Chapter 6: MICROSCOPIC EXAMINATION OF URINE Commercial Systems
 System provide a variety of options including capped,
Purpose: to detect and identify insoluble materials present in the urine
A. Macroscopic Screening
 Abnormalities in the physical and chemical portions of
urinalysis play a primary role in the decision to perform a
microscopic analysis.
 Protocols of consideration on population under macroscopic
screening: Pregnant women, pediatric, geriatric, diabetic,
immunocompromised and renal patients.
*(Refer to Table 6-1 “Macroscopic Screening and Microscopic
Correlations”)*
Specimen Preparation
 Specimen should be examined while fresh or adequately
preserved
 Formed elements- RBCs, WBCs and hyaline casts-
disintegrate rapidly, particularly in dilute alkaline urine
 Refrigeration- may cause precipitation of amorphous urates
and phosphates and other non-pathologic crystals that can
obscure other elements in the urine sediments
 Warming the specimen to 37 oC- can dissolve some of the
crystals
 Midstream clean-catch specimen- minimizes external
contamination of the sediment
 Dilute random specimen- cause false negative readings
Specimen Volume
 Standard amount of urine: between 10 and 15 mL, is
centrifuged in conical tube-provides an adequate volume of
the elements present in the specimen
 12-mL volume-frequently used because multiparameter
reagent strips are easily immersed ad capped centrifuge
tubes often calibrated to this volume
 If obtaining a 12 mL is not possible- for pediatric patients,
it should be noted on the report form
Centrifugation
 Centrifugation time: 5 minutes
Centrifugal force: 400 RCF
Effect: optimum amount of sediment with least chance of
damaging elements
 Use of braking mechanism to slow centrifuge: cause
disruption of the sediment prior to decantation and should
not be used.
 To prevent biohazardous aerosols: all specimens must be
centrifuged in capped bottles
Sediment Preparation
 Uniform amount of sediment and urine should remain in the
tube after decantation
 Frequently used volume: 0.5 and 1.0 mL
 Concentration Factor- volume of urine centrifuged divided
by the sediment volume
 To maintain a uniform sediment concentration factor, urine
should be aspirated off rather than poured off, unless
otherwise specified by the commercial system in use
 The sediment must be thoroughly resuspended by gentle
agitation.
 Vigorous agitation should be avoided
 Thorough resuspension is essential to provide equal
distribution of elements in the microscopic fields
Volumes of Sediment Examined
 Volume of sediment place in the microscope slide should be
consistent for each specimen
 Conventional glass slide method: recommended volume
is 20 uL covered by a 22x22 mm glass cover slip- which will
allow the specimen to flow outside the cover slip- result in
the loss of heavier elements such as casts
 Commercial Systems: control the volume of sediment
examined by providing slides with chambers capable of
containing specified volume
 Cholesterol: do not stain but capable of polarization
calibrated centrifuge tubes; decanting pipettes to control the
sediment volume; and slides that control the amount of
sediment examined; produce a consistent monolayer of
sediment for examination, and provide calibrated grids for
more consistent quantitation.
 Cen-Slide and R/s Workstations do not require manual
loading of the centrifuged specimen onto a slide and are
considered closed systems that minimize the exposure to the
specimen
 Cen-Slide provides a specially designed tube that permits
direct reading of the urine sediment
 R/S Workstations consist of glass flow cell into which urine
sediment is pumped, microscopically examined, and then
flushed from the system
Examining the Sediment
 Microscopic examination must be performed in a consistent
manner and include observation of a minimum 10 fields
under both low (10x) and high (40x)
 The slide is first examined in low power to detect cast then if
it is identified changed to high power
 Bright filed microscopy: essential for unstained sediment
Reporting the Microscopic Examination
 Casts: reported as the average number per lpf following the
examination f 10 fields
 RBCs and WBCs: as the average number per 10 hpf
 Epithelial cells, crystals and other elements: reported in
semiquantitative terms such as rare, few, moderate, and
many, or as 1+, 2+, 3+, and 4+ following lpf or hpf formatwas
use
Correlating Results
 Microscopic results should be correlated with the physical
and chemical finings to ensure the accuracy of the report
 Specimens in which the results do not correlate must be
rechecked for both technical and clerical errors
*(Refer to table 6-2 “Routine Urinalysis Correlations”)*
B. Sediment Examination Techniques
 Many factors can influence the appearance of urinary
sediments including cells and cats, distortion of cells and
crystals by the chemical content of specimen and
contamination of artifacts.
 Identification can be enhanced through the use of sediment
stains and different types of microscopy
 *(Refer to table 6-3 “Urine Sediment Stain Characteristics”)*
Sediment Stains
 Staining increases the overall visibility of sediment elements
being examined in bright field microscopy by changing their
refractive index
 Sternheimer-Malbin Stain: most frequentlyused stain which
is consisting of crystal violet and safranin O.
-The stain is available commercially under a variety of
names, including Sedi-Stain and KOVA stain
 0.5% solution of toluidine blue a metachromatic stain that
provides enhancement of a nuclear detail.
