ANALYSIS OF URINE AND BODY FLUIDS

MIDTERM (1ST SEMESTER - 2021-2022)

LECTURE SESSION
Lecture 1: Microscopic Examination of Urine Sediment Part 1

TOPIC OUTLINE Leukocyte esterase WBCs intact or lysed,

MICROSCOPIC EXAMINATION OF URINE SEDIMENT PART 1 WBC casts (Bacteria in
I. Correlation UTI)
II. Specimen Preparation Nitrite Bacteria (WBCs in UTI)
III. Specimen Volume Protein Increased casts and
IV. Centrifugation cells
V. Sediment Preparation
VI. Commercial system: Cen Slide
VII. Examination of the sediment è If positive for blood:
VIII. Manner of reporting • Clear = Hemoglobin or Myoglobinuria
IX. Reference intervals • Cloudy or Turbid = intact red blood cells
X. Conversion è Yeast – grows best at low pH with an increased
XI. Urine sediment stains concentration of glucose
XII. Microscopy è If true UTI = Bacteria and WBCs are seen in urine
è Bacteria with no WBC – usually contamination
only
è Some laboratories (especially outside the è WBC only without bacteria – usually
Philippines) don’t perform microscopic inflammation / inflammatory condition
examination if the urine is clear and all the tests è Bacteria that can be seen in the urine that
in the reagent strip are negative. But in the causes UTI usually can convert nitrate to nitrite
Philippines, if it is routine urinalysis physical, è Casts are made up of Tamm-Horsfall protein
chemical and microscopic examination of urine Many casts = positive for protein| RBC or WBC may be positive for protein
are all done. (though it is clear, or negative for
reagent strip)
è According to CLSI, you can skip microscopic
examination EXCEPT if: if konti, will not be
turbid or red
1. There is a request from the physician. --> Hgb na lang
natira so not
2. If the person is under a specified seen
laboratory population like pregnant infection: --wont be seen under
women, person with kidney disease, microscope
geriatrics, pediatrics.
3. If the person’s physical and chemical
examinations are abnormal.

Correlation: Macroscopic Screening, Chemical and

Microscopic Examinations

Screening Test Significance

Color (red), cloudy or Blood (intact RBC)
turbid
Clarity: • Squamous EC
1 2
Cloudy or Turbid (NP ), WBCs (P )
è Increased WBC – turbid or milky
• Confirm pathologic è If increased bacteria and infection, pH
or non-pathologic
becomes alkaline
causes of turbidity
è Color – e.g. bilirubin – amber yellow; when
Blood RBCs / RBC casts
you shake, presence of yellow foam
Glucose Yeast

