeting that took place on the subject. Chapter
lead authors were chosen and contributing authors were recruited. Subsequent
meetings to clarify the details of this classification were held at the ASC meeting
in Orlando, FL, in November 2013; the USCAP meeting in San Diego, CA, in
March 2014; the European Congress of Cytopathology, Geneva, Switzerland, in
September 2014; and the ASC meeting in Dallas, TX, in November 2014. During
these meetings, the participants discussed the details of the classification and the
clinical significance of the cytologic diagnosis. After considering the results of
web surveys, additional discussions, and teleconferences, the authors decided on
the final diagnostic categories, their definition, and diagnostic criteria. Finally,
these were included in a book authored by 49 cytopathologists, cytotechnologists,
surgical pathologists, and urologists from 10 countries. The Paris System for
Reporting Urinary Cytology was edited by Drs. Rosenthal, Wojcik, and Kurtycz
[166], with an introduction by Drs. Ritu Nayar and Philippe Vielh and a very
thoughtful prologue written by Dr. William M. Murphy [166], one of the pioneers
in urinary cytology. The Paris System was well received and was adopted by most
institutions in the United States and many institutions throughout the world. One
of the most important outcomes was the reduction of the “atypical” diagnoses in
almost all institutions that have adopted The Paris System.
Another of the important features of the first edition TPS was a call to research.
A host of questions needed to be answered. In the afterword to The Paris System
book, the editors admitted that much of the published information on which the
reporting system was built was based on poorly defined, inadequately studied, unin-
vestigated, and/or anecdotal evidence. They also hoped that the publication of TPS
would stimulate investigations that would fill gaps in our knowledge base. At the
time of the publication, there was no or only scant evidence on some of the issues
covered by TPS, especially regarding the use of the recommended criteria in differ-
ent types of preparation and the applicability of the diagnostic criteria for urothelial
carcinoma variants. It was also obvious that one of the most important issues regard-
ing this classification was assigning a risk of malignancy to every diagnostic cate-
gory to aid clinical management. Given that the Paris categories were transformative
and had not been used before in the manner recommended by the Paris system,
studies had to be done after the implementation of TPS to define these risks. As
such, from the beginning, it was decided that would be revised as soon as more
information became available. Preparations for the second edition of The Paris
System (2.0) were started by Drs. Wojcik, Rosenthal, and Kurtycz in 2019, and
meetings were held at the ASC meeting in Salt Lake City in November 2019 and
USCAP meeting in Los Angeles in March 2020.
In the midst of the COVID-19 pandemic, the authors of TPS 2.0 have been com-
mitted to completing this volume despite the additional demands on their professional
time and energies. The gratifying and near universal acceptance of the thinking behind
TPS continues the centuries-old tradition of turning to urine to unravel many of the
mysteries of the human body. TPS will continue to be modified by new evidence and
evolving concepts of urothelial neoplasia. The authors and editors of TPS look for-
ward to advances in genetics and associated biomarkers that will help the laboratory
refine accuracy and advance precision in the decades ahead (Fig. 13.27).
Fig. 13.27 The participants at the initial meeting in Paris 2013 from left to right: Upper row –
Dorothy L Rosenthal, Eva M. Wojcik, Rana Hoda, Momin T. Siddiqui, Stefan E. Pambuccian. Lower
picture – Lukas Bubendorf, Margareta Strojan Fležar, Güliz A. Barkan, Eric Piaton, and Marcus Quek
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fterword – Honoring the Past, Anticipating
A
the Future
My introduction to Cytology was as a medical student, brief but intriguing. During

my internship (1967), I was privileged to be taught by a student of Dr. Papanicolaou,
Dr. Elizabeth McGrew, at the University of Illinois. That was the beginning of my
love affair with cytomorphology, and that passion has grown over the years. In the
1970s the specialty was an orphan. No one in pathology wanted to claim it. A few
gynecologists recognized its value, but not until the 1980s did it become more sci-
ence than witchcraft, nurtured and promoted by George Wied, Leopold Koss,
Stanley and Florence Patten, and John Frost. As cytopathology became generalized,
there was nothing more thrilling than for a clinician to declare “You’ve changed the
way we practice medicine!” Of course, the “you” meant our discipline, not me per-
sonally. That was true for all the specialties, especially gynecology, pulmonology,
endocrinology, oncology … all, that is, except for urology.
Many of the early cytopathologists tried very hard to translate the morphologic
changes seen in histologic sections into cells seen in voided urines. Since the low-
grade lesions are far more common than the high grade, cytopathology struggled to
match the sensitivity of histopathology. But we were proving Einstein’s definition
of insanity. No matter how hard we tried, we could not reliably identify cells of low-
grade urothelial carcinoma on urinary samples. What we could reliably diagnose
were the high-grade life-threatening lesions that are often flat and not obvious tar-
gets during cystoscopy.
Drs. Wojcik’s and Kurtycz’s Foreword describes the journey of The Paris System
founders from idea to widely accepted concept. Included in the philosophy was the
commitment to have the approach and criteria based on consensus resulting from
robust evidence. Because TPS was a paradigm shift, consensus was reached without
having studied the effects of the approach on the diagnosis of urinary samples. TPS
1.0 raised more questions than answers. At the end of that volume, the Afterword
proposed studies that needed to be done. Some have been completed, some are
ongoing, and others have yet to be conducted. The Wish List below results from the
experiences of the authors of each chapter. We hope you will participate as research-
ers in your own practice to provide the evidence that our specialty needs to insure
the viability of urinary cytology.
TPS 2.0 is my final contribution to Cytopathology. I cherish the experiences and
friendships that have enriched my life over the past five decades. Working with the