-It can be useful in the differentiation between WBCs and
renal tubular epithelial cells and is also used in examination
of cells from other body fluids
 0.2 % acetic acid: alsoenhance the nuclear detail
-This method is cannot be used for initial sediment because
RBCs lyses on acetic acid
*(Refer to table 6-4 “Expected Staining Reactions of Urine
Sediment Constituents”)*
Lipid Stains
 Passage of lipids across he glomerular membrane results in
the appearance of free droplets and lipid containing cells and
casts in the urinary sediment
 Sudan III and Oil Red O are lipid stains and Polarizing
microscopy can be used to detect the presence of the
lipids.
 Triglycerides and neutral fats: stains orange-red
Gram Stain
  Primarily used in microbiology section for differentiation
between gram positive (blue) and gram negative (red)
bacteria.
 In routine analysis it is limited in determining bacterial casts,
which can easily be confused with granular casts
 To perform gram staining, a dried, heat fixed preparation is
used
Hansel Stain
 The preferred stain for urinary eosinophils is Hansel stain,
consisting of Methylene Blue and eosin Y.
 Wright’s stains: it can also be used on a dried smear of the
centrifuged specimen or a centrifuged preparation of
sediments
Prussian Blue Stain
 Due to episodes of hemoglobinuria, yellow brown granules
may be seen in renal tubular epithelial cells and casts or free
floating in the urine sediment.
 Confirmation of Hemosideran in granules is used and the
resulting color is blue
Cytodiagnostic Urine Testing
 Frequently performed independently of routine urinalysis for
detecting the malignancies of the lower urinary tract.
 A voided first morning urine specimen is recommended for
testing
 Provides more definitive information about real tubular
changes associated with transplant rejection; viral, fungal
and parasitic infections; cellular inclusions; pathologic casts;
and inflammatory conditions
Microscopy
 Bright field microscopy: most common type of microscopy
performed in urinalysis laboratory.
 The type of microscopy used depends on the specimen type,
the refractive index of the object, and the ability to image
unstained living cells
 All microscopes are designed to magnify small objects to
such a degree that the details of their structure can be
analyzed
*(Refer to table 6-5 “Urinalysis Microscopic Technique”)*
The Microscope
 Lens system: primary components are the oculars,
objective and the coarse and fine adjustment knobs.
 Illuminationsystem: contains the light source, condenser
and field and iris diaphragm
 Mechanical stage: platform where objects to be examined
are placed
 Compound bright field microscope: used primarily in
urinalysis laboratory and consist of two lens system
combined with a light source
First lens system: located in theobjective and is adjusted
near the specimen
Second lens system: the ocular lens is located in the
eyepiece
 Ocular or Eyepiece: located at the top of the body;
designed to further magnify the object that has been
enhanced by the objectives for viewing
 Objectives:contained in the revolving nose piece located
above the mechanical stage
 Resolution:ability to visualize fine details; ability of the lens
to distinguish two small objects that are specific distance
apart
-Objectives used in urinalysis are 10x and 40x
 Final Magnification: product of the objective magnification
times the ocular magnification
 Numerical aperture number: represents the refractive
index of the material between the slide and the outer lens
and the angle of light passing through it
 Coarse and fine focusing knob: the distance between the
slide and the objective
 Coarse knob- initial focusing is performed
 Fine focusing knob- to sharpen the image
 Condenser- located below the stage then focuses the light
on the specimen and controls the light for uniform
illumination
 Aperture diaphragm- controls the amount of light and the
angle of light rays that passes the specimen
 Birefringent uric acid crystals -- cystine crystals
TYPES OF MICROSCOPY
 Bright-Field Microscopy
o Most frequently used in the clinical laboratory
o Objects appear dark against a light background
o Has a light source emitting light in the visible
wavelength range
o For use in examination of urine sediments:
 Must be examined using decreased
light(adjust the rheostat, not the condenser)
*Sediment with low refractive index –
overlooked when subjected to high light
intensity
 Staining of sediment – increases the
visualization of the elements
 Phase-Contrast Microscopy
o Phase difference – light rays that pass through an
object are slowed in comparison to the rays passing
through the air, producing increased light intensity and
contrast
- Affected by:
 thickness of the object
 refractive index
 other light absorbance properties
o Provide best contrast by:
 shifting one quarter of a wavelength, of the
light that does not pass through the
specimen, and compare it with the phase
difference of the specimen
o Adaptation of a bright-field microscope with a phase-
contrast objective lens and a matching condenser
*Image has best contrast when the background isdarkest
o Advantageous in identifying:
 Low refractive hyaline casts or mixed cellular
casts
 Mucous threads
 Polarizing Microscopy
o Aids in identification of crystals and lipids
 Crystals and lipids
– have the ability to rotate the path of the
unidirectional polarized light beam to
produce:
 Characteristic colors – crystals
 Maltese cross formation – lipids
– Birefringent (a property indicating that the
element can refract light in two
dimensions at 90 degrees to each other)
