MICROSCOPIC

EXAMINATION
OF
URINE
 MACROSCOPIC SCREENING
 To enhance the cost-effectiveness of urinalysis, many laboratories have developed protocol
whereby a microscopic examination of the urine sediments is performed on the specimens meeting
specified criteria.
 Abnormalities in the physical and chemical portions of the urinalysis play a primary role in the
decision to perform a microscopic analysis thus the use of the term “macroscopic screening”.

Macroscopic Screening and Microscopic Correlations

Screening test Significance
Color Blood
Clarity Hematuria versus hemoglobinuria/ myoglobinuria
Confirm pathologic or nonpathologic cause of turbidity
Blood RBCs, RBC casts
Protein Casts, Cells
Nitrite Bacteria, WBCs
Leukocyte WBCs, WBC casts, Bacteria
esterase
Glucose Yeast

SPECIMEN PREPARATION
 Specimen should be examined while fresh or adequately preserved
o Formed elements-primarily RBCs, WBCs and hyaline casts- disintegrate rapidly,
particularly in dilute alkaline urine.
 Refrigeration may cause precipitation of amorphous urates and phosphates and other non-
pathologic crystals that can obscure other elements in the urine.
 Warming the specimen to 37°C prior to centrifugation may dissolve some of the crystals.
 The midstream clean catch specimen minimizes external contamination of the sediments
o As with the physical and chemical analysis, dilute random specimens may cause false-
negative readings.
 Care must be taken to thoroughly mix the specimen prior to decanting a portion into a centrifuge
tube.

SPECIMEN VOLUME
 A standard amount of urine, usually between 10 – 15 mL, is centrifuged in a conical tube.
 A 12 mL volume is frequently used because multiparameter reagent strips are easily immersed in
this volume, and capped centrifuge tubes are often calibrated to this volume.
 If obtaining a 12 mL specimen is not possible, as with pediatric patients, the volume of the
specimen used should be noted on the report form.

CENTRIFUGATION
 Speed of the centrifuge and the length of time the specimen is centrifuged should be consistent.
 Centrifugation of 5 mins at a relative centrifugal force (RCF) of 400 produces an optimum amount
of sediment with the least chance of damaging the elements.
 The RPM value shown on the centrifuge tachometer can be converted to RCF using nomograms
available in many laboratory manuals or by using the formula:
RCF = 1.118 x 10¯⁵ x radius in centimetre x RPM²
  Centrifugation calibration should be routinely performed.
 Use of the breaking mechanism to slow the centrifuge causes disruption of the sediment prior to
decantation and should not be used.
 To prevent biohazardous aerosols; all specimens must be centrifuged in capped tubes.

SEDIMENTATION PREPARATION
 A uniform amount of urine and sediment should remain in the tube after decantation.
 Volume of 0.5 and 1.0 mL are frequently used.
 The volume of urine centrifuge divided by the sediment volume equals the concentration factor.
 The sediments concentration factor relates to the probability of detecting elements present in low
quantities and is used when quantitating the number of elements present per millilitre.
 To maintain a uniform sediment concentration factor, urine should be aspirated off rather than
poured off, unless otherwise specified by the commercial system in use. Some systems provide
pipettes for this purpose.
 The sediment must be thoroughly resuspended by gentle agitation. This can be performed using a
commercial-system pipette or by repeatedly tapping the tipoff the tube with the finger.
 Vigorous agitation should be avoided, as it may disrupt some cellular elements.
 Thorough resuspension is essential to provide equal distribution of elements in the microscopic
examination fields.

VOLUME OF SEDIMENT EXAMINED

 The volume of sediment placed on the microscope slide should be consistent for each specimen.
 When using the conventional glass-slide method, the recommended volume is 20μL (0.02mL)
covered by a 22 x 22 mm glass cover slip.
o Allowing the specimen to flow outside of the cover slip may result in the loss of heavier
elements such as casts.
 Commercial systems control the volume of the sediment examined by providing slides with
chambers capable of containing a specified volume.
 Care must be taken to ensure the chambers are completely filled.
 Product literature supplies the chamber volume, size of the viewing area, and approximate number
of low-power and high-power viewing areas, based on the area of the field of view using standard
microscope. This information, together with the sediment concentration factor, is necessary to
quantitate cellular elements per millilitre of urine.