https://doi.org/10.1007/978-3-030-88686-8
318 Afterword – Honoring the Past, Anticipating the Future
authors of this book has been the most gratifying collaboration in my 50-year career.
They have devoted enormous time, energy, and expertise during the devastating
COVID pandemic, despite the demands of their roles as essential workers develop-
ing and providing new tests for patient care. Their dedication to our field is my
legacy. I couldn’t be more proud of them.
July 2021.
Dorothy L. Rosenthal, MD.
Emerita Professor of Pathology/Cytopathology.
The Johns Hopkins School of Medicine.
Baltimore, Maryland, USA
Future Clinical and Research Needs
Chapters 1 and 9: Pathogenesis and ancillary tests are intertwined by their shared
molecular bases.
Chapter. 1: Pathogenesis
1. In TPS 1.0: Will the suspected distinction between the hyperplasia and dysplasia
pathways remain following more intense molecular studies?
2. In TPS 2.0: The molecular classification of The Cancer Genome Atlas (TCGA)
(2014) and subsequent studies largely upheld the distinction between the precur-
sors of Low-Grade Urothelial Carcinoma (LGUC) (TCGA Cluster I) and of
High-Grade Urothelial Carcinoma (HGUC) (predominantly TCGA Cluster II).
Development of invasive carcinoma from a LGUC remains rare.
3. In TPS 3.0: Further studies will refine the molecular basis of HGUC and identify
biomarkers with the potential for high clinical utility in AUC and SHGUC cases.
In order to relate morphologic classifications of cytology and histology to
defined molecular groups, large-scale detailed studies are needed. Results would
influence clinical management decision.
Chapter 9: Ancillary Tests
1. Prospectively compare the performance of novel tests on the sensitivity and neg-
ative predictive value of AUC and SHGUC categories.
2. Explore to what degree the correlation between morphological features and tar-
geted FISH analysis can improve the diagnostic skills of cytopathologists in
resolving AUC and SHGUC categories.
3. Investigate if the results of ancillary testing could be added to the defining crite-
ria of specific TPS categories in addition to morphological features.
4. Determine whether surveillance guidelines can be changed using currently
approved ancillary tests (e.g., U-FISH and uCyt) for patients with urothelial car-
cinoma depending on individual risk factors.
Afterword – Honoring the Past, Anticipating the Future 319
5. Establish the cost-effectiveness of ancillary testing across different countries and

health care systems.
Chapters 2 and 10: Specimen adequacy and preparatory methods are aimed at
the same goal: providing a sample that is optimized to provide the platform on
which a reliable diagnosis can be based.
Chapter 2: Adequacy
1. In TPS 1.0: What is adequate volume and cellularity in the context of various
collection and preparation methods?
2. Questions answered in TPS 2.0: Volume recommendation for voided urine sam-
ples in the context of the adequacy algorithm.
3. Questions still unanswered for TPS 3.0: What is adequate cellularity in the con-
text of various collection and preparation methods?
Chapter 10: Cytopreparation Methods
1. Determine whether time, temperature, and chemical composition of urine impact

collection and processing.
2. Establish evidence-based recommendations for collection of urine samples for
optimal morphology.
3. Explore cell block urine preparation methods for best practices.
Chapters 3 through 8: The diagnostic categories define the criteria for placing
cytomorphologic changes into logical and reproducible bins.
Chapter 3: Negative for HGUC (NHGUC)
1. Define the cytomorphologic features most predictive for low-grade urothelial

neoplasms (LGUN), particularly the predictive value of true papillary fragments
for LGUN when features of HGUC are absent in a specimen.
2. Establish improved criteria for defining whether urothelial tissue fragments are
benign or atypical.
3. Determine instances, if any, in which specimens containing polyomavirus should
be classified outside of the NHGUC category.
Chapter 4: Atypical Urothelial Cells (AUC)
1. Construct studies to further refine morphologic criteria (in different preparation

