What can a urinalysis do for you in practice?
Karen Jackson BVSc, MANZCVSc, Dipl. ACVP
IDEXX Laboratories – Sydney
Unit 20, 38-46 South St, Rydalmere, NSW, 2116
A urinalysis is an essential component of baseline pathology data in veterinary
patients. At the most basic end of the urinary clinical pathology spectrum, the USG
alone is paramount in differentiating pre-renal from renal causes for azotaemia. At the
more advanced end of the urinary clinical pathology spectrum, researchers are
investigating the significance of certain urinary biomarkers which can help differentiate
glomerular from tubular renal disease and will hopefully allow for better prognostication
in patients affected by acute and chronic renal disease.
The urinalysis is essential in the diagnosis of urinary tract disease, inflammation, renal
tubular dysfunction, and glomerular disease. Non-renal disease (e.g. liver disease,
myopathies, diabetes, and haemolytic diseases) also causes significant diagnostic
abnormalities in the urinalysis. Urinalysis should always be assessed in combination
with the history, physical examination findings, and haematology and chemistry results
because alone it is relative insensitive and may not show significant abnormalities until
2/3-3/4 of the kidney is damaged.
The focus of this lecture will be on maximizing the diagnostic yield of urine in your
practice by discussing:
1. Specimen procurement and handling
2. Urinalysis
a. Physical properties
b. USG measurement
c. Chemical properties (i.e. dipstick use and pitfalls),
d. The importance of a sediment examination
3. The utility of a urine protein to creatinine ratio (UPC).
4. Urine cytology – when is this useful
Urine Collection and Handling
A. Method of Collection
1. Voided – ideally mid-stream to reduce the following:
Increased numbers of epithelial cells
Increased WBCs
Bacterial contamination from the distal urogenital tract
+/- Contamination from hair, faeces, litter, pollen, dust etc.
2. Catheterised
Increased numbers of epithelial cells
With trauma increased RBCs
Bacterial contamination from the distal urogenital tract
3. Cystocentesis
Ideal sample for culture and sensitivity
Most representative of bladder contents unless inadvertent
contamination due to aspiration of capillary RBCs/GI contents
B. Timing of Collection
1. Random
Most common sample type evaluated
Need to take into account: recent dietary/water intake, physical activity, total
time urine has resided in the bladder, and recent therapeutic interventions
2. First morning/8-hour = urine collected immediately upon awakening
Presumed that the animal has neither voided nor ingested food/water during
the previous 8 hours
Most concentrated – so best single urine sample assessment of renal
tubular concentrating ability. Also better concentration of cells, casts, and
bacteria.
Poor cellular morphology – prolonged contact with urine compared to other
samples
3. Fasting/second morning
Collected after a period of food deprivation and should not contain
metabolites from dietary ingestion
4. Timed/24h specimen:
24-h excretion studies are the most definitive method for determining
urinary excretion of a specific analyte (e.g. electrolyte/protein). As 24-h
studies are difficult to impossible to perform in veterinary practice, fractional
excretions are more often used (urine/plasma analyte compared to
urine/plasma creatinine).
C. Sample handling
Analysis within 60mins is preferred to avoid pH changes (alkalinisation of urine),
bacterial growth, cell lysis, cast fragmentation, and crystal formation.
If this cannot occur, refrigeration is required prior to and during transport to
reduce bacterial growth and aid in preservation of cellular and biochemical
constituents
Urinalysis
A. Physical properties
1. Colour
Yellow: normal
Red-red/brown: haematuria (check sediment), haemoglobinuria (check
serum/plasma colour and PCV), or myoglobinuria (check CK)
Brown-black: severe myoglobinuria, methaemoglobinuria
Yellow-brown/orange: bilirubin/highly concentrated
Green: drugs, bacteria
White: pyuria, crystalluria, mucin, epithelial cells, spermatozoa
NOTE: Highly coloured urine can alter interpretation of dipstick
measurements both by machines and human eyes.
2. Turbidity
Clear: normal in dogs and cats
Cloudy: cells, crystals, mucus, microorganisms, casts, sperm, lipid, contrast
media
B. Refractometry
Urine is approximately 95% water and 5% solutes. Specific gravity via
refractometer is the most common method of measuring urine solutes with urine
osmolality the less common but more accurate method that is rarely clinically
required or available. Urinary solute concentration is affected by hydration
status of the patient, the solutes being filtered, and underlying renal tubular
concentrating and diluting ability. Interpretation of the USG measurement is
therefore based strongly on history (i.e. is there a history of PU/PD?), hydration
status of the patient, concurrent medications, and knowledge of any azotaemia.
