The role of cytology in urinalysis of dogs and cats

Francesco Cian
Batt Laboratories/LABOKLIN
Coventry
United Kingdom

Urinalysis is an important part of the minimum database for routine health screen and is a crucial test to
asses and monitor renal function. It is recommended in the presence of clinical signs related to the urinary
tract (e.g. polydipsia, polyuria, pollakiuria, pain, discoloured urine, dysuria), to assess an animal with
systemic disease, and in the presence of mass lesions or other abnormalities discovered by palpation or
imaging in the urinary tract.

Routine urinalysis includes assessment of the physical properties of urine (e.g. colour, clarity, and USG),
dipstick (chemical) examination (e.g. glucose, bilirubin, urobilinogen, ketones, blood, pH, and protein), and
finally, microscopic examination of the urine sediment. The focus of this lecture is on the cytologic
examination of urine sediment for evidence of inflammation, infection, and/or neoplasia.

SAMPLE COLLECTION
Sample collection method should be known when analysing a urine sample because it can influence
dramatically the interpretation of results. There are three main sampling methods, which are indicated in the
table below, together with their advantages and disadvantages. Cystocentesis is overall the preferred
method of urine collection when evaluating the significance of cells or bacteria in the urine or obtaining urine
for culture.

FREE CATCH URINE by normal voiding or manual compression

Advantages
• No risks and pet owner can collect the sample
Disadvantages
•Samples are often contaminated and are not suitable for culture testing
•Manual compression may be traumatic
•Unable to localise the inflammation/infection, when present, which may involve any tract of the
genitourinary tract
CATHETERISATION
Advantages
• Bladder does not have to be distended to perform the sampling
Disadvantages
• Contamination of the sample may still occur, risk of iatrogenic infections
• Traumatic technique it may result in iatrogenic haematuria and release of excess of epithelial
cells
CYSTOCENTESIS
Advantages
• Less risk for iatrogenic infections and contaminants
• Advisable method when culture testing is required
• Allows better localisation of the inflammation/infection
Disadvantages
• Requires trained personnel and bladder to have sufficient amounts of urine in order to be
effective
• Possible risk for patients with severe coagulopathy, risk for microscopic haematuria
• Minimal risk of urine leak in the abdomen

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SAMPLE HANDLING AND PREPARATION FOR IN HOUSE EXAMINATION
For most laboratory testing, urine should be collected in a sterile, plain tube. EDTA sample may be
considered for cytologic evaluation of urine sediment and boric acid preserved urine transportation tubes for
culture purposes.

Once a sample is collected, urinalysis should be performed within the hour in order to avoid artifactual
changes as casts and cell deteriorate and there may be in vitro crystal formation or dissolution, depending
on type and urine pH; with time, bacterial overgrowth may also occur. Urine sample should also be protected
from sunlight to avoid spurious decrease in bilirubin. If the urine cannot be examined in house and will have
to be shipped to an external laboratory, the slide of the container should be rightly secured to avoid sample
evaporation and the sample should be refrigerated to slow the rate of artifactual changes. This means
storing the sample in the fridge and, when possible, shipping the sample with a cold pack. Urinalysis should
still be performed within 12-24 hours and urine should be mixed well and brought back to room temperature
before analysis.

MICROSCOPIC EXAMINATION OF URINE SEDIMENT

Accurate results depend on a correct microscopic examination of the sample but first of all on a proper and
standardised procedure of preparation of the urine sediment. The procedure is described below:
a) A standard amount of urine (e.g. 5ml) should be placed into a clean conic tube and centrifugated at
low speed (e.g. 1500rmp) for a set amounts of time (e.g. 5min)
b) After centrifugation, sediment may or may not be visible at the bottom of the tube. Most of the
supernatant (e.g. 4.5ml) should be removed. The fluid that remains should be remix with the
sediment by using a pipette or gently shaking the tube.
c) Using a disposable pipette, one drop of the sediment should then be transferred on a clean slide and
a coverslip should be placed on top of it.

Microscopic examination of unstained sediment should be performed for all urine samples. Supravital stains
are available but present some limitations. Since the urine sediment is unstained, it is important that the
condenser of the microscope is lowered in order to increase the contrast and better visualise cells and other
elements. Slide should be initially examined at low power (10x) in order to be able to quantify the material
present and its distribution. At this magnification, crystals are visible and often recognisable. Examination at
high power (40x) enables the examiner to better identify casts and crystals and to evaluate cell numbers and
morphology. Red blood cells (rbcs) and white blood cells (wbcs) should be counted by averaging the number
of elements in 10 fields. Epithelial cells, casts and crystals are reported in a semiquantitative manner (low,
moderate or high numbers). At high magnification, it is also possible to appreciate other elements (e.g.
sperm, contaminants) and infectious agents, in particular bacteria.