and specimen types), and report the institutional rates of AUC following TPS2.0.
2. Investigate the utility of the AUC rate as a quality improvement metric by com-
paring the rate among laboratories of various sizes and risk levels of patients.
3. Establish patient management guidelines for this category based on follow-up of
patients with a diagnosis of AUC.
320 Afterword – Honoring the Past, Anticipating the Future
Chapter 5: Suspicious for High-Grade Urothelial Carcinoma (SHGUC)
1. Determine whether the same quantitative and qualitative criteria of the “suspi-
cious for HGUC” category should be used in different specimen types (upper
versus lower tract, voided versus instrumented).
2. Determine whether the association of the “suspicious for HGUC” and “positive
for HGUC” cytological categories with a histologically confirmed HGUC is sta-
tistically different enough to justify keeping those two categories separate.
Chapter 6: High-Grade Urothelial Carcinoma (HGUC)
1. Establish or refute, based on robust evidence, that a minimum of five to ten

malignant cells are sufficient to diagnose HGUC on different cytologic
preparations.
2. Study the impact of degenerative changes on the diagnosis of HGUC. How can
we address this issue?
3. Explore the application of digital image processing and artificial intelligence for
diagnosing HGUC, especially for measuring N/C ratio.
4. Is an N/C ratio of 0.7 a mandatory criterion for HGUC?
Chapter 7: Upper Urinary Tract Cytology (UTUC)
1. Data are needed from more institutions regarding the performance of TPS for
upper tract lesions.
2. Determine whether upper tract lesions have any distinctly different cytomorpho-
logic features compared to their bladder counterparts, being careful to exclude
method of procurement (e.g., instrumentation) as a cause for these differences.
3. Define improved criteria for making a diagnosis of HGUC in the upper tract vs.
the bladder, particularly whether the ten-cell cut-off is appropriate for upper
tract HGUC.
Chapter 8: Non-Urothelial Malignancies (NUM)
1. Evaluate clinical data from major academic centers to assess the success of mor-
phology and immunohistochemistry for cytological detection of NUM of the
urinary tract.
2. Investigate application of innovative molecular and genetic tests to aid in the
identification of sources of non-urothelial cancers of the urinary tract as well as
determine the cell of origin in primary non-urothelial tumors.
3. Correlate cytologic diagnoses with clinical data to evaluate the clinical signifi-
cance of rendering a specific diagnosis of NUM.
4. Determine risk of malignancy associated with the diagnosis of NUM.
Afterword – Honoring the Past, Anticipating the Future 321
5. Further study the significance of “atypical squamous cells” in urinary cytology,

and correlate the degree of atypia with risk of malignancy.
Chapters 11 and 12: Risk of Malignancy (ROHM) and Clinical Management:

The reason TPS exists is to provide clinicians with rational and tested cytologic
results for safe and effective patient management.
Chapter 11: ROHM
1. Assess ROHM for all categories based on the novel recommendations by the
second edition.
2. Compare new metrics such as likelihood ratios and pre-test probability (based on
clinical and epidemiological parameters) to classical ROHM, in order to assess
a more personalized post-test probability of having malignancy.
3. Assess potential bias in ROHM according to age, gender, and ethnical
distribution.
Chapter 12: Clinical Management
1. Since the first edition of TPS, clinical trials have been performed demonstrating
improved accuracy with fluorescence-assisted cystoscopy in bladder cancer
diagnoses and in performing a more complete resection, thereby decreasing
recurrences.
2. For the future:
• Plan clinical trials integrating these new techniques with cytology results.
• Explore the role of novel urine-based molecular marker assays to see how
these tests can be optimally integrated with cytology to enhance diagnostic
accuracy and surveillance.
Index
A urothelial cell content, 13

Acute bacterial infections, 46 in voided and instrumented specimens, 10
Adenocarcinoma, 187, 188 volume and adequacy, 10–13
Adenocarcinoma with signet ring cell volume factors, 10
features, 188 Anaplastic large-cell lymphoma (ALCL), 181
Adequacy algorithm for voided urines, 13 Ancillary testing in upper tract urothelial
Adequacy of urine specimens carcinoma, 136–137
adequacy criteria, 10 Aneuploidy, 303
adequacy recommendations in TPS, 9 Aristolochic acid (AA), 117
algorithm, 9 Atypia, 66
biopsy/surgical excision, 12 Atypical glandular cells, 159
cytomorphological findings, 9 Atypical parakeratosis (dyskeratosis), 152
for cytopathologists, 8 Atypical spindle cells, 189
decision about specimen adequacy, 9 Atypical squamous cells (ASC), 151, 152
diagnostic accuracy, 8 and categorization, 74
diagnostic discrepancies, 7 definition of, 153
evidence-based, and consensus-determined diagnosis of, 152
cutoff values for volume and explanatory note, 153
cellularity, 9 with LSIL, 152
false negative diagnoses, 7 with mild atypia, 187
instrumented urine specimen, 16 in urine cytology, 143
“less-than-optimal” adequacy, 16, 17 Atypical urothelial cells (AUC), 4, 68–73,
over-interpreting cellular degeneration 78, 91–93
artifact, 7 abnormal ploidy associated with neoplastic
physical and sexual activity of the growth, 79
patient, 8 atypical cytology interpretations, 66
pre-analytical variables, 7 AUC diagnosis, 77
preparation-dependent cutoffs, 9 cellular changes, 77
prescriptive qualities, 16 cellular degeneration, 64, 76
prevalence of malignant and suspicious cytologic changes, 66
diagnosis, 12 cytologic method, 76
quantitative results of validation, 16 cytologic reports, 80
satisfactory for evaluation, 17 definitive diagnosis, 66
specimen characteristics, 9 designation, 21
in TPS, 16 diagnostic category, 66
unsatisfactory for evaluation, 17 diagnostic reality, 65
unsatisfactory/non-diagnostic specimen, 15 diagnostic testing, 64
urine specimen types, 12 domain of atypia, 64
urine volume, 10 enlarged nuclei, 79