Note the USG will also influence interpretation of other chemical properties of
the urine (i.e. 2+ proteinuria is much more significant in urine with a USG 1.008
than urine with a USG of 1.055).
Causes of false elevations:
a. Protein – 1g/dL increases USG by ~0.003-0.005
b. Glucose – 1g/dL increases USG by ~0.004-0.005
c. Temperature – cold urine can falsely increase USG – always measure at
room temperature
C. Chemical properties (i.e. dipstick)
Urine pH, protein, haem protein, bilirubin, urobilinogen, glucose, and ketones
are generally considered the important measurements.
Leukocyte esterase, nitrites, and specific gravity are generally regarded as
unreliable as they were developed for human samples and have not shown the
same accuracy in veterinary medicine.
Important methodology to remember for accurate results:
1. Dipstick measurements, in general, are reliant on colour change by a
specified time therefore MUST TIME THE TEST.
2. Mixing of reagents can cause false reactions so either:
a. Remove excess urine after dipping in urine
b. Preferably place a single drop of urine on each pad
3. Reagents should be a room temp so warm urine to room temp
4. Reused sediment before applying to dipstick
5. Ensure strips are within expiry date and have been stored in an airtight
container.
Dipstick reactions
1. pH
Normal carnivore urine is acidic: 5.5-7.5
Alkaline urine: post-prandial, delayed analysis with bacteria in urine, urine
retention with bacteriuria, UTI, metabolic/respiratory alkalosis
Acidic: Metabolic/respiratory acidosis, fever
NOTE: pH affects crystalluria and urolith formation
2. Protein
Protein can be pre-glomerular (e.g. fever, hypertension,
hyperglobulinaemia, intravascular haemolysis), glomerular/tubular (renal
disease), or post-renal (i.e. UTI, voided sample). Most people use urine
protein to evaluate for renal disease so pre-glomerular and post-renal
causes should be RULED OUT before interpreting the USG and this
requires a sediment examination and assessment of the patient for
concurrent illness (e.g. fever, hypertension, intravascular haemolysis).
Note the dipstick measures predominantly ALBUMIN but can detect
globulins when in high concentrations.
A more accurate quantification of urinary protein that normalizes for urinary
concentration (as creatinine is produced in relatively stable amounts from
muscle breakdown) is the urine protein to creatinine ratio (UPC). IRIS
staging criteria indicate that a stable UPC >0.5 in a dog and >0.4 in a cat is
considered clinically significant proteinuria. With the range 0.2-0.5 and 0.20.4 considered borderline proteinuric in dogs and cats respectively.
Of renal proteinuria causes, glomerular losses cause higher values for the
UPC than tubular losses. This is because in health the proximal tubule
absorbs almost all the protein lost by the glomerulus; however, when the
glomerulus is injured and “leaky” the massive amount of filtered protein
exceeds the absorptive ability of the proximal tubule. Proteinuria due to
glomerular disease is predominantly albumin due to its small size, so is
easy to detect with the dipstick. Tubular proteinuria is often of a lower
magnitude as it reflects the inability of the proximal tubule to reabsorb only
the small amount of protein that the glomerulus usually filters +/- loss
directly through the proximal tubule. Most often, the proteinuria from renal
disease represents a combination of glomerular and tubular loss.
Interpretation of a positive dipstick protein is based on:
a. Collection technique
Voided/catheterised samples can have increased protein due to lower
urinary tract/reproductive tract contamination
b. USG
Low USG samples with protein on the dipstick are much more significant
than high USG samples
c. Cellularity (SEDIMENT required)
Haematuria and pyuria will increase protein in the urine due to postrenal disease (i.e. blood contamination, UTI) and so when the sediment
is active an elevated protein should not be interpreted as renal disease.
If there is no haematuria or pyuria but there are casts with increased
protein consider tubular disease
Alkaline pH can cause a false proteinuria on the dipstick. This should
always be checked with UPC.
d. Chronicity
Proteinuria is often transient when pre-glomerular and post-renal causes
are identified and this is why the IRIS staging system requires
proteinuria on 2 separate samples with negative sediment if the UPC is
<2.0 to confirm persistent proteinuria.
Chronic proteinuria with negative sediment suggests renal disease (i.e.
glomerular or tubular cause).
3. Haem protein (blood)
The occult blood test will react with the haem molecules found in
erythrocytes, haemoglobin, or myoglobin and for this reason a positive
result can be seen with haematuria, haemoglobinuria, and myoglobinuria. It
is termed occult because it the test is much more sensitive than visual
detection (50-100 x more sensitive).
