Fig. 1. Schematic drawing of urothelium of urinary tract: Main source of exfoliated cells which can be examined by urinary cytology.

H.J. deVoogt PRathert M. E. Beyer-Boon

00 Phase Contrast Microscopy and Analysis of Stained Smears

Foreword by L.G. Koss

79 Mostly (blored Figures in 327 Separate Jllustrations

Springer-Verlag Berlin Heidelberg New York 1977

Herman]. de Voogt, MD, PhD Lecturer in Urology, Academisch Ziekenhuis. Department of Urology, Leiden, The Netherlands

Privatdozent Dr. med. Peter Rathert Facharzt fiir Urologie, Cytologisches Labor der Abteilung Urologie, Medizinische Fakultiit, Rheinisch- Westfiilische Technische Hochschule Aachen, Germany Head of the Department of Urology, Krankenanstalten Diiren, Germany

Mathilde E. Beyer-Boon, MD, PhD Pathologist-Cytopathologist, Head of the Department of Cytopathology, Pathology Laboratory, Academisch Ziekenhuis, Leiden, The Netherlands Director of Het Leids Cytologisch Laboratorium, Head of the School of Cytotechnology, Leiden, The Netherlands

ISBN-13 978-3-642-96390-2 DOT: 10.1007/978-3-642-96388-9

e-ISBN-l3: 978-3-642-96388-9

Library of Congress Cataloging in Publication Data Voogt. H..I. de, 1925-Atlas of urinary cytology. Includes bibliographical references and index. I. Urine- Examination -Atlases. 2. Diagnosis, Cytologic - Atlases. I. Rathert, Peter, joint author. II. Beyer-Boon. Mathilde E., 1941-joint author. III. Title. RB53.V66 616.07'582 77-7021

The use of general descriptive names, trade names, trade marks, etc. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone. This work was subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under ~ 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher.

by Springer-Verlag, Berlin· Heidelberg 1977

Softcover reprint of the hardcover 1 st edition 1977

Foreword

Cytologic diagnosis of cancer has its roots in clinical micro­scopy as it was shaped during the first half of the 19th century. In reviewing some of the early writing on this subject, one is amazed at the accuracy of the descriptions and soundness of the observations. Cytology of the urine is no exception: in 1864 Sanders described fragments of cancerous tissue in the urine of a patient with bladder cancer (Edinburgh Med. J. 111, 273). This observation was confirmed by Dickinson in 1869 (Tr. Path. Soc. London, 20, 233). It is a source of special pride to me that in 1892 a New York pathologist, Frank Ferguson, advocated the examination of the urinary sediment as a best means of diagnosing bladder cancer, short of cystoscopy. Papanico­laou freely acknowledged these contributions while estab­lishing sound scientific bases for continuation and spread of this work. Papanicolaou's work in the area of the urinary tract has not fallen on dead ears. He documented to several urologists who were within his sphere of personal influence, mainly Dr. Victor Marshall, Professor of Urology at Cor­nell University Medical School, that urinary tract cytology was a reliable tool in the diagnosis of urothelial carcinoma. Some of us who have attempted to spread the master's word had their share of success within institutions with which we were associated. Perhaps the most important contribution of urinary tract cytology has been in the identi­fication of non-papillary carcinoma in situ, a key lesion in the assessment or prognosis of urothelial neoplasia. Yet, the authors of this fine Atlas of Urinary Cytology are quite right when they imply that the vast majority of urologists are either not aware or are skeptical of this method of diagnosis. There are many reasons for this, perhaps the most important of which are its limitations. Well-differen-

