CHAPTER 6 MICROSCOPIC EXAMINATION OF URINE

Urine Analysis

Under The Microscope

Urine analysis is the term used to refer to the test used to evaluate a urine sample. Typically, this test is used for the
purposes of assessing a wide range of disorders, which may include kidney disease, urinary tract infection (UTI)
dehydration as well as diabetes. The test will involve an examination of the appearance, concentration as well as
content of the urine sample.

In microscopy, a sample of urine is centrifuged to obtain some sediment, which can then used to examine the presence
of crystals, casts, white and/or red blood cells or bacteria/yeast infection. While the appearance or coloration can give
some indication of the problem, microscopy allows for a deeper urine analysis, which would prove useful for diagnosis
and prognosis. For the purposes of microscopic urine analysis, the first morning specimen is the recommended
specimen of choice. This due to the fact that it is generally more concentrated because of the amount of time it
remained in the bladder. As such, the sample would contain relatively higher amounts of such analytes as proteins or
other cellular elements if at all they are present. To prevent any form of contamination, a midstream clean catch
specimen is recommended.

Here, the patient/participant will be required to start by cleansing the urethral area using a castile soap towelette (or
simply cleansed and rinsed well) A small amount of urine should then be voided in to the toilet in order to reduce the
changes of contaminants from entering in to the collecting container before collecting the rest midstream in to the clean
container. The third part of routine urinalysis, after physical and chemical examination, is the microscopic examination
of the urinary sediment. Its purpose is to detect and to identify insoluble materials present in the urine. The blood,
kidney, lower genitourinary tract, and external contamination all contribute formed elements to the urine. These include
red blood cells (RBCs), white blood cells (WBCs), epithelial cells, casts, bacteria, yeast, parasites, mucus, spermatozoa,
crystals, and artifacts. For urine analysis, the sediment should first be observed under low power when observing for
crystals, casts, squamous cells or other larger objects. When making a report, the number of casts seen under the
microscope is usually reported as the number of each type per low power field. Moreover, low power allows for a wider
view, which allows for clear observation of the number of casts seen.

To observe and identify cells, crystals and bacteria, high power is used. In this case, the types of cells will also be
described as the number of each type found per the high power field.

Note** - When observing the slide under low power, low light source should be used. This is because of the fact that too
much light would make it more difficult to see he cellular and crystalline elements.

Macroscopic Screening
 To enhance the cost-effectiveness of urinalysis, many laboratories have developed protocols whereby
microscopic examination of the urine sediment is performed only on specimens meeting specified criteria.
 Abnormalities in the physical and chemical portions of the urinalysis play a primary role in the decision to
perform a microscopic analysis, thus the use of the term “macroscopic screening.”
  Parameters considered significant vary among laboratories but usually include color, clarity, blood, protein,
nitrite, leukocyte esterase, and possibly glucose.

TABLE 6-1 Macroscopic Screening and Microscopic

Correlations
Screening Test Significance
Color Blood
Clarity Hematuria versus
hemoglobinuria/myoglobinuria
Blood RBCs, RBC casts
Protein Casts, cells
Nitrite Bacteria, WBCs
Leukocyte esterase WBCs, WBC casts, bacteria
Glucose Yeast

Specimen Preparation

 Specimens should be examined while fresh or adequately preserved. Formed elements—primarily RBCs, WBCs,
and hyaline casts—disintegrate rapidly, particularly in dilute alkaline urine.
 Refrigeration may cause precipitation of amorphous urates and phosphates and other non-pathologic crystals
that can obscure other elements in the urine sediment.
 Warming the specimen to 37°C prior to centrifuging may dissolve some of these crystals.
 The midstream clean-catch specimen minimizes external contamination of the sediment. As with the physical
and chemical analyses, dilute random specimens may cause false-negative readings.

Specimen Volume

 A standard amount of urine, usually between 10 and 15 mL, is centrifuged in a conical tube. This provides an
adequate volume from which to obtain a representative sample of the elements present in the specimen.
 A 12-mL volume is frequently used because multi-parameter reagent strips are easily immersed in this volume,
and capped centrifuge tubes are often calibrated to this volume.