Differentiation of whether haematuria, haemoglobinuria, or myoglobinuria is
the cause for pigmenturia is important due to the very different clinical
entities causing these abnormalities. Sediment examination can diagnose
haematuria as the centrifugation will clear the colour from the urine and the
sediment will show erythrocytes. If haematuria is ruled out, assessment of
serum/plasma colour will differentiate haemoglobinuria (serum/plasma is
pink/red) from myoglobinuria (serum is clear). Haemoglobin is scavenged
in the blood stream by binding with haptoglobin and so is too large to filter
through to glomerulus – leaving the plasma appearing pink/red.
Haemoglobinuria is only noted once this system has been overwhelmed.
Also note, correlation with other CBC/biochemical findings makes this
differentiation easier (i.e. PCV, blood smear evaluation, CK/AST
measurement).
Haematuria: urinary and extra-urinary (e.g. reproductive tract)
haemorrhage, iatrogenic (e.g. cystocentesis/catheterization) or pathologic
urinary tract haemorrhage (e.g. trauma, inflammation, neoplasia, inherited
and acquired primary and secondary haemostatic disorders, and idiopathic
disorders).
Haemoglobinuria: RBC lysis after haematuria (e.g. alkaline/dilute urine) and
intravascular haemolysis (e.g. IMHA, haemic parasites,
hypophosphataemia, inherited enzyme deficiencies).
Myoglobinuria: muscle damage/necrosis (e.g. exertional rhabdomyolysis,
snake bite, ischaemic myopathy). Myoglobin can cause renal tubular injury
so accurate detection is essential.
False positives: oxidizing agents (hydrogen peroxide, hypochlorite), marked
bilirubinuria, large amounts of bromide/iodide, bacterial/leucocyte
peroxidases
4. Bilirubin
Bilirubin is a breakdown product of haemoglobin metabolism. Indirect or
unconjugated bilirubin is bound to albumin and therefore is not filtered
through a healthy glomerulus and even if the glomerulus is damaged the
dipstick is relatively insensitive to unconjugated bilirubin, so may not be
detected even if present. Once conjugated to glucuronic acid (by the liver) it
becomes water-soluble and unbound, so is now freely filtered. Bilirubin
tubular resorption varies depends on the species and in most species there
is no bilirubin in the urine as most/all is resorbed in health; however, the
canine renal tubules do not reabsorb much filtered bilirubin, so small
amounts (trace – 1+) can be seen in concentrated canine urine in health.
Even trace bilirubin is considered significant in the cat and as the dipstick is
very sensitive for conjugated bilirubin, occasionally bilirubinuria can be
detected before hyperbilirubinaemia.
Bilirubinuria can be seen with: haemolytic crises (with time for haemoglobin
breakdown), hepatic cholestasis, and post-hepatic cholestasis.
False positives: etodolac (NSAID), deeply pigmented urine, indican
(intestinal bacterial metabolite), haemoglobinaemia (tubular formation of
bilirubin in the dog)
5. Urobilinogen
Conjugated bilirubin is delivered to the intestine where intestinal bacteria
convert it to urobilinogen for excretion. Small amounts of this urobilinogen
is reabsorbed into the portal blood and removed by the liver or excreted
through the kidney. It can be produced in high quantities during haemolytic
crises in dogs and theoretically in patients with hepatic dysfunction;
however, most investigators agree that interpretation of this analyte in the
urine of veterinary species is unreliable.
6. Glucose
Glucosuria in the vast majority of patients is caused by hyperglycaemia;
however, as filtered glucose is absorbed by both active and facilitated
processes in the proximal renal tubule, renal tubular dysfunction (e.g. acute
renal tubular injury, Fanconi- syndrome) can be a rare cause of glucosuria
and is diagnosed by noting a concurrent euglycaemia. To cause glucosuria
with adequate renal tubular function, the hyperglycaemia must exceed the
renal tubular threshold, which differs depending on the species:
Dogs: 10-12mmol/L
Cats: 15.5-16mmol/L
Glucosuria usually results in polyuria through osmotic diuresis. This is
either noticed alone or in combination with polydipsia.
False positives: Hydrogen peroxide, hypochlorite (i.e. oxidizing cleaning
products) – so beware glucose positive samples collected from
benches/floors.
7. Ketones
Three ketones are produced when energy is required from fat metabolism
(i.e. usually when carbohydrate metabolism has been exhausted e.g.
diabetes mellitus, starvation): acetone, acetoacetate, and betahydroxybutyrate (BOHB). The dipstick can ONLY detect acetone and
acetoacetate and preferentially detects acetoacetate. All 3 ketones are
readily filtered by the glomerulus with acetone the only ketone able to be
reabsorbed. Ketonuria can therefore be seen with diabetic ketosis,
starvation, and less commonly with renal tubular dysfunction. Note that
beta-hydroxybutyrate is the most commonly generated ketone in diabetic
ketosis and as the dipstick cannot detect this ketone, ketosis can be missed
in diabetic patients without blood measurement of beta-hydroxybutyrate.
