URINE CYTOLOGY

5th Annual EFCS Tutorial,Trondheim, Norway 28th May 1st June 2012

Dubrava University HospitalZagreb, Croatia

Prof. KARMEN TRUTIN OSTOVI, M.I.A.C.Head of Centre of clinical cytology and cytometry

URINARY TRACT

Upper urinary tract Kidneys Ureters

Lower urinary tract Urinary bladder Urethra

(Prostate)

I. EXFOLIATIVE CYTOLOGY URINE

Early morning voided Catheterised Ileal conduit

WASHING (epithelial surface) Urinary bladder Ureters left and right separately

BRUSHING (epithelial surface) Ureter Urinary bladder Urethra

II. FNAC (solid and cystic lesions): Kidney Prostate Suprarenal glands Retroperitoneum (lymph nodes ...)

III. IMPRINTS Intraoperative tumours

Ref. Koss Diagnostic Cytology 2006

PROCESSING TECHNIQUES1. SPECIMEN COLLECTION

2. SPECIMEN PREPARATION

3. FIXATION

4. STAINING

5. ANALYSIS /EVALUATION/

6. DIAGNOSIS

7. ARCHIVING

1. SPECIMEN COLLECTI0NVOIDED URINE INSTRUCTIONS for the patients

Give the voided urine for 3 consecutive days

Take one gramme of C-vitamin the night before urine analysis

Drink litre of water 30 minutes prior to urination

Wash genital tract by clean-catch technique

Give the second mornings voided urine at the cytology department (do not bring urine from home!)

2. SPECIMEN PREPARATION

Immediately preparation (the best time is within half of an hour after urination)

Sediments for native microscopy (for voided urine)

Sediments for stained cytological slides

Samples for ancillary tests (cytochemistry, flow cytometry, immunocytochemistry,FISH,PCR...)

After 3 hours

Within 30 minutes

NATIVE URINE SEDIMENTS

Voided urine 10 20 ml of urine is centrifuged at 3000 rpm for 5 minutes The supernatant is decanted from conical centrifuge tube A drop of sediment mixed with 0.1% of saphranine is dropped

onto the slide

CYTOSPIN SEDIMENTS

Voided urine Couple of drops (depending of clarity) are cytocentrifuged at

1000 rpm for 5 min in cytocentrifuge

2 - 4 slides per one sample of urine are prepared for staining

3. FIXATION 4. STAINING

1. Pappenheim staining (MGG) Drying on the air (2 hours)

2. Papanicolaou staining (PAPA) Diving wet slide in 96% ethyl

alcohol (24 hours)or

Spraying wet slide with hair-spray

3. Haemacolor rapid staining Drying on the air (1-2 min)

3

5. ANALYSIS /EVALUATION/

Native urine analysis Qulitative Semiquantitative

Analysis of stained slides Qualitative Semiquantitative

NATIVE URINE ANALYSIS

QUALITATIVE

a) Casts (hyaline)b) Crystals (ca oxalate)c) (Glomerular erythrocytes)

ab

NATIVE URINE ANALYSIS

QUALITATIVE

a) Casts (hyaline)b) Crystals (ca oxalate)c) (Glomerular erythrocytes)

QUALITATIVE ANDSEMIQUANTITATIVE

Erythrocytes1. Smooth 2. Dysmorphic

ab

21From: Chronolab, Atlas sedimenta mokrae , Split 2004.)

NATIVE URINE ANALYSIS SEMIQUANTITATIVE

Percentage limits of erythrocytes (E) to determine the place of bleeding

(in voided urine only)

> 70% dysmorphic E - haematuria from the upper urinary tract

> 70% smooth E - haematuria from the lower urinary tract

dysmorphic E : smooth E = 50% : 50% - mixed haematuria

ANALYSIS OF STAINED SLIDESQUALITATIVE ANALYSIS

Background Cellularity Epithelial cells

Urothelial cells Squamous cells Columnar cells Tubular renal cells

Non-epithelial cells Leukocytes Histiocytes Macrophages

Non-cellular elements Casts Crystals Contaminants

QUALITATIVE ANALYSIS non-cellular elements

Casts Normal urine:

Hyaline c., Granular c.

