AUBF BS MLS

URINALYSIS AND BODY FLUIDS / STRASINGER 3rd Year

TRANS UNIT VI: MICROSCOPIC EXAMINATION OF URINE

o Laboratory-designated criteria can also be programed
into automated instruments
OUTLINE  Patient population must be considered when developing
Macroscopic Screening protocols for macroscopic screening
Specimen Preparation
o CLSI recommends that microscopic examination be
Specimen Volume
Centrifugation performed when requested by physician, when lab
Sediment Preparation specified patient population is being tested, or when
Volume of Sediment Examined any abnormal physical or chemical result is obtained
Commercial Systems
Examining the Sediment Table No. 6-1. Macroscopic Screening and Microscopic
Reporting the Microscopic Examination Correlations
Correlating Results SCREENING TEST SIGNIFICANCE
Sediment Examination Techniques
Sediment Stains
Color Blood
Cytodiagnostic Urine Testing Clarity Hematuria versus hemoglobinuria/
Microscopy myoglobinuria
Types of Microscopy Confirm pathologic or
Urine Sediment Constituents nonpathologic cause of turbidity
Red Blood Cells Blood RBCs, RBC casts
White Blood Cells
Protein Casts, cells
Epithelial Cells
Bacteria Nitrite Bacteria, WBCs
Yeast Leukocyte esterase WBCs, WBC casts, bacteria
Parasites Glucose Yeast
Spermatozoa
Mucus SPECIMEN PREPARATION
Casts
Urinary Crystals  Specimens should be examined while fresh or adequately
Urinary Sediment Artifacts preserved
 Formed elements (RBCs, WBCs & hyaline casts)
disintegrate rapidly, particularly in dilute alkaline urine
INTRODUCTION  Refrigeration may cause precipitation of amorphous urates
 Third part of routine urinalysis, after physical & chemical & phosphates and other non-pathologic crystals that can
examination obscure other elements in urine sediment
 Purpose: detect & to identify insoluble materials present in o Warming specimen to 37°C before centrifuging may
urine dissolve some of crystals
 Blood, kidney, lower genitourinary tract & external  Midstream clean-catch specimen minimizes external
contamination contribute formed elements to the urine contamination of sediment
o Include red blood cells (RBCs), white blood cells  As w/ physical & chemical analyses, dilute random
(WBCs), epithelial cells, casts, bacteria, yeast, specimens may cause false-negative readings
parasites, mucus, spermatozoa, crystals, and  Care must be taken to thoroughly mix specimen before
artifacts decanting portion into centrifuge tube
 Examination of urinary sediment must include identification
& quantitation of elements present. SPECIMEN VOLUME
o It is since some of components have no clinical  Standard urine amount: bet. 10 & 15 mL as a volume of
significance & considered normal unless they are specimen
present in increased amounts o It is centrifuged in conical tube and provides adequate
volume from w/ch to obtain representative sample of
 Microscopic analysis is subject to several procedural
elements present in specimen
variations
 12-mL volume is frequently used
 Protocols have been developed to increase standardization
o Since multiparameter reagent strips are easily
& cost-effectiveness of microscopic urinalysis
immersed in this volume & capped centrifuge tubes are
often calibrated to this volume
MACROSCOPIC SCREENING o If obtaining such is not possible, as with pediatric
 Many labs developed protocols where microscopic patients, volume of specimen used should be noted on
examination of urine sediment is performed only when report form and allows physician to correct results, if
meeting specified criteria to enhance cost – effectiveness indicated
 Abnormalities in physical & chemical portions of urinalysis  Some labs choose to make correction before
play primary role in decision to perform microscopic reporting (e.g. 6 mL of urine is centrifuged, results
analysis are multiplied by 2)
o The use of term “macroscopic screening”
 Parameters considered significant vary among laboratories
but usually include color, clarity, blood, protein, nitrite,
leukocyte esterase, and possibly glucose

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 1

 TRANS: MICROSCOPIC EXAMINATION OF URINE
CENTRIFUGATION
 Speed of centrifuge & length of time specimen is
centrifuged should be consistent
 Centrifugation for 5 mins at relative centrifugal force (RCF)
of 400 produces optimum amount of sediment w/ least
chance of damaging elements
 In correcting differences in diameter of centrifuge heads,
RCF rather than revolutions per minute (RPM) is used
 RPM value shown on centrifuge tachometer can be
converted to RCF using nomograms available in many lab
manuals or by using formula
RCF = 1.118 × 10–5 × radius in centimeters × RPM2
 Centrifugation calibration should be routinely performed
 Use of braking mechanism to slow centrifuge causes
disruption of sediment prior to decantation & should not be
used
 To prevent biohazardous aerosols, all specimens must be
centrifuged in capped tubes
SEDIMENT PREPARATION
 Uniform amount of urine & sediment should remain in tube
after decantation
 0.5 & 1.0 mL: volumes frequently used
 Volume of urine centrifuged divided by sediment volume
equals concentration factor
 Sediment concentration factor relates to probability of
detecting elements present in low quantities and is used
when quantitating number of elements present per milliliter
 To maintain uniform sediment concentration factor, urine
should be aspirated off rather than poured off
o It is unless specified by commercial system in use
o Some systems provide pipettes for this purpose
o Pipettes are used for sediment resuspension &
transferring specimens to slide
 Sediment must be thoroughly resuspended by gentle
agitation
o It can be performed using commercial-system
pipette or by repeatedly tapping tip of the tube w/
finger
o Vigorous agitation should be avoided, may disrupt
some cellular elements
o Thorough resuspension is essential to provide equal
distribution of elements in microscopic examination
field
VOLUME OF SEDIMENT EXAMINED
 When using conventional glass-slide method,
recommended volume is 20 m L (0.02 mL) covered by 22 ×
22 mm glass cover slip
 Allowing specimen to flow outside of cover slip may result
to heavier elements loss such as casts
 Commercial systems control volume of sediment examined
by providing slides w/ chambers capable of containing
specified volume
 Care must be taken to ensure chambers are completely
filled
 Product literature supplies chamber volume, size of viewing
area & approximate number of low-power and high-power
viewing areas, based on area of field of view using standard
microscope
o This information w/ sediment concentration factor is
necessary to quantitate cellular elements per
milliliter of urine
COMMERCIAL SYSTEMS
 Commercial slide systems have substantially improved
conventional method of placing drop of centrifuged urine on
DELA CRUZ, GALANTO
glass slide, adding cover slip & examining microscopically
through commercial slide systems use
o CLSI recommends their use with standardization of
all phases of methodology, including conventional
method
o Systems provide variety of options including capped,
calibrated centrifuge tubes, decanting pipettes to
control sediment volume & slides controlling amount
of sediment examined, producing consistent
monolayer of sediment for examination & provide
calibrated grids for more consistent quantitation
 Systems currently available: KOVA, Urisystem, Count-10,
Quick-Prep Urinalysis System, CenSlide 2000 Urinalysis
System and R/S Workstations 1000, 2000, 2003
 Cen-Slide & R/S Workstations don’t require manual loading
of centrifuged specimen onto slide and considered closed
systems minimizing specimen exposure
o Cen-Slide provides specially designed tube
permitting direct reading of urine sediment
o R/S Workstations consist of glass flow cell into w/ch
urine sediment is pumped, microscopically
examined then flushed from system
EXAMINING THE SEDIMENT
 Microscopic examination must be performed in consistent
manner including observation of minimum of 10 fields under
low (10×) & high (40×) power
 Slide is first examined under low power detecting casts & to
ascertain general composition of sediment
o When elements like casts requiring identification are
encountered, setting is changed to high power
 If conventional glass-slide method is being used, casts
have tendency to locate near edges of cover slip
o Low-power scanning of cover-slip perimeter is
recommended
 This does not occur when using standardized commercial
systems
 When sediment is examined unstained, many sediment
constituents have refractive index similar to urine
o It is essential that sediments be examined under
reduced light when using bright-field microscopy
 Initial focusing can be difficult w/ fluid specimen, and care
must be taken to ensure that examination is being
performed in correct plane
 Focusing on artifacts should be avoided
o They are often larger than regular sediment
elements and cause microscopist to examine objects
in wrong plane
 Continuous focusing w/ fine adjustment aids in obtaining
complete representation of sediment constituents
REPORTING THE MICROSCOPIC EXAMINATION
 Routinely, casts are reported as average number per low-
power field (lpf) following examination of 10 fields & RBCs
and WBCs as average number/10 high-power fields (hpf)
 Epithelial cells, crystals & other elements are reported in
semiquantitative terms (rare, few, moderate, and many, or
as 1+, 2+, 3+, and 4+)
 Following laboratory format as to lpf or hpf use, lab must
determine particular reference values based on sediment
concentration factor in use
o Example: Urisystem, with concentration factor of 30,
states reference value for WBCs of zero to eight/hpf,
as opposed to conventional value of zero to five/hpf
used w/concentration factor of 12
CSU BS MLS 3RD YEAR 2
 TRANS: MICROSCOPIC EXAMINATION OF URINE

 Converting average number of elements per lpf or hpf to the Bacteria Turbidity ↑ pH Number and type
number per milliliter provides standardization among
various techniques in use + Nitrite
 Steps include the following: + Leukocytes
EXAMPLE
1. Calculating the area of an lpf or hpf for the microscope in use Crystals Turbidity pH Number and type
using the manufacturer-supplied field of view diameter and the + Bilirubin
formula πr2 = area.
Diameter of hpf = 0.35 mm 3.14 × 0.1752 = 0.096 mm2 SEDIMENT EXAMINATION TECHNIQUE
2. Calculating the maximum number of lpfs or hpfs in the viewing  Many factors can influence appearance of urinary sediment
area. o Includes cells & casts in various stages of
Area under a 22 mm × 22 mm cover slip = 484 mm2 development & degeneration, distortion of cells &
484 = 5040 hpfs crystals by chemical content of specimen, presence
.096 of inclusions in cells & casts & contamination by
3. Calculating the number of hpfs per milliliter of urine tested artifacts
using the concentration factor and the volume of sediment  Identification can sometimes be difficult even for
examined. experienced laboratory personnel
5040 = 5040 = 21,000 hpf/mL of urine o Identification can be enhanced through sediment
0.02 mL × 12 .24 stains use and different types of microscopy
4. Calculating the number of formed elements per milliliter of
urine by multiplying the number of hpfs per milliliter by the
SEDIMENT STAINS
average number of formed elements per field
4 WBC/hpf × 21,000 = 84,000 WBC/mL  Staining increases overall visibility of sediment elements
being examined using bright-field microscopy
o Done by changing refractive index
 Provided the same microscope & volume of sediment
examined are used, number of lpfs and hpfs per milliliter of  Elements like hyaline casts have refractive index very
urine remains same, simplifying calculation similar to urine
 Labs should evaluate advantages and disadvantages of  Staining imparts identifying characteristics to cellular
adding additional calculation step to microscopic structures, like nuclei, cytoplasm & inclusions
examination  Most frequently used stain in urinalysis: Sternheimer-
 CLSI states that all decisions w/ regard to reporting of Malbin stain
microscopic should be based on needs of individual lab o It consists of crystal violet & safranin O
 Procedures should be completely documented and  Stain is available commercially
followed by all personnel o Sedi-Stain & KOVA stain
 Commercial brands contain stabilizing chemicals
CORRELATING RESULTS preventing precipitation occurring w/ original stain
 Microscopic results should be correlated w/ physical &  Dye is absorbed well by WBCs, epithelial cells, and casts
chemical findings to ensure accuracy of report o Provides clearer delineation of structure &
 Specimens in w/ch results do not correlate must be contrasting colors of nucleus & cytoplasm
rechecked for technical & clerical errors  0.5% solution of toluidine blue
o Metachromatic stain
ADDIS COUNT o Provides enhancement of nuclear detail
 First procedure to standardize quantitation of formed o Useful in differentiation bet. WBCs & renal tubular
elements in urine microscopic analysis was developed by epithelial cells
Addis in 1926 o Used in examination of cells from other body fluids
 It used hemocytometer to count number of RBCs, WBCs,
casts, and epithelial cells present in 12-hour specimen Table No. 6-3. Urine Sediment Stain Characteristics
 Normal values have wide range & approx. 0 to 500,000 STAIN ACTION FUNCTION
RBCs, 0 to 1,800,000 WBCs and epithelial cells, and 0 to Sternheimer- Delineates structure Identifies WBCs, epithelial
5000 hyaline casts Malbin and contrasting colors cells, and casts
 It was used primarily to monitor course of diagnosed cases of the nucleus and
of renal disease cytoplasm
 It has been replaced by various standardized commercial Toluidine Enhances nuclear Differentiates WBCs and
systems for preparation, examination & quantitation of blue detail renal tubular epithelial
formed elements in nontimed specimens (RTE) cells
2% acetic Lyses RBCs and Distinguishes RBCs from
Table No. 6-2. Routine Urinalysis Correlations acid enhances nuclei of WBCs, yeast, oil droplets,
MICRO- PHYSICAL CHEMICAL EXCEPTIONS WBCs and crystals
SCOPIC Lipid stains: Stain triglycerides and Identify free fat droplets
ELEMENTS Oil Red O neutral fats orange-red and lipid-containing cells
RBCs Turbidity + Blood Number and Sudan III Do not stain cholesterol and casts
Red color + Protein Hemolysis Gram stain Differentiates gram- Identifies bacterial casts
WBCs Turbidity + Protein Number positive and gram-
+ Nitrite Lysis negative bacteria
+ LE Hansel stain Methylene blue and Identifies urinary
Epithelial Turbidity Number eosin Y stains eosinophils
Cells eosinophilic granules
Casts + Protein Number