v

VI

tiated papillary lesions of the bladder, such as papilloma and papillary carcinoma, grade I, are unlikely to yield diag­nostic cells. Thus, the expectations of the urologists that any bladder tumor will be reliably diagnosed by cytology is false. Similar mistakes are committed by the pathologists and cytopathologists who often fail to recognize the limita­tions of the method and in attempting to diagnose too much make major mistakes of judgment and are left with the mistrust of their clinicaL colleagues. Urinary tract cytol­ogy is difficult and is full of pitfalls and distressing sources of diagnostic mistakes. It cannot be learned casually but requires many years of experience and close cooperation between the pathologist and the urologist. This atlas should contribute to the popularization of this important method of diagnosis which admirably complements but does not replace clinical judgment and the biopsy. The goal of these efforts is a relatively simple one: to offer the patient with cancer of the lower urinary tract the best possible chance for an early diagnosis resulting in a cure or at least contain­ment of the disease and as comfortable a life as possible if a cure is not possible. To this goal urinary cytology may contribute in a very significant fashion by identifying the patients at a very high risk for invasive cancer whose urinary sediment contains obvious cancer cells. For such patients radical treatment of the diseased urothelium prior to the development of metastases may be the best and some­times only change of salvation. Drs. Beyer-Boon, de Voogt, and Rathert should be congra­tulated on this fine atlas. It should contribute substantially to the clarification and education of both urologists and pathologists who are interested in cancer of the lower uri­nary tract.

LEOPOLD G. Koss Professor and Chairman Department of Pathology Albert Einstein College of Medicine at Montefiore Hospital and Medical Center Bronx, New York 10467

Contents

Preface . . . . . . . . . . . . . . . . . . I I. Clinical Application of Urinary Cytology 5

2.

2.1.

H.i. de Voogt

Preparatory Techniques M.E. Beyer-Boon Collection of Material .

7

7 2. I. I. Urine . . . . . . . . 7 2.1.2. Bladder and Renal Pie vis Washings 7 2.1.3. Brushing Techniques .... 8 2.1.4. Prefixation.... . . . . . 8 2.2. Cell Concentration Techniques 8 2.3. Smear Preparation Techniques 9 2.3.1. Smears for Phase Contrast Microscopy and

Methylene Blue Staining (Non-Permanent). 9 2.3.2. Smears for the Papanicolaou Stain (Per-

manent) . . . . . . . . . . . . 10 2.3.2.1. Smears form Freshly Voided Urine 10 2.3.2.2. Smears from Prefixed Urine II 2.3.2.3. Smears for the MGG Method (Permanent) 11 2.4. Staining Methods . . II 2.4.1. Methylene Blue Stain II 2.4.2. Papanicolaou Stain 12 2.4.3. May-Gruenwald Giesma Staining Method 12 2.5. Pitfalls..... 12 2.5.1. Cell Degeneration . . . . . . . 12 2.5.2. Formalin Effect . . . . . . . . 12 2.5.3. The Damaging Effect of Hypertonic Urine 12 2.5.4. Cell Loss During Staining 13 2.5.5. Overstaining . . . . . . . . 13 2.5.6. Cellular and Nuclear Shrinkage

3. Urinary Cytology and its Relationship to His-tology of the Urinary Tract. . . 15 M.E. Beyer-Boon

3.1. Normal Structure of Urothelium 15

VII

VIII

3.1.1. 3.1.2. 3.1.3. 3.2. 3.3. 3.3.1. 3.3.1.1. 3.3.1.2. 3.3.1.3. 3.3.1.4. 3.3.2. 3.3.3. 3.3.4. 3.3.5. 3.3.6. 3.3.7. 3.3.8. 3.4.

Histology of Normal Urothelium Epithelial Variants. . . . . . . Cytology of Normal Urothelium Epithelial Contamination Benign U rothelial Lesions Inflammatory Changes Bacterial Infections Viral Infections . . Parasitic Infections Mycotic Infections Malakoplakia. . . Squamous Metaplasia Glandular Cystitis . . Urinary Calculi . . . Hyperplasia of the Urothelium Atypical Hyperplasia of the Urothelium Condylomata Acuminata Urothelial Tumors ...... .