https://doi.org/10.1007/978-3-030-88686-8
324 Index
Atypical urothelial cells (AUC) (cont.) Cell clusters, 37

exclusion of reactive/reparative causes, 80 Cellient® cell block, 237
high-grade malignancy, 63 Cellular aggregates, 37
major (required) criterion, 77 Cellular degeneration secondary to stasis, 30
management, 259, 260 Cellularity of instrumented urinary tract
minor criteria, 77 specimens, 13
morphologic abnormality, 64 Cellular/volume adequacy,
morphological definition, 76 overestimation of, 7
morphology of normal basal urothelial Centrifugation, 239
cells, 77 equipment, 240
multifactorial and probabilistic, 76 procedure, 240
N/C ratio, 77 responsibilities, 240
non-neoplastic changes and conditions, 67 Centrifugation sediment cell block, 235, 236
nuclear enlargement, 79 Cercariform cells derived from LGUN, 130
nuclear to cytoplasmic (N/C) ratio, 66 CertNDx, 305
Paris system, 80 Cervical cancer, 302
performance characteristics, 63 Chemotherapy, 54
quality and quantity of, 77 Clear cell AdCa, 157
range of values/morphologic features, 64 Clear cell carcinoma, 160
reparative phenomena, 64 Clinical management
reported atypia rate, 63 for AUC, 259, 260
sensitivity and specificity, 79 for HGUC, positive for, 260, 261
Atypical urothelial tissue fragments (AUTF), for LGUN, 261, 262
34, 37, 43 for NHGUC, 258, 259
for non-urothelial tumors, 262, 263
for suspicious for HGUC (SHGUC), 260, 261
B for unsatisfactory specimen, 263
BD Totalys™ Multiprocessor, 230, 231 Cluster I of TCGA, 2
BD Totalys™ SlidePrep, 230, 231 Cluster II of the TCGA, 2
Benign glandular cells, 26, 27 Cluster III and IV, 4
endometriosis, 26 Colorectal AdCa, 166, 167
Benign squamous cells, 25 COVID-19 pandemic, 307
Benign superficial (umbrella) urothelial Cyclin-dependent kinase inhibitor 2A
cells, 22–24 (CDKN2A), 2
Benign urothelial cells, 67 Cystitis cystica/glandularis, 27
Benign urothelial tissue fragments (BUTF), and intestinal metaplasia, 26
34, 35, 37, 43, 127–129 Cystourethroscopy, 260
in instrumented urine, 43 Cytocentrifugation, 226–228
in voided urine, 36 Cytocentrifuge cell block standard process,
Bilharzial SqCC (B-SqCC), 146 245, 246
Biopsy-proven HGUC, 79 Cytomorphological findings, 221
Bladder cancer, 286, 290, 294 Cytopreparation, 221, 223, 239
Bladder diversion specimens, 55–58 assessment, 222
Bladder tumor, 289 preparation methods, 223, 224
Bladder washings, 13 BD Totalys™ multiprocessor and BD
Brand, Henning, 276 Totalys™ SlidePrep, 230, 231
Breast AdCa, 171, 172 cell block preparation, 233, 234
Burkitt’s and mantle cell lymphomas, 181 Cellient® Cedsll Block, 237
centrifugation sediment cell block,
235, 236
C cytocentrifugation, 226–228
Carcinoma in situ (CIS), 115 direct smear preparation, 229
Cell block (CB), 134, 135, 137, 233, 234 Millipore filtration, 232
preparation containing HGUC, 135 Shandon Cytoblock™ cell block,
preparation containing LGUC, 136 236, 237
Index 325
ThinPrep® liquid based, 224 Granulomatous reaction following BCG