NP – non-pathologic
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SPECIMEN PREPARATION
1. Fresh and properly preserved specimen
- Very hard to identify RBCs and WBCs in the
microscope from old specimen
2. Bring the specimen to room temperature before
centrifugation
- Amorphous materials and their crystals
usually precipitate in cool temperature
- Room temperature = dissolve
3. Mix specimen well before transferring into a
centrifuge tube
- RBCs ad WBCs settle at the bottom of the
tube = false negative result if not properly
mixed
SPECIMEN VOLUME
è 10-15 mL; usually 12 mL
è Less volume in pediatrics patients
- Note the volume on the report form
è Or perform correction
Example:
6mL of urine is centrifuged multiply results by 2
CENTRIFUGATION
è Centrifugation Time: 5 minutes
è Speed of the Centrifuge:
- 400 RCF (Relative Centrifugal Force
- 1,500-2,500 RPM
- RCF is better because RPM depends on the
diameter of centrifuge
è Convert RPM to RCF
RCF = (1.118 X 10-5) (radius in cm) (RPM2)
è Braking of the centrifuge: False negative
- If you push the brake, the centrifuge will
suddenly stop (DO NOT DO THIS)
- Doing this could disturb or agitate the
sediments = False Negative
- If you decant, the sediments will be included
è Centrifuge urine in capped tubes
- If no cap, use paraffin seal
- Because it forms biohazardous aerosols
SEDIMENT PREPARATION
è 0.5-1 mL should remain in the tube after
decantation
- “Bottom’s up” – technique used in
decantation
- Book says it is better to pipette off
o Example: 10mL is measured and then
0.5 sediments remained, therefore,
pipette off the remaining 9.5
o Pipetting have the tendency to disturb
the sediments (sasama ang
sediments)
o Re-suspend with 0.5-1 mL
LECTURE
è Gently tap repeatedly the lower part of the tube
with the finger to mix the sediment
!"#$%& "( $)*+& ,&+-)*($.&/
Concentration factor =
0&/*%&+- 1"#$%&
è Concentration factor (CF) using 10 mL urine; 1 mL
sediment = CF is 10
è Concentration factor (CF) using 10 mL urine; 0.5
mL sediment = CF is 20
è 0.02 mL (20uL) – volume of sediment examined
*Exam Question: How many mL is centrifuged?
- 10-15 mL; average of 12
- How long? 5 minutes
- Decant (bottom’s up)
- Flick the Tube
- 0.5 – 1 ml should remain
- put one drop on a slide – should be measured
and should be around 0.02 mL (20uL) =
volume of sediment examined
è In actual practice, they only put 2/3 of urine in the
tube, centrifuge for 5 minutes, decant without
measuring amount of sediment left, flick the
tube, and no longer measure amount put in the
coverslip.
COMMERCIAL SYSTEM: CEN SLIDE
è Closed system
- Does not require manual loading of the
centrifuge specimen in the slide
è Fast, clean, accurate, microscopic urine analysis
è Specially designed tube
- Plastic tube with cover
- Put urine up to the mark
- Centrifuge it, in less than 1 minute
è Direct reading of the urine sediment
è No decanting, re-suspending, pipetting,
sediment transfer, microscope slides and
coverslips
- After centrifugation, the sediment will go to
the narrow part of the tube and then put it in
the slide holder, then examine directly under
the microscope
- Plastic tube is not that clear (vague)
compared to the glass slide
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- If sediments are too many, it clumps in one è Reported quantitatively – bibilangin talaga;
place when using plastic tube; not distributed report number
equally è In other lab, their manner of reporting follows the
range (from lowest to highest)
EXAMINATION OF THE SEDIMENT Example:
è 10-20 fields low (10x) and high (40x) power 1-6; 2-8 = like they are overlapping in terms of
- one peak in the microscope = one field their values
- 10 fields = change or transfer view 10
times RBCs/WBCs / HPF
- in most laboratories, usually only è Average number per 10 high power fields (hpf)
examines 10 fields
- not use oil immersion None
è low power to detect casts (near the edges of 0-2
the coverslip) 2-5
- usually casts can be seen near edges of 5-10
coverslip so you need to examine the 10-25
sides, especially is positive for protein. 25-50
è high power to identify the type of cast 50-100
- Because if you use low power, you >100 (TNTC)
cannot see if it is RBC cast or WBC cast è TNTC – too numerous to count
- RBC cannot be identified under the low è Quantitative
power è If medications depend on number; adjust dosage
è bright field microscopy: subdued light depending on how many are present
è examine the sediment in the correct plane è Proper mixing is very important especially when
- wrong plane = more artifacts there are outliers
- use an epithelial cell as a point of - If Vortex Mixer is available to mix the
reference (Squamous epithelial cells – sediment, it is better to use it to achieve
can easily be seen in females than in equal distribution of the microscopic
males) elements
- If you already see a squamous epithelial
cell, do not leave that area anymore; do CRYSTALS / HPF
not move the coarse adjustment knob None 0
anymore, rather only move the fine Rare 0-2
adjustment knob. Few 2-5
Moderate 5-20
MANNER OF REPORTING Many >20

Squamous Epithelial Cells / LPF è ABNORMAL CRYSTALS average / LPF

è Semi-quantitative
None 0 è If Crystal only – report semi-quantitatively per HPF
Rare 0-5 è If abnormal crystals (usually big), they are
Few 5-20 reported per LPF, average number
Moderate 20-100
Many >100 BACTERIA / HPF
None 0
è Squamous are big, therefore use low power field Rare 0-10
è If small (like RBC and WBC), use high power field Few 10-50
è Reported Semi-quantitatively (None, rare, few, Moderate 50-200
moderate, many) Many >200
è If quantitatively, provide number
è Hard to count so usually estimation (because
Casts / LPF they are moving
None è They are small; therefore, HPF
0-2 è Rare means they are present in some field, and
2-5 absent in the other fields
5-10
>10

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REPORTING Example: Diameter of HPF is 0.35mm

