MLT-270

CLINICAL PATHOLOGY AND CYTOLOGY

UNIT-01

Urinary tract specimens

URINARY TRACT
Composition of Urine

Urine is the fluid containing water-soluble waste products excreted from the blood via the

kidneys. Urine is mainly composed of 95% water and the rest is made up of urea, uric acid,

creatinine, sodium, potassium, chloride, calcium, phosphates, etc.

 The composition of urine varies a great deal and is affected by three factors:

(i) Nutritional status of the individual

(ii) State of metabolic processes

(iii) Ability of the kidneys to selectively handle the material presented to them.
Properties of Normal Urine
Dry weight 55-70
pH 4.5-8.6(mean 6.1)
specific gravity
Neonates 1.012
Infants 1.002-1.006
Adults 1.003-1.030
Volume per day :-

Newborns (0–28 days):

• Normal volume:-------------15–60 mL/day.

• Urine output rate: 1–2 mL/kg/hour.

Infants (1 month–1 year):

• Normal volume: ----------------100–300 mL/day.

• Urine output rate: ~1–2 mL/kg/hour

Children (1–10 years):

• Normal volume: ---------------500–1200 mL/day.

Adolescents (10–18 years):

• Normal volume: 800–1500 mL/day.

• Urine output rate: 0.5–1 mL/kg/hour

Adults (18–65 years):

• Normal volume: 800–2000 mL/day.

• Urine output rate: 0.5–1 mL/kg/hour (average ~50–70 mL/hour).

Older Adults (65+ years):

• Normal volume: ~800–2000 mL/day (similar to adults, but may vary).

Organic constituents/ 24 hours
Urea: 15-30 g
Creatinine 1.0-1.8 g
Uric acid: 0.3-0.6 g
Protein (Albumin): 0-0.1 g
Glucose: < 0.5 g
 Introduction of Urine cytology

• George Papanicolaou and Marshal first time introduced urinary cytology for detection of
malignancy.

• Later on, it was considered as a well-established technique for the detection of malignancy
in the urinary tract.

• Despite of its low sensitivity, urinary cytological examination is still considered as a useful
adjunct to the cystoscopy.

• The major disadvantages of urinary cytology are its low detection rate and failure to
ascertain the exact anatomical localization of the lesion.
The major indications of urinary cytology are:

• To investigate the cause of hematuria

• Screening of recurrence of bladder cancer

• To screen asymptomatic high-risk cases for bladder cancer.

The most common presentation of bladder carcinoma is hematuria.

• A simple examination of urine sample can readily detect malignant cells.

• These cases should be further confirmed by cystoscopy and cystoscopic-guided biopsy.

• Urine examination is particularly helpful in follow-up of patients with bladder cancer.

• High-grade urothelial carcinoma (UC) is prone to recur.

• Cystoscopy may be helpful in the detection of the recurrent cases.

• However, urine examination is particularly helpful in case of tumors that are not visualized by
cystoscopy.

• It is a simple and inexpensive test for bladder cancer in asymptomatic people.

• Urine examination can be done for the high-risk asymptomatic people such as persons who
are exposed to aniline dyes or cyclophosphamide therapy.
ANATOMY

The lower urinary tract acts as a passage to transmit and store urine. It consists of renal pelvis, ureters, urinary
bladder and urethra.

Renal Pelvis

• The renal pelvis is a funnel-like dilated portion of the proximal end of the ureters within the kidney.

• It receives the final product of urine from the renal calyces.

Ureter

• The ureter is a muscular, tube-like, long, slender structure which transmits urine from the kidney to the
bladder.

• Each ureter is 25–30 cm long and 3–4 mm in diameter.

• The ureter is a retroperitoneal structure and passes through the abdominal cavity and then opens in the back
of the urinary bladder
• Ureter has three layers:

1. Inner mucosal layer—Mucosa: Mucosa of the ureter is lined by 3–5 layers of

transitional epithelium and is rested on dense fibroconnective tissue, known as
lamina propria.

2. Muscular layer: Muscle layer consists of inner longitudinal, middle circular

and outer longitudinal layer.