processing, 222 immunotherapy, 53
specimen collection, 222 Guar bean, 58
Cytopreparatory techniques, 16 Gynecologic malignancies, 168, 169
Cytospin (CS) centrifugal method, 145
Cytospin method, 112
Cytospin preparations, 14 H
of instrumented urine cytology Hematopoietic malignancies, 181
specimens, 14 lymphoma, 181, 182
plasmacytoma, 183
Hematoxylin, 300
D Herpes simplex virus (HSV), 47
Decoy cells, 47 High-grade carcinoma with squamous
Decoy ducks, 47 features, 187
Degenerated benign intermediate urothelial High-grade prostatic adenocarcinoma, 188
cells, 30 High-grade urothelial carcinomas (HGUC), 1,
Degenerated umbrella cells, 32 21, 33, 66, 97, 98, 113, 115, 120,
Degeneration, 31, 34, 74 125, 143
of benign cells, 33 cytomorphologic criteria of malignancy
Degenerative changes, 30–34, 107 for, 103
Diffuse large B-cell lymphoma cytomorphologic features, 103
(DLBCL), 181 cytopreparation on cytomorphology of, 112
Direct smear preparation, 229 degenerative changes, 107
DNA synthesis, 79 extremely dark chromatin, 108
in fragments, 124
with glandular differentiation, 109
E histologic variants of, 109–112
Effusion cytology, 10 Hypochromasia, 106
EGFR, 4 N/C ratio, 103, 106
Enteric AdCa, 155, 156, 159 in pelvic washing with cell-in-cell
Enteric cells from a urinary diversion forms, 120
post-cystectomy, 56 positive for, 260, 261
ERBB2 enrichment, 4 with prominent cercariform cell
Extremely dark chromatin, 108 formation, 119
in renal pelvis, 118
with squamous differentiation, 109
F TPS criteria, 102, 103
Failed therapy, 54 High-power fields (HPF), 14
FGFR3 alterations, 2, 4 High-risk HPV (hrHPV) testing, 152
FGFR3/RAF/RAS signaling pathway, 2 Histone modification pathway, 4
Fibrovascular cores, 112 History of urinary cytology
Fluorescent in situ hybridization ancient history, 267–278, 280
(FISH), 304 HRAS/KRAS/NRAS mutations, 2
Fragment of HGUC from the ureter, 122 Human polyoma viruses, 47
Fragments of low-grade urothelial neoplasm Hygienic collection policies, 7–8
without fibrovascular cores, 126 Hyperchromasia, 34, 75, 90, 103
Hypochromasia, 106, 123
Hypochromatic HGUC cells, 123
G
Galenic theory, 272, 277
Gastric AdCa, 172, 173 I
Glandular cells, 26, 27, 30 Immunohistochemistry (IHC), 145
Glandular epithelium, 26 Inadequate for diagnosis, 8
Glandular type cells from urinary tract, 26 Indeterminate cells in voided urine, 134
Granulomatous change caused by BCG, 53 Infiltrating neutrophils, 46
326 Index
Instrumentation of urinary tract, 13 Mullerian lesions, 159

Instrumented urinary specimens, 13 Mullerian rests (Mullerianosis), 26
Intermediate (“parabasal-like”) urothelial Multinucleated umbrella cells, 73
cells, 22, 24
with increased N/C ratio, 90
Intestinal metaplasia, 26 N
Intraurothelial neoplasia, low grade, 38 Negative for high grade urothelial carcinoma
Intravesical BCG immunotherapy, 53 (NHGUC), 4, 94
Intravesical mitomycin and thiotepa, 54 bladder washings, 60
Intravesicular tumor seeding, 116 category, 59, 67
Irregular nuclear contours, 79 cytology reporting systems, 21
definition of, 22
diagnoses, 59
K management, 258, 259
Keratinizing squamous cell carcinoma, 186 urine cytology (voided), 59, 60
Koss, Leopold, 304 Nephrogenic adenoma (NA), 185
Nested variant of urothelial carcinoma, 110
Neuroendocrine carcinomas (NEC), 161
L Neuroendocrine (NE) tumors, 161, 162, 262
Lambl, Wilhelm, 287 criteria, 162, 163
Large-cell NEC (LCNEC), 161, 162 explanatory note, 164
Large pulsating groin tumor, 285 Non-basal urothelial cells, 66
Leiomyosarcoma, 176 Non-bilharzial SqCC (NB-SqCC), 146
Liquid-based preparations (LBPs), 145 Non-epithelial malignancies, 176
Lithiasis, 44 hematopoietic malignancies, 181
Low grade noninvasive bladder cancer lymphoma, 181, 182
(LGUC), 260 plasmacytoma, 183
Low grade papillary urothelial carcinoma malignant melanoma, 179–181
(LGPUC), 38 sarcomas, 176–178
Low-grade squamous intraepithelial lesion Non-urothelial malignancies (NUM), 143
(LSIL), 152 clinical history of, 144
Low-grade urothelial carcinoma (LGUC), 115 non-epithelial malignancies, 176
Low-grade urothelial neoplasms (LGUN), 1, hematopoietic malignancies, 181–183
38–42, 249, 261, 262 malignant melanoma, 179–181
cells with plasmacytoid morphology, 131 sarcomas, 176–178
Lung carcinoma, 173 primary squamous cell carcinoma (see
Lymphoma, 166, 181, 182 Primary squamous cell carcinoma)
secondary epithelial malignancies (see
Secondary epithelial malignancies)
M urine cytology findings of, 144
Malignant melanoma, 179–181 Non-urothelial tumors, management of,
Matula, 273 262, 263
Melamed-Wolinska bodies, 32, 33, 74 Nuclear cytoplasmic (N/C) ratio as a
Memorial Sloan Kettering Cancer Center, 47 criterion, 74–75
Metastatic ovarian AdCa, 169 Nuclear hyperchromasia, 79
Micropapillary urothelial carcinoma, 110 Nuclear hypochromasia with UTHGUC, 123
Millipore filtration, 232 Nuclear matrix protein 22 (NMP22), 304
Molecular analysis on paired primary UTUC
and recurrent UCB tissues, 116
Molecular pathways of neoplastic P
transformation of urothelium, 2–4 Papanicolaou staining, 112
Mucinous AdCa, 155, 156 Papanicolaou, George Nicholas, 300–302
Mucosa-associated lymphoid tissue Papillary urothelial neoplasm of low malignant
(MALT), 181 potential (PUNLMP), 38
Index 327
Papillomas, 301 RB mutations, 2