COMMERCIAL SYSTEMS
 The CLSI recommends the use of commercial system together with standardization of all phases of
the methodology, including the conventional method.
 Systems currently available include:
o KOVA (Hycor Biomedical, Inc., Garden Grove, CA)
o Urisystem (ThermoFisher Scientific, Waltham, MA)
o Count-10 (V-Tech, Inc., Pomona, CA)
o Quick-Prep Urinalysis System(Globe Scientific , Paramus, NJ)
o CenSlide 2000 Urinalysis System (International Remote Imaging System, Norwood, MA)
o R/S Workstation 1000, 2000, 3000 (DioSys, Waterbury, CA)
 The systems provide a variety of options including:
o Capped, calibrated centrifuge tubes
o Decanting pipettes to control sedimentation volume
 o Slider that control the amount of sedimentation for examination
 The Cen-Slide and R/S Workstations do not require manual loading of the centrifuged specimens
onto a slide and are considered closed systems that minimize exposure to the specimen.
 Cen-Slide provides a specially designed tube that permits direct reading of the urine sediments.
 The R/S Workstations consist of a glass flow cell into which urine sediment is pumped,
microscopically examined, and then flushed from the system.

EXAMINING THE SEDIMENTS

 Microscopic examination should be performed In a consistent manner and include observation of a
minimum of 10 fields under both low (10x) and high (40x) power.
 The slide is first examined under low power to detect casts and ascertain the general composition
of the sediments.
 When elements such as casts that require identification are encountered, the setting is changed to
high power.
 If the conventional glass –slide method is being used, casts have a tendency to locate near the
edges of the cover slip; therefore, low power scanning of the cover slip perimeter is recommended.
 This does not occur when using standardized commercial systems.
 When the sediments is examined unstained, many sediment constituents have a refractive index
similar to urine. Therefore it is essential that sediments be examined under reduced light when
using bright-field microscopy.
 Initial focusing can be difficult with a fluid specimen and care must be taken to ensure that the
examination is being performed in the correct plane.
 Often an epithelial cell will be present to provide point of reference.
 Focusing on artefacts should be avoided, because they are often larger than the regular sediment
elements and cause the microscopist to examine object in the wrong plane.
 Continuous focusing with the fine adjustment aids in obtaining a complete representation of the
sediment constituents.

REPORTING THE MICROSCOPIC EXAMINATION

The terminology and methods of reporting may differ slightly among laboratories but must be
consistent within a particular laboratory system.

 Routinely, casts are reported as the average number per low-power field (lpf) following examination
of 10 fields, and RBCs and WBCs, as the average number per 10 high-power fields (hpf).
 Epithelial cells, crystals and other elements are frequently reported in semi quantitative terms such
as, rare, few, moderate, and many, or as 1+, 2+, 3+, and 4+, following laboratory format as to lpf or
hpf use.
 Laboratories must also determine their particular reference values based on the sediment
concentration factor in use.
 Converting the average number of elements per lpf or hpf to the number per millilitre provides
standardization among the various technique in use.

CORRELATING RESULTS
Microscopic results should be correlated with the physical and chemical findings to ensure the accuracy of
the report.

 Specimens in which the results do not correlate must be rechecked for both technical and clerical
errors.
  The amount of formed elements or chemicals must also be considered, as must the possibility of
interference with chemical tests and the age of the specimen.

Routine Urinalysis Correlations

Microscopic elements Physical Chemical Exceptions
RBCs Turbidity +Blood Number
Red color +Protein Hemolysis
WBCs Turbidity +Protein Number
+Nitrite Lysis
+LE
Epithelial Cells Turbidity Number
Casts +Protein Number
Bacteria Turbidity ↑pH Number and type
+Nitrite
+Leukocytes
Crystals Turbidity pH Number and type
Color +Bilirubin

URINE SEDIMENT CONSTITUENTS

CLINICAL
SEDIMENT APPEARANCE DESCRIPTION
SIGNIFICANCE
REPORTING

Damage to the glomerular

Smooth, non-
membrane or vascular
nucleated,
injury within genitourinary
biconcave disks Average
tract
(7mm diameter) number seen in
Macroscopic
HEMATURIA: cloudy with 10 hps
Hypersthenuric: and color
red to brown color,
cells shrink due
advanced glomerular
to loss of water May be
damage or caused by
and may appear reported as
Red Blood trauma, acute infection or
crenated or >100 per hpf
Cells irregularly
inflammation and
coagulation disorders
shaped COMPLETE
Macroscopic
Hyposthenuric: URINALYSIS
HEMATURIA: early
cells absorbs CORRELATIONS
diagnosis of glomerular : color in reagent
water, swell, and
disorders and malignancy strip blood
lyse rapidly,
of urinary tract and to reaction
releasing
confirm presence of rena
hemogloin
canaliculi, strenuous
“Ghost cells”
exercise
 Neutrophil:
average
Predominant is number seen
the Neutrophil- in 10hpfs
easier to Neutrophil: no clinical Eosinophil:
identify, contain significance finding >1% is
White Blood granules and significant
Cells multilobed Eosinophil: drug-
nuclei induced nephritis, UTI, COMPLETE
Eosinophils: renal transplant rejection URINALYSIS
CORRELATIONS
Hansel stain : Leukocytes
esterase, nitrite
specific gravity
and pH