Pathological: Erythrocyte c. Leukocyte c. Epithelial c. Waxy c. Mixed cell c.

Crystals Contaminants

QUALITATIVE ANALYSIS non-cellular elements

Casts Normal urine:

Hyaline c., Granular c.

Pathological: Erythrocyte c. Leukocyte c. Epithelial c. Waxy c. Mixed cell c.

Crystals Normal urine:

Uric acid Ca oxalate Triple phosphatase

Pathological: Cystine Tyrosine Leucine Bilirubin Drugs (laxative, sulfa

drugs...)

Contaminants

QUALITATIVE ANALYSIS non-cellular elements

Casts Normal urine:

Hyaline c., Granular c.

Pathological: Erythrocyte c. Leukocyte c. Epithelial c. Waxy c. Mixed cell c.

Crystals Normal urine:

Uric acid Ca oxalate Triple phosphatase

Pathological: Cystine Tyrosine Leucine Bilirubin, Drugs (laxative, sulfa

drugs...)

Contaminants- Powder - Alternaria - Cotton - CorporaAmylacea

- Mucus- Fibrin - Lubricant

QUALITATIVE ANALYSISNON-CELLULAR ELEMENTS

QUALITATIVE ANALYSISNON-CELLULAR ELEMENTS

Enterobius vermicularis

AlternariaUric acid crystal

Powder

Granular cast and lubricant

Drugs crystal

From: Bardales RH: Practical Urologic Cytopathology, Oxford 2002

QUALITATIVE ANALYSISEpithelial cells

Urothelial (transitional) cells Pelvis Ureters Urinary bladder Urethra

Non-cornifying squamous cells Trigone of bladder (female) Urethra

Columnar cells Trigone of bladder Urethra (male)

Renal tubular cells Kidney

UROTHELIAL CELLS Variation in shape

Cell shape depends upon bladder extensiona) Distended transitional cellsb) Non-distended

Variation in size

They shed singly (1) or in clusters (2,3)1. Superficial (umbrella, luminal) cell2. Intermediate (piriform) cells3. Basal (deep) cells 1 2

3

1

1

2

2

3

3

a)

b)

Ref. Koss Diagnostic Cytology 2006

EPITHELIAL CELLS1. Urothelial superficial (umbrella) cells

Mononucleated Multinucleated

2,10 or more nuclei of variable sizes2. Urothelial intermediate and basal cells

Rare in voided urine (usually after instrumentation)

3. Squamous cells Cells from trigone and urethra respond to

hormonal changes in both sexes Be careful: difficult differentiation from

contaminant squamous cells (use catheterised urine!)

4. Columnar cells Rare in voided urine

5. Renal tubular cells Rare in voided urine

(

RENAL TUBULAR CELLS CORTEX

15 60 m Oval or round cells Usually single Ill-defined cytoplasm Granular cytoplasm Pyknotic nucleus

MEDULLA 15 60 m Cuboid or cylindric cells Single or in clusters Well-defined cytoplasm Fine granular cytoplasm Nucleus is not pyknotic

ANALYSIS OF STAINED SLIDESQUALITATIVE

Background Cellularity Epithelial cells

Urothelial cells Squamous cells Columnar cells Tubular renal cells

QUALITATIVE AND SEMIQUANTITATIVE Erythrocytes

1. Glomerular2. Non-glomerular

Non-epithelial cells Leukocytes Histiocytes Macrophages

Non-cellular elements Casts Crystals Contaminants

QUALITATIVE AND SEMIQUANTITATIVE ANALYSIS

ErythrocytesNon-glomerular

a) Spiked forms

b) Discoid forms (like normal E butlarger)

c) Creased forms with Mercedes star patternin central zone orobliquely (cap shape)

d) Double-rim formsd

ca

b

From: Rathert P.et al: Urinary Cytology, 1993

QUALITATIVE AND SEMIQUANTITATIVE ANALYSIS

Erythrocytes Glomerular

1. Ring forms (doughnut E)

2. Vesicular forms

3. Combination of vesicular and ring forms

4. Ruined forms (grotesque shape)

2

4

3 3

1

1

2

Percentage limits of glomerular erythrocytes (E)(synonims: G1 dysmorphic E or G1 erythrocytes (Tomita M)

to point the damage of glomerulus(in voided urine only)