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 3

 TRANS: MICROSCOPIC EXAMINATION OF URINE

Prussian Stains structures Identifies yellow-brown Bacteria Motile: do not stain Motile
blue stain containing iron granules of hemosiderin in Nonmotile: stain organisms are
cells and casts purple not impaired
Trichomonas Light blue-green Motility is
Table No. 6-4 Expected Staining Reactions of Urine Sediment vaginalis unimpaired in
Constituents fresh
ELEMENTS IN USUAL COMMENTS specimens
URINARY DISTINGUISHING when
SEDIMENT COLOR OF recommende
STAINED d volumes of
ELEMENTS stain are
RBCs Neutral—pink to used;
purple immobile
Acid—pink organisms
(unstained) Cytoplasm also
Alkaline—purple identifiable
Nuclei Mucus Pale pink or pale
WBCs (dark- Purple Purple blue
staining cells) granules Background Pale pink or pale
Glitter cells Colorless or light Pale blue or Some glitter purple
(Sternheimer blue gray cells exhibit
Malbin brownian LIPID STAINS
positive cells) movement  Lipid (triglycerides, neutral fats & cholesterol) passage
Renal tubular Dark shade of Light shade across glomerular membrane leads to appearance of free
epithelial blue-purple of blue- fat droplets & lipid-containing cells & casts in urinary
cells purple sediment
Bladder Blue-purple Light purple o Used in confirming presence of elements: lipid
tubular stains, Oil Red O, Sudan III & polarizing microscopy
epithelial
 Triglycerides & neutral fats stain orange-red in presence of
cells
stain
Squamous Dark shade of Light purple
 Cholesterol doesn’t stain but capable of polarization
epithelial orange-purple or blue
cells Inclusions and  Three lipids are usually present concurrently in sediment
Matrix o Permits staining or polarization use for confirmation
Hyaline casts Pale pink or pale Very uniform
purple color; slightly GRAM STAIN
darker than  Used primarily in microbiology section
mucous o Differentiation bet. gram-positive (blue) &
threads gramnegative (red) bacteria
Coarse Dark purple  Its role in routine urinalysis is limited to identification of
granular granules in purple bacterial casts that can easily be confused w/ granular casts
inclusion matrix  In performing staining, dried, heat-fixed preparation of urine
casts sediment must be used
Finely Fine dark purple
granular granules in pale HANSEL STAIN
inclusion pink or pale purple
casts matrix  Polynuclear WBCs seen in urinary sediment are almost
always neutrophils associated w/ microbial infection
Waxy casts Pale pink or pale Darker than
o In cases of drug-induced allergic reaction producing
purple hyaline casts,
but of a pale renal interstitium inflammation, eosinophils are
even color; present in sediment
distinct  This stain is the preferred stain for urinary eosinophils
broken ends o Consisting of methylene blue and eosin Y
Fat inclusion Fat globules Rare; o Wright’s stain can also be used
casts unstained in a pink presence is  Staining is performed on dried smear of centrifuged
matrix confirmed if specimen or cytocentrifuged preparation of sediment
examination
under PRUSSIAN BLUE STAIN
polarized light  Yellow-brown granules may be seen in renal tubular
indicates epithelial cells and casts or free floating in urine sediment
double after episodes of hemoglobinuria
refraction o Prussian blue stain for iron is used to confirm the
Red cell Pink to orange-red Intact cells
granules as hemosiderin and also stains it a blue
inclusion can be seen in
color
casts matrix
Blood Orange-red No intact cells
(hemoglobin) CYTODIAGNOSTIC URINE TESTING
casts  Frequently performed independently of routine urinalysis for
detection of malignancies of lower urinary tract
 Voided first morning specimen is recommended for testing