Lymphocytes: early
stages of renal transplant
rejection
Monocytes,
macrophages, and
histiocytes: appear
Lymphocytes, Normal urine:
vacuolated or contain
Mononuclear monocytes, <5 leukocytes
inclusions
Cells macrophages, per hpf (higher
Pyuria: increased
and histiocytes in females)
urinary WBCs- infection
or inflammation of
genitourinary system,
glomerulonephritis, LPE,
interstitial nephritis and
tumors
Epithelial
Cells
Squamous Squamous Squamous Epithelial Squamous
Epithelial Epithelial Cells: normal cellular Epithelial
Cells Cells: Largest, sloughing in female Cells: Rare,
with abundant urethra, no pathologic few, moderate
irregular significance or many per
cytoplasm and “Clue cell”- vaginal lpf
prominent infection by the COMPLETE
URINALYSIS
nuclei bacterium Gardnerella CORRELATIONS
Transitional Transitional vaginalis : Clarity
Cells Cells: Transitional Transitional
Spherical, Cells:Increased numbers Cells: Rare,
polyhedral, or singly, in pairs or in few, moderate
caudate with clumps (syncytia) or many per
central nucleus present ff. invasive hpf
RTE Cells: urologic procedures COMPLETE
Rectangular, (catherization), increased URINALYSIS
RTE Cells columnar, exhibiting vacuoles and CORRELATIONS
: Clarity, Blood if
round, oval or irregular nuclei= malignancy
cuboidal with malignancy or viral associated
eccentric infection RTE Cells:
nucleus RTE Cells:increased Average
Appear deep amount= necrosis of the number per 10
yellow in viral renal tubules hpfs
 hepatitis (RTE Oval Fat Bodies: COMPLETE
cells absorb Lipiduria- damage of the URINALYSIS
CORRELATIONS
bilirubin present glomerulus caused by : Leukocyte
in filtrate as a nephrotic syndrome, esterase and
result of viral severe tubular necrosis, nitrite, color,
hepatitis) diabetes mellitus and clarity, protein,
Oval Fat trauma (bone marrow) biluribin, blood
Bodies: Highly “bubble cells” – appear Oval Fat
refractile RTE to represent injured cells Bodies:Avera
Oval Fat Cells in which the endoplasmic ge number per
Bodies reticulum has dilated hpf
COMPLETE
prior to cell death. URINALYSIS
CORRELATIONS
:Clarity, protein,
blood, free fat
droplets

May be a contamination
Few,
Cocci or bacilli, Lower or upper UTI-
Bacteria moderate, or
small size accompanied with WBCs
many per hpf
(for quantitative culture)

Candida albicans-
Small, refractile
diabetic patients (acidic,
oval structures,
glucose containing
may contain
urine), Rare ,few,
bud or not
Yeast immunocompromised moderate, or
In severe
patients and women with many per hpf
infections:
vaginal monoliasis.
branched,
Should be accompanied
mycelial forms
with WBCs
 Trichomonas
vaginalis- pear
shaped
flagellate with
an undulating
membrane,
rapid darting Presence of
movement STD with vaginal ova of the
Parasites
Schistosoma inflammation parasites
haematobium-
ova of the
bladder parasite
Most common
contaminant-
Enterobius
vermicularis

Urine is toxic to
spermatozoa,
ff. sexual intercourse, Presence or
Oval, slightly masturbation, or none
tapered heads nocturnal emission (depending on
Spermatozoa
and long, Male infertility or laboratory
flagella like tails retrograde ejaculation in protocols,
which sperm is expelled varies)
into the bladder instead
of urethra

Frequently in female
Thread like urine specimen, no
structures with clinical significance Rare, few,
Mucus a low refractive (produced by glands and moderate or
index, irregular epithelial cells of lower many per lpf
appearance genitourinary tract and
RTE cells)
 CASTS
 The only elements found in the urinary sediments that are unique to the kidney. They are formed
within the lumens of the distal convoluted tubules and the collecting ducts, providing a microscopic
view of the conditions within the nephron. Their shape is representative of the tubular lumen, with
parallel sides and somewhat rounded ends, and they may contain additional elements present in
the filter.