o Halogen quartz lamp – produces light rays of many
different waves. [Each wave has its (1) Distinct direction
and (2) Vibration perpendicular to its direction]
 Normal or unpolarized light - vibrates in equal
intensity in all directions
 Polarized light - vibrates in the same plane or
direction
o Birefringent substance–substance from which light
passes through as it splits into two beams
o Isotropic substances– (ex. Blood cells) light passes
through this substance unchanged; does not have
refractive property
o A substance that rotates the plane of polarized light 90
degrees clockwise direction have positive
birefringence
o A substance that rotates the plane in a
counterclockwisedirection has negative birefringence
o Polarized light – obtained by using two polarizing filters
 The light emerging from one filter vibrates in one
plane
 Second filter – placed at 90-degree angle and
blocks all incoming light
*The filters are in opposite directions called
“crossed configuration”
o Bright-field microscopes can be adapted for polarizing
microscopy
o Used in urinalysis to confirm identification of:
 Fat droplets
 Oval fat bodies
 Fatty casts – produce Maltese cross pattern
o For distinctionbetween the following by their polarizing
characteristics:
 Monohydrate calcium oxalate crystals -- nonpolarizing
 RBCs
 Calcium phosphate crystals-- nonpolarizing bacteria
 Interference-Contrast Microscopy
o Provides a three-dimensional image showing very fine
structural detail by splitting the light ray so that beams pass
through different areas of the specimen
o Object appears bright against a dark background w/o the
diffraction halo associated with phase-contrast microscopy
o Not routinely used in the urinalysis laboratory
o Two types: [provide (1) Layer-by-layer imaging of the
specimen and (2) enhance detail for specimens w/ either low
or high refractive index]
 Modulation contrast (Hoffman)
- Polarized light rays pass through a split
aperture to the various areas of the specimen
and to the modulator where they are
converted to variations of light intensity
producing a 3-D image
- 3 zones of light transmission of the modulator:
 Dark zone – transmits 1% of light
 Gray zone – transmits 15% of light
 Clear zone – transmits 100%of light
 Differential-interference contrast (Nomarski)
- Uses prism
- Two-layered Nomarski-modified Wollaston
prism (required to separate individual rays of
light into pairs
 Dark-Field Micriscopy
o Enhance visualization of specimens hat cannot be seen
easily viewed with a bright-field microscope
o For unstained specimens
o Identify the spirochete Treponema pallidum
o Specimen appear light against black back-ground or
dark-field
o Bright-Field microscope is easily adapted for dark-field
microscopy by:
 Replacing the condenser w/ a dark-field condenser
that contains an opaque disk
 Fluorescence Microscopy
o Detect bacteria and viruses within cells and tissues
through immunofluorescence
o Visualization of naturally fluorescent substances or
those that are stained with fluorochrome or fluorophore
o Fluorescence –property by which some atoms absorb
light at a particular wavelength and subsequently emit
fluorescence lifetime
o Fluorescence lifetime – light of a longer wavelength
o Fluorescent substances – absorb energy and emit a
longer wavelength of light and is visualized with the use
of special filters:
 Excitation filter –selects the excitation
wavelength of light from a light source
 Emission filter – selects a specific wavelength of
emitted light from the specimen to become visible
o Dichroic mirror – reflects the excitation light to the
specimen and transmits the emitted light to the
emission filter
o Object observed as bright against a dark background
with high contrast when ultraviolet light source is used
o Powerful light sources: Mercury or xenon arc lamps
URINE SEDIMENT CONSTITUENTS
o Normal urine sediment may contain variety of formed
elements:
 Pathologically significant RBCs, WBCS and casts
 Rare epithelial cell or mucous strand
o Urine sediment preparation methods – determine the
actual (1) concentration of the sediment and (2)
number of elements that may be present in a
microscopic field
o Commonly listed values:
 0-2 or 3 RBCs/hpf
 0-5-8 WBCs/hpf
 0-2 hyaline casts/Ipf
*Wright’s stain can also be used
*Percentage of eosinophils in 100 to 500
 RED BLOOD CELLS
o Smooth, non-nucleated and biconcave disks
o Identified in high-power (40x) objective
o Reported as average number seen in 10 hpfs
o Concentrated urine (hypersthenuric)
– Cells shrink (loss of water) and may appear
crenated or irregularly shaped
o Dilute urine (hyposthenuric)
– Cells swell (absorb water) and lyse rapidly,,
releasing their hemoglobin and leaving only cell
membrane
– Ghost cells–large empty cells
o Most difficult for students to recongnize due to RBCs’:
 Lack of characteristic structures
 Variations in size
 Close resemblance to other urine sediment
constituents
o Confused with: (sources of error)
 yeast cells
 oil droplets
 air bubbles
o Dysmorphic RBCs – RBCs that vary in size, have
cellular protrusions or are fragmented
– Primarily associated with glomerular bleeding
– found with nonglomerular hematuria
– ↑after strenuous exercise
– use of Wright’s stained prep – shows cells to
be hypochromic and better delineates the
presence of cellular blebs and protrusions
* Acanthocyte with multiple protrusions – the
dysmorphic cell most closely associated with
glomerular bleeding
o Clinical Significance
Presence of RBCs in urine is associated with:
 Glomerular membrane or vascular injury within
genitourinary tract
No. of cells present
