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Journal of Feline Medicine and Surgery (2016) 18, 373–385

CLINICAL review

Practical urinalysis in the cat

2: Urine microscopic
examination ‘tips and traps’
George Reppas and Susan F Foster

INTRODUCTION Series outline: This is the second

article in a two-part series on urinalysis
Urine microscopic analysis is usually performed to aid in the diagnosis in the cat. The specific focus is urine
and monitoring of diseases of the urinary tract. Common clinical indi- microscopic examination. Part 1,
cations for this procedure in feline practice were outlined in Part 1.1 which appeared in the March 2016
Microscopic examination of urine involves the enumeration and issue, discussed urine macroscopic
identification of urine insoluble particles. The latter should be relative- examination.
ly straightforward for the experienced microscopist and can be Practical relevance: Urinalysis is an essential
performed on wet preparations, air-dried smears of urine or, ideally, a procedure in feline medicine but often
combination of the two. Enumeration of urine insoluble particles is little attention is paid to optimising the data yielded
more complex and, in veterinary medicine, this aspect of the urinalysis or minimising factors that can affect the results.
has often been problematic. Consequently results of urine sediment Clinical challenges: For the best results,
particle enumeration are poorly reproducible unless attention appropriately collected urine should be prepared
is directed towards using promptly by specialist laboratory personnel for
a standardised technique that the relevant tests and assessed by a clinical
Microscopic examination of
enables a more critical assess- pathologist. This is invariably impractical in clinical
urine involves the enumeration ment of the semiquantitative settings but careful attention can minimise artefacts
findings.2–5 and allow maximum useful information to be
and identification of in human medicine, urine obtained from this seemingly simple process.
urine insoluble particles. insoluble particle enumera- Audience: Clinical pathologists would be familiar
tion has been simplified with with the information provided in this article,
the use of automated urine but it is rarely available to general or specialist
cell counters. Such machines practitioners, and both groups can potentially
are currently being validated for use in veterinary medicine as well. benefit.
Factors that may affect urine insoluble particle enumeration and Equipment: Most of the required equipment
microscopic identification are outlined in the box on page 374. is routinely available to veterinarians. However,
instructions have been provided to give practical
URINE SAMPLE COLLECTION AND alternatives for specialist procedures in some
PREPARATION TECHNIQUES instances.
Evidence base: The evidence base for feline
Fresh urine (<60 mins post-collection) is the ideal sample for micro- microscopic urinalysis is quite poor and information
scopic examination, as the constituents of urine are not stable. Casts has largely been extrapolated from the human
and cells deteriorate rapidly, crystals may dissolve or form, and bac- literature. Information from feline studies has been
teria may die or proliferate. The method of urine collection will also included where available. In addition, practical
influence the cellular composition of the sample: a voided sample may clinicopathological and clinical observations
contain cells and bacteria from the genital tract; a cystocentesis sample are provided.
may have increased numbers of red blood cells (RBCs); and catheteri-
sation may result in increased numbers of urothelial cells.1,5

George Reppas
BVSc DipVetPath FANZCVS Dipl ECVP*
Susan F Foster
BVSc MVetClinStud FANZCVS
Vetnostics, 60 Waterloo Road,
North Ryde, NSW 2113, Australia
*Corresponding author:
george.reppas@vetnostics.com.au

doi: 10.1177/1098612X16643249
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R E V I E W / Practical urinalysis – microscopic examination

Fa c t o r s t h a t m ay a f fe c t m i c ro s c o p i c ex a m i n a t i o n
of urine and insoluble particle enumeration
< Volume of urine formed
< Method of urine collection
< Method of urine preservation
< Sample contamination
< Varying volumes of urine submitted to the laboratory for
testing
< Failure to thoroughly mix urine samples before centrifugation
and also before resuspension of the urine sediment
< Failure to use a constant volume of centrifuged urine for each
sample examination
< Failure to standardise test tubes and transfer pipettes
< Failure to centrifuge urine at standardised relative centrifugal
force (thus affecting the sedimentation force)
< Allowing urine sediment to dry on the microscope slide
< Inadequate resuspension or reconstitution of sediment after
centrifugation
< Size of drop of urine sediment solution transferred to
microscope slide
< Use of dirty or scratched microscope slides or coverslips
< Failure to use a standardised weight and size of coverslip,
resulting in urine films of varying thickness
< Use of low power or high power magnification for
microscopic examination
< Too much bright light during microscopic examination
of urine sediment
< Failure to standardise the number of microscope fields
examined to count urine insoluble particles
< Counting discrepancies due to the diameter of microscope
ocular lenses (25 mm vs 20 mm ocular lenses)
< Failure to consider the effect of dilution, as a result of
addition of stains to the sediment, on the number of urine
insoluble particles detected
< Failure to use calibrated disposable urine analysis chambers
for counting urine insoluble particles
< Lysis of some urine insoluble particles (eg, cells, crystals,
casts) due to prolonged storage and/or changes in pH or
urine specific gravity (eg, RBCs may lyse in hyposthenuric
urine and crystals may dissolve/precipitate as pH changes)
< Ability to recognise and differentiate between various urine
insoluble particle structures