Paraganglioma, 183, 184 Reactive urothelial cells, 94
Parasites, 50, 52 Referral centers, 59
Plasmacytoid urothelial carcinoma, 111, 159 Renal cell carcinoma (RCC), 174, 294
Plasmacytoma, 183 Renal tubular cells (RTC), 29
Polyoma infected cells, 47 Renal tubular epithelial cells (RTCs), 26, 30
Polyoma virus, 48 Reported risk of malignancy (ROM) for
Polyoma virus infected cells, 47 UTUC based on UUT cytology for
Polyomavirus, 49, 67 each TPS category, 132
Polyomavirus infected cells, 49 Reporting system, 1
Positive for high grade urothelial carcinoma Rhabdomyosarcomatous differentiation, 176
(HGUC), 260, 261 Risk of high grade malignancy (ROHM),
Primary adenocarcinomas, 155, 159, 160, 262 249, 253
criteria, 156, 157 current evidence for, 249–251
definition of, 155 in low-grade neoplasms, 251, 252
enteric AdCa, 155 TPS, performance of, 253
mucinous AdCa, 155 Risk of malignancy (ROM), 249
Primary epithelial malignancies RNA sequencing, 2
atypical squamous cells (ASC), 151, 152 RTK/RAS/PI3K pathway, 4
definition of, 153
diagnosis of, 152
explanatory note, 153 S
with LSIL, 152 Sarcomas, 176–178
neuroendocrine tumors, 161, 162 Schistosoma haematobium, 50
criteria, 162, 163 Secondary epithelial malignancies
explanatory note, 164 breast AdCa, 171, 172
primary adenocarcinoma, 155, 159, 160 colorectal AdCa, 166, 167
criteria, 156, 157 cytohistologic appearance of, 166
definition of, 155 gastric AdCa, 172, 173
enteric AdCa, 155 gynecologic malignancies, 168, 169
mucinous AdCa, 155 lung carcinoma, 173
primary squamous cell carcinoma, 146 prostate AdCa, 167, 168
cytologic criteria, 147 renal cell carcinoma, 174
definition of, 146 Secondary tumors, 145
explanatory notes, 150 Seminal vesicle cells, 54, 55
Primary epithelial NUM, 143, 144 Shandon Cytoblock™ Cell Block, 236, 237
Primary lymphoma, 181 Small-cell carcinoma (SmCC), 161
Primary polyoma virus infections, 47 definition of, 162
Primary squamous cell carcinoma, 146 Small-cell (high-grade neuroendocrine)
cytologic criteria, 147 carcinoma, 188
definition of, 146 Small fragments of HGUC, 119
explanatory notes, 150 Specimen contamination during
Prostate AdCa, 167, 168 collection, 46
Squamous cell carcinoma, 262
Squamous epithelial cells, superficial and
Q intermediate, 25–26
Quantitative adequacy criteria for specimen Stone atypia, 43–46
cellularity, 14 Superficial cells, 22
Superficial urothelial (umbrella) cells, 23
SurePath (SP), 145
R SurePath® preparation, 12, 243, 244
Radiation changes, 52 Surgical excision methods to bladder washing
Radiation-induced cytomorphologic specimens, 14
changes, 52 Suspicious for adenocarcinoma, 159
328 Index
Suspicious for high grade urothelial carcinoma U