< 20% glomerular E - no glomerulopathia

20 - 50% glomerular E possible glomerulopathia

50 - 75% glomerular E possible glomerulonephritis

> 80% glomerular E - glomerulonephritis

STAINED SLIDES semiquantitative analysis

CYTOLOGY OF THE URINE

NORMAL URINE

PATHOLOGICAL LESIONS

NORMAL VOIDED URINE FEW EPITHELIAL CELLS

Urothelial Squamous cells Columnar cells Tubular renal cells

FEW NON-EPITHELIAL CELLS Erythrocytes (nonglomerular) Leukocytes

Neutrophiiic granulocytes Lymphocytes

NON-CELLULAR ELEMENTS (OCCASI0NAL) Hyaline and finely granular casts Corpora amylacea (male)

CONTAMINATION Female (from vagina and vulva)

1.Squamous cells2.Doderlein bacillus

Male3. Seminal vesicle cells

Be careful: similar to malignant cells!4. Spermatozoa

43

1, 2

CYTOLOGY OF THE URINE

BENIGN LESIONS Infections Inflammatory conditions Haematuria Urolithiasis

PREMALIGNANT LESIONS Squamous metaplasia Dyskariosis

SUSPICIOUS FOR MALIGNANCY NON-CLASSIFIED LESIONS

Reactive changes Polymorphia Atypia

TUMOURS Urothelial tumours Papillary tumours

Non-papillary

Non-urothelial tumours Squamous Adenocarcinoma

Metastatic tumours

INADEQUATE

PATHOLOGICAL LESIONS (VOIDED URINE)

HAEMATURIA

Upper urinary tract(more than 70% dysmorphic E) Kidney neoplasm Acute glomerulonephritis Tuberculosis Tumours of ureters Pyelonephritis Nephrolithiasis Cistyc kidney Trauma of kidney Infarct of kidney

Essential Phisiological Ill effects to anticoagulation therapy and radiation Systemic disease (Mb. Bechterew, hemophilia, leukaemia)

Lower urinary tract(more than 70% smooth E) Tumors of bladder Tumors of urethra Hemorrhagic cystitis Cystitis Bladder lithiasis Diverticulum Urethritis Trauma of urethra

ERYTHROCYTES

NATIVESEDIMENT

STAINEDSMEARS

DYSMORPHIC

SMOOTH

GLOMERULAR

NON-GLOMERULAR

GLOMERULONEPHRITIS

>80% glomerular erythrocytes

Numerous renal tubular cells

Lots of casts Erythrocytic Coarsely granular

Urothelial cells (often reactive)

GLOMERULONEPHRITISBe careful:use high magnification for analysis of erythrocytes, do not misdiagnose ruined forms of GE with damaged erythrocytes ruined forms have intact memebrane!

Perform semiquantitative analysis on 4 different parts of sediment

The cytological diagnosis should be confirmed by electron microscopic (EM) analysis

FOCAL SEGMENTAL NECROTISING GLOMERULONEPHRITIS

IgA nephropathy IgA positive

Immune deposits in mesangial cells

UROLITHIASIS Calculi may be present in all urinary

tract (the most common in pelvis and bladder)

Polymorfism of urothelial cells Dyskaryosis (dysplasia) Reactive urothelial cells, usually

umbrella Squamous metaplasia Numerous erythrocytes (non-glomerular)