Information based on reference 2

Traditionally, urine insoluble particle enu-

meration and identification in cats has been
Urine preservatives

based on the microscopic examination of wet

To preserve casts and other urine insoluble particles for wet-prep examination,

preparations comprising resuspended urine

chemical preservatives such as neutral buffered formalin can be added to the

sediments following centrifugation of a con-

urine. The addition of chemical preservatives should be noted on the urine

sistent volume of urine (typically 3–5 ml,

container’s label, as formalin-treated urine precludes dipstick analysis,

depending on the individual laboratory,

microbial culture and air-dried smear examination.1,3

although lesser volumes of urine may be

adequate). With some modification, this tech-
nique may also be used to prepare air-dried
tube. Standardising the volume of remaining

smears for subsequent staining and cytologi-

supernatant in which to resuspend the

cal examination.
A standardised protocol for preparation of
urine sediment samples for microscopic
examination using conventional centrifuga-
tion (wet preparations or air-dried smears) is
described below.3,5,6–9
Standardised protocol for
preparing urine sediment samples
< Thoroughly, but gently, mix the urine
sample before removing and placing an
aliquot (ideally 3–5 ml) into a conical
centrifuge tube.
< Centrifuge the urine aliquot at
approximately 1000–1500 revolutions
per minute (rpm) (or 450 x g) for 3–5 mins
(note that a longer duration or higher rpm
may damage casts and cells).
< Decant most of the supernatant, leaving
approximately 10% of the original aliquot
volume above the sediment pellet that has
deposited at the bottom of the centrifuge
sediment (ie, for 10% of an original 5 ml urine
aliquot, removing 4.5 ml of supernatant after
centrifugation) aids interpretation of results
and comparisons between urine samples
collected from the same patient at different
time points.9
< Gently resuspend the sediment pellet
in the remaining urine supernatant.
The microscopist’s level of expertise in
examining urine wet preparations for the pur-
poses of insoluble particle identification and
enumeration will dictate whether staining
will be used during this process (see box on
page 375). Note, however, that semiquantita-
tive results should always be determined
using an unstained wet preparation, to avoid
the dilutional effects of adding stain. Also,
any crystals or microorganisms identified in a
stained wet preparation should be confirmed
on an unstained wet preparation.9
Fresh urine (<60 mins post-collection)
is the ideal sample for microscopic examination.

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R E V I E W / Practical urinalysis – microscopic examination

When to stain the wet preparation for microscopic examination

< Experienced microscopists do not often require stained wet preparations of urine samples for microscopic examination.
Place a drop of resuspended urine sediment solution on a clean glass slide and apply a coverslip. In veterinary laboratories,
phase contrast microscopy is used to examine unstained urine.
< Less experienced microscopists will be aided by stained wet
preparations of urine samples for microscopic examination (but see
page 374 for exceptions). Place two drops of resuspended urine sample
at one end of a clean glass slide and apply a coverslip. At the other end
of the glass slide place one drop of urine (Figure 1), add a drop of either
Sternheimer-Malbin stain (Sedi-Stain; Becton Dickinson) or 0.5% new
methylene blue (NMB), and apply a coverslip to the mixture.9 The use
of water soluble supravital stains like Sedi-Stain or NMB may help Figure 1 Wet preparation of a urine sample. Two drops of
differentiate white blood cells (WBCs) from epithelial cells until sufficient unstained urine and a drop of urine to which a drop of new
methylene blue stain has been added are placed side by side
experience has been gained to proceed without the use of staining on the same glass microscope slide, with a coverslip applied
during wet-prep examination of urine (Figure 2). to each

PERFORMING AND ocular lens, then only 10 WBCs/HPF would

INTERPRETING THE
MICROSCOPIC ANALYSIS
Enumeration of urine insoluble
particles
insoluble particles in urine may be counted
using conventional (semiquantitative) meth-
ods employed during routine wet-prep
microscopic examination or using counting
chamber methods.
Conventional method
The conventional method of
counting urine insoluble parti-
cles in urine sediment prepa-
rations involves quantifying
the number of structures
seen per microscopic field
(expressed as the number of
insoluble particles per low
power field [LPF] or high
power field [HPF] when
viewed under a x 10 or x 40
microscope objective, respec-
tively).
This semiquantitative analy-
sis may be problematic,
especially as the width of the
viewed microscopic ocular
field varies between micro-
scopes. Some microscopes use
25 mm ocular lenses, while
older microscopes tend to
have 20 mm ocular lenses.
The area viewed through
the former is approximately
50% larger than the area
viewed through the latter.
Consequently, if 15 WBCs/
HPF were seen with a 25 mm
Figure 2 Wet-prep
examination. (a) Unstained
urine sample with numerous
red blood cells and scant
white blood cells (arrows).
(b) Urine sample to which a
drop of new methylene blue
stain has been added,
highlighting the neutrophils
a cellular detail. At low power
b insoluble particles.3,8
be seen through a 20 mm ocular lens on the
same sample.5
A coverslip is essential, particularly when
examining a specimen under a x 40 micro-
scope objective; not only to provide optimal
microscopic resolution, but to avoid inadver-
tently placing the tip of the x 40 objective
nosepiece in the specimen. The visibility of
urine insoluble particles can be improved by
lowering the microscope condenser or closing
the substage iris diaphragm to increase the
contrast. Focusing up and down on the slide,
while systematically scanning
it, enhances the examination of
magnification (x 10), the edges
of the coverslip need to be
checked for casts (and other
heavier urine insoluble parti-
cles) that may have floated
to the periphery of the field.
At high power magnification
(x 40), RBCs, WBCs and epithe-
lial cells need to be counted,
in addition to identifying the
type of casts, small crystals,
bacteria, sperm, lipid droplets,
yeasts, parasitic ova and other
With the conventional
method, the centrifugation
step and removal of super-
natant are important for
concentrating urine insoluble
particles in the specimen being
examined, but are also a major
source of error, especially
when urine volumes available
for testing vary. Consequently
there is no precise reproducible
information regarding the