(SHGUC), 4, 34, 74, 86–89, 121 Unsatisfactory specimen, management of, 263
altered cellular morphology, 91 Upper tract high-grade urothelial carcinoma
cellular degeneration, 91 (UTHGUC), 115, 116
criteria, 86–90 in instrumented specimens, 137
definitive diagnosis, 85 vs. upper tract LGUC, 117
diagnosis, 85 in voided urine specimens, 125, 137
management, 95, 260, 261 Upper tract low-grade urothelial neoplasms
N/C ratio, 90, 91 (LGUN), 125–131
nuclear membrane irregularity, 90 modern performance of urinary
prevalence, 94 cytology, 133
qualitative morphological criteria, 89 Upper tract urothelial carcinoma (UTUC)
risk of high-grade malignancy, 94 adequacy, 136
Suspicious for malignancy, 187, 188 circumspection, 121
Suspicious for NUM, 186 clinical management, 133
SWI/SNF complex pathway, 4 coarse chromatin and bizarre single
Synchronous UTUC and urothelial carcinomas cells, 122
of bladder (UCB), 116 criteria for, 117–131
Systemic cyclophosphamide (Cytoxan), 54 cytomorphologic features of, 122
environmental factors, 117
etiology, 116
T historical performance of urinary
The Bethesda System for Reporting cytology, 131–132
Gynecologic Cytology, 21 in instrumented specimens, 122–125
The Cancer Genome Atlas (TCGA), 2 in instrumented urinary tract
differential gene expression by RNA specimens, 123
sequencing, 2 modern performance of urinary cytology
pathway analysis based on mutation and for, 132–133
copy-number alterations, 2 molecular biology of, 116–117
The Paris System (TPS), 1, 4, 63, 64, 115, specimen collection, 133–134
257, 293, 306 tissue biopsy and/or UUT cytology, 133
atypical urothelial cells (AUC) category, 63 tissue biopsy specimen, 116
classification of urine cytology, 94 vs. UCB, 116–117
criteria, 75 UUT cytology, 134
cytomorphological criteria for HGUC, 117 on washing/barbotage specimens, 116
performance of, 253 Urinary casts, 284
recommendation, 11 Urinary cytology, 1
The Paris System survey (2020), 76 Urinary diversion specimens, 55, 57
ThinPrep (TP)®/Cytospin systems, 14–15, Urinary lining, 8
112, 145 Urine collection, 239
with histologic follow up, 123 Urine cytology, 1, 257–259, 280–285, 295,
NonGynecologic Processing, 241–243 297–300, 303
preparations of urinary bladder wash bladder cancer in urine, 289
cytology, 16 cellblock preparation, 292
processing devices, 14 centrifugalisation, 293
specimens, 136 daguerreotype of urinary sediment, 283
Thurneysser, Leonard, 276 Lambl, Wilhelm, 287
Tissue fragments, 37 Moore, Charles, 286
Totalys™ multiprocessor, 243, 244 papillary tumor of the bladder, 291
TP53 mutations, 2, 116 Quensel, Ulrik, 296
TP53/RB pathway, 4 urinary sediment cells, 292
True papillary tissue fragments arising from urine sediment findings in papilloma and
LGUC, 130 papillary carcinoma, 293
Index 329
Vogel, Julius, 288 Urothelial tissue fragments

Widal and Dr. Ravaut, 296 (UTFs), 34–46
Urine processing, 222 in voided urine, 42–43
Urine specimens from acute bacterial
infections, 46
Urine specimens from neobladders, 55 V
Urine volume, 11 van Leeuwenhoek, Anton, 278
Urolithiasis, 43–46, 79 Variation in cellularity among urinary tract
Uroscopy, 272 samples, 8
Urothelial carcinoma and squamous cell Visually striking vegetable material, 57
carcinoma, 46 Voided urine, 17, 44
Urothelial cells, 71, 74 Volume-independent adequacy criteria, 14
in voided urine specimen, 13
Urothelial cells lining the bladder, 8
Urothelial changes associated with treatment W
effects, 52–54 Well-differentiated neuroendocrine tumors
Urothelial changes characteristic of infectious (WDNET), 161, 162
processes, 46–52 Willis, Thomas, 267
Urothelial degeneration, 73–74 2004/2016 World Health Organization/
Urothelial proliferation of unknown malignant International Society of Urologic
potential (UPUMP), 38 Pathologists (WHO/ISUP), 38

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Eva M. Wojcik, Daniel F.I. Kurtycz, Dorothy L. Rosenthal - The Paris System For Reporting Urinary Cytology-Springer (2022)

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The

The Paris System

ISBN 978-3-030-88685-1 ISBN 978-3-030-88686-8 (eBook)

© Springer Nature Switzerland AG 2022

Maywood, IL, USA Eva M. Wojcik MD

“Few” ~ 5-10 cells “Many” ~ 10 cells

NEGATIVE FOR HGUC ATYPICAL SUSPICIOUS FOR HGUC HGUC

2. Graphic illustration of visual differences in N/C ratios.

ML Zhang, Cancer Cytopathol 2016;124:669-77

3. Essential data for each diagnostic category.

Unsatisfactory Voided urine–volume (>30ml) 0 - 5% 0 - 16%

ROHM –Risk of High Grade Malignancy

NHGUC Negative for high-grade urothelial carcinoma

1 Pathogenesis of Urothelial Carcinoma ���������������������������������������������������� 1

9 Ancillary Studies in Urinary Cytology���������������������������������������������������� 193

Afterword – Honoring the Past, Anticipating the Future������������������������������ 317