2 2
è Note presence of 3.14 X 0.175 = 0.096 mm
• Budding yeast 2
• Mycelial elements 0.175 is half the value of 0.35
• Trichomonas
2. Compute for the maximum number of LPFs or
• Sperm**
HPFs in the viewing area (coverslip).
- In some lab, they do not report presence
- under a 22 mm x 22 mm cover slip = 484
of sperm cells especially for female, but 2
mm (area of cover slip)
reported when male 232
= 5040 hpfs in one cover slip
4.467
NOTES TO REMEMBER! 0.096=area of high power field
è Quantitatively (0-2, 2-5, 5-10 …) 3. Compute for the number of HPFs per mL of urine
- RBCs, WBCs, Renal Tubular Epithelial Cells tested using the concentration factor (volume
(RTE), and casts centrifuged/sediment left) and the volume of
- Very important sediment examined.
8424
è Semi-quantitatively (Rare, few, moderate, and = 21,000 hpfs/mL of urine
4.49 %: ; <9
plenty, or 1+, 2+, 3+, $+)
- Squamous epithelial cells, crystals, yeast, 0.02 – amount of sediment you put on the slide
bacteria 12 – concentration factor
è HPF
- RBCs, WBCs, Crystals, Renal Tubular 4. Calculation of the number of formed elements
Epithelial Cells (RTEs), yeast, and bacteria per milliliter of urine by multiplying the number
- Small elements of hpfs per milliliter by the average number of
formed elements per field.
è LPF 4 WBC/HPF x 21,000 = 84,000 WBC/mL
- Squamous epithelial cells, Transitional EC,
and casts

ü Some laboratories report in mL rather than LPF

or HPF

REFERENCE INTERVALS

COMPONENT NUMBER MAGNIFICATION

RBC 0-2 HPF
WBC 0-5 HPF
Casts (hyaline) 0-2 LPF
Squamous EC Few LPF
Transitional Few HPF
Renal EC 0-2 HPF
Bacteria & Yeast Negative HPF

è 0-2 RENAL EC = still normal

è Transitional EC can also be LPF URINE SEDIMENT STAINS
è It is very difficult to identify elements if it is under
CONVERSION wet preparation because they are unstained
è Usually, in routine urinalysis, no stains are used;
Average number / LPF or HPF to the number /mL only wet smear

1. Calculation of the area of an LPF or HPF Sternheimer Malbin Stain

Area = πr2 è Uses Crystal violet & Safranin
π (pi) = 3.14 è Staining microscopic elements in the urine
r = Radius; half of the diameter è Delineates/outlines the structure and contrasting
è colors of the nucleus and cytoplasm.
è RBC

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• Neutral- pink to purple Cytodiagnostic Urine Testing

• Acid – pink è Usually related to cancer cells/abnormal cells
• Alkaline - purple è Cytocentrifugation
è Staining with Papanicolaou stain
è Method for detecting and monitoring renal
disease/ malignancy
è First morning specimen

è WBC MICROSCOPY
• Cytoplasm – purple granules
• Nuclei- purple
è Squamous
• Nucleus – orange-purple
• Cytoplasm – light purple or blue

è Bacteria
• (motile) – unstained
• (non-motile) – purple
- Board Exam: Which of the following may be
unstained using Sternheimer-Malbin?
o Bacteria when alive resist stain PARTS OF A MICROSCOPE

0.5% Toluidine Blue Lens system

è Enhances nuclear detail
è Differentiates WBCs & RTEs (sometimes same
size)
è After staining, compare the nucleus of the two
Oil Red O & Sudan III
è For lipids and/or fats
• Triglycerides/Neutral Fats – orange red (they
are directly stained by these two stains)
• Cholesterol – not stained directly (needs to
heat to take up stain)
Hansel Eosinophils
è Uses Methylene blue & Eosin Y
è Oculars/Eyepiece
è Field of View
- Circular field observed thru a microscope
- One peak thru the lens is equivalent to one
Field of View (FOV)
FOV = Field Number / M
Field number = diameter of the view field in mm
M = magnification of the objective
è Ex: Field number = 18mm
- FOV in LPO= 18/10 = 1.8 mm
- FOV in HPO =18/40 = 0.45 mm
- FOV in OIO= 18/100 = 0.18 mm

Prussian Blue
è Stains structures containing iron yellow-brown
granules of hemosiderin in cells and casts
è Stains hemosiderin – blue