 Indicative of the extent of damage or injury
Hematuria
 Macroscopic hematuria:Urine appears cloudy with
a red to brown color
 Microscopic: reported in terms of greater than 100
per hpf or as specified by laboratory protocol
Refer to SUMMARY 6-1 (Microscopic RBCs)
Observation of microscopic hematuria
 Critical in the early diagnosis of glomerular
disorders and malignancy of the urinary tract to
confirm the presence of renal calculi
RBCs, hyaline, granular and RBC casts in urine
 Seen after strenuous exercise
 Nonpathologic and disappear after rest
*Presence or absence of RBCs in the urine cannot
always be correlated with specimen color or a positive
chemicaltest result for clood (Ex. Hemoglobin in urine –
Red urine – (+) chemical test in absence of hematuria)
 WHITE BLOOD CELLS
o Larger than RBCs
o Neutrophil – predominant WBC found in urine
 Much easier to identify than RBCs
 Contain granules with multilobed nuclei
 Reported as average number seen in 10 hpfs
 Lyse rapidly in dilute alkaline urine
 Brownian mov’t of granules within this cell
produces a sparkling appearance referred to
as “glitter cells”(no pathologic significance)
 Glitter cells – large cells that stain light blue with
Sternheinmer-Malbin stain as opposed to the
violet color usually seen with neutrophils
o Eosinophils
 Primarily associated with drug-induced interstitial
nephritis
 Small numbers in (1) Urinary tract infections (UTI)
and (2) Renal transplant rejection
 Concentrated and stained urine sediment –
required for urinary eosinophil test
 Centrifugation/cytocentrifugation –for
concentration of urine sediment
 Hansel – preferred eosinophil stain
cells is determined
 Not normally seen in urine, finding >1% is
 significant
o Mononuclear cells – usually not identifiedin the wet
preparation urine microscopic analysis
 Lymphocytes – the smallest WBCs and may
resemble RBCs
– ↑in early stages of renal transplant rejection
 Monocytes, macrophages and histiocytes – large
cells that appear vacuolated or contain inclusions
* Cytodiagnostic urine testing – referred to if
specimens containing an increased amt of mononuclear
cells cannot be identified as epithelial cells
 Source of error:
 Renal tubular epithelial (RTE) cells – larger
than WBCs with an eccentrically located
nucleus
– may be difficult to distinguish
from WBCs in the process of
ameboid motion because of their
irregular shape
* Supravital stainingor addition of acetic acid –
can be used to enhance nuclear detail if
necessary
 ↓than 5 leukocytes per hpf are found in normal
urine, but ↑numbers in females
 Pyuria - ↑in urinary WBCs
– Indicates the presence of an infection or
inflammation in the genitourinary
system
– Can cause bacterial infections
(pyelonephritis, cystitis, prostatitis and
urethritis)
* Refer to SUMMARY 6-2 (Microscopic WBCs)
 EPITHELIAL CELLS
o Represent normal sloughing of old cells
o Derived from the linings of the genitourinary system
o Not unusual to find in the urine unless found in large or
abnormal numbers
3 TYPES OF EPITHELIAL CELLS
o Squamous Epithelial Cells
 Largest cells found in the urine sediment
 Contain abundant, irregular cytoplasm and a
prominent nucleus about a size of an RBC
 First structures observed under low-power
magnification
 Good reference for focusing the microscope
 Reported in terms of: rare, few, moderate, or
many
 Originate from linings of the vagina and female
urethra and lower portion of male urethra
 ↑in female patients
 Midstream clean-catch specimens – less
aquamous cell contamination
 Clue cell –variation of the squamous epithelial cell
– Indicative of vaginal infection by
Gardnerella vaginalis
– Bacteria should cover most of the cell
surface and extend beyond the edges of
the cell to be considered to be a clue
cell
– Vaginal wet preparation (examined
during routine testing for clue cell)
o Transitional Epithelial (Urothelial) Cells
- smaller than squamous cells
- several forms ( because of ability to absorb water):
o Spherical (in contact with urine; larger than
polyhedral & caudate)
o polyhedral
o caudate
- all forms with centrally located nucleus
- originate from lining of the renal pelvis, calyces, ureters, and
bladder, upper portion of male urethra
- present in small numbers in normal urine=normal cellular
sloughing
- ↑ TEC singly/ pairs /clumps (syncytia) = catheterization (
no clin.significance)
Sources of error: Confirm with fat stains and polarized microscopy
- ↑ TECw/ vacuoles & irregular nuclei= malignancy or viral
infection
- identified and enumerated using high-power magnification
Reporting:rare, few, moderate, or many per hpf
Sources of error:
- spherical forms resemble RTE cells ( w/ eccentrically
loc. nucleus)
o RTE Cells
- rectangular, columnar, round, oval or, cuboidal w/ an
eccentric nucleus possibly bilirubin stained or hemosiderin-
laden
- size and shape varies depending on the area of the renal
tubules from which they originate
 Cells from proximal convoluted tubule (PCT):
o larger than other RTE cells
o rectangular (columnar /convoluted cells)
Sources of error:
- granular casts (no nucleus)
 Cells from distal convoluted tubule (DCT):
o smaller than PCT
o oval/ round (columnar /convoluted cells)
Sources of error:
- WBCs
- spherical transitional cells
 Collecting duct RTE cells:
o cuboidal and are neverround.
o eccentrically placed nucleus,
o presenceof at least one straight edge
differentiates them fromspherical and polyhedral
transitional cells .
o renal fragments:
 cells from the collecting duct that
appear in groups of three or more
 indication of severe tubular injury
w/ basement mem. disruption
o They are frequentlyseen as large sheets of cells.