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R E V I E W / Practical urinalysis – microscopic examination

Counting chamber method

Table 1 Microscopic urine sediment examination findings Enumeration of particles in urine is more
in cats utilising conventional measuring techniques
accurately performed using a counting
Insoluble urine Normal chamber method on an uncentrifuged urine
particles findings* Action/considerations sample. In human laboratories, this is also the
Red blood cells 0–5/HPF < Interpret in the light of urine collection method, reference measurement method used to mon-
(RBCs) as manual expression, catheterisation and itor the performance of automated urine cell
cystocentesis may all induce microscopic
haematuria
analysers. Quantitative estimations are based
< Consider genital tract disease conditions as on direct counting and the results are
possible sources of RBCs in voided feline urine expressed as particles per litre (rather than per
samples low or high power field).12,13
< Female cats postpartum may have an increased
RBC component contaminating the urine Some veterinary laboratories have adopted
< Centrifugation may result in artefactually the routine use of disposable plastic counting
decreased numbers due to rupture of some RBCs chambers with counting grids (Vetriplast or
White blood 0–5/HPF < Consider genital tract disease conditions as KOVA Glasstic Slide 10) to facilitate the
cells (WBCs) possible sources of WBCs in voided feline urine enumeration of insoluble particles in uncen-
samples trifuged urine samples (Figures 3–5).9
< When the origin of pyuria is questionable,
cystocentesis may help in localisation Counting techniques and calculations
< WBC numbers may be underestimated in wet
preparations if the cells are clumped
< Centrifugation may result in artefactually
decreased numbers due to rupture of some WBCs
Epithelial cells Variable/HPF < Depends on the urine collection method.
A few epithelial cells may normally be seen in
cystocentesis samples, while increased numbers
may be present in catheterised or voided samples
< Any epithelial cells displaying cytological criteria of
malignancy are significant (although this is better
assessed on air-dried stained smears)
Crystals Variable/LPF < See Table 4
Casts 0 to few/LPF < <2/LPF hyaline or fine granular or fatty casts may
be found in normal feline urine
< Any cellular cast (RBC, WBC, epithelial) should be
regarded as abnormal; the presence of RBCs or
WBCs in casts helps localise their source to at
least the level of the renal tubules
< Waxy, haemoglobin/myoglobin or bilirubin casts
are not found in normal feline urine
Figure 3 Disposable plastic
Spermatozoa Variable/LPF < During microscopic examination of urine from counting chambers
entire males, qualitatively assess as none, few, (KOVA Glasstic Slide 10)
with counting grids to
moderate or many facilitate the enumeration
Microorganisms None < Bacteria and fungi should not be present in of urine insoluble particles
in uncentrifuged urine
normal feline urine collected by cystocentesis
< Voided urine samples are often contaminated
samples

with bacteria and/or fungi

Lipid droplets Variable/LPF < Common in cats
< Need to be distinguished from RBCs a
< Variably sized, refractile and often float above the
focal plane
Parasites None < During microscopic examination, qualitatively
assess as none, few, moderate or many
Information based on references 2–11
*Expressed as an average across several microscopic fields
HPF = high power field; LPF = low power field

Figure 4 (a)
Vetriplast slide
chambers and
upper limit of normal for many insoluble (b) appearance of
the Vetriplast slide
particles found in urine sediment using chamber grid under
conventional counting techniques. Typical a x 2 objective lens
semiquantitative results (based on centrifuga-
tion of 5 ml aliquots of urine, with a drop of
resuspended urine sediment solution placed
on a clean glass slide, and a coverslip applied) b
are listed in Table 1.2

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R E V I E W / Practical urinalysis – microscopic examination

a b

Figure 5 Vetriplast slide chamber grid lines viewed under

the microscope using (a) x 20 and (b) x 40 objective lenses.
Struvite crystals (circled) and white blood cells (arrows) are
evident in (a). Struvite crystals (circled) and a transitional
epithelial cell (arrow) can be seen in (b)

required for the use of such plastic counting
chambers are included in the product insert
literature and are beyond the scope of this
article.
Identification of urine insoluble
particles in wet preparations
insoluble particles within feline urine include
Closer
epithelial cells seen per HPF) on wet-prep
cytological
examination should prompt closer cytological assessment
assessment of the epithelial cell component.
For this purpose, a stained air-dried urine of the
smear is often required to help differentiate
between malignant, dysplastic and hyperplas-
epithelial cell
tic epithelial cells (the latter two cell types component
being common in urine from animals with
cystitis and reactive hyperplasia). often requires