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 4

 TRANS: MICROSCOPIC EXAMINATION OF URINE
o Performed by cytology laboratory
 Such test provides more definitive information about renal
tubular changes associated w/ transplant rejection, viral,
fungal, parasitic infections, cellular inclusions, pathologic
casts & inflammatory conditions
 Not part of routine examination of urine sediment but
preparation of permanent slide w/ cytocentrifugation
followed by staining w/ Papanicolaou stain provides
additional method for detecting & monitoring renal disease
 Urinalysis laboratory should refer specimens w/ unusual
cellular findings to pathologist for further examination
MICROSCOPY
 Microscopic examination of urine is best performed when
laboratorian is knowledgeable about types of microscopes
available, primary characteristics & proper use and
maintenance of such
 Bright-field microscopy: most common type of microscopy
performed in urinalysis laboratory
 Other types of microscopy useful for examining urine
sediment: phase contrast, polarizing, dark field,
fluorescence, and interference contrast
 Type of microscopy used depends on specimen type,
refractive index of the object, ability to image unstained
living cells
 All microscopes are designed to magnify small objects to
such degree that details of their structure can be analysed
o They do this by employing variety of lenses & light
sources
THE MICROSCOPE
Figure 6–1. Parts of the binocular microscope
 All types of microscopes has lens system, illumination
system & body consisting base, body tube & nosepiece
o Primary components of lens system: oculars,
objectives, coarse & fine adjustment knobs
o Illumination system: light source, condenser, field &
iris diaphragms
 Objects to be examined: placed on platform, referred as
mechanical stage
 Compound bright-field microscope: used primarily in
urinalysis lab consisting of two-lens system combined w/
light source
o First lens system: located in objective, adjusted to
be near specimen
o Second lens system, ocular lens: located in
eyepiece
 Path of light passes through specimen up to eyepiece
DELA CRUZ, GALANTO
 Oculars or eyepieces of microscope: located at top of body
tube
o Designed to further magnify object enhanced by
objectives for viewing
o Lab microscope normally contain such capable of
increasing the magnification 10 times (10x)
o For optimal viewing conditions, oculars can be
adjusted horizontally to adapt to differences in
interpupillary distance bet. operators
 Diopter adjustment knob on oculars can be rotated
compensating for variations in vision bet. operators’ eyes
 Clinical laboratory microscopes are binocular, allows
examination to be performed using eyes providing more
complete visualization
 Field of view
o Determined by eyepiece
o Diameter of circle of view when looking through
oculars
o Varies with field number engraved on eyepiece and
magnification of objective
 The higher the magnification, the smaller the field of view
 Sediment constituents are reported as number per
microscopic field (number per hpf or lpf)
 Objectives
o Contained in revolving nosepiece above
mechanical stage
o Adjusted to be near the specimen
o Performs initial magnification of the object on
mechanical stage
 Image passes oculars for further resolution
o Resolution: ability of lens to distinguish two small
objects that are a specific distance apart
 Resolving power is best when distance bet. two objects is
small
o Dependent on wavelength of light and numerical
aperture of lens
 Shorter wavelength of light, greater resolving power
of microscope
 Routinely used objectives in clin lab: 10× (low power, dry),
40× (high power, dry) & 100× (oil immersion)
 Objectives used for examination of urine sediment: 10× and
40×
 Final magnification of object: product of objective
magnification times ocular magnification
o Using 10× ocular & 10× objective provides total
magnification of 100×
 In urinalysis is lpf observation
o 10× ocular & 40× objective provides magnification
of 400× for hpf observations
 Objectives are also inscribed w/ information describing their
characteristics including type of objective, magnification,
numerical aperture, microscope tube length, coverslip
thickness to be used
o Numerical aperture: represents refractive index of
material bet. slide and outer lens (air or oil) and
angle of light passing through
 Higher numerical aperture, better light-gathering
capability of lens yielding to greater resolving
power
 Higher numerical aperture, closer the lens is to the
object
o Length of objectives attached to nosepiece varies
w/ magnification
 Changing distance bet. lens & slide when they are
rotated
 Most of microscopes are designed to be parfocal
CSU BS MLS 3RD YEAR 5
 TRANS: MICROSCOPIC EXAMINATION OF URINE
o Indicates they require only minimum adjustment
when switching among objectives
 Distance bet. slide & objective is controlled by coarse- and
fine-focusing knobs located on body tube
 Initial focusing is performed using coarse knob moving
mechanical stage noticeably up & down until object comes
to view
o Followed by adjustment using fine-focusing knob to
sharpen image
 When using parfocal microscope, only fine knob should be
used for adjustment when changing magnifications
 Illumination for modern microscope is provided by light
source located in the base of microscope
o Light source is equipped w/ rheostat regulating
intensity of light
 Filters may also be placed on light source to vary
illumination & wavelengths of emitted light
 Field diaphragm contained in light source controls
diameter of light beam reaching slide and is
adjusted for optimal illumination
 Condenser located below stage focuses light on specimen
and controls light for uniform illumination
o Normal position of condenser is almost completely
up w/ front lens of condenser near slide but not
touching it
o Condenser adjustment (focus) knob moves
condenser up and down to focus light on object
o Aperture diaphragm in condenser controls amount of
light and angle of light rays passing the specimen
and lens that affects resolution, contrast & depth of
field of image
 By adjusting it to 75% of numerical aperture of
objective, maximum resolution is achieved
 It should not be used to reduce light intensity
since it decreases resolution
 Microscope lamp rheostat is used for this
adjustment
Table No. 6-5. Urinalysis Microscopic Techniques
TECHNIQUE FUNCTION
Bright-field Used for routine urinalysis
microscopy
Phase-contrast Enhances visualization of elements with low
microscopy refractive indices, such as hyaline casts, mixed
cellular casts, mucous threads, and
Trichomonas
Polarizing Aids in identification of cholesterol in oval fat
microscopy bodies, fatty casts, and crystals
Dark-field Aids in identification of Treponema pallidum
microscopy
Fluorescence Allows visualization of naturally fluorescent
microscopy microorganisms or those stained by a
fluorescent dye including labeled antigens and
antibodies
Interference- Produces a three-dimensional microscopy
contrast image and layerby-layer imaging of a specimen
PROCEDURE 6-1
CARE OF THE MICROSCOPE
1. Carry microscope with two hands, supporting the base with
one hand.
2. Always hold the microscope in a vertical position.
3. Clean optical surfaces only with a good quality lens tissue and
commercial lens cleaner.
4. Do not use the 10× and 40× objectives with oil.
5. Clean the oil immersion lens after use.
6. Always remove slides with the low-power objective raised.
DELA CRUZ, GALANTO
7. Store the microscope with the low-power objective in position
and the stage centered.
KOHLER ILLUMINATION
 Two adjustments to condenser—centering & Köhler
illumination—provide optimal viewing of illuminated field
 They should be performed whenever objective is changed
 To center condenser & obtain Köhler illumination, take the
following steps:
• Place a slide on the stage and focus the object using the
low-power objective with the condenser raised.
• Close the field diaphragm.
• Lower the condenser until the edges of the field diaphragm
are sharply focused.
• Center the image of the field diaphragm with the
condenser centering screws
• Open the field diaphragm until its image is at the edge of
the field.
• Remove an eyepiece and look down through the eyepiece
tube.
• Adjust the aperture diaphragm until approximately 75% of
the field is visible
• Replace the eyepiece.
 Additional focusing of object should be performed w/
adjustment knobs & rheostat on light source
 Routine preventive maintenance procedures on
microscope ensure good optical performance
 Microscope should always be covered when not in use
protecting it from dust
o If any optical surface becomes coated w/ dust, it
must be carefully removed w/ camel-hair brush
 Optical surfaces should be cleaned w/ lens paper
 Clean any contaminated lens immediately w/ commercial
lens cleaner
 Oil immersion lens must be wiped free of oil & cleaned after
each use
 Fingerprints and oil smears impair sharpness of image
 Annual professional cleaning for microscope is
recommended
 Light sources are replaced as necessary
Figure 6–2. Centering the condenser and Köhler illumination.
TYPES OF MICROSCOPY
BRIGHT-FIELD MICROSCOPY
 Most frequently used in clin lab
 Object appear dark against a light background
 Technique employs basic microscope described earlier with
light source emitting light in visible wave length range
 Use of the microscope for urine sediment examination can
present problems when amount of light reaching specimen
is not properly controlled
 Sediment constituents w/ low refractive index will be
overlooked when subjected to light of high intensity
o Sediments are then examined w/ decreased light
controlled by adjusting rheostat on light source
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 TRANS: MICROSCOPIC EXAMINATION OF URINE
 Sediment staining increases visualization of elements when
using bright-field microscopy
PHASE-CONTRAST MICROSCOPY
 Phase difference
o As light pass through an object, they are slowed in
comparison to rays passing through air (media) &
decreases intensity of light & producing contrast
o Affected by thickness of object, refractive index &
other light-absorbance properties
 Best contrast is obtained when light not passing through
specimen is shifted one quarter of wavelength & compared
w/ phase difference of specimen
o Phase-contrast microscopy provides such contrast
 This is accomplished by adaptation of bright-field
microscope w/ phase-contrast objective lens & matching
condenser
o Two phase rings appearing “targets” are placed in
condenser & objective
o One phase ring is placed in condenser or below it to
permit light to pass through central clear circular
area only
o Second phase-shifting ring w/ central circular area
retarding light by one quarter wavelength is placed
in objective
 Phase rings must match, thus is important to
check that objective & condenser mode are same
 Ring diameter varies w/ magnification
 Image has best contrast when background is darkest
o Phase-contrast rings must be adjusted to have
maximum contrast
 Two rings are adjusted to make them concentric
 Adjustment steps are as follows:
• Focus the microscope in bright-field with a specimen slide.
• Select a low-power phase condenser ring.
• Select the corresponding ring objective.
• Remove an ocular, insert the adjustment telescope, and
look through the telescope.
• Observe the dark and light rings (annuli).
• With the adjusting screw on the telescope, center the light
annulus (condenser) over the dark annulus
• Replace the ocular.
 Light passes to specimen through clear circle in phase ring
in condenser
o Forms halo of light around specimen
 Diffracted light enters central circle of phase-shifting ring &
all other light is moved one quarter of wavelength out of
phase
 Variations of contrast in specimen image due to various
refractive indexes in object are observed as light rays
merge together
o Enhances visualization and detail
 Advantage: for identifying low refractive hyaline casts or
mixed cellular casts and mucous threads
POLARIZING MICROSCOPY
 Polarized light use: aids in crystals & lipids identification
o Such substances can rotate path of unidirectional
polarized light beam producing colors in crystals &
Maltese cross formation in lipids
 Elements seen under such microscopy are
birefringent, property indicating element can refract
light in two dimensions at 90 degrees to each other
 Halogen quartz lamp in microscope produces light rays of
many different waves
o Each wave has distinct direction and vibration
perpendicular to its direction
DELA CRUZ, GALANTO
 Normal or unpolarized light vibrates in equal intensity in all
directions
 Polarized light vibrates in same plane or direction
 As light passes through birefringent substance, it splits into
two beams
o One beam rotated 90 degrees to the other
 Isotropic substances like blood cells don’t have refractive
property & light passes through unchanged
 Substance rotating plane of polarized light 90 degrees in
clockwise direction have positive birefringence
o In contrast, substance rotating plane in
counterclockwise direction has negative
birefringence
 Polarized light is obtained by using two polarizing filters
o Light emerging from one filter vibrates in one plane
o Second filter placed at 90-degree angle blocks all
incoming light
 Except that rotated by birefringent substance
o Filters are in opposite directions called “crossed
configuration”
o Between cross-polarizing filters, birefringent crystals
are visible in characteristic patterns
 Such microscope can be adapted from bright-field
microscopes
o Two polarizing filters must be installed in crossed
configuration
 First filter (polarizing filter): placed in condenser
filter holder
 Second filter (analyzer): placed in head bet.
objectives & ocular
 Polarizing filter is rotated to allow light vibrating in one
direction only to reach object
o If object doesn’t have birefringent properties, no light
will reach analyzer filter and object to appear black
 Refracted rays from birefringent object to reach analyzer to
cause object to appear white or colored against black
background
 Red compensated polarizing filter: additional filter that can
be added to the microscope & divides light entering
microscope into slow & fast vibrations
o Crystals can be easily identified by aligning them w/
slow vibration and observe blue or yellow color they
produce
 Polarizing microscopy is used in urinalysis to confirm fat
droplets, oval fat bodies and fatty casts’ identification
producing Maltese cross pattern character
 Birefringent uric acid crystals can be distinguished from
cystine crystals, monohydrate calcium oxalate crystals from
nonpolarizing RBCs & calcium phosphate crystals
differentiated from nonpolarizing bacteria by their polarizing
characteristics
Figure 6–3. Phase-contrast ring adjustment.
INTERFERENCE-CONTRAST MICROSCOPY
 Provides three-dimensional image showing very fine
structural detail
CSU BS MLS 3RD YEAR 7
 TRANS: MICROSCOPIC EXAMINATION OF URINE
o Splitting light ray so beams pass through different
areas of specimen
 Light interference produced by varied depths of specimen
is compared & three-dimensional image is visualized
 Advantage: object appearing bright against dark
background but w/out diffraction halo associated w/ phase-
contrast microscopy
 More extensive modifications to bright-field microscope are
required to perform this technique
o Not routinely used in urinalysis laboratory
 Two types of interference-contrast microscopy: modulation
contrast (Hoffman) and differential-interference contrast
(Nomarski)
o Bright-field microscopes can be adapted for such
 Modulation-contrast microscope
o Split aperture is placed below condenser, polarizer
is placed below split aperture and amplitude filter is
placed in back of each objective
o Modulator has three zones of light transmission: dark
zone transmitting 1% of light, gray zone transmitting
15% of light % clear zone transmitting 100% of light
o Polarized light ray pass through split aperture to
various areas of specimen and to modulator where
they are converted into variations of light intensity
producing three-dimensional image
 Differential interference contrast microscope
o Uses prisms & polarizing filter to output plane-
polarized light is placed bet. light source &
condenser
o Two-layered Nomarski-modified Wollaston prism
separating individual rays of light ray pairs is
required
o Lower Wollaston prism is built into condenser
o Upper prism is placed bet. objective and eyepiece
recombining rays
o Above top Wollaston prism, another polarizing filter
is placed causing wave interference to occur
producing three-dimensional image
 These two types of microscopy provide layer-by-
layer imaging of specimen & enhanced detail for
specimens with low or high refractive index
Figure 6–4. Diagram of polarized light.
Figure 6–5. Differential interference-contrast (Nomarski)
microscopy
DELA CRUZ, GALANTO
DARK FIELD MICROSCOPY
 Technique used in clin lab enhancing visualization of
specimens that can’t be seen easily viewed w/ bright-field
microscopy
 Often used for unstained specimens
o Identifies spirochete Treponema pallidum
 It easily adapts bright-field microscopy
o Replaces condenser w/ dark-field condenser
containing opaque disk
 Disk blocks light from entering directly the
objective and field of view is black
 As light rays pass through specimen at oblique angles, light
scatters, diffracts or reflects of specimen and captured by
objective lens
 Specimen appears light against black background or dark-
field
Figure 6–6. Dark-field microscopy.
FLUORESCENCE MICROSCOPY
 Rapidly expanding technique used in medical field today
 Used to detect bacteria & viruses w/in cells & tissues
through technique called immunofluorescence
 Fluorescence: property by w/ch some atoms absorb light at
particular wavelength & subsequently emit light of longer
wavelength, termed fluorescence lifetime
 Practical application in lab: allows visualization of naturally
fluorescent substances or those having been stained with
fluorochrome or fluorophore (fluorescent dyes) to produce
image
 Specimen is illuminated with light of specific wavelength
 Fluorescent substances absorb energy & emit longer
wavelength of light visualized w/ use of special filters called
excitation filter and emission filter
o Excitation filter: selects excitation wavelength of light
from light source
o Emission filter: selects specific wavelength of
emitted light from specimen to become visible
o Filters are chosen to match excitation & emission
wavelengths of fluorophore used to label specimen
 Dichroic mirror reflects excitation light to specimen &
transmits emitted light to emission filter collected w/
objective & imaged by detector
 Fluorescent substance can be observed in fluorescent
microscope a bright object against dark background w/ high
contrast when ultraviolet light source is used
 Powerful light sources are required & are usually mercury
or xenon arc lamps
CSU BS MLS 3RD YEAR 8
 TRANS: MICROSCOPIC EXAMINATION OF URINE
Figure 6–7. Fluorescent microscopy.
URINE SEDIMENT CONSTITUENTS
 Normal urine sediment may contain variety of formed
elements
 Appearance of small numbers of pathologically significant
RBCs, WBCs & casts can be normal
 Many routine urine specimens contain nothing more than
rare epithelial cell or mucous strand
o Students find hard time adjusting to such because
urine sediments containing abnormalities & multiple
elements are stressed in classroom setting but it
should be learned to trust observation after looking
at recommended number of fields
 Cellular elements are easily distorted by widely varying
concentration, pH & metabolite presence in urine
o Makes identification more difficult
 Actual normal numerical values are not clearly defined
 Urine sediment preparation methods determine actual
concentration of sediment
o Therefore, number of elements that may be present
in microscopic field
 Commonly listed values: zero to two or three RBCs/hpf,
zero to five to eight WBCs/hpf & zero to two hyaline casts/lpf
o Even such figures must be taken in context w/ other
factors like recent stress, exercise, menstrual
contamination & presence of other urine sediment
constituents
RED BLOOD CELLS
 They appear as smooth, non-nucleated, biconcave disks
measuring approx. 7 mm in diameter in urine
o Identified using high-power objective
 Routinely reported as average number seen in 10 hpfs
 Concentrated (hypersthenuric) urine: cells shrink due to
water loss & may appear crenated or irregularly shaped
 Dilute (hyposthenuria) urine: cells absorb water, swell &
lyse rapidly, releasing hemoglobin & leaving cell membrane
o These large empty cells are called ghost cells &
can be easily missed if specimens aren’t examined
under reduced light
 RBCs are most difficult for students to recognize of all urine
sediment elements
o Reason: RBCs lack characteristic structures, size
variation & close resemblance to other urine
sediment constituents
 RBCs are confused w/ yeast cells, oil droplets & air bubbles
o Yeast cells exhibit budding
o Oil droplets and air bubbles are highly refractile
when fine adjustment is focused up and down and
DELA CRUZ, GALANTO
can appear in different plane than other sediment
constituents
 Rough appearance of crenated RBCs can resemble
granules seen in WBCs much smaller than WBCs
 If identification continue to be doubtful, adding acetic acid
to a portion of sediment lyses RBCs, leaving yeast, oil
droplets & WBCs intact
o Supra-vital staining can also be helpful
 Studies focused on morphology of urinary RBCs as aid in
determining site of renal bleeding
 Dysmoprhic: RBCs varying in size having cellular
protrusions or fragmented & associated primarily w/
glomerular bleeding
 Number & appearance of dysmorphic cells must be
considered since abnormal urine concentration affects RBC
appearance
o Small number of dysmorphic cells are found w/
nonglomerular & hematuria
 Dysmorphic RBCs is demonstrated after strenuous
exercise w/ch indicates glomerular origin of the
phenomenon
 Dysmorphic cells are closely associated w/ glomerular
bleeding and appears to be acanthocyte w/ multiple
protusions
o Can be difficult to observe under bright-field
microscopy
 Further sediment analysis containing dysmorphic RBCs w/
Wright’s stained preparation shows cells to be hypochromic
and better delineates cellular blebs presence and
protrusions
Figure 6-8 Figure 6-9
Figure 6-10 Figure 6-11
Figure 6-12 Figure 6-13
Figure 6–8. Normal RBCs (×400).
Figure 6–9. Microcytic and crenated RBCs (×100).
Figure 6–10. Yeast. The presence of budding forms aid in
distinguishing from RBCs (×400).
Figure 6–11. KOVA-stained squamous epithelial cells and oil
droplets (×400). Notice how the oil droplet (arrow) resembles an
RBC.
Figure 6–12. Air bubble. Notice no formed elements are in
focus (×100).
Figure 6–13. Dysmorphic RBCs (×400). Notice the smaller size
and fragmentation.
CSU BS MLS 3RD YEAR 9
 TRANS: MICROSCOPIC EXAMINATION OF URINE