(native urine analysis of erythrocytes) Many leukocytes Crystals

Calcium oxalate Uric acid

Haematuria Cytologist (cytopathologist) - at the crossroad

suggesting to the patient to go to: Nephrologist:

Dysmorphic E >70% Glomerular E >20%

Urologist (usually): Smooth E >70% Non-glomerular E

Nephrologist and urologist: 50% smooth E and 50% dysmorphic E Non-glomerular E

Biopsy: >50% glomerular E

INFECTIONS Increased exfoliation of cells Non-specific morphological changes of

epithelial cells (bacterial) Specific morphological changes of epithelial

cells (viral DNA viruses) Identification of:

Parasites Fungal spores and hyphal forms

(microbiological identification) Bacteria (microbiological identification)

BACTERIAL INFECTION

1. Reactive urothelial cells

Enlargement of the cell Enlargement of the

nucleus Enlargement of the

nucleolus Nuclear-cytoplasmic

ratio is normal Prominent nucleolus

11

1

1

Inflammation?

Inflammation? No - Urothelial carcinoma

VIRAL INFECTIONSPOLYOMA VIRUS

In immunosuppressed patients and renal allograft recipients after reactivation of virus

1. Inclusion stage Single, centrally located irregular

hyperchromatic nucleus with large homogeneous, basophilic intranuclear inclusions

2. Postinclusion stage Enlarged nucleus with empty, pale

appearance with distinct network of chromatin filaments fishnet-stocking pattern

Immunocytochemistry: antigen to SV40 virus

Be careful: decoy cells are similar to malignant cells

1

1

2

PARASITIC INFECTIONSTrichomonas (TV)

Rare (urethritis, cystitis) Marked acute inflammation

Numerous neutrophilic granulocytes Squamous metaplastic cells Few urothelial cells Erythrocytes Necrosis is absent

A light gray, pear-shaped protozoan with dark eccentric nucleus

Be careful: do not misdiagnose trichomonas as destroyed cell

MALAKOPLAKIA Uncommon granulomatous cystitis

or urethritis or ureteritis Affects middle-aged female A yellow plaque with central depression Lots of cells

Histiocytes with single or multiple Michaelis-Gutmann bodies (PAS

and iron-positive) Few plasma cells, macrophages Reactive urothelial cells

The background is granular with nectrotic debris

Be careful: not misdiagnose with engulfed erythrocytes within macrophage

DYSKARYOSIS Synonims:

Atypical urothelial cells Dysplasia

It is the putative precursor of ca in situ and invasive urothelial carcinoma (occurs in 15 % of the patients within 5 years after detection)

Cytollogically follow up every 3 6 months Dyskaryotic cells

Increased uniform basal cells Irregular hyperchromatic nucleus Slight shape variation of the nucleus Slightly increased nuclear-cytoplasmic

ratio Scanty cytoplasm Single cells or in papillay clusters

Be careful: do not misdiagnose dyskariotic cells with single basal urothelial cells use highe magnification

WHO CLASSIFICATION OF TUMOURS (2004) UROTHELIAL TUMOURS Infiltrating urothelial carcinoma

With squamous differentiation With glandular differentiation With trophoblastic differentiation Nested Microcystic Micropapillary Lymphoepithelioma-like Lymphoma-like Plamsacytoid Giant cell Undifferentiated

Non-invasive urothelial neoplasias Urothelial ca in situ Non-invasive papillary ca high grade Non-invasive papillary ca low grade Urothelial papilloma

SQUAMOUS NEOPLASMS Squamous cell carcinoma Verrucous carcinoma Squamous papilloma

HAEMATOPOIETIC AND LYMPHOID TUMOURS Lymphoma Plasmacytoma

GLANDULAR NEOPLASMS Adenocarcinoma

Enteric Mucinous Signet-ring cell Clear cell

Villous adenoma NEUROENDOCRINE TUMOURS Small cell carcinoma Carcinoid Paraganglioma MELANOCYTIC TUMOURS Malignant melanoma Nevus MESENCHYMAL TUMOURS

Rhabdomyosarcoma Leiomyosarcoma Angiosarcoma Osteosarcoma Malignangt fibous histiocytoma Lleiomyoma Haemangioma.....

