AUBF Lec Week#6 (1) Intro To Microscopic Examination

MODULE 6: MICROSCOPIC EXAMINATION OF URINE
This module aims to focus on the third phase of urine examination which is the
microscopic examination of urine. Preparation of urine sample and different techniques
applicable for each type examination will be discussed. Identification of different urine
sediments and crystals and its clinical significance will also be covered.
At the end of the module, the students will be able to:
1. List the physical and chemical parameters included in macroscopic urine

screening, and state their significance.
2. Describe the recommended methods for standardizing specimen preparation and
volume; centrifugation; sediment preparation, volume, and examination; and
reporting results
3. Describe the basic principles of bright-field, phase-contrast, polarizing, dark-field,
fluorescence, and interference-contrast microscopy and their relationship to
sediment examination.
4. Differentiate between normal and abnormal sediment constituents.
5. Describe the clinical significance of red blood cells and white blood cells in urine
sediment
6. Name, describe, and give the origin and significance of the three types of
epithelial cells found in urine sediment
7. Describe the process of cast formation.
8. Describe and discuss the significance of hyaline, RBC, WBC, bacterial, epithelial
cell, granular, waxy, fatty, and broad casts
9. Identify the normal crystals found in acidic and alkaline urine.
10. Describe and state the significance of cystine, cholesterol, leucine, tyrosine,
bilirubin, sulfonamide, radiographic dye, and ampicillin crystals.
11. Differentiate between actual sediment constituents and artifacts.
12. Correlate physical and chemical urinalysis results with microscopic observations
and recognize discrepancies.
Lesson 1: Macroscopic Screening and Sediment Examination Techniques
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.
The Clinical and Laboratory Standards Institute (CLSI) recommends that microscopic
examination be performed when:
✔ Requested by a physician
✔ When a laboratory specified patient population is being tested
✔ When any abnormal physical or chemical result is obtained
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.
● Care must be taken to thoroughly mix the specimen prior to decanting a portion
into a centrifuge tube.
Specimen Volume
● A standard amount of urine, usually between 10 and 15 mL, is centrifuged in a

conical tube.
o 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.
● If obtaining a 12-mL specimen is not possible, as with pediatric patients, the
volume of the specimen used should be noted on the report form. This allows the
physician to correct the results, if indicated.
▪ Some laboratories choose to make this correction prior to reporting. For
example, if 6 mL of urine is centrifuged, the results are multiplied by 2.
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.
▪ To correct for differences in the diameter of centrifuge heads, RCF rather
than revolutions per minute (RPM) is used. The RPM value shown on the
centrifuge tachometer can be converted to RCF using nomograms
available in many laboratory manuals or by using the formula:
RCF = 1.118 × 10–5 × radius in centimeters × RPM2
● Centrifugation calibration should be routinely performed. Use of the braking
mechanism to slow the centrifuge causes disruption of the sediment prior to
decantation and should not be used.
● To prevent biohazardous aerosols, all specimens must be centrifuged in capped
tubes.
Sediment Preparation & Volume of Sediment Examined
● 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 sediment concentration factor relates to the probability of detecting elements
present in low quantities and is used when quantitating the number of elements
present per milliliter.
● 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.
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.
● If the conventional glass-slide method is being used, casts have a tendency to
locate near the edges of the cover slip.
▪ Therefore, low-power scanning of the cover-slip perimeter is
recommended.
● 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.
● Initial focusing can be difficult with a fluid specimen, and care must be taken to
ensure that the examination is being performed in the correct plane. Often an
epithelial cell will be present to provide a point of reference.
● Focusing on artifacts should be avoided, because they are often larger than the
regular sediment elements and cause the microscopist to examine objects in the
wrong plane.
▪ Continuous focusing with the fine adjustment aids in obtaining a complete
representation of the sediment constituents.
Reporting the Microscopic Examination
The terminology and methods of reporting may differ slightly among laboratories but
must be consistent within a particular laboratory system.
Casts Average number per 10 low-power field (LPF)
RBCs and Average number per 10 high-power fields (HPF)
WBCs
Epithelial cells, Reported in semi-quantitative terms such as:
Crystals, ▪ rare, few, moderate, and many
Other elements ▪ 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. However, the amount of formed
elements or chemicals must also be considered, as must the possibility of interference
with chemical tests and the age of the specimen.
● 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 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.
1. Lipid Stains
● The passage of lipids (triglycerides, neutral fats, and cholesterol) across
the glomerular membrane results in the appearance of free fat droplets
and lipid-containing cells and casts in the urinary sediment.
● The lipid stains, Oil Red O and Sudan III, and polarizing microscopy can
be used to confirm the presence of these elements.
● Triglycerides and neutral fats stain orange-red in the presence of stain,
whereas cholesterol does not stain but is capable of polarization.
● The three lipids are usually present concurrently in the sediment, thereby
permitting use of either staining or polarization for their confirmation.
2. Gram Stain
● The Gram stain is used primarily in the microbiology section for the
differentiation between gram-positive (blue) and gram negative (red)
bacteria.
● Its role in routine urinalysis is limited to the identification of bacterial casts,
which can easily be confused with granular casts.
● To perform Gram staining, a dried, heat-fixed preparation of the urine
sediment must be used.
3. Hansel Stain
● Polynuclear WBCs seen in the urinary sediment are almost always
neutrophils associated with microbial infection.
▪ However, in cases of a drug-induced allergic reaction producing
inflammation of the renal interstitium, eosinophils are present in the
sediment.
● The preferred stain for urinary eosinophils is Hansel stain, consisting of
methylene blue and eosin Y (Lide Labs, Inc., Florissant, MO); however,
Wright’s stain can also be used.
● Staining is performed on a dried smear of the centrifuged specimen or a
cytocentrifuged preparation of the sediment.
4. Prussian Blue Stain
● After episodes of hemoglobinuria, yellow-brown granules may be seen in
renal tubular epithelial cells and casts or free-floating in the urine
sediment.
● To confirm that these granules are hemosiderin, the Prussian blue stain for
iron is used and stains the hemosiderin granules a blue color.
Cytodiagnostic Urine Testing
● Not a part of the routine examination of the urine sediment