Gram Stain

Bacterial Casts
è Important in differentiating bacterial casts from è It is important to use both eyes because using
granular casts one eye may cause strain.
è Use dried, heat-fixed preparation of the urine
sediment Objectives
è Examination of urine sediment:
- 10× and 40×

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è Coarse knob: initial focusing
è Fine-focusing knob: sharpen the image
2 Features of Objective Lenses___________________
1. Parcentered
- Ability to retain the central FOV (when the
user switches from one objective to another,
it still stays in the center)
2. Parfocal
- Ability of the objective to remain in focus
regardless of the objective used
- Only minor adjustments by fine adjustment
knob
è Light waves pass through the specimen and
enter the objective in an inverted cone.
è The higher the numerical aperture:
• the closer the lens is to the object
• the better the light-gathering capability of
the lens
• greater resolving power
Illumination system
Light source
- Base of the microscope

Working Distance_____________________________
- Distance between the objective & the coverslip
on the slide
- Decreases as magnification of the objective
increases
- If you are using OIO, don’t use the coarse
adjustment knob because it may possibly break
the slides. Considering that the distance of
working distance is already at its minimum, and
moving the coarse adjustment would only move
it even more near.

Rheostat
- Regulate the intensity of the light

Field Diaphragm
- Controls the diameter of the light beam
Total Magnification___________________________
reaching the slide
è Objective magnification x Ocular Magnification
è Ex: Ocular mag = 10 (pag walang binigay 10 parin Condenser
gagamitin mo) - Below the stage
è LPO = 10x10 = 100 - Focuses the light on the specimen and
è HPO = 40x10 = 400 controls the light for uniform illumination
è OIO = 100x10 = 1000 - Focuses the light towards the specimen
- Needs to be adjusted to achieve optimum
visualization

Aperture diaphragm
- in the condenser controls the amount of light and
the angle of light rays that pass to the specimen
and lens
- Needs to be adjusted to achieve optimum
visualization

Numerical Aperture___________________________
è Has opening where light travels
è Refractive index of the material between the
slide and the outer lens (air or oil) and the angle
of the light passing through it.

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è Best percentage is 75%, not too bright, not too Other Ways (Not Recommended)
dim 1. Remove an eyepiece and look down through the
eyepiece tube.
Centering the condenser and Kohler illumination 2. Adjust the aperture diaphragm until
- Provides optimal viewing of the illuminated field approximately 75% of the field is visible
- The condenser and field diaphragms must be 3. Replace the eyepiece
adjusted each time the microscope is used and - In order skip this step, just estimate it to 75%,
each time the objective is changed. because it would still be a hassle to remove it.
- Alignments are adjusted to achieve optimum
visualization Body
è base
è body tube
è nose piece

Mechanical Stage
- Holds the slide on place

Routine preventive maintenance_________________

• Cover the microscope when not used to protect
it from dust
• Remove dust from the optical surfaces with a
camel-hair brush (not too hard)
• Optical surfaces should be cleaned with lens
paper
• An oil immersion lens must be wiped free of oil
and cleaned after each use; oils can damage the
objectives so it must be cleaned after use.
• An annual professional cleaning for the
microscope is recommended.

TYPES OF MICROSCOPY TECHNIQUES

Brightfield Microscopy

è Most frequently used in the clinical laboratory

routine urinalysis
è Subdued light
• For translucent structures in urine
• Hyaline cast & mucus threads
è Adjust the rheostat on the light source
è Point of reference - epithelial cell
è Avoid focusing on artifacts
è Do not examine objects in the wrong plane

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Phase Contrast Microscopy è Phase difference

- When light rays pass through an object,
they are slowed compared to the light
passing through the air (media) so the
intensity of light is decreased producing a
contrast.

è Defracted light
- Light that hit the specimen
- Slight bending occurs
è For detection of more translucent/ transparent è Background light
or low- refractile formed elements and living cells - Light that did not hit the specimen
like:
• Hyaline casts è Best contrast
• Mucous threads - When the light that does not pass through
• Mixed cellular casts the specimen is shifted one quarter of a
• Trichomonas wavelength and compared with the phase
• Sperm cells difference of the specimen

è Hardens the outlines of formed elements è Shifted one quater of a wavelength means that
enhances contrast and improves the visibility bumabagal yung light or nagdadarken ng konti
and definition of structures having a refractive
index similar to that of the urine
è Forms a halo of light around the specimen
è Uses condenser ring and phase objective ring
è Hyaline casts (blue picture - under brightfield)
- They are hard to identify
- Center pic: Under brightfield
- Right: Under phase contrast
è The specimens darken even without the stain
because the light weakens.

