PLAN OF THESIS

For M.D. (Pathology)

“ATYPICAL MALIGNANT CELLS IN URINE

CYTOLOGY:A DIAGNOSTIC DILEMMA”

Dr. Anshul Singla(Batch 2010-2013)

GOVT. MEDICAL COLLEGE, PATIALA

“ A typical malignant cells in urine cytology: A Diagnostic Dilemma”

Name of the Candidate Dr. Anshul Singla

(Batch 2010-2013)

Name of the Supervisor Dr.Ish Kumar

Name of the Co-Supervisors Dr Manjit Singh Bal

ABSTRACT

This present study aims to screen the patient’s urine for atypical cytology in patients

with suspected lesions of urinary tract.The study will take into account the urine

specimens for cytological analysis in the Pathology Department,Government Medical

College,Patiala. The specimens will be analysed after centrifugation and staining with

Papinacolau stain.The normal findings or any variation (neoplastic,inflammatory,

chemotherapy,radiotherapy induced) will be noted down.The clinical,therapeutic as

well as cytological findings will be complied on a performa and subjected to stastical

analysis.

AIM

The aim of this study was to find out the characteristic morphology of malignant atypical

cells which were missed on routine cytology of urine.

MATERIALS AND METHODS

In this study, we will examine the detailed cytomorphology of atypical urinary cytology

which are missed on routine examination and are further proved on histopathology as

transitional cell carcinoma (TCC) of bladder. The cytological features of these cases are

compared with the cases of benign urine samples.

SAMPLE COLLECTION

Voided urine is the specimen of choice for all screening and diagnostic studies in male

patients. Catherized urine is preffered in female patients.

In our study we take into consideration three random mid stream urine samples on three

consecutive days. The sample will be collected in a clean,dry container and preferably

examined in two hours.

Bladder washings with normal saline or ringer lactate solution ,yield a highly cellular

specimen that contain more cell clusters and large superficial cells.

PREPARATION AND FIXATION OF SMEARS

(a) Macroscopic examination of collected material will be made.

(b) The Laboratory serial number of the specimen under examination will be written at

the end of the slide so that the number is parallel to the short edge.

(c) Fixation with equal volume of 50% ethyl alcohol will be done.

(d) Centrifugation of specimen will be done.(1200rpm for 9min)

(e) Supernatant after centrifugation will be discarded,and the urinary sediment at the

bottom of the test tube will be transferred to the slide,care being taken to ensure that

material will be placed in the centre of the slide and evenly spread to cover

approximately l/3rd of the slide in the center, with a fairly thick uniform smear.

(f) The lid of the specimen container will be closed thereafter.

(g) The slides will be air dried. The slides will never be stacked or stored one above the

other and the smears should not come in contact with each other.

(h) Specimens have to be processed with the staining methods given below:

PAPANICOLAOU METHOD (PAP) (Bancroft and Gamble, 2002)

Method

1. Fix slides in acetic/alcohol fixative for 15 minutes.

2. Absolute alcohol 2 minutes.

3. 70% alcohol 2 minutes.

4. 50% alcohol 2 minutes.

5. Tap water 2 minutes.

6. Stain in haematoxylin 4 minutes.

7. Rinse in tap water briefly.

8. Differentiate in acid alcohol 5 seconds.

9. Blue in tap water.

10. Dehydrate in absolute alcohol x2.

11. Stain in orange G 10 seconds.

12. Rinse in absolute alcohol x2.

13. Stain in E.A. 50 2 minutes.

14. Rinse in absolute alcohol x2.

15. Clear in xylene x3.

16. Mount sections in DPX

Result

1. Cellularity

Grading of cellularity will be done according to following table:

Score Name Microscopy

+1 Low cellularity An average of <10 epithelial cells

+2 Moderate cellularity

+3 High cellularity

(without performing formal counts)

2.Cell clusters

Cell clusters will be examined for patterns and numbers.A cluster

Score clusters

+1 1_3 clusters

+2 4-9 clusters

+3 >9 clusters

3. Papillae, Comet cells ,India ink cells

4. Necrosis, Apoptosis

5. N/C Ratio, Cytoplasmic detail

6. Nuclear size , pleomorphism , margins , hyperchromasia and chromatin abnormalities

INTRODUCTION

The urinary system involves the kidneys,the urters,the bladder and the uretha.(,urinary

tract).