3. Outer fibroconnective tissue coat.

Urethra

• Urethra is the tube that connects the urinary bladder to the outside of the body.

• In females, it is used for urination and in male urethra serves as a conduit of semen and also

urine.

• Male urethra extends from the internal urethral orifice of the bladder to the external urethral

orifice of the penis. It is about 17–20 cm long.

• The female urethra is a membranous tube 4–5 cm long and extends from the internal urethral

orifice of the urinary bladder to the external genitalia in between the clitoris and vagina.
Urinary Bladder :-

• The urinary bladder is the muscular bag that receives urine from the two ureters and
temporarily stores urine.

• It is superior to the prostate. In the male, the urinary bladder is situated in between the rectum
posteriorly and pubic bone anteriorly.

• In females, the bladder is situated in between vagina posteriorly and pubic bone anteriorly.
Urine exits from bladder via the urethra.
Urinary bladder has three layers:

(1) inner mucosal layer,

(2) submucosal layer.

(3) muscular layer.

1. Inner mucosal layer:

• Mucosa of the urinary bladder is lined by 5–6 layers of transitional epithelial cells.

• There are three different zones of cells: basal cell layer, intermediate cell layer and surface
cell layer.

• The basal layer is composed of one cell layer of cuboidal epithelial cells.

• The intermediate cell layers are about five layers thick and the cells are more columnar with
their nuclei orientated at right angles to the basement membrane
• The superficial layer is only one layer thick.

• These cells are also known as umbrella cells or dome cells and have the unique
characteristics that allow them to maintain impermeability to urine to mucosal layer.

• The cells are large and oval in shape having abundant eosinophilic cytoplasm and round
centrally placed nuclei.

• The surface outline of the cell is fuzzy and indistinct.

• Many cells are binucleated and a few are multinucleated.

2. Submucosal layer: Mucosal membrane of the bladder rests on the submucosal layer. It
consists of connective tissue and dense capillary plexus.

This capillary plexus serves not only as vascular supply but it also acts as a urinary-blood
barrier.

3. Muscular layer: The muscle layer consists of innermost longitudinal, middle circular and
outermost longitudinal layers that are often difficult to distinguish.
Role of the Urinary Tract
• Waste Removal: Eliminates urea, a byproduct of protein metabolism.
• Fluid Balance: Maintains proper hydration levels in the body.
• Electrolyte Regulation: Balances minerals and salts.
• pH Regulation: Helps maintain acid-base balance in the blood.
Common Urinary Tract Issues
• Urinary Tract Infections (UTIs): Infections in any part of the urinary tract, more common in
females due to the shorter urethra.
• Kidney Stones: Crystallized minerals that form in the kidneys and can cause pain as they
pass through the urinary tract.
• Bladder Disorders: Conditions like overactive bladder or incontinence.
• Kidney Diseases: Chronic kidney disease (CKD) or acute kidney injuries can impair function
Kidneys
• Function: The kidneys are two bean-shaped organs located on either side of the spine, just below the ribcage. They filter blood to
remove waste products, toxins, and excess water, forming urine.
• Additional Role: They help regulate blood pressure, produce hormones, and maintain a balance of electrolytes like sodium and
potassium.
• 2. Ureters
• Function: The ureters are two thin tubes that transport urine from the kidneys to the bladder. Muscular contractions in the ureter walls
help move urine downward.
• 3. Bladder
• Function: The bladder is a hollow, muscular organ located in the pelvis. It stores urine until it is ready to be excreted.
• Capacity: A healthy bladder can typically hold about 400-600 mL of urine.
• 4. Urethra
• Function: The urethra is a tube that carries urine from the bladder to the outside of the body during urination.
• Length Differences: In males, the urethra is longer and passes through the penis, serving as a pathway for both urine and semen. In
females, the urethra is shorter and located above the vaginal opening.
 Methods of Collection of Urine

Urine should always be collected in a clean dry container. The container should be without additives or

detergent.

Sterilized containers are required for bacteriological examination. A wide-mouth container with a lid

supplied by the laboratory is to be used by the patient

• Random collection: Taken at any time of day with no precautions regarding contamination. The

urine may be dilute, isotonic, or hypertonic and may contain white cells, bacteria, and squamous

epithelium as contaminants. In females, the specimen may contain vaginal contaminants as

trichomonads, yeast, and during menses, red cells.