Centrifugation

 The speed of the centrifuge and the length of time the specimen is centrifuged should be consistent.
Centrifugation for 5 minutes at a relative centrifugal force (RCF) of 400 produces an optimum amount of
sediment with the least chance of damaging the elements.

Sediment Preparation

 A uniform amount of urine and sediment should remain in the tube after decantation. Volumes of 0.5 and
1.0 mL are frequently used.
 The volume of urine centrifuged divided by the sediment volume equals the concentration factor, which in
the preceding examples are 24 and 12, respectively.
 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.

Volume of Sediment Examined

  The volume of sediment placed on the microscope slide should be consistent for each specimen. When
using the conventional glass-slide method, the recommended volume is 20 µ L (0.02 mL) covered by a 22
× 22 mm glass cover slip.
 Allowing the specimen to flow outside of the cover slip may result in the loss of heavier elements such
as casts.

Commercial Systems

 The conventional method of placing a drop of centrifuged urine on a glass slide, adding a cover slip, and
examining microscopically has been substantially improved through the use of commercial slide
systems.
 The CLSI recommends their use together with standardization of all phases of the methodology, including
the conventional method, as discussed in the following sections.

Examining the Sediment

 Microscopic examination should be performed in a consistent manner and include observation of a

minimum of 10 fields under both low (10×) and high (40×) power. The slide is first examined under low
power to detect casts and to ascertain the general composition of the sediment. When elements such as
casts that require identification are encountered, the setting is changed to high power.
 When the sediment is examined unstained, many sediment constituents have a refractive index similar to
urine. Therefore, it is essential that sediments be examined under reduced light when using bright-field
microscopy.

Reporting the Microscopic Examination

 The terminology and methods of reporting may differ slightly among laboratories but must be
consistent within a particular laboratory system.
Routinely, casts are reported as the average number per low-power field (lpf) following examination of 10
fields, and RBCs and WBCs, as the average number per 10 high-power fields (hpf). Epithelial cells, crystals, and
other elements are frequently reported in semiquantitative terms such as, rare, few, moderate, and many, or as
1+, 2+, 3+, and 4+, following laboratory format as to lpf or hpf use.

Correlating Results

Microscopic results should be correlated with the physical and chemical findings to ensure the accuracy of the
report. Specimens in which the results do not correlate must be rechecked for both technical and clerical errors.

TABLE 6-2 Routine Urinalysis Correlations

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

Sediment Examination Techniques

 Many factors can influence the appearance of the urinary sediment, including cells and casts in various
stages of development and degeneration, distortion of cells and crystals by the chemical content of the
specimen, the presence of inclusions in cells and casts, and contamination by artifacts.
 Therefore, identification can sometimes be difficult even for experienced laboratory personnel.
Identification can be enhanced through the use of sediment stains (Table 6–3) and different types of
microscopy.

Sediment Stains

 Staining increases the overall visibility of sediment elements being examined using bright-field microscopy by
changing their refractive index. As mentioned, elements such as hyaline casts have a refractive index very similar
to that of urine. Staining also imparts identifying characteristics to cellular structures, such as the nuclei,
cytoplasm, and inclusions.
 The most frequently used stain in urinalysis is the Sternheimer-Malbin stain, which consists of crystal violet and
safranin O.