THE URINE HAS BEEN REFERRED TO AS A LIQUID BIOPSY OF URINARY

TRACT-PAINLESSLY OBTAINED.(HENRY TODD.380) It yields a great deal of

information quickly and economically.Like any other laboratory procedure the urine tests

need to carefully performed and properly controlled..

The presence of single renal epithelial casts,cell casts, or tissue fragments has been

recognised for many decades as an important finding in the evaluation of patients.().The

kidneys have a little bearing on the cytology of the urinary tract.Renal tubular epithelial

cells are not present in most of the urine specimens,and tumour cells exfoliate from renal

adenocarcinoma only in advanced stages of disease.(bibo,William h. Kern,urinary tract)

Urothelial cells are present in all urine specimens and exfoliate readily from the tumours of

urothelial linning.Urine cytology is therefore an important primary methd in diagnosing

urothelial tumours,and in combination with cystoscopy and biopsy,it is used as an adjunct.

Cytological examination of urine is performed in screening programms of asymptomatic but

high risk patients for case finding,in the diagnostic evaluation of symptomatic patients, and

in the follow up and monitoring of patients with known and treated disease.

Normal voided urine contains very few cells.since urine is not isotonic,but hypertonic,all

freshly desquamated cells display a measure of degeneration.as a rule the epithelial cells ars

flat,cuboidal or oval.the cytoplasm is farily abundant,and the nuclei frequently have a

peripheral ring of chromatin and acentre that appears clear.the occasional squamous cells of

superficial type noted are all of urethral origin in male,in females there may be some

admixed of squamous cell types,some of which represent vaginal contamination.(koss)

Transitional cells in urine occur singly, or in the form of cohesive clusters or sheets,varying

in dia from 9µm to 40µm. The cytoplasm is opaque,granular or vacuolated.The described

three cell types of the urothelium-namely,the large,often multinucleate superficial cells,the

intermediate pyramidal cells,and the cuboidal cells adjacent to the basement membrane.

Ellidi and Paten reported that the basal cells have a mean cytoplasmic area of

82µm,predominantly oval,with basophilic,dense cytoplasm anda single nuclei.The

pyramidal,are,larger,with a mean area of 229µm,predominantly oval,basophilic and well

defined cell borders.The large superficial cells or binucleate cells(19%),often have a large

nuclei with salt and pepper appearance.Nucleoli are more prominent.They are particularlt

prominent in catherised specimens.

Urine cytology for screening of transitional cell carcinoma (TCC) has been used for long

time. Despite the advent of several newer techniques for screening and diagnosis of

urothelial malignancies, cytomorphology still remains an important tool."Atypical cells" in

urine have been recognized and studied time and again. The accurate interpretation of the

character of "Atypical cell" in urine is a major challenge for cytopathologists. In this present

paper we analyzed the characteristic morphology of malignant atypical cells which were

missed on routine cytology of urine.