Condenser Ring
è Light annulus
è Placed on the condenser
è Phase objective ring
- phase plate or dark annulus
è Annulus means area between two concentric
circles
è Placed on the objectives
è These two will be aligned.

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Interference Contrast
è Difference in Phase Conrast Miscroscopy is that
in Interference Contrasts, you would distinguish
its thickness, unlike in PCM
è 3D image
è Layer by layer imaging
è Bright against a dark background without halo

Two Types___________________________________
1. Modulation Contrast (Hoffman)
- Uses split aperture, polarizer, filter
2. Differential-interference contrast DIC
Nomarski)
è Uses: Wollaston Prism and Polarizing filter
Analyzer
- Placed between the objectives and the ocular.
Polarized Microscopy
è For lipids (cholesterol) and birefringent crystals
è The lipids/fats are hard to identify when Polarizing filter
unstained, so that’s why stain and Polarized - Placed on the condenser
microscopy are used. - When added with polarizing filter, light will travel
è Dito tinitignan is cholesterol and sa stain is in only one direction from the light source
triglycerides. - Horizontal

è Triglycerides, cells and casts are not seen by

using this microscope

ü Birefringence: ability of a substance to refract

light in two directions (at 90 degrees)

è Uses a polarizer and an analyzer (these 2 are

filters)
è Not all elements could be seen in polarized
microscope, only those that have the ability to
rotate the path of the unidirectional polarized
light beam like crystals and lipids.
è Light from light source travels in several
directions (unpolarized), but once it passed
through the polarizing filter, its orientation will
become horizontal, therefore, travels only in a
single direction.
è If the specimens have the ability to rotate the
light, they would be seen
è There is another filter which is the analyzer that
is oriented at 90 degrees or vertical, which
blocks the light = dark background.
è When the object in the specimen is birefringent,
it will reach the analyzer.

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and subsequently emit light of a longer

wavelength.
è Detects specific wavelengths of light emitted
from objects

Dark-field Microscopy
ISOTROPIC è Light against a black background
è Properties of a material are the same in all è For elements that cannot be easily stained
directions è Spirochetes like Treponema pallidum
è Since they are the same, they are not rotating, so è Condenser of bright-field microscope is replaced
they are not seen under polarized microscope with a dark-field condenser that contains an
opaque disk
ANISOTROPIC è Indirect light is reflected off the object
è When the properties of a material vary with
different orientation
è Could be seen under polarizer
- Positive birefringence

è If a substance rotates the plane of polarized light

90 degrees in a clockwise direction
- Negative birefringence

è If a substance rotates the plane in a

counterclockwise direction

Type of Microscope Function Features

Bright-field For routine
urinalysis
Phase-contrast Elements with low Phase
RI Transparent objective ring,
Condenser
ring
Polarizing Microscopy Anisotropic Polarizer and
elements Analyzer
birefringent
crystals
Dark-field T palluidum Dark-field
condenser
Fluorescence Fluorescent Fluorescent
microorganisms dye, Special
Fluorescence Microscopy filters
è Imaging of innately fluorescent microorganisms Differential Interference 3D image layer by Wollaston
(bacteria and viruses) or those stained by a Contrast layer image prims,
fluorescent dye (fluorophore or fluorochrome) Polarizing
è Fluorescence is the property by which some filter
atoms absorb light at a particular wavelength

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ADDIS COUNT
è Estimate of the formed elements
è 12-hour urine specimen
è You are going to count the elements using
hemocytometer
è First procedure to standardize the quantitation
of formed elements in the urine microscopic
analysis

Normal values
RBCs 0 to 500,000
WBCs and Epithelial 0 to 1,800,000
Cells
Hyaline Casts 0 to 5,000

REFERENCES
NOTES FROM THE DISCUSSION OF:
DEAN MARIA BENILDA DE GUZMAN

DATE: SEPTEMBER 15, 2021

VIDEO LECTURE LINK:

https://www.youtube.com/watch?v=rPzdfBNCKuo&t=1s

RD
3 YEAR MEDICAL LABORATORY SCIENCE
FAR EASTERN UNIVERISTY – NICANOR REYEES MEDICAL FOUNDATION

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