Erythrocytes (RBCs)
cells (epithelial cells, RBCs, WBCs and neo- a stained
plastic cells), casts, microorganisms, crystals,
lipid droplets, spermatozoa (gender specific), Erythrocytes tend to be the smallest cellular air-dried
mucin and artefacts. Some insoluble particles constituents in urine when they are present
are more difficult to identify in wet prepara- urine smear.
tions of urine than in air-dried blood or
cytology smears, as these particles may be
subjected to varying periods of exposure to
osmotic and pH changes as well as bacterial 3–
Ty p e s o f e p i t h e l i a l c e l l s
toxins, with resultant changes in their size,
structure and transparency.2 Renal epithelial cells moderately high nucleus to
< Derived from the renal tubules cytoplasm ratio
< Small round cells (in suspension), < Typically granular cytoplasm
Epithelial cells
Cells
usually degenerate, are < Increased numbers may be
Epithelial cells can be found in low numbers indistinguishable from leukocytes present with inflammatory
in the urine sediment of healthy cats because or small transitional cells in disorders and neoplasia
they are constantly exfoliating into the urinary unstained wet smears (especially transitional cell
tract lumen as they are replaced by new cells. < When stained, nucleus is round carcinoma [TCC]); and may also
Accurate data regarding the number of and centrally positioned be associated with traumatic
epithelial cells normally present in the urine < Cytoplasm is granular and may catheterisation technique
of cats is not available.3 contain a few vacuoles
Three main types of epithelial cells may be < Only reliable as indicators of Squamous epithelial cells
found in urine, depending on their origin renal disease when incorporated < Line the distal urethra, vagina,
along the urinary tract – renal, transitional into casts vulva or prepuce
and squamous (see box). Some laboratories < Largest cells in the urine
report these epithelial cell populations sepa- Transitional epithelial cells < Large flat cells with a single
rately. However, this is often problematic on (urothelial cells) nucleus and abundant cytoplasm
wet preparations, particularly as urine sam- < Line the urinary tract from the < Most often found in voided
ples sent to the laboratory are often not fresh. renal pelvis to approximately samples or with catheterisation
Consequently, all epithelial cell types may be the distal portion of the urethra < Usually of little or no pathological
counted under the one general epithelial cell < Medium-sized cells with a significance
category. A high total epithelial cell count
Information based on references 3–5 and 8
(usually enumerated as a range or mean of

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R E V I E W / Practical urinalysis – microscopic examination
(Figure 2). Their appearance may be affected
by urine specific gravity (USG) and urine
pH:2,3
< if USG is 1.010–1.020, RBCs appear small
(approximately half the size of WBCs),
round, uniform in size, moderately refractile,
and pale yellow to orange in colour.
< if urine is concentrated, RBCs can become
crenated and appear granular.
< in hypotonic (especially if USG <1.006) or
alkaline urine, RBCs swell, appear as balloons
(with smooth edges and pale yellow
cytoplasm) or colourless rings (ghost or
shadow cells), or completely lyse. osmotic lysis
of erythrocytes can be complete within 2 h.
Leukocytes (WBCs)
Leukocytes in fresh urine generally appear as
round cells with granular cytoplasm (Figure
2), intermediate in size between RBCs and
transitional epithelial cells. However, their
appearance may be affected by a number of
factors:2,3
< Nuclei may or may not be discernible
using reduced illumination, bright-field
microscopy but can be readily demonstrated
in stained smears.
< Crenation or swelling depends on USG
and urine pH.
< if kept at room temperature for an hour
or longer, leukocytes may appear degenerate
with foamy cytoplasm and mild karyorrhexis,
pyknosis or karyolysis.
< Up to 50% of WBCs may lyse within an hour
at room temperature in alkaline dilute urine.
< WBC numbers may be underestimated in
smears if the WBCs are clumped.
Neoplastic cells
Neoplastic cells are rarely detectable on
routine wet-prep urine sediment examination.
Swelling and degeneration of cells can mimic
Significance of casts in feline urine (cylindruria)
< Increased numbers of casts in urine
indicate pathology at the level of the
renal tubules or collecting ducts
< < Leukocyte casts reflect
Cast numbers do not reliably
correlate with the degree of
pathology at the level of the renal
tubules or collecting ducts
<
< Occasional hyaline and granular casts
Hyaline casts in increased numbers
may be seen in animals with
< No cellular casts should be observed in
glomerular proteinuria
< Epithelial casts suggest active
tubular degeneration or necrosis
(eg, gentamicin induced)
< Granular casts result from cellular degeneration
and increased numbers reflect tubular degeneration,
Information based on references 3 and 5
378 JFMS CLINICAL PRACTICE
malignancy, particularly when fresh urine
samples have not been used. if exclusion of
Examination neoplasia in the urinary tract is required, a
wet-prep sediment or, preferably, several air-
of freshly dried smears must be prepared for examina-
tion from freshly formed urine (ie, not the first
formed urine morning sample) immediately after centrifu-
is required for gation. Air-dried smears can be stained with a
rapid Romanowsky stain and assessed more
exclusion of closely for the presence of cytological criteria
of malignancy (see page 383).
neoplasia in
difficulties may be encountered when
the urinary trying to differentiate hyperplastic, dysplastic
and neoplastic changes within epithelial cells
tract. – particularly in the presence of significant
inflammation and/or infection.2,3,5 in these
instances, it would be prudent to refer the
slides to an experienced veterinary
cytopathologist for examination.
Casts
Casts are elongate, parallel-walled structures
that form in the acidic and concentrated lumi-
nal environment of the ascending limb of the
loop of Henle, distal renal tubules and collect-
ing ducts. They are cylindrical moulds of
the renal tubules formed on a matrix of
Tamm–Horsfall mucoproteins secreted by the
epithelial cells lining these tubules and ducts.
RBCs, WBCs, epithelial cells, haemoglobin,
myoglobin, lipid or bilirubin may be incorpo-
rated during their formation.2,3
ideally, cast examination should be per-
formed on unpreserved urine immediately
after collection. if chemicals (eg, formalin) are
used to preserve cast morphology, this should
Cast examination should ideally be undertaken
on unpreserved urine immediately after collection.
Supravital stains may assist identification.
necrosis and/or inflammation
< Erythrocyte casts indicate
Of particular note:
< Fatty casts are the most common type
glomerular or tubular haemorrhage
of casts in cats due to the physiological
storage of lipid in feline tubular epithelial
inflammation involving renal tubules
cells; increased numbers are seen in
(eg, pyelonephritis)
< Waxy casts suggest chronic
cases of nephrotic syndrome and
diabetes mellitus
renal pathology and intrarenal
stasis
<
per low power field are considered normal
Haemoglobin and myoglobin
sediment from normal cat urine
casts may be seen with
haemoglobinuria and
myoglobinuria, respectively
< Large numbers of casts indicate the presence of active
renal disease/damage
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R E V I E W / Practical urinalysis – microscopic examination

be noted on the laboratory submission form.