REVIEW OF LITERATURE

Brown FM(Urol Clin North Am. 2000;27(1):25-37.) after his study concluded thatExfoliate

Cytology is still a valuable test in detecting bladder cancer. Patients with positive urine

cytology are more likely to have higher stage and grade disease. Hence, investigations in

these patients need to be more exhaustive. Despite new diagnostic methods ,urine cytology

still has a role to play in the detection of more advanced and higher grade urothelial cancers,

early detection of which is likely to affect long-term prognosis

García Castro MA, Fernández Fernández E ET AL ALSO suggested that thePatients

with positive urine cytology and tumours not confirmed at cystoscopy should be carefully

followed up si

nce a hidden bladder or upper urinary tract tumour could be missed. Urine cytology has a

significant role as the gold standard for bladder cancer screening

Although The study by Feifer AH, Steinberg J, Tanguay S, Aprikian AG, Brimo F, Kassouf

W.(Department of Surgery (Urology), McGill University, Montreal, Quebec,

Canada.)supports evaluating patients with AMH because a significant percentage of patients

will have UC, voided urine cytology added a significant cost without any diagnostic benefit

in the work-up of low-risk patients with AMH.

Brimo F, Vollmer RT, Case B, Aprikian A, Kassouf W, Auger M(Department of Pathology,

McGill University, Montreal, Quebec H3A 2B4, Canada.) suggested that dividing atypical

cases into 2 categories based on the level of cytologic suspicion of cancer does not add

clinically relevant information within the atypical category. They also raise the question of

the significance of the a typical category altogether.

Nakamura K, Kasraeian A, Iczkowski KA, Chang M, Pendleton J, Anai S, Rosser CJ.

(Division of Urology, The University of Florida, Jacksonville, Florida, USA. Kogenta)

found out that Because of this low prevalence of bladder cancer in patients presenting with

microscopic hematuria and the low sensitivity of detecting bladder cancers, the utility of

urinary cytology in the initial evaluation of patients with hematuria may be minimal. The

exact role of urinary cytology in the evaluation of hematuria is unknown.

Ordon M, Boerner S, Zlotta AR, Jewett MA, Fleshner N.(Department of Pathology,

University Health Network, Toronto, Canada.) AFTER THEIR EXTENSIVE STUDY

found that All patients who had a previous history of, or developed, urothelial carcinoma

during the follow-up. There were 41 instances (16.3%) in which bladder cancer was evident

at the time of the UUCyt and 29% of these tumours were high-grade. There were another 44

instances (17.5%) in which new or recurrent bladder cancer developed in the subsequent

year after a UUCyt test, and many (38.6%) of these tumours were high-grade.Thus

concluded that The incidence of urothelial carcinoma after a UUCyt was high (33.9%) with

a substantial number of high-grade (34%) tumours, implying that a UUCyt result cannot be

interpreted as negative for malignancy. Therefore, in these cases, the urologist must depend

on cystoscopy to make a diagnosis.

Kapur U, Venkataraman G, Wojcik EM.(Department of Pathology, Loyola University

Medical Center, 2160 S. First Ave., Maywood, IL 60153) concluded thatan AU diagnosis is

more predictive of a subsequent adverse biopsy diagnosis in voided urine specimens

compared with instrumented urines. In the absence of a benchmark for the atypia rate, it is

prudent to keep the atypia rate low to keep it more meaningful. This important category

should be used by the pathologist to convey concern and recognize the difficulty in

interpretation of specimens that may require close follow-up.

Bibiliography:

Koss' diagnostic cytology and its histopathologic bases, Volume 1 - Page 775

Leopold G. Koss, Myron R. Melamed - Medical - 2006 - 1752 pages

(letter to the Editor) Comparison of Thinl'rep and conventional preparations:

Urine cytology evaluation. Diagn Cytopa- thol 21:364-366, 1999. ...

Limited preview - About this book - More editions

Bladder cancer: current diagnosis and treatment - Page 135

Michael J. Droller - Medical - 2001 - 454 pages

Cytology/DNA Ploidy Cytologic evaluation of urine specimens and bladder ...

Bancroft JD and Gamble M.

Theory and Practice of Histological Techniques, 5th edition, Churchill Livingstone; 2002. 631-32, 642-

43, 650-51, 654-55.