CLINICAL SIGNIFICANCE
 RBC Presence in urine is associated w/ damage to
glomerular membrane or vascular injury w/in genitourinary
tract
o Number of cells present: indicative of extent of
damage or injury
 Patient histories mention presence of macroscopic versus
microscopic hematuria
 Macroscopic hematuria presence Figure 6-14
o Urine appears cloudy w/ red to brown color
o Associated w/ advanced glomerular damage
o Also seen w/ damage to vascular integrity of urinary
tract caused by trauma, acute infection or
inflammation & coagulation disorders
 Microscopic analysis may be reported in terms of > 100/hpf
or as specified by lab protocol
 Observation of microscopic hematuria can be critical to
A B
early diagnosis of glomerular disorders & malignancy of
Figure 6-15
urinary tract and confirm renal calculi presence
 Presence of not only RBCs but also hyaline, granular & Figure 6–14. RBCs and one WBC (×400). Notice the larger size
RBC casts may be seen following strenuous exercise and granules in the WBC.
o Such abnormalities are nonpathologic & disappear Figure 6–15. WBCs. A. One segmented and one
after rest nonsegmented WBC (×400). B. Notice the multilobed nucleoli
 Possibility of menstrual contamination must also be (×400).
considered in specimens from female patients
 Hemoglobin presence that was filtered by glomerulus EOSINOPHILS
produces red urine w/ positive chemical test result for blood
in absence of microscopic hematuria  Urinary eosinophil presence: associated w/ drug-induced
 Specimen appearing macroscopically normal can contain interstitial nephritis
small but pathologically significant number of RBCs when  Small numbers of eosinophils may be seen w/ urinary tract
examined microscopically infection (UTI) & renal transplant rejection
 To perform urinary eosinophil test, evaluation of
SUMMARY 6-1. Microscopic RBCs concentrated, stained urine sediment is required
Appearance Non-nucleated biconcave disks  Urine sediment may be concentrated by routine
Crenated in hypertonic urine centrifugation alone or w/ cytocentrifugation
Ghost cells in hypotonic urine  Hansel is preferred eosinophil stain but Wright’s stain can
Dysmorphic with glomerular also be used
membrane damage  Eosinophil percentage in 100 to 500 cells is determined
Sources of identification Yeast cells  Eosinophils are not normally seen in urine
error Oil droplets o Finding more than 1% is considered significant
Air bubbles
Reporting Average number per 10 hpfs
Complete urinalysis Color
correlations Reagent strip blood reaction

WHITE BLOOD CELLS

 Larger than RBCs
 Measures an average of about 12 mm in diameter
 Neutrophil
o Predominant WBC found in urine sediment
o Much easier to identify than RBC due to granules
they contain & multilobed nuclei
o Identified w/ high-power microscopy
o Reported as average number seen in 10 hpfs
o Lyse rapidly in dilute alkaline urine & begin to lose
nuclear detail
o Exposed to hypotonic urine absorb water & swell
 Brownian movement of granules w/in these larger cells
produces sparkling appearance referred as glitter cells
o Glitter cells do not have pathological significance
 When stained w/ Sternheimer-Mabin stain, these larger
cells stain light blue as opposed to violet color usually seen
w/ neutrophils
Figure 6-16 Figure 6-17
Figure 6–16. Glitter cells (×400). Observe the very noticeable
granules.
Figure 6–17. Hansel-stained eosinophils (×400).
MONONUCLEAR CELLS
 Lymphocytes, monocytes, macrophages & histiocytes may
be present in small numbers
o Usually not identified in wet preparation urine
microscopic analysis
 Lymphocytes may resemble RBCs since they are the
smallest WBCs
o Can be seen in increased numbers in early stages of
renal transplant rejection
 Monocytes, macrophages & histiocytes: large cells & may
appear vacuolated or contain inclusions
 Specimens w/ increased mononuclear cells w/ch can’t be
identified as epithelial cells are referred for cytodiagnostic
urine testing

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 10

 TRANS: MICROSCOPIC EXAMINATION OF URINE

 Primary concern in WBCs identification: mononuclear cells  Usually at least few squamous epithelial cells are present
disintegration & disintegrating neutrophils from round renal in urine sediment
tubular epithelial (RTE) cells o Serve as good reference for focusing of microscope
o RTE cells are usually larger than WBCs w/  After examination of appropriate number of fields, these
eccentrically located nucleus cells are commonly reported in terms of rare, few,
 WBCs in process of ameboid motion may be difficult to moderate, or many
distinguish from epithelial cells due to its irregular shape o Reported in terms of low-power or high-power
 Supravital staining or addition of acetic acid can be used to magnification based on lab protocol
enhance nuclear detail  Difficulty identifying squamous cells is rare
 Fewer than 5 leukocytes/hpf are found in normal urine o May occasionally appear folded, possibly resembling
o Higher numbers may be present for females cast & begin to disintegrate in urine that isn’t fresh
 Leukocytes are capable of ameboid migration through  In urine sediments containing large amounts of squamous
tissues to site of infection or inflammation aside from its cells, clumps of cells may make it more difficult to
ability to enter urine through glomerular or capillary trauma enumerate smaller pathologic elements like RBCs & WBCs
like RBCs o They should be carefully examined
 Pyuria  These cells originate from linings of vagina & female urethra
o Increased urinary WBCs and lower portion of male urethra
o Indicates presence of infection or inflammation in  Represent normal cellular sloughing & have no pathologic
genitourinary system significance
o Frequent causes of pyuria: bacterial infections  Increased amounts are more frequently seen in urine from
(pyelonephritis, cystitis, prostatitis & urethritis) female patients
o It is also present in nonbacterial disorders  Specimens collected using midstream clean-catch
(glomerulonephritis, lupus erythematosus, interstitial technique contain less squamous cell contamination
nephritis & tumors)  Clue cells
 Reporting bacteria presence in specimens containing o Variation of squamous epithelial cells having
leukocytes is important pathologic significance
o Indicative of vaginal infection by Gardnerella
vaginalis bacterium
o Appearing squamous epithelial cells covered with
Gardnerella coccobacillus
o To be considered as such, bacteria should cover
most of cell surface extending beyond edges of cell
 This gives the cell granular, irregular appearance
 Routine testing for clue cells
o Examining vaginal wet preparation for presence of
Figure 6–18. WBCs with acetic acid nuclear enhancement. characteristic cells
Notice the ameboid shape in some of the WBCs.  Small number of clue cells may be present in urinary
sediment
SUMMARY 6-2. Microscopic WBCs o Microscopists should remain alert for their presence,
Appearance Larger than RBCs as urinalysis may be the first test performed on patient
Granulated, multilobed
neutrophils
Glitter cells in hypotonic urine
Mononuclear cells with
abundant cytoplasm
Sources of identification Renal tubular epithelial cells
error
Reporting Average number per 10 hpfs
Complete urinalysis Leukocyte esterase
correlations Nitrite Figure 6-19 A B
Specific gravity Figure 6-20
pH

EPITHELIAL CELLS
 It is not unusual to find epithelial cells in urine since such
are derived from linings of genitourinary system
 Represents normal sloughing of old cells unless they are in
large number or abnormal forms
 Three types seen in urine: squamous, transitional
(urothelial) & renal tubular Figure 6-21 Figure 6-22 Figure 6-23
 Classified according to site of origin w/in genitourinary
Figure 6–19. Sediment-containing squamous, caudate
system
transitional, and RTE cells (×400).
Figure 6–20. A. Squamous epithelial cells identifiable under low
SQUAMOUS EPITHELIAL CELLS power (×100). B. KOVA-stained squamous epithelial cells
 Largest cells found in urine sediment (×400). Compare the size of the nucleus with the RBCs in Figure
 Contain abundant, irregular cytoplasm & prominent nucleus 6-8.
(about size of RBC) Figure 6–21. Phenazopyridine-stained sediment showing
 Often first structures observed when urine sediment is squamous epithelial cells and phenazopyridine crystals formed
examined under low-power magnification following refrigeration (×400).
DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 11
 TRANS: MICROSCOPIC EXAMINATION OF URINE
Figure 6–22. Clump of squamous epithelial cells (×400).
Figure 6–23. Clump of squamous epithelial cells with folded
forms (×400).
TRANSITIONAL EPITHELIAL (UROTHELIUM) CELLS
 Smaller than squamous cells
 Appear in several forms (spherical, polyhedral & caudate)
 Such differences are caused by ability of transitional
epithelial cells to absorb large amounts of water
o Cells in direct contact w/ urine absorb water,
becoming spherical in form & much larger than
polyhedral and caudate cells
 All forms have distinct, centrally located nuclei
 Transitional cells: identified & enumerated w/ high-power
magnification
o Reported as rare, few, moderate, or many following
lab protocol
 Spherical forms of these cells are sometimes difficult to
distinguish from RTE cells
o Presence of centrally located rather than
eccentrically placed nucleus & supravital staining aid
in differentiation
 Such cells originate from lining of renal pelvis, calyces,
ureters & bladder and from upper portion of male urethra
 Usually present in small numbers in normal urine,
representing normal cellular sloughing
 Increased numbers of transitional cells seen singly, in pairs,
or in clumps (syncytia): present following invasive urologic
procedures (catheterization) & are of no clinical significance
 Transitional cells increase exhibits abnormal morphology
like vacuoles & irregular nuclei may be indicative of
malignancy or viral infection
o In such cases, specimen should be referred to
pathologist
Figure 6-24 Figure 6-25
Figure 6-26 Figure 6-27
Figure 6–24. Transitional epithelial cells.
Figure 6–25. KOVA-stained spherical transitional epithelial cells
(×400).
Figure 6–26. Caudate transitional epithelial cells (×400).
Figure 6–27. Syncytia of transitional epithelial cells from
catheterized specimen (×400).
RENAL TUBULAR EPITHELIAL CELLS
 Vary in size & shape depending on area of renal tubules
from w/ch they originate
 Cells from proximal convoluted tubule (PCT) are larger than
other RTE cells
 They tend to have rectangular shape & referred as
columnar or convoluted cells
 Cytoplasm: coarsely granular
DELA CRUZ, GALANTO
o RTE cells often resemble casts
 They should be closely examined for presence of nucleus
o Nucleus would not be present in cast
 Cells from distal convoluted tubule (DCT) are smaller than
PCT and are round or oval
o Can be mistaken for WBCs & spherical transitional
epithelial cells
 Observation of eccentrically placed round nucleus aids in
differentiating them from spherical transitional cells
 Collecting duct RTE cells: cuboidal & never round
o Along w/ eccentric nucleus, presence of at least one
straight edge differentiates them from spherical and
polyhedral transitional cells
 Nucleus is not easily visible in unstained sedument since
RTE cells present as result of tissue destruction (necrosis)
 Renal fragments: cells from collecting duct appearing in
groups of 3 or more & frequently seen as large sheet of cells
 PCT & DCT cells aren’t seen in large sheet of cells
 RTE cells are identified & enumerated w/ high-power
magnification
o Reported as rare, few, moderate, or many, or as
actual number per high-power field depending on lab
protocol
 RTE cells classification as to site of origin is not considered
part of routine sediment analysis & often requires special
staining techniques
 Presence of more than 2 RTE cells/hpf: tubular injury
o Specimen is referred for cytologic urine testing
Figure 6-28 Figure 6-29
Figure 6-30 Figure 6-31
Figure 6–28. RTE cell. Columnar proximal convoluted tubule
cell with granules and attached fat globules (×400). N, nucleus.
Figure 6–29. RTE cells. Oval distal convoluted tubule cells.
Notice the eccentrically placed nuclei (×400).
Figure 6–30. RTE cells, cuboidal from the collecting duct
(×400).
Figure 6–31. Fragment of RTE cells from the collecting duct
under phase microscopy (×400).
CLINICAL SIGNIFICANCE
 RTE cells: most clinically significant of epithelial cells
o Increased amounts: indicative of necrosis of renal
tubules w/ possibility of affecting overall renal
function
 Conditions producing tubular necrosis: exposure to heavy
metals, drug-induced toxicity, hemoglobin & myoglobin
toxicity, viral infections (hepatitis B), pyelonephritis, allergic
reactions, malignant infiltrations, salicylate poisoning &
acute allogenic transplant rejection
 RTE cells can be seen as secondary effects of glomerular
disorders
CSU BS MLS 3RD YEAR 12
 TRANS: MICROSCOPIC EXAMINATION OF URINE