Table 2 Factors that may affect detection of bacteria
Use of supravital stains may aid in the correct
during microscopic examination of urine sediment
identification of casts, which can be difficult in preparations
unstained smears.2,3
Several types of casts may be identified False positives – causes False negatives – causes
on routine wet-prep urine sediment examina- Sample contamination during collection, Small numbers of bacteria
centrifugation or staining
tion. These structures are classified according Recent antibiotic therapy
to their appearance, which reflects their Allowing urine to stand at room temperature
content:2,3,5 for longer than 2 h Diuresis/dilute sample
< Hyaline casts are primarily composed
Misidentification of bacteria in urine sediment Intermittent shedding of bacteria
of Tamm–Horsfall mucoproteins. They are
difficult to see, dissolve rapidly in dilute or Information based on references 2, 3 and 10
alkaline urine and are commonly found with
renal diseases associated with proteinuria.
< Cellular casts include WBC, RBC and
a
sediment. Single cocci
epithelial cell casts. The presence of WBC are difficult to detect.
casts should prompt urine culture. it can be Coliform rods may form
difficult to distinguish WBC casts from chains, which need to be
epithelial cell casts. However, epithelial cell distinguished from fun-
casts contain highly refractile tubular gal hyphae (air-dried
epithelial cells that have not yet smears allow differentia-
disintegrated. A popular but unproven tion). Stained wet prepa-
hypothesis is that renal tubular epithelial cell, rations have been shown
fatty, granular and waxy casts represent to have an unacceptably
different stages of degeneration of epithelial high false-positive rate
cells in casts. for bacteria.10
< Fatty casts contain many small, round,
b
Examination of modi-
highly refractile lipid droplets. fied Wright-stained (or
< Granular casts are produced once cells other rapid Romanow-
degenerate; casts may take on a coarse sky stained) air-dried
granular appearance or, with more smears of urinary sedi-
degeneration, a fine granular appearance. ment at high magnifica-
< Waxy casts represent the final stage of tion (high dry or oil
degeneration of granular casts and are immersion, Figure 6)
relatively stable. significantly improves
< Pigmented casts include haemoglobin the sensitivity, specifici-
(yellow–brown) and myoglobin (red–brown) ty and efficiency of
casts. Figure 6 (a,b) Air-dried microscopic detection
cytospin smear of urine from
a cat with an Escherichia and classification of bacteriuria compared

Bacteria
Microorganisms
detection of bacteria in the urine (bacteriuria)
depends on the skill and experience of the
observer and the numbers present. More than
104 bacterial rods/ml or 105 bacterial cocci/ml
need to be present before they can be readily
detected in the unstained sediment.2
Bacteriuria is not a finding in healthy cats.14
it was also thought to be rare in idiopathic
feline lower urinary tract disease, although
two studies have challenged this notion –
with bacteriuria detected in 22% of
catheterised samples in one15 and 23% of cysto-
centesis samples in the other.16 Bacterial
urinary tract infections are more common in
older cats. Pyuria should not be a criterion
for determining the presence or absence of
bacteriuria.10
if examining wet preparations of urine sedi-
ment, this should be performed under low
light intensity to increase contrast. Bacteria
usually refract light, and bob and quiver in the
coli urinary tract infection. with the wet-unstained method.10
Note the short bacterial
rods, located both intra- and Factors that may affect the detection of bac-
extracellularly, accompanied teria during microscopic examination of urine
by numerous degenerating
neutrophils. Urine was sediment preparations are outlined in Table 2.
obtained by cystocentesis Factors that may result in poor correlation
and the smear was prepared
using a cytocentrifuge and between microscopy and urine culture results
stained with Diff Quik are listed in Table 3.
Table 3 Factors that may affect culture of samples in which
bacteriuria is correctly identified or ruled out on
microscopy
Negative culture with bacteriuria Positive culture with no bacteriuria
Non-viable bacteria in urine at time of collection Small numbers of bacteria
fail to be cultured due to antimicrobial drug effects
Sample contamination with oxidants (eg, bleach)
Death of fastidious bacteria between time of
sample collection and urine culture. For culture,
there is no significant change in refrigerated
samples for up to 6 h; however, >6 h can result
in false-negative cultures
Chemical preservation of urine sample
Information based on references 2, 3 and 10

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Table 4 Commonly encountered crystals and their significance in feline urine (continued on page 381)