TABLE 6-3 Urine Sediment Stain Characteristics

Stain Action Function
Sternheimer- Delineates structure Identifies WBCs,
Malbin and contrasting epithelial cells,
colors of the and casts
nucleus and
cytoplasm
Toluidine blue Enhances nuclear Differentiates
detail WBCs and renal
tubular epithelial
(RTE) cells
2% acetic acid Lyses RBCs and Distinguishes
enhances nuclei of RBCs from
WBCs WBCs, yeast, oil
droplets, and
crystals
Lipid stains: Stain triglycerides Identify free fat
Oil Red O and and neutral fats droplets and
Sudan III orange-red lipid-containing
Do not stain cells and casts
cholesterol
Gram stain Differentiates gram- Identifies
positive and gram- bacterial casts
negative bacteria
Hansel stain Methylene blue and Identifies urinary
 eosin Y stains eosinophils
eosinophilic
granules
Prussian blue Stains structures Identifies yellow-
stain containing iron brown granules
of hemosiderin
in cells and casts

TABLE 6-4 Expected Staining Reactions of Urine

Sediment Constituents
Elements Usual Comments
in Urinary Distinguishing
Sediment Color of
Stained
Elements
RBCs Neutral – pink
to purple
Acid – pink
(unstained)
Alkaline –
purple Cytoplas
Nuclei m
WBCs Purple Purple
(dark- granules
staining
cells)
Glitter cells Colorless or Pale Some glitter
(sternheim light blue blue or cells exhibit
er-Malbin gray brownian
positive movement
cells)
Renal Dark shape of Light
tubular blue-purple shade of
epithelial blue-
cells purple
Bladder Blue-purple Light
tubular purple
epithelial
cells
Squamous Dark shade of Light
epithelial orange-purple purple
cells or blue
Inclusions and
matrix
Hyaline Pale pink or Very uniform
casts pale purple color; slightly
darker than
mucous
threads
Coarse Dark purple
granular granules in
inclusion purple matrix
casts
Finely Fine dark
granular purple granules
inclusion in pale pink or
casts pale purple
matrix
Waxy casts Pale pink or Darken than
pale purple hyaline casts,
but of a pale
even color;
distinct
broken ends
Fat Fat globules Rare;
inclusion unstained in a presence is
casts pink matrix confirmed if
examination
under
polarized
light
indicates
double
refraction
Red cell Pink to orange- Intact cells
inclusion red can be seen
casts in matrix
Blood Orange-red No intact
(hemoglobi cells
n) casts
Bacteria Motile: do not Motile
stain organism are
Nonmotile: not impaired
stain purple
Trichomon Light blue- Motility is
as vaginalis green unimpaired
in fresh
specimens
when
recommende
d volumes of
stain are
used;
immobile
organism also
identifiable
Mucus Pale pink or
pale blue
Backgroun Pale pink or
d pale purple

Cytodiagnostic Urine Testing

  Cytodiagnostic urine testing is frequently performed independently of routine urinalysis for detection of
malignancies of the lower urinary tract. A voided first morning specimen is recommended for testing, which is
performed by the cytology laboratory.
 Cytodiagnostic urine testing also provides more definitive information about renal tubular changes associated
with transplant rejection; viral, fungal, and parasitic infections; cellular inclusions; pathologic casts; and
inflammatory conditions.

Microscopy

 Microscopic examination of urine is best performed when the laboratorian is knowledgeable about the types of
microscopes available, their primary characteristics, and the proper use and maintenance of these microscopes.
 Bright-field microscopy is the most common type of microscopy performed in the urinalysis laboratory. Other
types of microscopy that are useful for examining the urine sediment are phase contrast, polarizing, dark field,
fluorescence, and interference contrast.

TABLE 6-5 Urinalysis Microscopic Techniques

Technique Function
Bright-field Used for routine urinalysis
microscopy
Phase-contrast Enhances visualization of elements
microscopy with low refractive indices, such as
hyaline casts, mixed cellular casts,
mucous threads, and Trichomonas
Polarizing Aids in identification of Treponema
microscopy pallidum
Fluorescence Allows visualization of naturally
microscopy fluorescent microorganisms or those
stained by a fluorescent dye including
labeled antigens and antibodies
Interference- Produces a three-dimensional
contrast microscopy image and layer-by-layer
imaging of a specimen

PROCEDURE 6-1
Care of the Microscope
1. Carry microscope with two hands, supporting
the baste 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 10x and 40x objectives with oil.
5. Clean the oil immersion lens after use.
6. Always remove slides with the low-power
objective raised.
7. Store the microscope with the low-power
objective in position and the stage centered.
Types of Microscopy

Bright-Field Microscopy

o Bright-field microscopy, in which objects appear dark againsta light background, is most frequently used
in the clinical laboratory.