3.4.1. Introduction . . . . . . . . . . 3.4.2. Classification of Urothelial Tumors 3.4.2.1. Macroscopy 3.4.2.2. Microscopy ....... . 3.4.2.3. Stage . . . . . . . . 3.4.2.4. Clinical Classification (UICC) 3.4.3. Macroscopy and Histology of Pure Transi­

tional Cell Tumors 3.4.3.1. Papillary Tumors . . . . . . 3.4.3.2. Solid Tumors . . . . . . . . 3.4.3.3. Flat Intra-Epithelial Carcinomas (Carcinoma

in situ) ............... . 3.4.4. Cytology of Pure Urothelial Tumors. . . . 3.4.4.1. Papillary Tumors Grade 0 (Benign Papilloma)

and Grade I (Papilloma with Atypia) 3.4.4.2. Papillary Tumors, Grade 2, 3 and 4 (Carci-

nomas) ........ . 3.4.4.3. Solid Urothelial Carcinomas . . . . . . . 3.4.4.4. Carcinoma in situ . . . . . . . . . . . . 3.4.5. Squamous Differentiation of Transitional Cell

Carcinoma and Pure Squamous Cell Carci-

15 16 16 18 18 18 18 19 19 20 20 20 21 21 22 22 22 23 23 23 23 25 25 26

28 28 30

30 31

31

34 35 36

noma . . . . . . . . . . . . . . . .. 36 3.4.6. Adenomatous Differentiation of Transitional

Cell Carcinoma and Pure Adenocarcinoma. 37 3.5. Adenocarcinoma of the Prostate ..... 38 3.6. Infiltration of the Bladder or Ureter from Ad­

jacent Carcinomas and Metastasis of other Carcinoma . . . . . . . . . . . . . .. 38

3.7 3.8. 3.9.

Adenocarcinoma of the Kidney . . . Effect of Radiation on the Urothelium Effect of Cancer Drugs

4. Phase Contrast Microscopy of the Urinary Sediment .. HJ. de Voogt

39 40 40

43

5. Methylene Blue Stain of the Urinary Sediment 49 P. Rathert

6. Epidemiology and Etiology of Urothelial Tu-

7.

7.1.

7.2. 7.3.

7.4.

7.4.1. 7.4.2.

mors ... P. Rathert

Efficacy of Urinary Cytology in the Detection of Tumors of the Urinary Tract . . . . . . M.E. Beyer-Boon, HI. de Voogt, P. Rathert Diagnosis of Patients with positive Cytolog­ical Results . . . . . . . . . . . . . . . Diagnosis of Patients with Atypical Findings Sensitivity and Specificity of Urinary Cytol-ogy ................. . The Validity of the Provisional Contrast Mic­roscopy Diagnosis . . . . . . . . . . . . Phase Contrast Microscopy Underdiagnosis . Phase Contrast Microscopy Overdiagnosis

51

55

56 56

58

60 61 61

Acknowledgement 62 References. 63 Illustrations . . . 69

1. Normal Transitional Epithelium . . . . 71 2. Inflammatory Changes. . . . . . . . . 77 3. Non-Bacterial Inflammations and Contaminants 81 4. Atypical Hyperplasia . . . . . . . .. 87 5. Phase Contrast Microscopy: Criteria for Malig-

nancy . . . . . . . . . . . . . . 6. Grade 1 Tumors of the Bladder 7. Grade 2 Tumors of the Bladder, with and without

91 95

Infiltrative Growth . . . . . . . . . . . .. 99 8. Grade 2 Bladder Tumors and Grade 3 U roteral

Tumor. . . . . . . . . . . . . . . . . . . 103 9. Grade 2 Tumors of Bladder and Urethra with

Infiltrative Growth . . . . . . . . 107 10. Grade 3 Tumors of Bladder, Renal Pelvis and

Ureter. . . . . . . . . . . . . . III

IX

II. Grade 3 Tumors of Renal Pelvis, Ureter and Bladder 117

12. Grade 4 Bladder Tumors 121 13. Grade 4 Solid Carcinoma of the Bladder 123 14. Carcinoma in situ. 127 IS. Squamous Cell Carcinoma of the Bladder 135 16. Adenomatous Differentiation. 141 17. Adenocarcinoma . . . . . . . 147 18. Adenocarcinoma of Kidney lSI 19. Bladder Cancer and Prostate Cancer ISS 20. Cystitis Glandularis Combined with Squamous