Crystal Description Urine pH Significance Action/considerations

Magnesium Colourless, coffin-like
ammonium prisms. Often with three
phosphate to six (or more) sides that
(struvite) typically have oblique
or ‘triple ends.Sometimes seen
phosphate’ as ‘razorblade’ structures.
(Figure 7) Occasionally aggregate
into fern-like structures
Amorphous Amorphous granular
phosphates precipitate. May be a dull
(magnesium, brown colour
calcium)
Ammonium Yellow–brown, round
(bi)urate structures with horn-like
(Figure 8) projections of variable
length (‘thorn apples’).
These projections may
break off to leave small
brown crystals with fine
radiating lines
Amorphous Yellow to yellow–brown
urates amorphous granules
(calcium,
magnesium,
sodium,
potassium)
(Figure 9)
Uric acid Yellow to yellow–brown
pleomorphic diamonds
or prisms, oval plates
or rosettes
Calcium Colourless squares, of
oxalate varying size, displaying an
dihydrate octahedral or envelope
(weddelite) shape with corners
(Figure 10) connected by intersecting
diagonal lines
Common in slightly acidic
to alkaline urine.
Readily dissolved in urine
acidified by adding acetic
acid
Found in neutral to alkaline
urine (especially alkaline).
Readily dissolved in urine
acidified by adding acetic acid
Found in slightly acidic,
neutral and, less commonly,
alkaline urine. Insoluble in
acetic acid
Found in acidic to neutral
urine and insoluble in acetic
acid
Found in acidic urine
Found most commonly
in acidic to neutral urine,
although they can form
in alkaline urine
May be found in urine of cats that are
apparently healthy, in addition to those
with: (1) infection-induced struvite
uroliths; (2) sterile struvite uroliths;
(3) non-struvite uroliths; (4) urethral
plugs that contain struvite crystals;
and (5) urinary tract disease without
uroliths (eg, feline idiopathic cystitis)
May be found in normal urine. May also
be associated with in vitro precipitation
due to refrigeration
Uncommon in healthy cats. May be
associated with hepatic disease
(eg, portovascular anomalies) in some
cases. Specific cat breeds such as the
Egyptian Mau, Birman and Siamese
have been reported to be at increased
risk of urate urolith formation but the
pathogenesis is unclear18
Uncommon in healthy cats.
May be associated with in vitro
precipitation due to refrigeration
Uncommon in healthy cats
May be found in urine from normal cats Tendency for in vitro formation.
and cats with calcium oxalate uroliths.
May also be encountered in cases of
ethylene glycol intoxication, particularly
if seen in large numbers associated
with acute kidney injury and other
appropriate clinical signs; less common
in this toxicity than calcium oxalate
monohydrate crystals
In vitro formation may occur in
refrigerated stored samples or
samples that become alkaline
with storage. Verify true
presence of struvite crystals
on a freshly obtained urine
sample
No clinical significance. These
crystals may dissolve once urine
sample is returned to room
temperature before examination
When present, serum bile acid
testing is recommended to
exclude hepatic causes
These crystals may dissolve
once urine sample is returned
to room temperature before
examination
Crystals have the same
significance as described for
ammonium and amorphous
urates
Verify via analysis of a freshly
obtained urine sample

Yeasts/fungi
Fungi, including yeasts, are often
contaminants in urine but in an
appropriately handled cystocentesis
sample should be regarded as sig-
nificant and submitted for culture.
Yeasts may be difficult to distin-
guish from RBCs or lipid droplets
on microscopic examination of wet
preparations. Budding forms and
a double refractile wall may help
identify yeast organisms more
easily. Examination of air-dried
preparations after cytocentrifuga-
tion of urine will also help deter-
mine the presence of a yeast/fungal
infection.2,3
Fungal culture of appropriately
collected urine samples is
paramount if identification of a
particular yeast/fungus is required.
Parasites
Eggs of Dioctophyma renale (giant
kidney worm), Capillaria plica and
Capillaria feliscati (bladder worms)
may be present in urine.2,17
Crystals
Crystals are commonly found
b in cats on routine urinalysis
(Table 4). Further investigation is
required to assess the diagnostic
and clinical significance of the
crystalluria. The starting point
should always be microscopic
examination of a fresh (<1 h post-
collection), non-refrigerated urine
sample to avoid possible in vitro
artefacts (eg, formation or dis-
solution of crystals in urine asso-
Figure 7 Struvite crystals in (a) an unstained wet ciated with prolonged storage or
preparation (arrows) and (b) an air-dried smear stained
with Diff Quik
refrigeration).

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Table 4 Commonly encountered crystals and their significance in feline urine (continued from page 380)

Crystal Description Urine pH Significance Action/considerations

Calcium oxalate Vary in size and may have a spindle, Found most May be found in urine Tendency for in vitro formation. Verify via
monohydrate oval, dumbbell or hemp seed shape. commonly in from healthy cats, analysis of a freshly obtained urine sample.
(whewellite) Flat, elongated, six-sided crystals acidic to cats with calcium In oliguric acute kidney injury, the presence of
(‘picket fence post’ or ‘stake’ neutral urine, oxalate uroliths and these crystals is highly suggestive of ethylene
appearance) although they cats with ethylene glycol toxicity (though crystals may also be
can form in glycol toxicity absent in these cases)
alkaline urine
Bilirubin Yellow–gold to reddish brown needles Found in Absent in normal Presence of these crystals in cats
(Figure 11) or fine spicules arranged freely or in acidic urine cat urine may precede hyperbilirubinaemia. Investigate
bundles, or occasionally forming causes of hyperbilirubinaemia (pre-, intra- or
granules post-hepatic disease) more closely
Cholesterol Large, colourless, thin, flat rectangular Found in May be present in Significance is unknown but cholesterol may be
plates with distinct right angles and a acidic to normal cat urine found in the urine of animals with previous urinary
characteristic square notched corner neutral urine tract haemorrhage or degenerative disease
Cystine Colourless, flat hexagonal plates that Found in Absent in normal Cystinuria is a rare inherited metabolic
commonly aggregate acidic urine cat urine disease characterised by defective amino acid
reabsorption
Sulphonamide Yellow to yellow–brown crystals Found in Absent in normal Associated with sulphonamide administration in
in various arrangements: needles, acidic urine cat urine dehydrated animals. Identity of these crystals can
sheaves or spherical with radial be confirmed with the lignin test: a drop of urine
striations sediment on newspaper, with one or two drops
of dilute hydrochloric acid added, produces a
bright yellow–orange colour
Xanthine Yellow-brown amorphous granules, May Absent in normal Xanthinuria is a rare inborn error of metabolism
spheroids or oval structures that may precipitate in cat urine in cats
be impossible to distinguish from amorphous
amorphous urates or ammonium form in acidic
urates by light microscopy urine
Melamine/ Brown to green–brown circular crystals Acidic urine Absent in normal Documented in cats with acute kidney
cyanuric acid with radiating striations originating cat urine injury with a history of ingesting pet food
from the centre of the crystal contaminated with melamine and cyanuric acid
Information based on references 2–4, 6, 9, 11 and 17–20