D. Sediment analysis
This is the final step in the complete urinalysis. It requires centrifugation,
removal of the supernatant, and examination of the remaining sediment under
the microscope. For standardization the amount of urine used should be kept
consistent with at least 5ml recommended and 10ml considered gold standard.
No matter the volume used, this should be recorded. Also centrifugation speed
should be kept standard with 5mins centrifugation at 400 relative centrifugal
force (RCF) recommended to concentrate formed elements in urine. Most
centrifuges work on revolutions per minute (RPM) with the conversion below
used to determine the RPM on your centrifuge:
RPM = (square root [400/1.12R]) x 1000
(R=radius of centrifuge rotor in mm)
Removal of the supernatant should be with pipette, leaving 0.5-1.0mL for
sediment examination. Gentle resuspension is recommended. A small drop of
this well-mixed sediment is placed on a slide with a coverslip (20uL if a pipette
is available). Excessive sediment will result in larger formed elements (e.g.
casts) flowing off the edges of the coverslip. Whether to stain or not is a
personal preference.
Unstained urine should be examined under lower light with the condenser
lowered (to increase contrast and make visualization of the cellular elements of
urine easier). Urine sediment should be examined at 10x (low power field) and
40x (high power field). With casts and crystals enumerated at low power and
the remainder (i.e. rbcs, wbcs, epithelial cells, bacteria) enumerated at high
power.
Normal urine should have <5rbcs/hpf, <5wbcs/hpf, few epithelial cells
(transitional from bladder/urethra, squamous from distal urethra and vagina),
and occasional hyaline/granular casts, crystals, and fat droplets (particularly
cats). Note the concentration of formed elements should always be interpreted
in light of the USG, as with the dipstick analysis.
The sediment examination helps you to interpret the significance of the
chemical constituents. For example, if a sample has 3+ proteinuria with 50-100
wbcs/hpf, this is likely post-renal proteinuria from UTI/urinary tract inflammation
whereas if the sediment is negative, consider UPC to quantify and if persistent
proteinuria identified this is likely renal in origin.
Haematuria (>5rbcs/hpf): Haemorrhage from kidneys to bladder (cystocentesis)
or urethra/genital tract (voided). Haematuria, if not procedural, can be
secondary to trauma, inflammation, necrosis, neoplasia, or inherited/acquired
primary/secondary coagulation defects.
Pyuria (>5wbcs/hpf): Inflammation from kidneys to bladder (cystocentesis) or
urethra/genital tract (voided). Most often pyuria is secondary to bacterial
infection; however, it can also be seen with neoplasia and urolithiasis.
Epithelial cells: Their presence is rarely a diagnostic finding; however, if there
are large numbers of epithelial cells found in a sediment from a patient
suspected to have transitional cell carcinoma, consider cytology to further
characterize their morphology. Other causes of increased numbers of epithelial
cells include samples collected via catheterisation, urine from patients with
urolithiasis or chronic inflammation.
Casts: Cylindrical structures composed of mucoproteins and associated cellular
material that forms within renal tubules (so forming the parallel wall shape). Up
to 2 hyaline/granular casts per low power field are considered normal with
increased numbers suggesting renal tubular disease. Shedding is intermittent
so may be missed on single sediment exams.
Bacteria: Bacteria must be present in large numbers in urine before they can be
detected microscopically. As the distal urethra and vagina normally contain
bacteria, only bacteria from a cystocentesis sample can definitively be used to
diagnose a UTI.
Crystals: Formation of crystals is dependent on the temperature, pH, and
concentration of solutes in the urine and is often not a pathologic finding. If
there is a concurrent urolith present, the type of crystals present on UA are not
always indicative of the composition of the urolith with X-ray crystallography
recommended to determine the cause for the urolith formation (i.e. the nidus of
the stone). Significant crystals to note on UAs: tyrosine, leucine, and
ammonium biurate (hepatic dysfunction); cystine (metabolic defect); bilirubin
(associated with bilirubinuria). Crystals form at low temperatures so making a
sediment before refrigeration or letting the urine warm to room temperature
before making the sediment can help to evaluate for clinically significant crystal
formation.
References
Sink, C; Weinstein, N (2012) Practical Veterinary Urinalysis. Ames: Wiley-Blackwell.
Stockham, S; Scott, M (2008) Fundamentals of Veterinary Clinical Pathology 2nd ed.
Ames: Wiley-Blackwell.