 Renal fragments: indication of severe tubular injury w/  In acute tubular necrosis, RTE cells containing large,
basement membrane disruption nonlipid-filled vacuoles may be seen w/ normal renal tubular
 Single cuboidal cells are noticeable in salicylate poisoning cells & oval fat bodies
 It usual that RTE cells contain substance from filtrate since  Bubble cells: appear to represent injured cells in w/ch
one of its function is reabsorption of glomerular filtrate endoplasmic reticulum has dilated before cell death
 These cells absorb bilirubin present in filtrate as result of
liver damage, such as occurs w/ viral hepatitis appearing
deep yellow color
 Hemoglobin present in filtrate is absorbed by RTE cells &
converted to hemosiderin
 Following episodes of hemoglobinuria (transfusion
reactions, paroxysmal nocturnal hemoglobinuria, etc.), RTE
cells contain characteristic yellow-brown hemosiderin
granules
o Granules can be seen free-floating in urine sediment Figure 6-33 Figure 6-34 Figure 6-35
 Confirmation of presence of hemosiderin is performed by
staining urine sediment w/ Prussian blue Figure 6–33. Oval fat body (×400).
 Iron-containing hemosiderin granules stain blue Figure 6–34. Sudan III-stained oval fat body (×400).
Figure 6–35. Oval fat body under bright-field (left) and polarized
(right) microscopy. Notice the Maltese cross formation (arrow)
(×400).

SUMMARY 6-3. Epithelial Cells

SQUAMOUS CELLS
Appearance Largest cells in the sediment
with abundant, irregular
cytoplasm and prominent
Figure 6–32. Prussian blue–stained hemosiderin granules. nuclei
Sources of error Rarely encountered, folded
OVAL FAT BODIES cells may resemble casts
Reporting Rare, few, moderate, or many
 RTE cells absorb lipids present in glomerular filtrate per lpf
o They then appear highly refractile & nucleus may be
Complete urinalysis Clarity
more difficult to observe
correlations
o Oval fat bodies: lipid-containing RTE cells
TRANSITIONAL CELLS
 Oval fat bodies are usually seen in conjunction w/ free-
Appearance Spherical, polyhedral, or
floating fat droplets
caudate with centrally located
 Identification: confirmed by staining urine sediment w/
nucleus
Sudan III or Oil Red O fat stains & examining sediment w/
Sources of error Spherical forms resemble RTE
polarized microscopy
cells
 Droplets are composed of triglycerides, neutral fats &
Reporting Rare, few, moderate, or many
cholesterol
per hpf
 Fat stains stain triglycerides & neutral fats
Complete urinalysis Clarity
o Produces orange-red droplets
correlations Blood, if malignancy-
 Examination of urine sediment w/ polarized light: associated
appearance of characteristic Maltese cross formations in
RTE CELLS
droplets containing cholesterol
Appearance Rectangular, columnar, round,
 Urine sediments negative for fat after staining must be
oval or, cuboidal with an
checked w/ polarized light if only cholesterol is present
eccentric nucleus possibly
 Staining must be performed on urine sediments negative bilirubin-stained or
under polarized light hemosiderin-laden
 Oval fat bodies: reported as average number per hpf Sources of error Spherical transitional cells
 Free-floating fat droplets stain or polarize depending on Granular casts
composition Reporting Average number per 10 hpfs
o May be observed floating on top of specimen Complete urinalysis Leukocyte esterase and nitrite
 Care must be taken not to confuse droplets w/ starch & correlations (pyelonephritis)
crystal particles that polarize Color
 Specimen contamination by vaginal preparations & Clarity
lubricants used in specimen collection is considered when Protein Bilirubin (hepatitis)
only free-floating fat droplets are present Blood
 Lipiduria: associated w/ glomerulus damage due to OVAL FAT BODIES
nephrotic syndrome Appearance Highly refractile RTE cells
o Also seen w/ severe tubular necrosis, diabetes Sources of error Confirm with fat stains and
mellitus & in trauma cases causing release of bone polarized microscopy
marrow fat from long bones Reporting Average number per hpf
 In lipid-storage diseases, large fat-laden histiocytes may Complete urinalysis Clarity
also be present correlations Blood
o Differentiated from oval fat bodies by their large size
Protein
Free fat droplets/fatty casts

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 13

 TRANS: MICROSCOPIC EXAMINATION OF URINE
BACTERIA
 Not normally present in urine
 Few bacteria can be present if specimens are collected
under sterile conditions (catheterization) due to vaginal,
urethral, external genitalia or collection-container
contamination
o Such contaminants multiply rapidly in specimens
remaining at room temperature for extended periods,
but have no clinical significance
o Contaminants can produce positive nitrite test result
& result in pH above 8, indicates unacceptable
specimen
 They can be present in form of cocci (spherical) or bacilli
(rods)
 Bacteria are observed w/ high-power magnification due to
their small size
 Reported as few, moderate, or many per hpf
 Bacteria must be accompanied by WBCs to be considered
significant for UTI
 Some labs report bacteria only if observed in fresh
specimens with WBCs
 Presence of motile organisms in drop of fresh urine
collected under sterile conditions correlates well w/ positive
urine culture
 Observing bacteria for motility is useful in differentiating
them from similarly appearing amorphous phosphates &
urates
 Use of phase microscopy aids in visualization of bacteria
 Bacteria presence can be indicative of lower or upper UTI
 Specimens w/ increased bacteria & leukocytes are routinely
followed up w/ specimen for quantitative urine culture
 Bacteria most frequently associated with UTI:
Enterobacteriaceae (referred to as gramnegative rods)
o Cocci-shaped Staphylococcus & Enterococcus can
also cause
 Actual bacteria producing UTI can’t be identified w/
microscopic examination
A B
Figure 6-36
A B
Figure 6-37
Figure 6–36. A. Rod-shaped bacteria often seen in urinary tract
infections. B. KOVA-stained bacteria and WBC (×400).
Figure 6–37. A. Budding yeast B. Yeast showing mycelial forms
(×400).
YEAST
 Yeast cells appear in urine as small, refractile oval structure
o May or may not contain a bud
 In severe infections, they can appear as branched, mycelial
forms
DELA CRUZ, GALANTO
 They are reported as rare, few, moderate or many per hpf
 Differentiation bet. yeast cells & RBCs can be difficult
o Careful observation for budding yeast cells should
be helpful
 Yeast cells, primarily Candida albicans: seen in urine of
diabetic, immunocompromised & women w/ vaginal
moniliasis
 Acidic, glucose-containing urine of diabetic patients
provides ideal medium for yeast growth
 As w/ bacteria, small amount of yeast entering specimen as
contaminant multiplies rapidly if specimen isn’t examined
while fresh
 True yeast infection must be accompanied by WBC
presence
PARASITES
 Trichomonas vaginanalis
o Most frequently encountered parasite in urine
o Trophozoite: pear shaped flagellate w/ undulating
membrane
o Easily identified in wet preparation of urine sediment
by rapid darting movement in microscopic field
o Usually reported as rare, few, moderate or many/hpf
o It is difficult to identify when not moving
 It can resemble WBC, transitional or RTE cells
o Phase microscopy can enhance visualization of
flagella and undulating membrane
o Sexually transmitted pathogen associated w/ vaginal
inflammation
o Infection of male urethra & prostate is asymptomatic
o Males are asymptomatic carriers
 Schistosoma haematobium
o Ova of such appears in urine
o Associated w/ bladder cancer in other countries
 Fecal contamination in urine specimen can result in
presence of ova from intestinal parasite in urine sediment
o Enterobius vermicularis ova: most common
contaminant
Figure 6-38 Figure 6-39
A B
Figure 6-40
Figure 6–38. Trichomonas vaginalis. Notice the flagella and
undulating membrane. (From Leventhal and Cheadle, Ed 6, p 87).
Figure 6–39. Schistosoma haematobium ova (×300). Eggs are often
contained in the last few drops of urine expelled from the bladder.
Figure 6–40. A. Enterobius vermicularis ova (×100) B. Enterobius
vermicularis ova (×400).
SPERMATOZOA
 Easily identified in urine sediment
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 TRANS: MICROSCOPIC EXAMINATION OF URINE

o Oval, slightly tapered heads & long, flagella-like tails Complete pH

 Urine is toxic to spermatozoa urinalysis Nitrite
o Thereby, they rarely exhibit motility observed when correlations LE
examining semen specimen WBCs
 Occasionally found in urine of men and women following YEAST
sexual intercourse, masturbation or nocturnal emission Appearance Small, oval, refractile structures with buds
 Rarely of clinical significance and/or mycelia
o Except in cases of male infertility or retrograde Sources of error RBCs
ejaculation where sperm is expelled in bladder Reporting Rare, few, moderate, or many per hpf, the
instead of urethra presence of WBCs may be required
 Positive reagent strip test for protein may be seen when Complete Glucose
increased amounts of semen are present urinalysis LE
 Lab protocols vary w/ regards to report or not the presence correlations WBCs
of spermatozoa in urine specimen TRICHOMONAS
o Labs not reporting its presence cite lack of clin Appearance Pear-shaped, motile, flagellated
significance and possible legal consequence Sources of error WBCs, renal tubular epithelial cells
o Labs supporting spermatozoa reporting cite possible Reporting Rare, few, moderate, or many per hpf
clinical significance and minimal possibility of legal Complete LE
consequences urinalysis WBCs
correlations
SPERMATOZOA
Appearance Tapered oval head with long, thin tail
Sources of error None
Reporting Present, based on laboratory protocol
Complete Protein
urinalysis
correlations
Figure 6–41. Spermatozoa (×400) MUCUS
Appearance Single or clumped threads with a low
MUCUS refractive index
 Protein material produced by glands & epithelial cells of Sources of error Hyaline casts
lower genitourinary tract & RTE cells Reporting Rare, few, moderate, or many per lpf
 Uromodulin Complete None
o Glycoprotein excreted by RTE cells of distal urinalysis
convoluted tubules & upper collecting ducts correlations
o Major constituent of mucus as showed by
immunologic analysis
CASTS
 Mucus appears microscopically as thread-like structures w/
 Only elements found in urinary sediment unique to kidney
low refractive index
 Formed w/in lumens of distal convoluted tubules &
 Subdued light is required when using bright-field
collecting ducts
microscopy
o Provides microscopic view of conditions w/in
 Care must be taken to not confuse clumps of mucus w/
nephron
hyaline casts
 Shape is representative of tubular lumen w/ parallel sides &
o Differentiation can be made by observing irregular
somewhat rounded ends & may contain additional elements
appearance of mucus threads
present in filtrate
 Mucus threads are reported as rare, few, moderate, or
 Examination of sediment for casts’ detection is done w/
many per lpf.
lower power magnification
 It is more frequently present in female urine specimens
 When glass cover-slip method is used, low-power scanning
 No clinical significance when present in female or male
is performed along edges of cover slip
urine
 Observation under subdued light is essential
o Since cast matrix has low refractive index
 Cast matrix dissolves quickly in dilute, alkaline urine like
other sediment constituents
 Casts must be further identified as composition w/ high-
power magnification once detected
 They are reported as average number per 10 lpfs