Clinical cytopathology and aspiration biopsy: fundamental principles and ... - Page 276

Ibrahim Ramzy - Medical - 2000 - 619 pages

Nemoto R, Kato T, Harada M, et al: Mass screening for urinary tract cancer with

urine cytology. J Cancer Res Clin Oncol 1982:104:155-159

Cytologic detection of urothelial lesions - Page 173

Dorothy L. Rosenthal, Stephen S. Raab - Medical - 2006 - 187 pages

Koss LG, Deitch D. Ramanathan R. and Sherman AB: Diagnostic value of cytology of

voided urine. Acta Cytol 1985; 29:810-816. Murphy WM. Crabtree WN. ...

Bladder Cancer: Diagnosis, Therapeutics, and Management - Page 17

Cheryl Lee, David P. Wood - Medical - 2009 - 332 pages

Urine cytology. It is still the gold standard for screening? Urol Clin North Am

2000 Feb;27(l):25-37. 61 . Schrag D, Hsieh LJ, Rabbani F, Bach PB, Herr H, ...

Blueprints Medicine - Page 120

Vincent B. Young, William A. Kormos, Davoren A. Chick, Allan H Goroll - Medical - 2009 - 448 pages

While urine cytology is fairly sensitive for bladder cancer, it is relatively

insensitive for detection of upper urinary tract tumors.

PROFORMA

Sr. No: Cytology No: Date:

Patient's name Age and Sex

Address

CR. No. /OPD. No.

Brief history

1..Fever 2.hematuria 3.pain while passage of urine

4.frequency of urination 5.urgency

6.stream of flow 7.others

Relevant Investigation (if any)

Hb., TLC, DLC, ESR , FBS etc

Radiological investigations

Gross appearance of urine

Cytological findings

CONSENT FORM

Whole study and its procedure has been well explained in the

language I can understand best.

I hereby consent voluntarily to participate as a study subject for

the purpose of thesis.

(Signature/Thumb Print of Patient / Relative)

Full Name ______________________________________

Relation with the patient _________________________

Date : __________________

Place : __________________

FORM OF APPLICATION FOR APPROVAL OF SUBJECT OF THESIS FOR M.D.

(PATHOLOGY)

1. Name of Candidate : Dr. Anshul Singla

2. Father's Name : Er.Gian Chand Singla

3 Address Postgraduate Student

Deptt. of Pathology,

Govt. Medical College, Patiala

4 Year and month of passing M.B.B.S : December 2006

5 Name of the University from which

graduated

:Maharastra University of Health

Sciences(Nashik)

6 Present occupation : Post-graduate Student,

Deptt. of Pathology,

Govt. Medical College, Patiala

7. Date of joining of PG course : June 2010

8. Proposed subject of thesis : “Screening of atypical malignant cells in

urine: A Diagnostic Dilemma”

9 Special subject : Pathology

10. Facilities for work on the subject of

thesis

: Available

(certificate of the Supervisors attached)

11. Detailed scheme according to which the

candidate proposes to work

: As per plan attached.

12. Name and address of the Supervisor : Dr. Ish Kumar

Deptt. of Pathology,

Govt. Medical College, Patiala.

13. Name and address of the Co Supervisors Dr. Harjinder Singh

Deptt. Of Urology,

Govt. Medical College, Patiala.

Dr. Manjit S. Bal

Professor and Head

Deptt. of Pathology,

Govt. Medical College, Patiala.

Place: Patiala

Dated: (Signature of the candidate)

Certificate of the SupervisorsWe certify that facilities for working on the subject of plan entitled “Atypical malignant cells in

urine:A Diagnostic Dilemma” exist in our department. We shall guide the candidate in her research work

and shall see that the data being included in the thesis is genuine and that research work is being done by the

candidate herself.

We certify that, the work on the subject of thesis has not been carried out earlier in this institution.

If approved as an internal examiner to evaluate the thesis, we are willing to act as such.

Dr.M.S.Bal

M.D.FIC PATH Proffessor and Head,

Deptt of pathology

Patiala.

Dr. Ish Kumar

Deptt of pathology

PATIALA

.