(PCT& DCT cells are not seen in large sheets of
cells)
- identified and enumerated using high-power magnification
- presence of more than 2 RTE cells = tubular injury and
specimens for cytologic urine testing
Reporting: average number per 10 hpfs
sediment
Sources of error:
- granular casts (no nucleus)
RTE cells - most clinically significant of the epithelial cells
- function for reabsorption, unusual to contain subs.
from filtrate
- absorb bilirubin present in the filtrate (viral hepatitis)
- absorb lipids
- absorb hemoglobin convert it to hemosiderin
- hemosiderin:
o yellow-brown granules.
o free-floating in the urine sediment
o Prussian blue - stain urine sediment to confirm
for presence of hemosiderin
*iron-containing hemosiderin granules = blue
↑ RTE cells= tubular necrosis
produced by:
o exposure to heavy metals, drug-induced toxicity,
hemoglobin and myoglobin toxicity, viral infections
(hepatitis B), pyelonephritis, allergic reactions,
malignant infiltrations, salicylate poisoning & acute
allogenic transplant rejection. Secondary effects of
glomerular disorders
o Oval Fat Bodies
- lipid-containing, highly refractile RTE cells, nucleus more
difficult to observe
- seen in conjunction w/ free-floating fat droplets
- Sudan III or Oil Red O fat stain- Identifies & confirms oval
fat bodies by staining urine sediment
- Maltese cross formations in droplets containing
cholesterol
-
Reporting:Average number per hpf
Lipiduria- most frequently associated with damage to the glomerulus
caused by the nephrotic syndrome
“bubble cells”- large, nonlipid-filled vacuoles may be seen along with
normal renal tubular cells and oval fat bodies.
Complete urinalysis correlations:Clarity, Blood, Protein, Free fat
droplets/fatty casts
 BACTERIA
- small spherical (cocci) and rod-shaped (bacilli) structures
- Enterobacteriaceae, Staphylococcus & Enterococcus
- not normally present in urine
- multiply rapidly at room temp. (no clin significance)
- produce + nitrite test result & result in pH >8 (unacceptable
specimen)
- observed & reported using high-power magnification
phase contrast microscopy
Reporting: Few, moderate, or many per hpf
- Bacteria accompanied by WBCs= indicative for UTI (upper/
lower)
- + urine culture =motile org. in drop of fresh urine
*Motility of bacteria differentiates it from amorphous
phosphates &urates
Complete urinalysis correlations: pH, Nitrite, LE, WBCs
 YEAST CELLS
- small, oval, refractile structures (may or may not contain a
bud) in branched, mycelial forms
*differentiation with RBCs is difficult, budding of yeast cells is helpful
Reporting: Rare, few, moderate, or many per hpf,
Candida albicans-seen in the urine of diabetic patients (acidic,
glucose-containing urine) immunocompromised patients & women w/
vaginal moniliasis
*presence of WBCs = true yeast inf.
Complete urinalysis correlations: Glucose, LE, WBCs
 PARASITES
Trichomonasvaginalis (trophozoite)-Pear-shaped, motile, flagellated,
w/ an undulating membrane with rapid darting motility( in wet prep.)
- sexually transmitted assoc. primarily w/ vaginal
inflammation. Inf.of the male urethra and prostate
is asymptomatic
- resembles WBCs, renal tubular epithelial cells differentiated
with presence of undulating membrane
- use phase microscopy
Reporting: rare, few, moderate, or many perhpf
Schistosomahaematobium (ova)- bladder parasite assoc. w/ bladder
cancer
Enterobiusvermicuralis (ova)- most common fecal contaminant
Complete urinalysis correlations: LE, WBCs
 SPERMATOZOA
- oval, slightly tapered heads and long,thin, flagella-like tails
- occasionally found in the urine of men & women ff.
sexual intercourse, masturbation, or nocturnal emission
male infertility or retrograde ejaculation- sperm is expelled into the
bladder instead of the urethra
+ reagent strip test for protein = ↑ amt. of semen
*Urine is toxic to spermatozoa
Width of the cast depends on size of tubule in w/c it is formed
Reporting: Present, based on laboratoryprotocol
 MUCUS
- protein material produced by glands & epithelial cells of
lower genitourinary tract & RTE cells
- Microscopically, single or clumped threads with alow
refractive index
- frequently present in female urine specimens
uromodulin– major constituent of mucus
- glycoprotein excreted by RTE cells of DCT and upper
collecting ducts
- confused with hyaline casts
Reporting: rare, few, moderate, or many per lpf
 CASTS
- only elements found in the urinary sediment , unique to the
kidney
- formed within the lumens of DCT and collecting ducts
- shape is representative of tubular lumen, with parallel sides
& somewhat rounded ends, & contain additional elements in
filtrate.
- use lower power magnification
- in glass cover-slip method, low-power scanning should be
performed along the edges of the cover slip
- subdued light is essential because cast matrix has
lowrefractive index.
- dissolves quickly in dilute, alkaline urine
- use high-power magnification for futher identification of
composition
Reporting: average number per 10 lpfs.