Eva M. Wojcik, MD Department of Pathology and Laboratory Medicine, Loyola

Derek B. Allison, MD Department of Pathology & Laboratory Medicine,

Ashish Chandra, MD FRCPath DipRCPath (Cytol) Department of Cellular

Ashish M. Kamat, MD, MBBS Department of Urologic Oncology (Surgery), The

Donna K. Russell, MEd, CT(ASCP)HT, CMIAC Department of Pathology and

Summary of Changes in the Second Edition

• Incorporation of The Cancer Genome Atlas molecular characterization into the

For any reporting system to be successfully applied in daily practice, it must be

© Springer Nature Switzerland AG 2022 1

an increased nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, coarse chroma-

Molecular Pathways of Neoplastic Transformation of Urothelium

Urothelial Carcinoma, Non-Invasive

Low Grade High Grade Carcinoma

FGFR, RAS FGFR, TP53 TP53, RB

Urothelial Carcinoma, Muscle Invasive

RTK/RAS/PI3K Histone SWI/SNF

Summary of Changes in the Second Edition

• More detailed discussions of urinary cytology adequacy

Although adequacy criteria have been established in some areas of cytopathology to

© Springer Nature Switzerland AG 2022 7

The Adequacy Algorithm

A common misperception is that cells in body fluids are solutes in a homogenous

YES Appropriate Benign

Non-Urothelial Features YES

YES Appropriate Benign

2.5 12.5 22.5 32.5 42.5 55 65 75 85 95 150

urine might achieve sufficient cellularity, a higher volume (30 mL) is recommended

The Less Than Optimal Adequacy Category

optimal” in urinary cytology specimens. Furthermore, it may be beneficial to incor-

Note: The interpretation is limited by low urothelial cellularity/obscuring inflam-

Note: The sample shows only rare benign urothelial/inflammatory/other cells in

Note: The interpretation is limited by scant cellularity/obscuring inflammation/

Note: The specimen is acellular or consists of only lubricant/blood/bacteria, etc.

Summary of Changes in the Second Edition

The following major changes have been made to this chapter:

• Clarification and streamlined discussion of atypical vs. benign-appearing urothe-

© Springer Nature Switzerland AG 2022 21

 efinition of Negative for High-Grade Urothelial

A sample of urine, either voided or instrumented (including washing specimens),

Criteria of Components of NHGUC

Benign Superficial (Umbrella) Urothelial Cells

Fig. 3.3 Deep urothelial

Squamous Epithelial Cells, Both Superficial and Intermediate

Fig. 3.4 Benign squamous

Fig. 3.7 Renal tubular

intracytoplasmic inclusions, so-called Melamed-Wolinska bodies (Fig. 3.10a).

Fig. 3.10 Degeneration of

Maywood, IL, USA Eva M. Wojcik MD

1 Pathogenesis of Urothelial Carcinoma �� 1

9 Ancillary Studies in Urinary Cytology�� 193

Afterword – Honoring the Past, Anticipating the Future�� 317

Summary of Changes in the Second Edition

Molecular Pathways of Neoplastic Transformation of Urothelium

Summary of Changes in the Second Edition

The Adequacy Algorithm

The Less Than Optimal Adequacy Category

Summary of Changes in the Second Edition

efinition of Negative for High-Grade Urothelial

Criteria of Components of NHGUC

Benign Superficial (Umbrella) Urothelial Cells

Squamous Epithelial Cells, Both Superficial and Intermediate

Urothelial Tissue Fragments

Benign Urothelial Tissue Fragments

Atypical Urothelial Tissue Fragments (AUTFs)

Low-Grade Urothelial Neoplasia (LGUN)

Urothelial Tissue Fragments (UTFs) in Voided Urine

Benign Urothelial Tissue Fragments (BUTFs) in Instrumented Urine

Instrumented urine specimens are often cellular, consisting of numerous benign-

Urolithiasis (“Stone Atypia”)

Urothelial Changes Characteristic of Infectious Processes

Acute Bacterial Infections

Characteristic Viral Cytopathic Effects

Urothelial Changes Associated with Treatment Effects

Seminal Vesicle Cells

Bladder Diversion Specimens

The Rate of Negative Samples in a Usual Laboratory Population

Summary of Changes in the Second Edition

larification of Issues Unresolved by the First Edition of TPS

Urothelial Degeneration: Does Degeneration Mean Atypia?

Atypical Squamous Cells (ASC) and Categorization

Nuclear Cytoplasmic (N/C) Ratio as a Criterion

PS Criteria Including Those for AUC Are Maintained for Upper

PS Criteria Including Those for AUC Are Currently Maintained

Updates from The Paris System Survey (2020)

Summary of Changes in the Second Edition