METASTATIC TUMOURS MISCELLANEOUS TUMOURS Carcinoma of glands

UROTHELIAL TUMOURS 95 % of bladder tumours are urothelial carcinoma

Per se In combination with squamous or glandular

differentiation In the time of initial diagnosis

75% of the patients have noninvasive urothelial carcinoma

20% have invasive carcinoma 5% have metastatic disease

60% of tumours are recurrent Most of carcinoma are papillary and multifocal

Be careful: after biopsy, if histological diagnosis is benign, it does not mean that patient has no carcinoma if cytological diagnosis is malignant!

UROTHELIAL TUMOURS

Papillary tumours Papilloma Papillary tumor grade I (papillary neoplasm of low malignant

potential) Papillary carcinoma grade II (papillary carcinoma, low grade) Papillary carcinoma grade III (papillary carcinoma, high

grade)

Nonpapillary tumours Flat carcinoma in situ Invasive carcinoma

UROTHELIAL TUMOURS

Papillary tumours Papilloma Papillary tumor grade I (papillary neoplasm of low malignant

potential) Papillary carcinoma grade II (papillary carcinoma, low grade) Papillary carcinoma grade III (papillary carcinoma, high

grade)

Nonpapillary tumours Flat carcinoma in situ Invasive carcinoma

Papillary tumoursPapilloma

Increased number of benign urothelial cells and erythrocytes

The diagnosis is histological

Note: when there are lots of reactive cells and papillary clusters of dyskaryotic cells in sediment, cytological diagnosis of possible papilloma can be put but biopsy is recommanded

Papillary tumoursPapillary tumour grade I

(papillary neoplasm of low malignant potential - PUNLMP)

The background is clean Dyskaryotic cells in three-dimensional

clusters Lots of reactive urothelial cells Malignant urothelial cells

In papillary clusters (can suggest the diagnosis)

Nuclear-cytoplasmic ratio is slightly high Cytoplasm is thin and semitransparent Nuclei are round or oval with powdery

chromatin, uniform pencil-drawn nuclear contours and small nucleoli

Cell blocks of urinary sediment enables the diagnosis immunocytochemistry (CK20)

Suspicious lesions Cytology of the urinary sediment

does not lend itself to the diagnosis dg. is usually suspicious

Pathohistology PUNLMP-s (papillary urothelial

neoplasm of low malignant potential)

Papillary tumoursPapillary carcinoma grade II

(papillary carcinoma, low-grade)

Few small clusters and single markly atypical or frankly malignant cells Enlarged cells Slightly irregular nuclear contour Moderate hypechromasia with reduced

nuclear transparency Prominent nucleoli Homogeneous cytoplasm Moderate increased the

nuclear-cytoplasmic ratio

Papillary tumoursPapillary carcinoma grade II (low-grade)

Cytological diagnosis can be: atypical or suspicious or probably malignant low-grade papillary carcinoma

FISH (fluorescent in situ hybridization) can helpDNA pattern analysis can help aneuploid DNA is strongly suggestive of carcinoma

Flow cytometry (FCM)

DNA analysis (ploidy and proliferative activity) Discriminates high-grade (aneuploid) and low-grade

(diploid) urothelial tumours It and detects urothelial carcinoma 12 to 18 months earlier

than cystoscopy

Be careful: FCM requires large number of abnormal cells to give the accurate value (45% of voided urine give satisfactory result)

Papillary tumoursPapillary carcinoma grade III (high grade)