Cast Composition and Formation
Cast matrix:
 uromodulin - major constituent of casts
 albumin
 immunoglobulins
stress and exercise= ↑rate of excretion
- account for the transient appearance of hyaline casts
protein gels more readily under conditions of:
 urine-flow stasis
 acidity
 presence of Na &Ca
↑urinary protein = presence of casts caused byunderlying renal
conditions
Scanning electron microscope studies, provided a step-by-step
analysis of the formation of the 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
*Cast forms= ↓urinary flow w/n tubule = lumen is blocked
*Wrinkled and convoluted app. of older hyaline casts
= dehydration of the protein fibrils and internal tension
Appearance of a cast influenced by
  materials present in the filtrate at time of its formation
 length of time it remains in the tubule
Elements present in the tubular filtrate (cells, bacteria, granules,
pigments, and crystals) become embedded in or attached to the cast
matrix
*Broad casts result from tubular distension
*Extreme urine stasis, from formation in the collecting ducts
*Formation of casts at junction of ascending loop of Henle&DCT
produce structures w/ tapered end (cylindroids)
cylindruria- presence of urinary casts
Hyaline Casts
- most frequently seen cast is the hyaline type, which
consistsalmost entirely of uromodulin
- morphology : varies, ( normal parallel sides & rounded ends,
cylindroid forms, &wrinkled or convoluted shapes that
indicate aging of the castmatrix)
- Presence of an occasional adhering cell or granule may also
be observed but does not change the cast classification.
- colorless in unstained sediments &have a refractive index
similar to urine
- normal = presence 0-2 hyalinecasts per lpf
- finding of: ↑ numbers ff. strenuous exercise,
dehydration,heat exposure, emotional stress
- ↑in acute glomerulonephritis, pyelonephritis,chronic renal
disease, & congestive heart failure
Sternheimer-Malbin stain
- produces a pinkcolor in hyaline casts
- increased visualizationcan be obtained by phase microscopy
Normal Crystals Seen in Acidic Urine
 Urates(composed of amorphous urates, uric acid, uric
urates, acid urates, and sodium urates), are the most
common crystals seen in acidic urine.
 Urates appear yellow to reddish brown microscopically
(only colored normal crystal in acidic urine).
 Amorphous uratesyellow-brown granules microscopically.
May resemble granular casts and attached to other
sediment struct. Frequently encountered in refrigerated
specimens then produced pink sediment (caused by
uroerythrinaccumulation on top). Found in acidic urine (pH
> 5)
 Uric acid crystals seen in variety of shapes (rhombic, four-
sided flat pates *whetstones, wedges, and rosettes). Usually
yellow-brown, but may also be colorless and six-sided (like
cystine crystals). They are highly birefringent (aids to
differentiate them from cystine crystals). Inc in amount is
associated with inc levels of purines and nucleic acids in
patients with leukemia who are under chemotherapy,
patients with Lesch-Nyhan syndrome, and sometimes in
gout.
 Acid Urates and Sodium Urates, rarely encountered and
seen in less acidic urine. Usually mixed with amorphous
urates but have little clinical significance. Acid urates are
larger and may have spicules (same with ammonium biurate
crystals in alkaline urine). Sodium urate crystals are needle-
shaped and seen I synovial fluid (in case of gout) but may
also appear in urine.
 Calcium oxalate crystals can also be found in neutral but
rarely in alkaline urine. Dihydrate calcium oxalate
crystals(most common form), is colorless, octahedral
envelope or as two pyramids joined by their bases.
Monohydrate calcium oxalate crystals (not frequently
seen form), oval or dumbbell-shaped. Both forms are
birefringent under polarized light (may help to distinguish
them from nonpolarizing RBCs. Calcium oxalate crystals
o Leucine Crystals are yellow-brown
spheres that demonstrate
may attach to mucous strands and be related to the
formation of renal canaliculi. Also associated with food high
in oxalic acid (tomatoes and asparagus). Cases of ethylene
glycol (antifreeze) poisoning is the primary pathologic
significance of the crystal (in monohydrate form).
Normal Crystals Seen in Alkaline Urine
 Phosphates (include amorphous phosphates, triple
phosphate, and calcium phosphate) are the majority
of crystals seen in an alkaline urine. There are also
calcium carbonate and ammonium biurate.
 Amorphous Phosphates are granular like amorphous
urates (can be distinguished by the urine pH and
sediment color). White precipitate ,which could not be
dissolved by warming, is formed when present in large
amt and refrigerated.
 Triple Phosphate ( ammonium magnesium
phosphate) crystals are identified by their prism shape
which resembles “coffin lid”. Develop feathery
appearance when disintegrated. Birefringent under
polarized light and of no significance but seen in highly
alkaline urine and associated with the presence of urea-
splitting bacteria.
 Calcium Phosphate Crystals are not frequently
encountered. They are colorless, flat rectangular plates
or thin prism often in rosette formations. The rosette
formation may be confused with sulfonamide crystals
when the urine is neutral in pH. Calcium phosphate
crystals dissolve in dilute acetic acid and sulfonamides
do not. No clinical significance but common constituent
of renal canaliculi.
 Calcium Carbonate crystals are small and colorless
with dumbbell or spherical shapes. They may resemble
amorphous materials but distinguished by their gas
formation after addition of acetic acid. They have no
clinical significance.