•
Midstream urine:

The first part of the urine stream is discarded, and then a sample of urine is

collected into a container and the remaining part of urine is voided again in the

toilet.

The midstream urine sample contains very few contaminating bacteria.

The sample can be measured immediately or stored for later measurements.

Catheterization:
Catheterization is occasionally used for some bacteriological tests performed
on urine.
This is usually done for unconscious patients and for patients who are
already having an indwelling catheter.
 First-morning sample:

The first-morning specimen is collected when the patient first wakes up in the morning, having

emptied the bladder before going to sleep.

Early morning samples are most frequently for analysis due to urine's day-to-day consistency.

It is the most concentrated of the urine samples and is used for qualitative analysis. It is also

essential for preventing false negative pregnancy tests and orthostatic proteinuria.

The morning sample of urine is preferred because a large volume of urine can be obtained.

The morning sample is suitable for hormone estimation but should be kept in the refrigerator

or icebox during transport to a distant laboratory.

Fasting specimen: Urine is voided after 6 or more hours of food intake and discarded.
A subsequent sample of urine represents a fasting specimen.
24 hours post-prandial: This specimen is collected 2 hours after the patient has eaten a meal
and requires only a clean container.
Specimens collected at different hours of the day show varying degrees of sugar content
excretion which is dependent on the absorption of the glucose from the diet.
The specimen is tested for glucose, and the results are used to monitor insulin therapy in
patients with diabetes mellitus.
Suprapubic aspiration of urine:
This method is used when a bedridden patient cannot be catheterized or a sterile specimen is
collected by needle aspiration through the abdominal wall into the bladder. ( urinary incontinence
(leakage), urinary retention (not being able to urinate), surgery that made a catheter necessary, or another health problem. )

Pediatric specimen:
For infants and small children, a special urine collection bag adheres to the skin surrounding
the urethral area.
Once the collection is completed, the urine is poured into an evacuated tube with a transfer
straw.
Urine collected from a diaper is not recommended for laboratory since contamination from
the diaper material may affect test results.
24 hours urine collection:

• Begin the 24 hours collection with the second urine of the morning Save all urine throughout

the day and night.

• Save the first urine of the second day (this provides complete 24 hours testing).

• It is used to measure 24 hours urine protein, sugar, microalbuminuria, creatinine clearance,

5-hydroxylcorticosteroids, metanephrine, hormones, For minerals, and other chemical

compounds.

• It is also used for the detection of AFB in urine.

Urine cytology:-

There are mainly three types of specimen collection techniques of urine:

1. Voided urine specimen .

2. Catheterized specimen.

3. Bladder wash and brush cytology.

Voided urine collection is the specimen of choice for screening in a population with specific
symptoms such as hematuria.

The urine sample should be collected 3–4 hours after the last micturition.

The first morning voided urine should better be avoided as it shows a considerable amount of
degeneration because of prolonged stagnation.

 Fresh randomly voided urine specimens should be collected in a clean container and should be
sent to a laboratory for immediate processing to avoid degenerative changes.

About 50 mL of urine is sufficient for cytological examination.

Voided urine is easy to collect; however, the urine sample shows sparse cellularity and
considerable degenerative changes. In addition, in female patients, there may be high chances of
Catheterized Urine

 A catheterized urine sample is used in cases of follow-up of a patient with known

neoplastic lesion of the lower urinary tract .

Catheterized urine sample yields better cellularity and shows less degenerative changes.
However, it may cause the breaking up of large tissue fragments that may simulate
malignancy.

Another important disadvantage of this technique is the introduction of urinary tract

infection by the tip of the catheter
Bladder Wash and Brush Cytology

Bladder wash and brush are indicated in cases of suspected urothelial neoplasm or follow-
up of a case of known neoplastic lesion.

 The bladder wash specimen is obtained by rinsing the bladder with 50 mL of sterile
normal saline 3–4 times. The collected fluid is processed.

Bladder brushing is done by direct supervision under cystoscopy.