A B CAST COMPOSITION AND FORMATION

Figure 6-42. A. Mucus threads (×400). B. Mucus clump (×400).
SUMMARY 6-4. Miscellaneous Structures
BACTERIA
Appearance Small spherical and rod-shaped structures
Sources of error Amorphous phosphates and urates
Reporting Few, moderate, or many per hpf, the
presence of WBCs may be required
 Uromodulin
o Major constituent of casts
o Excreted at relatively constant rate under normal
conditions
 Excretion rate appears to be increased under
conditions of stress & exercise, accounting for
transient appearance of hyaline casts when these
conditions are present
 Other proteins present in urinary filtrate like albumin &
immunoglobulins are incorporated into cast matrix

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 15

 TRANS: MICROSCOPIC EXAMINATION OF URINE
 Protein gels more readily under conditions of urine-flow
stasis, acidity & presence of sodium and calcium
 Extent of protein glycosylation is important
 Uromodulin protein is found in normal and abnormal urine
 It is not detected by reagent strip protein methods
o Thereby, increased urinary protein frequently
associated with presence of casts is caused by
underlying renal conditions
 Scanning electron microscope studies provided step-by-
step analysis of formation of uromodulin protein matrix:
1. Aggregation of uromodulin protein into individual protein
fibrils attached to the RTE cells
2. Interweaving of protein fibrils to form a loose fibrillar
network (urinary constituents may become enmeshed in the
network at this time)
3. Further protein fibril interweaving to form a solid structure
4. Possible attachment of urinary constituents to the solid
matrix
5. Detachment of protein fibrils from the epithelial cells
6. Excretion of the cast
 As cast forms, urinary flow w/in tubule decreases as lumen
becomes blocked
 Accompanying dehydration of protein fibrils & internal
tension may account for wrinkled & convoluted appearance
of older hyaline casts
 Cast width depends on tubule size being formed
 Broad casts may result from tubular distension or in case of
extreme urine stasis, from formation in collecting ducts
 Cast formation at junction of ascending loop of Henle &
distal convoluted tubule may produce structures w/ tapered
end
o Termed as cylindroids w/ same significance as casts
 Cylindruria: presence of urinary cast
o Appearance of cast is influenced by materials
present in filtrate at time of its formation & length of
time remaining in the tubule
 Any elements present in tubular filtrate including cells,
bacteria, granules, pigments & crystals may become
embedded in or attached to cast mix
 Types of casts found in sediment represent different clinical
conditions
HYALINE CASTS
 Most frequently seen cast
 Consists almost entirely of uromodulin
 Presence of 0 – 2 hyaline casts/lpf & finding of increased
numbers following strenuous exercise, dehydration, heat
exposure & emotional stress are considered normal
 Pathologically, these casts are increased in acute
glomerulonephritis, pyelonephritis, chronic renal disease &
congestive heart failure
 It appear colorless in unstained sediments & have refractive
index similar to urine
o Easily overlooked if specimens are not examined
under subdued light
 Sternheimer-Malbin stain produces pink color in hyaline
casts
 Increased visualization can be obtained by phase
microscopy
 Morphology of these casts is varied
o It consist of normal parallel sides & rounded ends,
cylindroid forms & wrinkled or convoluted shapes
indicating cast matrix aging
 Presence of occasional adhering cell or granule may be
observed
o It does not change cast classification
DELA CRUZ, GALANTO
Figure 6-43 Figure 6-44
A B
Figure 6-45
Figure 6-46 Figure 6-47
Figure 6–43. Hyaline casts under low power (×100).
Figure 6–44. Hyaline cast (A) and amorphous urates (B)
attached to mucus pseudocast (×100).
Figure 6–45. A. Hyaline cast (×400). B. Hyaline cast under
phase microscopy (×400).
Figure 6–46. Convoluted hyaline cast (×400).
Figure 6–47. Hyaline cast containing occasional granules (×400)
RBC CASTS
 RBCs finding in urine indicates bleeding from area w/in
genitourinary tract
o Presence of RBC casts is much more specific,
showing bleeding w/in nephron
 RBC casts are associated w/ damage to glomerulus
(glomerulonephritis) allowing passage of cells through
glomerular membrane
o Any damage to nephron capillary structure causes
their formation
 RBC casts associated w/ glomerular damage are
associated w/ proteinuria & dysmorphic erythrocytes
 RBC casts have been observed in healthy individuals
following participation in strenuous contact sports
 They are easily detected under low power by their orange-
red color
 More fragile than other casts and may exist as fragments or
have more irregular shape
o Result of tightly packed cells adhering to protein
matrix
 Examination under high-power magnification should
concentrate on determining that cast matrix is present
o Differentiating structure from clump of RBCs
 Actual RBCs’ presence must be verified, preventing
inaccurate reporting of nonexistent RBC cast due to its
serious diagnostic implication
 Highly improbable that RBC casts will be present in
absence of free-standing RBCs & positive reagent strip test
for blood
 As an RBC cast ages, cell lysis begins & cast develops
more homogenous appearance, retaining orange-red color
from released hemoglobin
CSU BS MLS 3RD YEAR 16
 TRANS: MICROSCOPIC EXAMINATION OF URINE

 These casts may be distinguished as blood casts, indicating o These structures must be carefully examined to
greater stasis of urine flow determine that cast matrix is present
o It is not necessary since all casts containing blood  WBCs frequently form clumps
have same clinical significance o Do not have same significance as casts
 Both types of casts are reported as number of RBC casts/lpf
 Homogenous orange-red or red-brown casts may be
observed in presence of massive hemoglobinuria or
myoglobinuria
 Granular, dirty, brown casts representing hemoglobin
degradation products like methemoglobin may be present
o Associated w/ acute tubular necrosis caused by toxic
effects of massive hemoglobinuria
 Leads to renal failure
 These dirty, brown casts must be present with other Figure 6-53 Figure 6-54
pathologic findings like RTE cells & positive reagent strip
test for blood

Figure 6-55 Figure 6-56

Figure 6–53. WBC cast. Notice the free WBCs to aid in

Figure 6-48 Figure 6-49 Figure 6-50 identification.
Figure 6–54. KOVA-stained WBC cast (×400).
Figure 6–55. Disintegrating WBC cast (×400).
Figure 6–56. WBC clump. Notice the absence of a cast matrix

BACTERIAL CASTS
 Such casts containing bacilli w/in & bound to protein matrix
are seen in pyelonephritis
 They may be pure bacterial casts or mixed w/ WBCs
Figure 6-51 Figure 6-52  Identification of these casts can be difficult
o Packed casts packed w/ bacteria can resemble
Figure 6–48. RBC cast (×400). granular casts
Figure 6–49. KOVA-stained RBC cast under phase microscopy
 Their presence is considered when WBC casts & many free
(×400).
WBCs and bacteria are seen in sediment
Figure 6–50. Disintegrating RBC cast. Notice the presence of
 Bacterial casts’ confirmation: best made by performing
free RBCs (arrows) to confirm identification.
Gram stain on dried or cytocentrifuged sediment
Figure 6–51. Cast containing hemoglobin pigment. A
comparison of RBCs (A) and yeast (B) also can be made (×400).
Figure 6–52. Granular, dirty, brown cast (×400). EPITHELIAL CELL CASTS
 Casts containing RTE cells represents advanced tubular
WBC CASTS destruction presence
o Produces urinary stasis along w/ disruption of tubular
 Its appearance in urine signifies infection or inflammation
linings
w/in nephron
 They are associated w/ heavy metal & chemical or drug-
 Most frequently associated w/ pyelonephritis
induced toxicity, viral infection & allograft rejection similar to
 Primary marker for distinguishing pyelonephritis (upper
RTE cells
UTI) from cystitis (lower UTI)
 They accompany WBC casts in cases of pyelonephritis
 They are present in nonbacterial inflammation like acute
 Fibrils of uromodulin protein making up cast matrix remain
interstitial nephritis & may accompany RBC casts in
attached to RTE cells producing them
glomerulonephritis
o Observation of occasional tubular cell attached to
 Visible under low-power magnification
hyaline cast can be expected
o Can also be positively identified w/ high power
 When tubular damage is present, some cells may be
 Most frequently, they are composed of neutrophils,
incorporated into cast matrix
therefore, they may appear granular & multilobed nuclei will
o The majority will be very noticeably attached to cast
be present unless disintegration has occurred
surface
o Supravital staining can demonstrate characteristic
 Cells visible on cast matrix are smaller, round & oval cells
nuclei
owing to cast formation in distal convoluted tubule
 Particularly helpful for differentiating WBC casts from
o They may be difficult to differentiate from WBCs
RTE casts
especially if degeneration occurred
 Observation of free WBCs in sediment is essential
 Staining & phase microscopy use can help to enhance
 Bacteria are present in cases of pyelonephritis but are not
nuclear detail for identification
present w/ acute interstitial nephritis
 Fragments of epithelial tissue may be attached to cast
o Eosinophil casts may be present in appropriately
matrix
stained specimens (Hansel & Wright’s stain)
 Bilirubin-stained RTE cells are seen in cases of hepatitis
 Casts tightly packed w/ WBCs may have irregular borders

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 TRANS: MICROSCOPIC EXAMINATION OF URINE
Figure 6-57 A B
Figure 6-58
Figure 6-59
Figure 6–57. RTE cell cast (×400).
Figure 6–58. A. KOVA-stained RTE cell cast (×400). B. KOVA-
stained RTE cell cast under phase microscopy (×400).
Figure 6–59. RTE cast with bilirubin-stained cells (×400).
FATTY CASTS
 Seen w/ oval fat bodies & free fat droplets in disorders
causing lipiduria
 Most frequently associated w/ nephrotic syndrome
o It can also be seen in toxic tubular necrosis, diabetes
mellitus & crush injuries
 Highly refractile under bright-field microscopy
 Cast matrix may contain few or many fat droplets & intact
oval fat bodies may be attached to matrix
 Confirmation of such is performed w/ polarized microscopy
& Sudan III or Oil Red O fat stains
 Cholesterol demonstrates Maltese cross formations under
polarized light & triglycerides & neutral fats stain orange w/
fat stains
 Fats do not stain with Sternheimer-Malbin stain
Figure 6-60 Figure 6-61 Figure 6-62
Figure 6–60. Fatty cast showing adherence of fat droplets
(arrows) to cast matrix (×400).
Figure 6–61. Fatty cast (×400).
Figure 6–62. Fatty cast under phase microscopy (×400).
MIXED CELLULAR CASTS
 Observing casts containing multiple cell types is common
o Considering that variety of cells may be present in
urinary filtrate
 Mixed cellular casts most frequently encountered: RBC and
WBC casts in glomerulonephritis & WBC & RTE cell casts,
or WBC & bacterial casts in pyelonephritis
 Mixed elements present in cast can make identification
more difficult
o Staining or phase microscopy aids in the
identification
 There must be homogenous cat of at least one of the cell
types when mixed casts are present
DELA CRUZ, GALANTO
o They will be the primary diagnostic marker
 Example: in glomerulonephritis, predominant casts will be
RBC & in pyelonephritis, predominant casts will be WBC
 Bacteria are often incorporated into WBC casts
o Provide little additional diagnostic significance
 Lab protocol should be followed in reporting of mixed
cellular casts
GRANULAR CASTS
 Coarsely & finely granular casts are frequently seen in
urinary sediment
o May be of pathologic or nonpathologic significance
 Not considered necessary to distinguish bet. coarsely &
finely granular casts
 Origin of granules in nonpathologic conditions appears to
be from lysosomes excreted by RTE cells during normal
metabolism
 It is usual to see hyaline casts containing one or two of
these granules
 Increased cellular metabolism occurring during periods of
strenuous exercise accounts for transient increased
granular casts accompanying increased hyaline casts
 In disease states, granules may represent disintegration of
cellular casts & tubule cells or protein aggregates filtered by
glomerulus
 Scanning electron microscope studies confirmed granular
casts are seen w/ WBC casts containing WBC granules of
varying sizes
 Urinary stasis that allows cast to remain in tubules must be
present for granules resulting from disintegration of cellular
casts
 Granular casts occurring as result of cellular disintegration
may contain occasional recognizable cell
 They are easily visualized under low-power microscopy
o Final identification must be performed using high
power to determine presence of cast matrix
 Artifacts like clumps of small crystals & fecal debris may
occur in shapes resembling casts
o It must be differentiated
 Columnar RTE cells may resemble granular casts &
staining for nuclear detail may be required
 If granular casts remain in tubules for extended periods,
granules further disintegrate & cast matrix develops waxy
appearance
o Structure becomes more rigid, ends of casts may
appear jagged or broken & diameter becomes
broader
Figure 6-63 Figure 6-64 Figure 6-65
Figure 6-66 Figure 6-67
Figure 6–63. Finely granular cast (A) and uric acid crystals (B)
(×400)
Figure 6–64. Granular cast formed at a tubular bend (×400).
CSU BS MLS 3RD YEAR 18
 TRANS: MICROSCOPIC EXAMINATION OF URINE