 Ammonium Biurate Crystals yellow-brown, and
described as “thorny apples” due to their appearance
as spicule-covered spheres. They dissolve in 600C and
convert to uric acid crystals when glacial acetic acid is
added. Almost always encountered in old specimens
and may be associated with the presence of urea-
splitting bacteria.
Abnormal Crystals Seen in Acidic Urine
 Abnormal urine crystals are found in acid urine or
rarely in neural urine.
 Cystine Crystals are found in the urine of
persons with cystinuria (metabolic disorder that
prevents reabsorption of cysteine by the renal
tubules). They are colorless, hexagonal plates,
and may be thick or thin. Disintegrating forms may
be seen in the presence of ammonia. Positive
confirmation of the crystals is made using the
cyanide-nitroprusside test.
 Cholesterol Crystals rarely seen unless urine is
refrigerated. They resemble a rectangular plat with
notch in one or more corners. They are associated
with disorders producing lipiduria, and are seen in
conjunction with fatty casts and oval fat bodies.
They are highly birefringent with polarized light.
 Radiographic Dye Crystals have a very similar
appearance with cholesterol crystals and are also
highly birefrigent. Differentiation is made by
comparison of other urinalysis results and patient
history. The specific gravity of a specimen with
radiographic contrast media is highly elevated
when measured by a refractometer.
 Crystals Associated with Liver Disorders
o Tyrosine crystals are fine colorless to
yellow needles that frequently form
clumps or rosettes. Usually seen
together with leucine crystals in
bilirubin positive specimens. The
crystals may also be encountered in
inherited disorders of amino acid
metabolism.
concentric circle and radial striations.
Less frequently seen and must be
 accompanied with tyrosine crystals.
o Bilirubin Crystals present in hepatic
disorders produsing a large amount of
bilirubin. They appear as clumped
needles or granules with the yellow
color of bilirubin. Positive bilirubin test is
expected.
 Sulfonamide Crystals inadequate patient
hydration is the primary cause of their presence.
The crystals’ appearance can suggest the
possibility of tubular damage I they are forming in
the nephron. Shapes are needles, rhombics,
whetstones, sheaves of wheat, and rosettes with
colors ranging from colorless to yellow-brown.
 Ampicillin Crystals form following the intake of a
massive dose of penicillin compound without
proper hydration. They appear as colorless
needles that tend to form bundles following
refrigeration.
Urinary Sediment Artifacts
 Most frequently encountered artifacts include starch, oil
droplets, air bubbles, pollen grains, fibers, and fecal
contamination.
 They are highly refractile, different than the true sediment
constituent.
 Starch granule contamination may occur when cornstarch
is in powdered gloves. They resemble fat droplets when
polarized, producing a maltese cross formation.
 Oil dropletsand air bubbles are highly refractile and may
resemble RBCs. Oil droplets may be due to contamination
by oil immersion or lotions and creams may be seen with
fecal contamination. Air bubbles occur when the specimen is
placed under the cover slip.
 Pollen grains are seasonal contaminants that appear as
spheres with a cell wall and occasional concentric
circles.
 Hair fiber from clothing and diapers may initially be mistaken
for casts.
 Fecal articactsmay appear as plant and meat fibers or as
brown amorphous material in a variety of sizes and
shapes.
RBC CAST
 more SPECIFIC
 Shows bleeding within the NEPHRON
 Associated with the damage to the glomerulus
(glomerulonephritis)
 RBC cast associated with glomerular damage are usually
associated with Proteinuria and dysmorphic
erythrocytes
 Orange red in color can be detected under low power
 More fragile than other cast, exist as fragments or a more
irregular shape
 presence of cast matrix under high power differentiates the
structure from a clump of RBC
 Actual presence of RBC must be verified to
prevent inaccurate reporting of nonexistent RBC
cast
 In the presence of massive hemoglobinuria or
myoglobinuria, homogenous orange-red or red brown cast
maybe observed
 granular,dirty,brown cast representing hemoglobin
degradation products such as methemoglobin may also be
present
 That is Associated with acute tubular necrosis often caused
by toxic effects of massive hemoglobinuria that leads to
renal failure
WBC CAST
 The appearance of WBC cast in the urine signifies infection
or inflammation of the nephron
MIXED CELLULAR CASTS
 Most frequently include RBC and WBC cast in
glomerulonephritis and WBC and RTE cell casts, or WBC
and bacterial cast in pyelonephritis
 Frequently associated with pyelonephritis and are a
primary marker for distinguishing pyelonephritis (upper
UTI) from cystitis (Lower UTI)
 also present in nonbacterial inflammations such as
acute interstitial nephritis and may accompany RBC cast
in glomerulonephritis
 WBC cast is visible under low-power magnification but must
be positively identified using high power.
 composed of neutrophils;appears granular and unless
disintegration has occurred multi lobed nuclei will be present
 Supravita staining may be necessary to demonstrate the
characteristic nuclei
 Bacteria are present in pyelonephritis,but are not present
with acute interstitial nephritis
 eosinophil cast may be present in in appropriately stained
specimen ( Hansel and Wright's stain)
 Cast tightly packed with WBC may have irregular borders
BACTERIAL CAST
 Bacterial cast containing bacilli both within and bound to the
protein matrix are seen in pyelonephritis ( they maybe pure
or bacterial cast or mixed with WBC's)
 identification of bacterial cast can be difficult , because
packed cast packed with bacteria can resemble granular
casts.