The bladder washing sample is obtained during or before cystoscopy which is an invasive
diagnostic procedure for the macroscopical evaluation of the bladder mucosa.

First the bladder should be emptied by a catheter. Then 50 to 100 ml of normal saline is
instilled and recovered and this procedure is repeated three times.
The main advantages of this technique are:

(1) Good cellularity, and

(2) Better preservation of the cells.

The major disadvantages are:

(1) Instrumental artifact,

(2) Invasive technique and

(3) Uncomfortable to the patient

Cystoscopy is a procedure that uses a cystoscope to look inside the urethra and bladder.
• A cystoscope is a long, thin optical instrument with an
eyepiece at one end, a rigid or flexible tube in the
middle, and a tiny lens and light at the other end of the tube.
A urologist fills the bladder with fluid and looks at
detailed images of the urethra and bladder linings
on a computer monitor.
Ureteroscopy is a procedure that uses a
ureteroscope to look inside the ureters and kidneys.
Like a cystoscope, a ureteroscope has an eyepiece at
one end, a rigid or flexible tube in the middle, and a tiny
lens and light at the other end of the tube.
However, a ureteroscope is longer and thinner than
a cystoscope so the urologist can see detailed images of the lining of the ureters and kidneys.
 Other sampling Techniques

Ureteric Sample:-

• Urine may also be collected by introducing a catheter to the ureter.

• Urine can be collected separately from each ureter to localize the specific pathology in the ureter or
kidney.

• Smear from each side can be compared for subtle cytological abnormalities.

Ileal Conduits:- An ileal conduit aims to divert urine produced from the upper urinary tract to a newly formed reservoir created from the terminal
ileum. The ureters are disconnected from the bladder and implanted into the conduit

• In case of total cystectomy, the surgeon makes a duplicate bladder with the help of the ileum to
provide a conduit of urine.

• Urine samples are often taken to examine the recurrence of bladder cancer. The sample from the
ileal conduit usually contains a lot of intestinal epithelial cells.
URINE PRESERVATIVES
During warm weather, there is rapid decomposition of urine, which interferes with all examinations.
Several changes like bacterial action, the precipitation of phosphates (if urine is in an alkaline state)
and crystallization of uric acid take place if the urine is kept without preservatives.
An ideal preservative should have the following properties:-
1. It should preserve the urine from bacterial decomposition and the development of molds or
other growths for considerable periods under average conditions.
2. It should not interfere either positively or negatively with any physical, chemical, or
microscopic tests in ordinary use.
3. It should be readily soluble.
4. It should not interfere to any marked extent with the normal action of the urine.
5. It should be solid, and it should be reasonable.
6. It should be cheap.
Types of Urine Preservatives
1. Refrigeration: If a refrigerator is available, samples may be kept in it until examined.
Samples for pregnancy tests must be so preserved. Avoid freezing. The best general method
of preservation up to 8 hours is refrigeration at 4°-6°C. Refrigerated specimens are warmed
to room temperature before performing the analysis. It prevents bacterial growth and there
is minimal chance of chemical alteration of urine.
2 Boric acid: It is used as a preservative for many hormones, e.g. 17-hydroxy corticosteroids,
aldosterone, and testosterone. 1 g is added to 100 ml urine or 50 g per 24 hours of urine . It will
not interfere with the examination for protein, or ketone bodies. It delays decomposition but
may interfere with sugar determination and precipitate rhombic crystals of acid. However, yeast
can still grow and uric acid crystals get precipitated.
3. HCl: 6N HCI (30 ml) is added per 24 hours urine If 12N HCl is used then add 10 ml per
24-hour collection.
• To prevent changes in urine concentrated HCl 10 ml is used as a preservative during the
collection of urine for urea, ammonia, total nitrogen, creatinine, uric acid, phosphates,
cortisol, and calcium estimation.
4. Toluene: It is all round good preservative, which is to be added in sufficient quantity to form
a thin layer on the surface of the urine.
• Toluene merely lies on the surface of the urine, forming a thin layer and acting as a physical
barrier to air and bacteria.
• However, it interferes with some qualitative chemical tests, and it should not be used when
the glucose concentration is to be determined. 30 ml of toluene added to 24-hour urine.
5. Thymol (10% isopropanol): Thymol is another general-purpose preservative. 10 g is
added per 24-hour urine collection. Thymol, if used, should not exceed 0.1 g per 100 ml of
urine. It interferes. with albumin determination. It is unsatisfactory when urine contains sugar,
acetone, or diacetic acid. .
6.Glacial acetic acid: It is mainly used as a preservative for adrenaline and nor-adrenaline.
• 15 ml is added per 24/hours of urine.
7. Alcohol: 50% alcohol is added for cytological examination.
8. Chloroform: It preserves cells and casts. well, used in a proportion of 1 drop per 15 ml of
urine or 50 drops per 24 hours of urine.
• It is the least satisfactory preservative as it interferes with sugar determination and
microscopic examination.
Formalin (10%): 10% formalin is an excellent preservative for the formed elements in urine. About 4 drops of
formalin may be used for every 100 ml of urine. However, it interferes with some qualitative chemical tests, and it
should not be used when the glucose concentration is to be determined. It can precipitate proteins and can reduce
Benedict's solution. An excess may interfere with tests for indican sugar and albumin. It preserves cells and casts.
The bacteriological examination cannot be done with this preservative as the bacteria die in formalin, also
quantitative estimation will not be satisfactory as the liquid form is used which changes the volume of urine.
Sodium fluoride: It may be used, as a preservative for urine samples when one is concern with glucose as it
prevents glycolysis. It is used in a concentration of 5 mg per 100 ml of urine.
Chlorhexidine/N-propyl gallate (PG): It can be used at ambient temperatures as a urine preservative.
Acetic acid: If the urine has been collected check for the presence of Schistosoma haematobium ova but if cannot
be examined for several hours, it should be acidified with a few drops of 10% acetic acid.
Camphor: A small piece of camphor may be used for the preservation of urine.
 PROCESSING