Background may be bloody, but necrosis is absent

Cellularity is high with numerous tight clusters and single malignant cells

Papillary tumoursPapillary carcinoma grade III

(papillary carcinoma, high grade) Pleomorphic malignant cells

Larger than normal The cytoplasm ranges from

homogeneous and scant to vacuolated and abundant

Variable nuclear-cytoplasmic ratio Pleomorphic hyperchromatic nuclei

with prominent nucleoli Chromatin is clumped, irregular or

vesicular Nuclear contour is irregular Cannibalism

The diagnosis is cytological

Nonpapillary tumoursFlat carcinoma in situ

Background is without necrosis, there are few erythrocytes and inflammatory cells

Cellularity is variable Usually monotonous population of

medium-sized or small malignant urothelial cells in small clusters or single

Ocasional a few larger or bizzaremalignant cells may occur

Pleomorphic cells with scanty basophiliccytoplasm in one third of the patientsdif.dg. High grade ca

Nuclei are large, hyperchromatic with irregular contours and chromatin is coarse and irregular or pyknotic

Markedly increased nuclear-cytoplasmic ratioThe diagnosis is cytological

Nonpapillary tumours - Flat carcinoma in situ

The cytological diagnosis of ca in situ may remain unconfirmed for many years in the absence of an aggresive approach to bladder biopsies

Nonpapillary tumoursInvasive nonpapillary carcinoma

It is a high grade carcinoma The prognosis is poor and the 5-year

survival is less than 50% even with therapy

Background is dirty Marked inflammation Marked bleeding Marked necrosis

Pleomorphic cells Variable sizes Variable nuclear-cytoplasmic ratio Anaplastic nuclei with irregular

chromatin and large, irregular nucleoli

Squamous or glandular differentiation is common

Nonpapillary tumours Invasive nonpapillary carcinoma

The diagnosis is cytological

Metastatic tumours I.

Directly spread from adjacent organs Squamous carcinoma of the

uterine cervix Endometrial carcinoma of the

uterine corpus in postmenopausel females

Colorectal carcinoma Immunocytochemistry: positive CEA

Adenocarcinoma of the prostate Immunocytochemistry: positive PSA

Implantation from the upper urinary tract Hypernephroma

Fairly large cells with finely vacuolated and granular cytoplasm

Cytochemistry: PAS, PASwith amylase digestion and oil-red or Sudan III positive PAS with amylase digestion

Metastatic tumours II.Lymphatic or hyematogenous spread from distant sites

1. Melanoma Pigment in malignant cells

Immunocytochemistry: positive HMB-45, S-100, Melan

2. Carcinoma Small cell ca (lung) dif. dg. small cell

undifferentiated urinary carcinoma Immunocytochemistry:

positive TTF1

3. Haematolymphoid malignancies(lymphoma, leukemia, multiple myeloma) Flow cytometry - immunophenotyping

Monoclonality3

DIAGNOSTIC VALUE OF URINE CYTOLOGY EXAMINATION

Diagnostic accuracy of urine cytology of bladder carcinoma diagnosis ranges between 26% and 100% Low and variable for low grade papillary neoplasms (accurate

in 1/3 of the cases) Highly accurate for high-grade tumours including invasive

carcinoma and carcinoma in situ (accurate in of the cases)

False-positive diagnosis range from 1.3% to 11.9% -high specificity

False-negative diagnosis range from 23% to 38% -low sensitivity

BIOMARKERS AND ANCILLARY TESTS FOR UROTHELIAL CARCINOMA

They have a potential role in the improvement of screening and detection of bladder carcinoma because they have

better sensitivity than conventional cytology

CONCLUSIONUrinary cytology

Is the only nonivasive method for detecting carcinoma in urinary tract

Has better specificity but lower sensitivity than ancillary tests and biomarkers

Remains the gold standard for the detection of urothelial carcinoma especially for the lesions inaccessible to cystoscopy or biopsy and those that are invisible by radiology and cystoscopy like dysplasia and flat tumours

Needs consensus of cytological classification system

DUBRAVA UNIVERSITY HOSPITAL, ZAGREB, CROATIACENTRE OF CLINICAL CYTOLOGY AND CYTOMETRY

Thank you

EUROPEAN CONGRESS OF CYTOLOGYCAVTAT - CROATIA - 2012