Phase-Contrast Microscopy

o As light rays pass through an object, they are slowed in comparison to the rays passing through air
(media), thereby decreasing the intensity of the light and producing contrast.

Polarizing Microscopy

The use of polarized light aids in the identification of crystals and lipids. Both substances have the ability to rotate
the path of the unidirectional polarized light beam to produce characteristic colors in crystals and Maltese cross
formation in lipids. These elements seen under polarized light microscopy are birefringent, a property indicating that
the element can refract light in two dimensions at 90 degrees to each other.

Interference-Contrast Microscopy

Interference-contrast microscopy provides a three-dimensional image showing very fine structural detail by
splitting the light ray so that the beams pass through different areas of the specimen.

Dark-Field Microscopy

A technique used in the clinical laboratory to enhance visualization of specimens that cannot be seen easily
viewed with a bright-field microscope. It is often used for unstained specimens, and, in particular, to identify
the spirochete Treponema pallidum.

Fluorescence Microscopy

A rapidly expanding technique used in the medical field today. It is used to detect bacteria and viruses within
cells and tissues through a technique called immunofluorescence.

Urine Sediment Constituents

The normal urine sediment may contain a variety of formed elements. Even the appearance of small numbers
of the usually pathologically significant RBCs, WBCs, and casts can be normal.
Likewise, many routine urine specimens contain nothing more than a rare epithelial cell or mucous strand.

Red Blood Cells

In the urine, RBCs appear as smooth, non-nucleated, biconcave disks measuring approximately 7 mm in
diameter (Fig. 6–8). They must be identified using high-power (40×) objective (×400 magnification). RBCs are
routinely reported as the average number seen in 10 hpfs.

SUMMARY 6-1 Microscopic RBCs

Appearance Non-nucleated biconcave
disks
Crenated in hypertonic
urine
 Ghost cells in hypotonic
urine
Dysmorphic with
glomerular membrane
damage
Sources of identification Yeast cells
error: Oil droplets
Air bubbles
Reporting: Average number per 10
hpfs
Complete urinalysis Color
correlations: Reagent strip blood
reaction

White Blood Cells

 WBCs are larger than RBCs, measuring an average of about 12 mm in diameter.

SUMMARY 6-2 Microscopic WBCs

Appearance: Larger than RBCs
Granulated, multilobed
neutrophils
Glitter cells in hypotonic
urine
Mononuclear cells with
abundant cytoplasm
Sources of identification Renal tubular epithelial
error: cells
Reporting: Average number per 10
hpfs
Complete urinalysis Leukocyte esterase
correlation: Nitrite
Specific gravity
pH

Epithelial Cells

 It is not unusual to find epithelial cells in the urine, because they are derived from the linings of the
genitourinary system. Unless they are present in large numbers or in abnormal forms, they represent normal
sloughing of old cells. Three types of epithelial cells are seen in urine: squamous, transitional (urothelial), and
renal tubular.

SUMMARY 6-3 Epithelial Cells

Squamous Cells
Appearance: Largest cells in the sediment with
abundance, irregular cytoplasm
and prominent nuclei
Sources of error: Rarely encountered, folded cells
may resemble casts
Reporting: Rare, few, moderate, or many per
lpf
Complete urinalysis Clarity
correlations:
Transitional Cells
Appearance: Spherical, polyhedral, or caudate
with centrally located nucleus
Sources of error: Spherical forms resemble RTE
cells
Reporting: Rare, few, moderate, or many per
hpf
Complete urinalysis Clarity
correlations: Blood, if malignancy-associated
RTE Cells
Appearance: Rectangular, columnar, round,
oval or, cuboidal with an
eccentric nucleus possibly
bilirubin-stained or hemosiderin-
laden
Sources of error: Spherical transitional celss
Granular casts
Reporting: Average number per 10 hpfs
Complete urinalysis Leukocyte esterase and nitrite
correlations: (pyelonephritis)
Color
Clarity
Protein
Bilirubin (hepatitis)
Blood
Oval Fat Bodies
Appearance: Highly refractile RTE cells
Sources of error: Confirm with fat stains and
polarized microscopy
Reporting: Average number per hpf
Complete urinalysis Clarity
correlations: Blood
Protein
Free fat droplets/fatty casts