● The preparation of permanent slides using cytocentrifugation followed by staining
with Papanicolaou stain provides an additional method for detecting and
monitoring renal disease.
● It 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.
● It 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
● All microscopes are designed to magnify small objects to such a degree that the
details of their structure can be analyzed. Basically, they do this by employing a
variety of lenses and light sources as described in the following section.
● Care of the Microscope
✔ Carry microscope with two hands, supporting the base with one hand.
✔ Always hold the microscope in a vertical position.
✔ Clean optical surfaces only with a good quality lens tissue and commercial
lens cleaner.
✔ Do not use the 10× and 40× objectives with oil.
✔ Clean the oil immersion lens after use.
✔ Always remove slides with the low-power objective raised.
✔ Store the microscope with the low-power objective in position and the
stage centered.
Bright-Field Microscopy
● Bright-field microscopy, in which objects appear dark against a light

background, is most frequently used in the clinical laboratory.
● This technique employs the basic microscope previously described with a
light source emitting light in the visible wavelength range.
● Sediment constituents with a low refractive index will be overlooked when
subjected to light of high intensity. Therefore, sediments must be
examined using decreased light controlled by adjusting the rheostat on the
light source, not by lowering the condenser.
● Staining of the sediment also increases the visualization of these elements
when using bright-field microscopy.

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MODULE 6: MICROSCOPIC EXAMINATION OF URINE

At the end of the module, the students will be able to:

1. List the physical and chemical parameters included in macroscopic urine

Lesson 1: Macroscopic Screening and Sediment Examination Techniques

To enhance the cost-effectiveness of urinalysis, many laboratories have developed

● Specimens should be examined while fresh or adequately preserved.

● A standard amount of urine, usually between 10 and 15 mL, is centrifuged in a

Sediment Preparation & Volume of Sediment Examined

● Microscopic examination should be performed in a consistent manner and

Reporting the Microscopic Examination

Following laboratory format as to LPF or HPF use

Cytodiagnostic Urine Testing

● Not a part of the routine examination of the urine sediment

● Microscopic examination of urine is best performed when the laboratorian is

● Bright-field microscopy, in which objects appear dark against a light

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