Metaplasia . . . . . . . . . . . . . . . . . 159 21. Effects of Radiation . . . . . . . . . . . . . 163 22. Effects of Cytostatic Drugs on Urothelial Cells 167 23. Cytological Changes Due to Urinary Calculi 173 24. Catheter Urine . . 179 25. Ileal Stomal Urine 183 26. Artifacts in PCM 187

Subject Index 191

x

Preface

In all probability urine contents have been observed since the microscope came into use for human pathology. Micro­scopic examination of unstained urinary sediment was rou­tine long before Papanicolaou (Papanicolaou and Mashall, 1945) introduced urinary cytology. Before that time red and white blood cells received the most attention as well as cylinders and crystals. Epithelial cells from the urinary tract (Fig. 1, p. I) were seen and the possibility of identifying malignant cells in urine was already thought of in the middle of the nineteenth century (Beale, 1858). Several investigators described with painstaking accuracy normal and abnormal cells in urine as well as in other body fluids. After Ehrlich introduced dried stained prepara­tions, such investigations increased but urinary cytology re­mained the work of individuals and they somehow did not succeed in introducing it as a routine diagnostic method (Deden, 1954). Following the pioneer work of Papanicolaou many dedi­cated cytologists modified and improved urinary cytology, but at the same time urologists criticized the method mainly because of its lack of accuracy as a diagnostic procedure. This controversy continues up to the present day and for a long time prevented urinary cytology from becoming a widely accepted routine diagnostic method. In our opinion another important cause is a lack of interest on the part of urologists for a simple diagnostic method, when they have such sophisticated tools for visualizing the urinary tract. To this may be added the fact that the Papanicolaou method cannot be done easily by the urologist himself dur­ing office hours. It is necessary to send fresh voided urine to the cytology laboratory where it has to be processed immediately if it is not prefixed. Staining, screening, and

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diagnosis have to be done by trained cytotechnologists and cytologists. The answer may take some time and when the practitioner or urologist is not familiar with the language of cytology and has his doubts about its reliability, it may very well be that he will not use the method very frequently. This, in turn, has a negative effect on cytologists in that they do not get enough material to keep up their routine practice and improve their ability in the special field of urinary cytology. How to break up this vicious circle? We thought that, in order to popularize urinary cytology, general practi­tioners and urologists ought to get more involved with the method. First we worked at making good smears of urinary sediment to improve the diagnostic efficacy for the cytolo­gist (de Voogt, 1972). Soon the advantages of interaction between urologist and cytologist became clear. The better the smears were, the more the enthusiasm and experience of the cytologists grew. But the real breakthrough for the urologist came with the redetection of phase-contrast micros­copy (PCM). Encouraged by the work of Stoll (1969) in vaginal cytology we soon learned that, mutatis mutandis, his principle of rapid screening during office examination of a patient was also applicable to urinary sediment. Every physician who is trained to examine urinary sediment for red and white blood cells and bacteria, can with simple additional equipment for PCM to his microscope see epithe­lial cells as well. In a period of 6-12 weeks he can, by applying simple rules, differentiate among normal, atypical, and malignant cells. From the same sediment smears can be made. After air-drying this smears can be stained by the May-Gruenwald Giemsa (MGG) method, adequate material for the cytologist is available (Lopes Cardozo, 1976). We found Esposti's fixative very useful in the Papani­colaou method, which makes the time-consuming methods with filters (Millipore or others) unnecessary. It had to be proved that PCM was reliable. Therefore every urinary specimen was examined, over a 5-year period by PCM as well as with the MGG and Papanicolaou method. In addition, in Aachen the monochromatic stain methylene blue was tried in the same way for reasons of rapid screening and compared with the other methods. The material, thus collected, was too valuable to be stowed away in archives. Therefore, we were greatful to Springer­Verlag that they edited the atlas in this outstanding quality. We hope that it may stimulate many practitioners and urol­ogists to use the microscope more intensively, that it may

help to guide them in the selection of proper treatment for their patients. By giving a survey of peM pictures, stained smears, and histology, we hope that cytotechnologists, pathologists and cytologists may profit from our experiences.

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