• The urine sample should be processed immediately after receiving the specimen.

• Urine is processed by any of these techniques depending on the amount and availability of
the technique in the laboratory:

(1) Centrifuge.

(2) Cytocentrifuge.

(3) Liquid-based preparation.

(4) Millipore technique.

(5) Cellblock.
Simple Centrifugation

• Approximately 50 mL of urine is centrifuged for 10 minutes with 1,200 rounds per minute.
Multiple smears are prepared from the sediment and alcohol-fixed smears are kept for
staining.

Centrifuge:

• The urine of a moderate amount (50– 100 ml) should be processed by centrifugation, e.g.
effusion fluid, turbid urine, lavage

• Put the fluid sample in a clean air-tight centrifuged tube.

• Rotate the tube at 1500 rounds per minute (RPM) for 10 min.

• Discard the supernatant liquid.

Cytocentrifuge:

• Small amount of clean fluid such as 0.5–1.0 ml is processed by cytocentrifuge, e.g. CSF,
ureteric urine, vitreous fluid, etc.

• Rotate the sample 1000 rounds per minute for 5 min.

• A thin layer of smear is formed on the glass slide.

• Fix the smears in 95% ethanol

There are two types of commercially available cytocentrifuge:

1. The cytocentrifuge removes the fluid during the time of sedimentation

2. The cytocentrifuge that retains the fluid

 Millipore Filtration

• The Millipore filtration technique is done for processing a large quantity of clear urine samples.

• Put the moistened Millipore filter paper with normal saline on the sieve.

• Attach the filter cup.

• Put the sample in the filter cup.

• Put on the suction process.

• The negative pressure at 100 mm mercury is created, and the fluid is drained into the bottle leaving
the cell on the filter paper

• Make multiple imprint smears on albumin-coated slides.

Cell Block

The cell block technique is mainly used for:

1. Immunocytochemistry

2. Cytochemistry

3. Preservation of archival tissue for future use

The cell block is made by the following steps:-

Collect the specimen in 10% neutral buffered formalin.

• Keep it 4 h in formalin to fix the cells.

• Centrifuge the sample at 1500 RPM for 10 min.

• Wash the sediment twice in PBS by centrifugation.

• Add 100 μl of plasma and 30 μl thrombin.