APPEARANCE CLINICAL
URINE CASTS DESCRIPTION
SIGNIFICANCE

Glomerulonephritis
Pyelonephritis
Colorless, Chronic Renal
Hyaline Casts homogenous Disease
matrix Congestive Heart
Failure
Stress and Exercise

Orange-red color,
Glomerulonephritis
RBC Casts cast matrix
Strenuous Exercise
containing RBCs
 Pyelonephritis
Cast matrix
WBC Casts Acute Intestinal
containing WBCs
Nephritis

Bacilli bound to
Bacterial Casts Pyelonephritis
protein matrix

RTE cells Renal Tubular

Epithelial
attached to Damage
Casts
protein matrix

Coarse and fine Glomerulonephritis

Granular Casts granules in a cast Pyelonephritis
matrix Stress and Exercise
 Highly refractile Stasis of urine flow
Waxy Casts cast with jagged Chronic Renal
ends and notches Failure

Nephrotic Syndrome
Fat droplets and
Toxic Tubular
oval fat bodies
Fatty Casts Necrosis
attached to
Diabetes Mellitus
protein matrix
Crush Injuries

Wider than normal Extreme urine stasis

Broad Casts
cast matrix Renal Failure
 URINARY CRYSTALS
CRYSTAL FORMATION

 formed by the precipitation of urine solutes, including inorganic salts, organic compounds, and
medications
 solutes precipitate more readily at room temperatures.
 Majority of crystal formation takes place in specimens that have remained at room temperature or been
refrigerated specimens and often present problems
 As the concentration of urinary solutes increases, their ability to remain in solution decreases, resulting
in urine formation
 Freshly voided urine with crystals= high specific gravity
 Organic and iatrogenic compounds crystalize easily in acidic pH
 Inorganic salts are less soluble in neutral or alkaline urine
*Calcium oxalate precipitates in both acidic and neutral urine.

NORMAL CRYSTALS SEEN IN ACIDIC URINE

CRYSTAL APPEARANCE DESCRIPTION

Uric acid Appear as large granules and
have spicules, yellow brown
(rosettes, wedges)

Amorphous urates Brick dust or yellow brown

Calcium oxalate Colorless, octahedral envelope

or dumbbell or as two pyramids
joined at their bases
 NORMAL CRYSTALS SEEN IN ALKALINE URINE

CRYSTAL APPEARANCE DESCRIPTION

Amorphous phosphates White- colorless, granular, in
large quatities ff. refrigeration
= white precipitate that does
not dissolve on warming

Calcium phosphate Colorless, flat rectangular

plates or thin prisms in rosette,
dissolve in dilute acetic acid

Triple phosphate Colorless, “coffin-lids”, as

disintegrate. May develop a
feathery appearance,
presence of urea-splitting
bacteria

Ammonium biurate Yellow-brown, thorny apples,

spicule covered spheres,
presence of ammonia by urea-
splitting bacteria

Calcium carbonate Colorless, dumbbell or

spherical shapes, formation of
gas after the addition of acetic
acid
 ABNORMAL URINARY CRYSTALS

CRYSTAL APPEARANCE DESCRIPTION CLINICAL

SIGNIFICANCE
Cysteine Colorless hexagonal Inherited
plates, + cystinuria:Metabolic
confirmation by disorder that
cyanide- prevents
nitropusside test reabsorption of
cysteine by renal
tubules,

Cholesterol Colorless notched Nephrotic syndrome,

plates, with fatty lipiduria
casts and oval fat
bodies

Leucine Yellow concentric Liver disease

circles and radial
striations

Tyrosine Colorless-yellow Liver disease,

needles or rosettes, inherited disorder of
+ for bilirubin amino acid
metabolism

Bilirubin Yellow clumped Liver disease:

needles or granules hepatic disorders,
viral hepatitis (casts)
 Sulfonamides Varied (needles, Infection treatment:
rhombics, UTI, inadequate
whetstones, shaves patient hydration,
of wheat, rosette, tubular damage in
colorless to yellow- fresh urine (crystals
brown in nephron)

Radiographic dye Colorless flat plates, Radiographic

markedly elevated procedures
specific gravity

Ampicillin Colorless needles to Infection treatment,

form bundles ff. massive dose of
refrigeration penicillin w/o
adequate hydration
 URINARY SEDIMENT ARTIFACTS

CRYSTAL APPEARANCE DESCRIPTION CONFUSED WITH

Starch Granules, highly RBCs
refractile spheres Fat droplets
with dimpled center
When cornstarch is
the powder used in
powdered gloves

Oil droplets Highly refractile, RBCs

contamination with
immersion oil or
lotions and creams
in fecal
contamination

Air bubbles Occurs when RBCs

specimen is placed
under cover slip

Pollen grains Spheres, with cell Their large sizes

wall and may cause out of
occasional focus of true
concentric circles sediment
constituents
Hair and fibers From clothing or Casts (do not
diaper, much polarize)
longer and more
refractile

Fecal Improperly Their large sizes

contamination collected may cause out of
specimens or focus of true
rarely the presence sediment
of fistula, appear constituents
as plant or meat
firbers as brown
amorphous
material