a b

Figure 8 Ammonium biurate crystals in cat urine, Figure 9 Amorphous urate crystals in unstained wet preparations; (a) with and (b) without polarisation
showing the classic ‘thorny apple’ appearance.
Image © Cornell University

a b

Figure 10 Calcium oxalate dihydrate crystals in a Diff Quik-stained smear viewed at (a) x 40
and (b) oil immersion x 100 Figure 11 Bilirubin crystals in a Diff Quik-stained smear

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R E V I E W / Practical urinalysis – microscopic examination

Other urine insoluble particles

Lipid droplets
< Usually of no pathological significance and common
in feline urine
< Appear as variably sized, green-tinged, round, highly
refractile droplets that float beneath the coverslip
out of the plane of focus of other sediment elements
< Product of normal exfoliation of aged tubular epithelial
cells or seen as a result of exfoliation of tubular cells that
have undergone sublethal fatty degeneration
< Also seen with diabetes mellitus and nephrotic syndrome
< A layer of white lipid is sometimes seen in normal feline urine
samples
Mucin
< Mucin strands appear as narrow, twisted ribbons and
homogeneous threads (not to be confused with casts)
Artefacts
< Include air bubbles, starch granules (from surgical gloves),
dust particles, hairs, faeces and bacteria
< Starch granules are sometimes confused with lipid droplets
but have scalloped margins, often have a central dimple and
are not refractile when viewed by bright light microscopy.
Under polarised light, they have a Maltese cross appearance
< Muscle fibres in samples obtained by cystocentesis

These various insoluble particles are occasionally seen on urine microscopic examination2,3

Preparation of air-dried smears some larger veterinary practices to prepare

for urine insoluble particle cytospin smears from urine samples. in gener-
identification al veterinary practice, preparing diagnostical-
ly useful smears from urine without the use of
When difficulty is encountered identifying cytocentrifuge machines can be challenging,
insoluble particles, or increased numbers of though several techniques can be used. direct
epithelial cells or abnormal cells are seen smears of the cell sediment can be prepared
on urine wet-prep examination, cytological by conventional centrifugation (as described
examination of an air-dried smear should also on page 375), although gravitational sedi-
be performed for further investigation. in mentation techniques or a combination of
veterinary practice, neoplastic cells in urinary conventional centrifugation and gravitational
sediment are best identified in air-dried sedimentation techniques may be more useful
smears.6,8,9 Liquid-based preparation tech- in producing adequate cellular smears for
niques such as ThinPrep and SurePath, which further cytological examination.

Cytocentrifugation technique
have gained popularity for processing human
urine specimens in the past few years, have
yet to be investigated for use in screening cats Cytocentrifuge machines such as the Shandon
and dogs with suspected urinary tract Cytospin (Figure 12) or Cyto–Tek are used to

of 250–500 µl of well mixed, resuspended

malignancies. prepare air-dried cytospin smears. An aliquot

Preparing air-dried smears for cytological urine solution (prepared as described above)
examination is placed in the cytocentrifuge’s cytospin
Air-dried smears may be prepared for chamber.
cytological examination using a variety
of techniques. These
attempt to preserve the
a b
cytomorphological fea-
tures of the generally low
numbers of nucleated
cells, while simulta-
neously concentrating
them onto a focal area of
the glass slide for further
assessment. The precise
preparation technique
will depend on the
resources and prefer-
ences of the veterinary
practice or veterinary
laboratory.
Cytocentrifugation is
routinely used in veteri-
nary laboratories and Figure 12 (a) A cytocentrifuge machine (Shandon Cytospin).
(b) Components of the cytocentrifuge chamber

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R E V I E W / Practical urinalysis – microscopic examination

a b

Figure 13 Cytospin smears of cat urine

prepared with a cytocentrifuge and stained
with Diff Quik. These show neoplastic
urothelial cells accompanied by
inflammatory cells and a mitotic figure
(arrow in [a]) and display some criteria
of malignancy (including prominent,
irregularly shaped nucleoli, anisokaryosis
and anisocytosis (arrows in [b])

Gravitational sedimentation may be helpful

Cytocentrifugation produces a small central
spot on the cytospin glass slide where cells are
concentrated. The superior cell morphological
preservation afforded by this technique makes
the detection of cytological criteria of malig-
nancy easier, particularly within epithelial cell
populations (Figure 13). The ability to concen-
trate low numbers of urine insoluble particles
into a central area on the slide also improves
the likelihood of detecting fungal infections,
as only scant fungal hyphae may be present
in urine.
for preparing adequate smears from
low cellular fluids such as urine.
then be stained and submitted for cytological
examination.
Simpler gravitational sedimentation cham-
bers may also be configured from syringe
barrels or perspex tubing (Figure 15) that have
been carefully cut, with one end dipped in
melted paraffin wax and mounted perpendic-

Gravitational
ularly on to a pre-warmed clean glass slide.