Figure 6–65. Granular disintegrating cellular cast (×400). Complete urinalysis Protein
Figure 6–66. Coarsely granular cast (A), squamous epithelial correlations Blood (exercise)
cell (B), and mucus (C) (×400). Color (exercise)
Figure 6–67. Granular cast degenerating into waxy cast (×400). Clinical Significance Glomerulonephritis
Pyelonephritis Chronic renal
WAXY CASTS disease Congestive heart
 Representative of extreme urine stasis, indicating chronic failure Stress and exercise
renal failure RBC
 Usually seen w/ other types of casts associated w/ Appearance Orange-red color, cast matrix
conditions that caused renal failure containing RBCs
 Brittle, highly refractive cast matrix from w/ch these casts Sources of error RBC clumps
derive their name is believed to be caused by degeneration Reporting Average number per lpf
of hyaline cast matrix & any cellular elements or granules Complete urinalysis RBCs
contained in matrix correlations Blood
 These casts are more easily visualized than hyaline casts Protein
due to their higher refractive index Clinical Significance Glomerulonephritis
 They often appear fragmented w/ jagged ends & have Strenuous exercise
notches in their sides as result of brittle consistency of cast WBC
matrix Appearance Cast matrix containing WBCs
 Waxy casts stain homogenous, dark pink w/ supravital Sources of error WBC clumps
stains Reporting Average number per lpf
Complete urinalysis WBCs
correlations Protein
LE
Clinical Significance Pyelonephritis
Acute interstitial nephritis
BACTERIAL
Appearance Bacilli bound to protein matrix
Sources of error Granular casts
Figure 6-68 Figure 6-69 Figure 6-70 Reporting Average number per lpf
Complete urinalysis WBC casts (pyelonephritis)
Figure 6–68. KOVA-stained waxy casts (×100). correlations WBCs
Figure 6–69. KOVA-stained waxy casts (×200). LE
Figure 6–70. KOVA-stained waxy cast (×400). Nitrite
Protein
BROAD CASTS Bacteria
Clinical Significance Pyelonephritis
 Often referred to as renal failure casts EPITHELIAL CELL
 Like waxy casts represent extreme urine stasis Appearance RTE cells attached to protein
 As mold of distal convoluted tubules, broad casts’ presence matrix
indicates destruction (widening) of tubular walls Sources of error WBC cast
 Casts form in the area & appear broad when flow of urine Reporting Average number per lpf
to larger collecting ducts becomes severely compromised Complete urinalysis Protein
 All types of casts may occur in broad form correlations RTE cells
o But considering accompanying urinary stasis, most Clinical Significance Renal tubular damage
commonly seen broad casts are granular & waxy GRANULAR
 Bile-stained broad, waxy casts are seen as result of tubular Appearance Coarse and fine granules in a
necrosis due to viral hepatitis
cast matrix
Sources of error Clumps of small crystals
Columnar RTE cells
Reporting Average number per lpf
Complete urinalysis Protein
correlations Cellular casts
RBCs
WBCs
Figure 6-71 Figure 6-72 Figure 6-73 Clinical Significance Glomerulonephritis
Pyelonephritis
Figure 6–71. KOVA-stained broad waxy cast (×400). Stress and exercise
Figure 6–72. Broad granular cast becoming waxy (×400). WAXY
Figure 6–73. Broad bile-stained waxy cast (×400). Appearance Highly refractile cast with
jagged ends and notches
SUMMARY 6-5. Urine Casts Sources of error Fibers and fecal material
HYALINE Reporting Average number per lpf
Appearance Colorless, homogenous matrix Complete urinalysis Protein
Sources of error Mucus, fibers, hair, increased correlations Cellular casts
lighting Granular casts
Reporting Average number per lpf WBCs
RBCs

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 19

 TRANS: MICROSCOPIC EXAMINATION OF URINE

Clinical Significance Stasis of urine flow GENERAL IDENTIFICATION TECHNIQUES

Chronic renal failure  Most commonly seen crystals have very characteristic
FATTY shapes & colors
Appearance Fat droplets and oval fat bodies o Variations occur & can present identification
attached to protein matrix problems, especially when they resemble abnormal
Sources of error Fecal debris crystals
Reporting Average number per lpf  Urine pH is the first consideration when identifying crystals
Complete urinalysis Protein  Crystals are routinely classified not only as normal &
correlations Free fat droplets abnormal, but also as to their appearance in acidic or
Oval fat bodies alkaline urine
Clinical Significance Nephrotic syndrome  All abnormal crystals are found in acidic urine
Toxic tubular necrosis  Additional aids in crystal identification: use of polarized
Diabetes mellitus microscopy & solubility characteristics of crystals
Crush injuries  Geometric shape of crystal determines its birefringence &
BROAD its ability to polarize light
Appearance Wider than normal cast matrix  Basic structure of crystals remains the same even size of
Sources of error Fecal material, fibers particular crystal may vary (slower crystallization produces
Reporting Average number per lpf larger crystals)
Complete urinalysis Protein o Polarization characteristics for a particular crystal
correlations WBCs are constant for identification purposes
RBCs  Reversal of changes can cause crystals to dissolve just as
Granular casts changes in temperature & pH contribute to crystal formation
Waxy casts o These solubility characteristics can be used to aid in
Clinical Significance Extreme urine stasis identification
Renal failure  Amorphous urates that usually form in refrigerated
specimens & obscure sediments may dissolve if specimen
URINARY CRYSTALS is warmed
 Crystals frequently found in urine are rarely of clinical  Amorphous phosphates needs acetic acid to dissolve
significance o This is not practical, as formed elements like RBCs
 May appear as true geometrically formed structures or as will be destroyed
amorphous material  When solubility characteristics are needed for identification,
 Primary reason for urinary crystals identification: detection sediment must be aliquoted preventing destruction of other
of presence of relatively few abnormal types that may elements
represent disorders as liver disease, inborn errors of
metabolism, or renal damage due to crystallization of NORMAL CRYSTALS SEEN IN ACIDIC URINE
medications compounds w/in tubules
 Urates
 Usually reported as rare, few, moderate, or many per hpf o Most common crystal seen in acidic urine
 Abnormal crystals may be averaged and reported per lpf o Consists of amorphous urates, uric acid, acid urates
& sodium urates
CRYSTAL FORMATION  Most urate crystals appear yellow to reddish brown
 Crystals are formed by precipitation of urine solutes microscopically and are the only normal crystals found in
including inorganic salts, organic compounds & acidic urine appearing colored
medications (iatrogenic compounds)  Amorphous urates
 Precipitation is subject to changes in temperature, solute o Microscopically appears as yellowbrown granules
concentration, & pH affecting solubility o May occur in clumps resembling granular casts &
 Solutes precipitate more readily at low temperatures attached to other sediment structures
o Majority of crystal formation takes place in o Frequently encoutered in specimens having been
specimens that have remained at room temperature refrigerated, producing very characteristic pink
or been refrigerated prior to testing sediment
 Crystals are extremely abundant in refrigerated specimen  Accumulation pigment uroerythrin causes the pink
o It often present problems since they obscure color
clinically significant sediment constituents o Found in acidic urine w/ pH greater than 5.5
 As urinary solutes concentration increases, its ability to  Uric acid crystals
remain in solution decreases o Appears when pH is lower
o Results in crystal formation o Seen in variety of shapes (includes rhombic, four-
 Crystals’ presence in freshly voided urine is the most sided flat plates (whetstones), wedges & rosettes)
frequently associated w/ concentrated (high specific o Usually appears yellow- brown but can be colorless
gravity) specimens & have six-sided shape similar to cystine crystals
 pH of specimen is a valuable aid in crystals’ identification o Highly birefringent under polarized light, aids in
o Determines type of chemicals precipitated distinguishing them from cystine crystals
 Organic and iatrogenic compounds crystallize more easily o Increased amount of such, particular to fresh urine
in acidic pH are associated w/ increased levels of purine &
 Inorganic salts are less soluble in neutral & alkaline solution nucleic acids & are seen in patients w/ leukemia
o Exception to calcium oxalate, where it precipitates in receiving chemotherapy, in patients w/ Lesch-Nyhan
acidic & neutral urine syndrom & sometimes in patients w/ gout
 Acid urates & sodium urates
o Rarely encountered & similar to amorphous urates,
seen in less acidic urine
o Frequently seen w/ amorphous urates
DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 20
 TRANS: MICROSCOPIC EXAMINATION OF URINE
o Have little clinical significance
o Acid urates appear as larger granules & may have
spicules like thaat of ammonium biurate crystals
seen in alkaline urine
o Sodium urate crystals are needle-shaped & seen in
synovial fluid during episodes of gout but can also
appear in urine
 Calcium oxalate crystals
o Frequently seen in acidic urine, but they can be
found in neutral urine & even rarely in alkaline urine
o Dihydrate: most common form of calcium oxalate
crystals that is easily recognized as a colorless,
octahedral envelope or as two pyramids joined at
their bases
o Monohydrate calcium oxalate crystals: less
characteristic and less frequently seen & are oval or
dumbbell shaped
o Both forms are birefringent under polarized light
 May be helpful in distinguishing monohydrate from
nonpolarizing RBCs
o Sometimes seen in clumps attached in clumps
attached to mucous strand and may resemble casts
o Clumps finding of these crystals in fresh urine can be
related to formation of renal calculi since majority of
renal calculi are composed of calcium oxalate
 They are also associated w/ food high in oxalic
acid like tomatoes and asparagus, & ascorbic acid
since oxalic acid is end product of ascorbic acid
metabolism
o Primary pathologic significance: very noticeable
presence of monohydrate form in cases of ethylene
glycol (antifreeze) poisoning
 Monohydrate form is most frequently seen in
children & pets since antifreeze tastes sweet &
uncovered containers left in garage can be very
tempting (massive amounts of crystals are
frequently produced in these cases)
Figure 6-74 Figure 6-75 Figure 6-76
Figure 6-77 A B
Figure 6-78
Figure 6-79 Figure 6-80
Figure 6–74. Amorphous urates (×400).
Figure 6–75. Amorphous urates attached to a fiber.
Figure 6–76. Uric acid crystals (×400).
DELA CRUZ, GALANTO
Figure 6–77. Clump of uric acid crystals (×400). Notice the
whetstone, not hexagonal, shape that differentiates uric acid
crystals from cystine crystals.
Figure 6–78. A. Uric acid crystals under polarized light (×100).
B. Uric acid crystals under polarized light (×400).
Figure 6–79. Classic dihydrate calcium oxalate crystals (×400)
Figure 6–80. Classic dihydrate calcium oxalate crystals under
phase microscopy (×400).
Figure 6-81 Figure 6-82
Figure 6–81. Attached classic dihydrate calcium oxalate
crystals (×400).
Figure 6–82. Monohydrate calcium oxalate crystals (×400)
NORMAL CRYSTALS SEEN IN ALKALINE URINE
 Phosphates represent majority of crystals seen in alkaline
urine including amorphous phosphate, triple phosphate &
calcium phosphate
 Other normal crystals associated w/ alkaline urine: calcium
carbonate & ammonium biurate
 Amorphous phosphates: granular in appearance like
amorphous urates
o When present in large quantities following specimen
refrigeration, they cause white precipitate that
doesn’t dissolve on warming
o Differentiated from amorphous urates by color of
sediment & urine pH
 Triple phosphate (ammonium magnesium phosphate)
crystals
o Commonly seen in alkaline urine
o In their routine form, they are easily identified by their
prism shape that frequently resembles “coffin lid”
o As they disintegrate, crystals may develop feathery
appearance
o Birefringent under polarized light
o No clinical significance but are often seen in highly
alkaline urine associated with presence of
ureasplitting bacteria
 Calcium phosphate crystals
o Not frequently encountered
o May appear as colorless, flat rectangular plates or
thin prisms often in rosette formations
 Rosette forms may be confused w/ sulfonamide
crystals when urine pH is in neutral range
o Dissolve in dilute acetic acid & sulfonamides do not
o No clinical significance even calcium phosphate is
common constituent of renal calculi
 Calcium carbonate crystals
o Small & colorless w/ dumbbell or spherical shapes
o May occur in clumps resembling amorphous
material, but they can be distinguished by formation
of gas after addition of acetic acid
o Birefringent which differentiates them from bacteria
o No clinical significance
 Ammonium biurate crystals
o Exhibit characteristic yellow-brown color of urate
crystals seen in acidic urine
o Frequently described as “thorny apples” due to their
appearance as spicule-covered spheres
o Except for their occurrence in alkaline urine, these
crystals resemble other urates in that they dissolve
CSU BS MLS 3RD YEAR 21
 TRANS: MICROSCOPIC EXAMINATION OF URINE
at 60°C & convert to uric acid crystals when glacial
acetic acid is added
o Almost always encountered in old specimens
o May be associated w/ presence of ammonia
produced by urea-splitting bacteria
Figure 6-83 Figure 6-84 Figure 6-85
Figure 6-86 Figure 6-87 Figure 6-88
A B Figure 6-90
Figure 6-89
Figure 6–83. Amorphous phosphates (×400). Urine pH 7.0.
Figure 6–84. Amorphous phosphates (×400).
Figure 6–85. Triple phosphate crystal (×400).
Figure 6–86. Triple phosphate crystals (arrow) and amorphous
phosphates (×400).
Figure 6–87. Calcium carbonate crystals (×400).
Figure 6–88. Ammonium biurate crystals (×400). Notice the “thorny
apple” appearance. (Courtesy of Kenneth L. McCoy, MD.)
Figure 6–89. Ammonium biurate crystals A. Ammonium biurate and
triple phosphate crystals (×100). Note thorn (arrow). B. Ammonium
biurate and triple phosphate crystals (×400).
Figure 6–90. Ammonium biurate crystals (×400). Note thorns (arrow).
ABNORMAL URINE CRYSTALS
 Found in acidic urine or rarely in neutral urine
 Most of them have very characteristic shapes
o Their identity can be confirmed by patient
information including disorders & medication
 Iatrogenic crystals can be caused by variety of compounds,
particularly when administered in high concentrations
 They may be of clinical significance when they precipitate
in renal tubules
CYSTINE CRYSTALS
 Found in urine of persons inheriting metabolic disorder
preventing reabsorption of cystine by renal tubules
(cystinuria)
o Persons w/ cystinuria have tendency to form renal
calculi, particularly at an early age
 Appear as colorless, hexagonal plates
 May be thick or thin
o Only thick cystine crystals have polarizing capability
 Disintegrating forms may be seen in presence of ammonia
 May be difficult to differentiate from colorless uric acid
crystals
DELA CRUZ, GALANTO
 Uric acid crystals: very birefringent under polarized
microscopy
 Positive confirmation of cystine crystals: made w/ cyanide-
nitroprusside test
Figure 6-91 Figure 6-92
Figure 6–91. Cystine crystals (×400).
Figure 6–92. Clump of cystine crystals (×400). Notice the
hexagonal shape still visible.
CHOLESTEROL CRYSTALS
 Rarely seen unless specimens have been refrigerated due
to lipids remaining in droplet form
o But when observed, they have most characteristic
appearance, resembling rectangular plate w/ notch
in one or more corners
 They are associated with disorders producing lipiduria like
nephrotic syndrome
 Seen w/ fatty casts & oval fat bodies
 Highly birefringent w/ polarized light
Figure 6-93 Figure 6-94
Figure 6–93. Cholesterol crystals. Notice the notched corners (×400).
Figure 6–94. Cholesterol crystals under polarized light (×400).
RADIOGRAPHIC DYE CRYSTALS
 Crystals of radiographic contrast media have very similar
appearance to cholesterol crystals and is highly birefringent
 Differentiation is best made by comparison of other
urinalysis results & patient history
 Specific gravity of specimen containing radiographic
contrast media is markedly elevated when measured by
refractometer
CRYSTALS ASSOCIATED WITH LIVER DISORDERS
 Three rarely seen crystals may be found in urine sediment
in presence of severe liver disorders: tyrosine, leucine &
bilirubin
 Tyrosine crystals
o Appear as fine colorless to yellow needles frequently
forming clumps or rosettes
o Usually seen w/ leucine crystals in specimens w/
positive chemical test result for bilirubin
o May be encountered in inherited disorders of amino
acid metabolism
Figure 6-95 Figure 6-96
CSU BS MLS 3RD YEAR 22
 TRANS: MICROSCOPIC EXAMINATION OF URINE