• Remove the clot and collect it on filter paper.

• Process the clot in the tissue processor as usual.

Liquid-based Preparation

• Liquid-based preparation may be used in urine specimen preparation.

• This technique provides better morphology and cell preservation along with
better cellularity.

• However, it may not be a cost-effective technique.

Normal cytology:-

• The normal voided urine sample shows scanty cells.

• The predominant cell population is transitional cells.

• However, the other cells may also be noted.

Transitional Cells:-

• The cells are usually present in discrete or in small loose clusters.

• Voided urine sample usually shows discrete superficial cells whereas, the
catheterized specimen shows clusters or papillary groups of cells containing
intermediate and parabasal cells.
NORMAL CELLS IN URINE
Superficial Cells (Umbrella Cells)

• Superficial cells are the largest epithelial cells in our body. They are also known as
umbrella cells as they protect the underneath cells from the toxic urine of bladder.

• These cells are about 100 micron in diameter.

• The cells are round to oval in shape with abundant eosinophilic finely granular
cytoplasm. The cytoplasmic margin of the cell is well-defined.

• The nucleus is round and central in position with very low nuclear-cytoplasmic (N/C)
ratio.

• The chromatin is granular with inconspicuous nucleoli.

• The umbrella cells are often bi or multinucleated and may show even 10–20 nuclei.
• At times, the superficial cells may show mild nuclear hyperchromasia and
pleomorphism.

• However, the cells maintain low N/C ratio and are usually easily recognizable from
neoplastic cells.

• Degenerated urothelial cells often contain large round eosinophilic cytoplasmic

inclusions that are known as Melamed-Wolinska bodies.

• These structures are seen more frequently in catheterized urine sample than voided
urine sample.
Intermediate Cells :-
• The basal/parabasal cells are small round to oval cells with well-defined cell
margin and scanty cytoplasm that often shows diffuse fine vacuoles.
• The nuclei are relatively larger with evenly distributed fine granular
chromatin.
• The intermediate cells are a bit larger compared to basal cells.
• The cells show basophilic cytoplasm with central round nuclei and
prominent nucleoli.
• Bi and multinucleated cells are infrequent as compared to superficial cells.
•
Reactive Transitional Cells:-

• Reactive transitional cells are commonly seen in bladder stone, viral or

bacterial cystitis, catheterization or other instrumentation and radiation or
drugs.

• The superficial transitional cells show nuclear enlargement, mild nuclear

pleomorphism, hyperchromasia and even coarse chromatin.

• Nuclear membrane may be thickened focally.

• There may be prominent nucleoli. Low-grade transitional cell carcinoma
(TCC) usually does not show prominent nucleoli.

• Murphy et al.2 have described such cells and emphasized to differentiate the
reactive transitional cells from high-grade TCC.

• The N/C ratio of the reactive transitional cells is usually not high compared to
high-grade TCC.

• The markedly coarse chromatin and hyperchromasia in TCC may also be

helpful cytological features to differentiate these two conditions
Squamous Cells:-

• Squamous cells are commonly seen in urine.

• Both superficial and intermediate types of squamous cells may be noted.

• The squamous cells are commonly exfoliated from squamous metaplasia of

trigone of the bladder.

• This is more frequent in females than males. In addition, the squamous cells
may come from the terminal part of urethra as this is lined by squamous
epithelium.

• The other sources of squamous cells are lithiasis, infections and inflammation
Columnar Cells

• The presence of columnar cells in voided urine is uncommon,

• However, these cells are frequently present in the catheterized urine sample.

• They are also present due to response to chronic irritation of the bladder.

• The other sources of columnar cells are cystitis cystica (reactive inflammatory change of the
bladder mucosa ), the lacunae of Morgagni(urethral lacunae) and the glands of Littre of the
urethra, seminal vesicles, prostatic epithelium, renal tubules and rarely from
endometrium in endometriosis.

• The cells are elongated, columnar in shape with moderate amount of cytoplasm and
basally placed nuclei. The cytoplasm of the cells may have small vacuoles.
(C) Many spermatozoa in urine
cytology smear

(D) Seminal vesicle cell in urine

cytology smear.
THANK YOU