Urine sediment can also be stained with Romanowsky stains (e.g. Diff Quik, Wright-Giemsa). This is
mandatory when evaluating epithelial cells and when there is suspicion for neoplasia. It may also help to
confirm infection as identification of bacteria on unstained sediment preparations can be challenging at
times. A study from 459 urine samples collected by cystocentesis from 441 dogs, showed that modified
Wright-stained preparations had significantly higher sensitivity and specificity (Se: 93.2% Sp: 99%) than
unstained urines (Se: 82.4%, Sp:76.4%). Similar results were also found in a study on 472 urinary samples
collected from 410 cats by cystocentesis. The procedure to obtain stained urine smears requires to remove
the coverslip from the wet mount preparation, air dry the slide and then stain it with Romanowsky stains.
Alternatively, a pull/squash preparation from the remaining sediment can be prepared and regularly stained.
If there is a cytospin machine, a small amount of urine can be spun to produce a concentrated preparation;
this technique has the advantage that is preserves cell morphology.

TRANSITIONAL CELL CARCINOMA

Transitional cell carcinoma (TCC) also referred to as urothelial carcinoma (UC), is the most common form of
malignant neoplasm of the urinary tract in dogs, accounting for 50% to 75% of reported cases of canine
urinary bladder cancer. This neoplasm is also reported in the feline species; however, its incidence is
significantly lower. The aetiology is not completely understood but is likely to be multifactorial and prevalence
appears to be higher in certain canine breeds, in particular Scottish Terrier dogs, which have a 21-fold
increased risk. Dogs usually present with lower urinary tract signs (e.g. haematuria, stranguria, pollakiuria,

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dysuria), and many have concurrent urinary tract infections (UTIs) that may initially delay the diagnosis.
Resolving the infection may temporarily alleviate the clinical signs, and patients often present with a several-
month history of lower urinary tract signs.

Grossly, TCC usually presents as a solitary papillary, polypoid, or sessile mass. The most common location
in dogs is the trigone of the urinary bladder, but this neoplasm can also arise from the renal pelvis, ureters,
urethra, or prostate. In a necropsy study of 137 dogs with TCC, nodal and distant metastases were found in
42% and 58% of dogs, respectively. The lung was the most common site of distant metastases, followed by
other abdominal organs, bones (e.g. lumbar spine) and skin.

From a diagnostic point of view, urine sediment examination reveals neoplastic cells in a limited percentage
of cases and is unlikely to provide a definitive diagnosis. Direct fine-needle aspiration of the mass is the
technique providing the highest cellular samples for analysis, although the potential for seeding neoplastic
cells is a consideration. Therefore, traumatic catheterization (also called suction biopsy) is often preferred.
When cells have a typical transitional morphology, exhibit marked criteria of malignancy and there is no
evidence of inflammation or infection, a diagnosis of TCC can be easily achieved by cytology. This may not
be possible when neoplastic cells are well differentiated or in the presence of concurrent inflammation since
morphology of well differentiated neoplastic cells and hyperplastic/dysplastic transitional cells may overlap.
In those cases, further diagnostic investigations should be considered and may include histopathology
and/or BRAF mutation testing.

ANCILLARY TESTS FOR THE DIAGNOSIS OF TRANSITIONAL CELL CARCINOMA

Conventionally, biopsy and histopathologic evaluation are required to confirm a diagnosis of canine TCC and
are particularly important when a definitive cytological diagnosis is not possible. More recently, new non-
invasive techniques have been described and have shown very promising results. These include molecular
testing for BRAF mutation, which is a PCR based assay that searches for a single mutation in exon 15 of the
canine BRAF gene within transitional epithelial cells. This mutation has been identified in up to 87% of TCC
cases and to date this has never been recorded in urine specimens neither from healthy dogs nor in patients
with inflammatory or dysplastic processes affecting the genitourinary tract. This means that finding the BRAF
mutation is confirmatory for TCC but the lack of it does not entirely rule it out, since false negative results
may occur.

One of the main advantages of this test is that it can be performed on any diagnostic sample that contains
transitional epithelial cells, including pre-stained cytology smears with equivocal diagnosis, which means no
additional samples are required. Molecular testing for BRAF mutation may also be considered as a
screening test in free-catch urine samples of healthy dogs with an increased risk of developing TCC, likely
enabling identification of these cases at early and even preclinical stages of the disease. Once a dog has
been diagnosed as positive for BRAF mutation and shown to have TCC, repeat analysis may also be used
over time to monitor changing levels of the mutational load detected in free-catch urine during treatment.
BRAF mutation testing may also help to identify metastases in cases of ambiguous cytology or
histopathology and could possibly assist target therapy with BRAF inhibitors.

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