Figure 6–95. Tyrosine crystals in fine needle clumps (×400).
Figure 6–96. Tyrosine crystals in rosette forms (×400).
 Leucine crystals
o Yellow-brown spheres demonstrating concentric
circles & radial striations
o Less frequently seen than tyrosine crystals
o When present, must be accompanied by tyrosine
crystals
Figure 6–99. Sulfa crystals in rosette form (×400).
Figure 6–100. Sulfa crystals, WBCs, and bacteria seen in UTI
(×400).
AMPICILLIN CRYSTALS
 Precipitation of antibiotics is not frequently encountered
except for rare observation of these crystals following
massive doses of this penicillin compound w/out adequate
hydration
 Appears colorless needles tending to form bundles
following refrigeration
 Knowledge of patient’s history can aid in identification

Figure 6–97. Leucine crystals (×400). Notice the concentric circles.

 Bilirubin crystals
o Present in hepatic disorders producing large amount
of bilirubin in urine A B
o Appear as clumped needles or granules w/ Figure 6-101. Ampicillin crystals. A. Nonrefrigerated ampicillin
characteristic yellow color of bilirubin crystals. (×400). B. Ampicillin crystals after refrigeration (×400).
o Positive chemical test result for bilirubin would be
expected Table No. 6-6. Major Characteristics of Normal Urinary
o In disorders producing renal tubular damage like Crystals
viral hepatitis, these crystals may be found CRYSTAL pH COLOR APPEARANCE
incorporated into matrix of casts Uric acid Acid Yellow-
brown
(rosettes,
wedges)
Amorphous Acid Brick dust
urates or yellow
brown
Calcium Acid/neutral Colorless
oxalate (alkaline) (envelopes,
oval,
Figure 6–98. Bilirubin crystals. Notice the classic bright yellow dumbbell)
color (×400). Amorphous Alkaline/ White–
phosphates neutral colorless
SULFONAMIDE CRYSTALS
 Findings of these crystals in urine of patients treated for UTI
was common before development of more soluble Calcium Alkaline/ Colorless
sulfonamides phosphate neutral
 Primary cause of sulfonamide crystallization: inadequate
patient hydration Triple Alkaline Colorless
 Appearance of such crystals in fresh urine suggests phosphate (“coffin
possibility of tubular damage if crystals are forming in lids”)
nephron Ammonium Alkaline Yellow-
 Variety of sulfonamide medications are on the market biurate brown
o One can expect to encounter variety of crystal (“thorny
shapes and colors apples”)
 Shapes: most frequently encountered include needles, Calcium Alkaline Colorless
rhombics, whetstones, sheaves of wheat & rosettes w/ carbonate (dumbbells)
colors ranging from colorless to yellow-brown
 Check of the patient’s medication history aids in
identification confirmation
Table No. 6-7. Major Characteristics of Abnormal Urinary
Crystals
CRYSTAL pH COLOR/ DIS- APPEARANCE
FORM ORDERS
Cystine Acid Colorless Inherited
(hexagon cystinuria
al plates)
Choles- Acid Colorless Nephrotic
terol (notched syndrome
Figure 6-99 Figure 6-100 plates)

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 23

 TRANS: MICROSCOPIC EXAMINATION OF URINE

Leucine Acid/ Yellow Liver  Improperly collected specimens or rarely fistula presence
neutral (concentri disease bet. intestinal & urinary tracts may produce fecal specimen
c circles) contamination
 Fecal artifacts may appear as plant & meat fibers or as
Tyrosine Acid/ Colorless Liver
brown amorphous material in variety of sizes & shapes
neutral –yellow disease
(needles)
Bilirubin Acid Yellow Liver
disease

Sulfona- Acid/ Varied Infection

mides neutral treatment
Figure 6-102 Figure 6-103
Radio- Acid Colorless Radiograph
graphic (flat ic
dye plates) procedure

Ampicillin Acid/ Colorless Infection

neutral (needles) treatment

URINARY SEDIMENT ARTIFACTS Figure 6-104 Figure 6-105

 Contaminants of all types can be found in urine
o Particular to specimens collected under improper
conditions or in dirty containers
 Most frequently encountered artifacts: starch, oil droplets,
air bubbles, pollen grains, fibers & fecal contamination
 Artifacts can present major problem to student since they
frequently resemble pathologic elements like RBCs & casts
o Often very highly refractile or occur in different
microscopic plane than the true sediment Figure 6-106 Figure 6-107
constituents
o Reporting of such is not necessary
 Starch granule
o Starch granule contamination may occur when
cornstarch is the powder used in powdered gloves
o Highly refractile spheres, usually w/ dimpled center
o Resemble fat droplets when polarized, producing
Maltese cross formation
o May occasionally be confused w/ RBCs
Figure 6-108
 Differentiation bet. starch & pathologic elements can be
made by considering other urinalysis results including Figure 6–102. Starch granules. Notice the dimpled center
chemical tests for blood or protein and presence of oval fat (×400).
bodies or fatty casts Figure 6–103. Fecal material and oil artifacts (×400).
 Oil droplets & air bubbles are highly refractile & may Figure 6–104. Pollen grain. Notice the concentric circles (×400).
resemble RBCs to inexperienced lab personnel Figure 6–105. Fiber and squamous epithelial cell (×400).
 Oil droplets may result from contamination by immersion oil Figure 6–106. Fiber under polarized light (×100).
or lotions & creams & maybe seen w/ fecal contamination Figure 6–107. Diaper fiber resembling a cast. Notice the
 Air bubbles occur when specimen is placed under cover slip refractility (×400).
o Presence of these artifacts should be considered in Figure 6–108. Vegetable fiber resembling waxy cast (×400).
context of other urinalysis results
 Pollen grains are seasonal contaminants appearing as
spheres w/ cell wall & occasional concentric circles
o Their large size may cause them to be out of focus REFERENCES
w/ true sediment constituents like many artifacts
 Hair & fibers from clothing & diapers may initially be
mistaken for casts Notes from the book by
o They are usually much longer and more refractile
 Examination under polarized light can frequently Urinalysis and Body Fluids, Sixth Edition Strasinger (2014)
differentiate between fibers and casts
o Fibers often polarize
o Casts, other than fatty casts, do not polarize

DELA CRUZ, GALANTO CSU BS MLS 3RD YEAR 24