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
Complete pH
urinalysis Nitrite
correlations: LE
WBCs
Yeast
Appearance: Small, oval, refractile structures
with buds and/or mycella
Sources of error: RBCs
Reporting: Rare, few, moderate or many per
hpf, presence of WBCs may be
required
Complete Glucose Spermatozoa
urinalysis LE Appearance: Tapered oval head with long, thin
correlations: WBCs tail
Trichomonas Sources of error: None
Appearance: Pear-shaped, motile, flagellated Reporting: Present, based on laboratory
Sources of error: WBCs, renal tubular epithelial cells protocol
Reporting: Rare, few, moderate, or many per Complete Protein
hpf urinalysis
Complete LE correlations:
urinalysis WBCs Mucus
correlations: Appearance: Single or clumped threads with a
low refractive index
Sources of error: Hyaline casts
SUMMARY 6-5 Urine Casts Reporting: Rare, few, moderate, or many per
Hyaline lpf
Appearance: Mucus, fibers, hair, increased Complete None
lightning urinalysis
Sources of error: Colorless, homogenous matrix correlations:
Reporting: Average number per lpf
Complete urinalysis Protein
correlations: Blood (exercise)
Color (exercise)
Clinical Significance: Glomerulonephritis
Pyelonephritis
Chronic renal disease
Congestive heart failure
Stress and exercise
RBC
Appearance: Orange-red color, cast matrix
containing RBCs
Sources of error: RBC clumps
Reporting: Average number per lpf
Complete urinalysis RBCs
correlations: Blood
Protein
Clinical Significance: Glomerulonephritis
Strenuous exercise

WBC
Appearance: Cast matrix containing WBCs
Sources of error: WBC clumps
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
Reporting: Average number per lpf
Complete urinalysis correlations: WBC casts (pyelonephritis)
WBCs
LE
Nitrite
Protein
Bacteria
Clinical Significance: Pyelonephritis
Epithelial Cell
Appearance: RTE cells attached to protein matrix
Sources of error: WBC cast
Reporting: Average number per lpf
Complete urinalysis correlations: Protein
RTE cells
Clinical Significance: Renal tubular damage
Granular
Appearance: Coarse and fine granules in cast matrix
Sources of error: Clumps of small crystals
Columnar RTE cells
Reporting: Average number per lpf
Complete urinalysis correlations: Protein
Cellular casts
RBCs
WBCs
Clinical Significance: Glomerulonephritis
Pyelonephritis
Stress and exercise
Waxy
Appearance: Highly refractile cast with jagged ends and notches
Sources of error: Fibers and fecal merial
Reporting: Average number per lpf
Complete urinalysis correlations: Protein
Cellular casts
Granular casts
WBCs
RBCs
Clinical Significance: Stasis of urine flow
Chronic renal failure
Fatty
Appearance: Fat droplets and oval fat bodies attached to protein matrix
Sources of error: Fecal debris
Reporting: Average numer per lpf
Complete urinalysis correlations: Protein
Free fat droplets
Oval fat bodies
Clinical Significance: Nephrotic syndrome
Toxic tubular necrosis
Diabete mellitus
 Crush injuries
Broad
Appearance: Wider than normal cast matrix
Sources of error: Fecal material, fibers
Reporting: Average number per lpf
Complete urinalysis correlations: Protein
WBCs
RBCs
Granular casts
Waxy casts
Clinical Significance: Extreme urine stasis
Renal failure