sedimentation
technique a c
Smears from low cel-
lular fluids, such as
urine, can also be
prepared using gravi-
tational sedimenta-
tion techniques.
Specialised sedi- b
mentation chambers
Figure 14 (a) Apparatus required for the gravitational sedimentation technique
(Figure 14) with a includes a sediment chamber, paper clips and glass slide. (b) Close-up of a
rubber flange at sediment chamber (PrepStain Settling Chamber; Becton Dickinson). (c) The fully
assembled chamber (c) is filled and left undisturbed for 30 mins. The technique
one end (PrepStain results in a cytospot which is air-dried and stained
Settling Chamber;
Becton dickinson),
are available. When
seated firmly on a glass slide using paper clips The slide should be allowed to cool prior to
to create an impervious seal (Figure 14c), they filling with the resuspended urine sample.
may be filled with up to 4 ml of urine and left The rest of the procedure is similar to the
undisturbed for approximately 30 mins to gravitational sedimentation smearing tech-
facilitate gravitational cell sedimentation. The nique described above.
supernatant may be carefully aspirated using
a pipette or, alternatively, tipped out by
briskly inverting the chamber/glass slide unit
contents before dismantling the chamber. The
remaining contents of the sediment chamber
adhering to the microscope slide can be rapid-
ly dried with a hairdryer (set at medium to
high for 15–30 s, or ceased sooner if the speci-
men on the slide appears dry), prior to unclip-
ping the sediment chamber from the glass
slide. The resultant smear is localised to a
focal area on the slide (Figure 14c) that can Figure 15 Simpler gravitational sediment chamber apparatus
for ‘in-house’ use with low cellular fluids such as urine

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R E V I E W / Practical urinalysis – microscopic examination

Figure 16 Line smear technique. Diagram 1

and glass slide (a) depict the position of the
resuspended drop of urine (labelled S) close
to one end of the frosted glass slide (which
is labelled in pencil with the patient’s details)
and the placement of the overlying spreader
slide; this spreader slide is drawn backwards
slowly to engage the resuspended drop of
urine. Diagram 2 shows the varying angles
at which the spreader slide can be held
while making the smear (dependent on the
desired thickness of the film to be produced).
Diagram 3 illustrates the abrupt lifting of
1 2 3 the spreader slide approximately 0.5 cm
from the non-frosted edge of the glass slide;
this action produces a dense ‘line’ at the end
of the smear (b,c) in which most of the larger
cells (including neoplastic epithelial cells)
will usually be present. A better ‘monolayer
effect’ may be achieved if, immediately after
a b line smearing, the glass slide is tilted slightly
with the ‘line’ uppermost, and rapidly dried
with a hairdryer

Direct line smear technique

direct smears may be prepared from the KEY Points
resuspended urine sediment pellet (see page < Microscopic examination of urine,
374) by using the line smear technique which includes urine sediment
described in Figure 16. examination and urine cytological
assessment, is more demanding
Staining urine air-dried smears for and time consuming than urine
cytological examination macroscopic examination.
< It involves greater skill and more
Slides prepared using one of the above-
discussed techniques can be left to air dry but
equipment to perform competently.
should preferably be dried rapidly using a
hairdryer (set at medium to high for 15–30 s, < Once the urine microscopic
or ceased sooner if the specimen on the slide examination technique has been
appears dry); this avoids slow-drying arte- mastered and the limitations
facts and helps ‘fix’ the cells to the glass slide, of the test have been considered,
reducing the likelihood of cells ‘washing off’ erroneous results and
the slide during staining. other heat fixation interpretations will be
methods are not encouraged as they are likely minimised.
to alter cell morphology.
Both direct and cytospin air-dried smears
submitted for microscopic examination can be
stained using rapid Romanowsky stains. in able at some specialist laboratories) may also
practice, one of the easiest, quickest and most be attempted on air-dried smears that reveal
cost-effective Romanowsky-type stains avail- increased numbers of fungal hyphae.21
able is diff Quik. Note that cytological stains must be replen-
As discussed earlier, air-dried smears ished regularly to avoid artefactual bacterial
stained with a rapid Romanowsky stain facili- presence in smears as a consequence of
tate checking for bacteria (see Figure 6). bacterial contamination and proliferation in
Stained air-dried smears have been shown to unchanged stain media. Stains must also be
have superior sensitivity (83%) and specificity renewed regularly and/or filtered to avoid
(98%) for bacterial detection compared with deposition of stain precipitate, which may
the wet unstained method.10 Air-dried smears mimic bacteria in smears.
can also be stained with a gram stain if bacteria
are identified. Panfungal PCR testing (avail- Acknowledgements

Figure 8 (ammonium biurate crystals) is used with

permission of Cornell University. The original image
Cytological stains must be replenished regularly is located on the ‘eClinPath’ website at Cornell
to avoid artefactual bacterial presence in smears. University. Cornell University retains full copyright.

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R E V I E W / Practical urinalysis – microscopic examination

Funding 10 Swenson CL, Boisvert AM, Gibbons-Burgener

SN, et al. Evaluation of modified Wright-
The authors received no financial support for the staining of dried urinary sediment as a
research, authorship and/or publication of this method for accurate detection of bacteriuria
article. in cats. Vet Clin Pathol 2011; 40: 256–264.
11 Chew dJ and diBartola SP. Sample handling,
Conflict of interest preparation, analysis and urinalysis interpre-
tation. in: interpretation of canine and feline
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est with respect to the research, authorship and/or handbook series, 1998, pp 9–21.
publication of this article. 12 Kouri T, Gyori A and Rowan RM. ISLH recom-
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