 Their presence should be considered when WBC cast and
many wbc and bacteria are seen in the sediment
 confirmation of bacterial cast is best made by performing
gram stain on dried or cytocentrifuged sediment
EPITHELIAL CELL CAST
 Associated with heavy metal and chemical or drug induced
toxicity,viral infections,and allograft rejection
 also accompany WBC cast in cases of pyelonephritis
 the cell visible on cast matrix are the smaller , round or
oval cells ; difficult to differentiate from WBC's
 Staining and use of phase microscopy can be helpful to
enhance the nuclear detail
 Bilirubin stained RTE cell are seen in cases of hepatitis
FATTY CAST
 seen in conjugation with oval fat bodies and free fat droplets
in disorder causing lipiduria
 associated with Nephrotic syndrome also seen in toxic
tubular necrosis, diabetes mellitus and crush injuries highly
refractive under bright microscope
 Cast matrix contains fat droplets and intact oval fat bodies
 Confirmation using polarized microscopy and Sudan III or
oil Red O fat stains
 Cholesterol Demonstrate characteristic Maltese cross
formation under polarized light neutral fat stains and
triglycerides stains orange with fat stains
 Fat do not stain with Sternheimer-malbin stains.
 staining or phase microscopy aids in the identification
 Primary diagnostic marker: presence of one homogenous
cast of at least one of the cell types Predominant Cast in
glomerulonephritis :
  RBC Predominant Cast in pyelonephritis: WBC
GRANULAR CAST
 coarsely and finely granular casts are frequently seen in
the urinary sediment may be pathologic or non pathologic
significance
 The origin of the granules in non pathologic conditions
appears to be from the lysosomes excreted by the RTE
cells during normal metabolism
 Increases cellular metabolism accounts for the Increase
of granular cast that accompany Increased hyaline
cast.....
WAXY CASTS
 representative of extreme urine stasis,indicating chronic
renal failure.
 the brittle, highly refractive cast matrix is believed to be
cause by degeneration of hyaline cast matrix.
 more easily visualized than hyaline casts because of their
higher refractive index often appear fragmented with jagged
ends and have notches on their sides
 with supravital stain, waxy cast stain a homogenous, dark
pink
BROAD CASTS
 Often referred to as Renal failure cast,
 broad cast like waxy cast represent extreme urine stasis
 presence of broad cast indicates destruction or widening
of the tubular walls
 also when the flow of urine to the larger ducts becomes
severely compromised,cast from this area and appear
broad
 All type of cast may occur in broad form -the most commonly
seen broad cast are granular and waxy
 bile-stained broad, waxy cast are seen as the result
of tubular necrosis caused by viral hepatitis ( fig 6-73)
URINARY CRYSTALS
 Crystals found in urine are rarely of clinical significance
 may appear as true geometrically formed structures or as
amorphous materials
 urinary crystals is to detect the presence of the relatively
abnormal types that may represents such disorders ( liver
disease, inborn errors of metabolism, or renal damage
caused by crystallization medication compounds within
the tubules)
 Crystals are reported as rare,few,moderate, many per hpf.
 Abnormal crystal maybe average and reported as lpf.
CRYSTAL FORMATION
 Crystals are formed by precipitation of urine solutes
including organic, inorganic and medications
( iatrogenic compounds)
 Precipitation is subject to changes in temperature,solute
concentration,and pH which affect solubility
 solutes precipitates at low temperatures
 Crystals are abundant in refrigerated specimen
 As the concentration of urinary solutes increases, their ability
to remain in solution decreases resulting in crystal formation
 Presence of crystals in freshly voided urine is associated
with concentrated ( high specific gravity) specimens
 a valuable aid in identification of crystals is the pH of the
specimen, this determines the type of chemicals
precipitated
 Organic and iatrogenic compounds crystalize more in acidic
ph
 Inorganic salts are less soluble in neutral and alkaline
solutions an
 Exemption is Calcium Oxalate , which precipitates in both
acidic and neutral urine.
GENERAL IDENTIFICATION TECHNIQUES
 all abnormal crystals are found in acidic urine
 polarized microscopy and solubility characteristics of crystals
aids in crystal identification geometric shape of crystal
determines birefringence and its ability to polarized light
polarization characteristics for a particular crystal are
constant for identification amorphous urinates may dissolved
if the specimen is warmed
 amorphous phosphate requires acetic acid to dissolve
 when solubility characteristics are needed for identification,
the sediment should be aliquoted to prevent destruction of
other elements .
(table 6.6 characteristic for the most commonly encountered
crystals)
NORMAL CRYSTAL SEEN IN URINE
 most common crystal seen in urine are Urates consisting of
amorphous urates, uric acid, and acid and sodium urates
 Microscopically most urate crystals appears yellow to
reddish brown and are the only normal crystals found in
acidic urine the appear colored
 Amorphous urates appear microscopically as yellow
brown granules, occurs in clumps resembling granular
casts and attached to sediment structures, produce a very
characteristic pink sediments because of accumulation of
pigment uroerythrin on the surface of the granules
 amorphous urates found in pH 5.5 ; uric acid crystals can
appear when the pH is lower