[TRANS] LABORATORY UNIT 1: LABORATORY SAFETY

LABORATORY SAFETY Table 1. Types of Safety Hazards

• Includes protocols or guidelines for waste disposal in

clinical microscopy sections TYPE SOURCE POSSIBLE INJURY
• Important in protecting the lives of employees, patients,
the laboratory equipment, facilities, and the environment Bacterial, fungal, viral,
Biologic Infectious agents
• Being aware of the basic safety rules and processes, or parasitic infections
Cuts, punctures, or
and understanding the basics of safety and biosafety Needles, lancets,
Sharps blood-borne pathogen
management issues are important when working with broken glass
exposure
toxic chemicals, biological samples, physical hazards, Exposure to toxic,
and when interacting with patients. Preservatives and
Chemical carcinogenic, or caustic
reagents
• In general, everyone in the laboratory is responsible for agents
safety Equipment and
Radioactive Radiation exposure
radioisotopes
SAFETY Ungrounded or wet
Electrical equipment; frayed Burns or shock
• “…laboratory personnel must learn what hazards exist, cords
the basic safety precautions associated with them and Open flames, Burns or
Fire/ explosive
how to apply the basic rules of common sense required organic chemicals dismemberment
for everyday safety of patients, co-workers, and Wet floors, heavy Falls, sprains, or
Physical
themselves…” boxes, patients strains
• Safety policies are mandated by:
o CDC – Centers for Disease Control
and Prevention Biologic Hazards
o OSHA – Occupational Safety and
• Laboratory acquired infections are not
Health Administration
infrequent in medical laboratories
o Laboratory personnel must strictly
adhere to these laboratory safety • Potentially harmful microorganisms are
guidelines always present especially in all
laboratory specimens
• Safety procedure manuals must be readily available in • All the specimens received in laboratory
the laboratory and updated and reviewed annually by are highly infectious/pathological
the laboratory director. • Chain of Infection: Understand how
o CLSI – Clinica l and Laboratory microorganisms are transmitted
Standards Institute o To prevent the diseases and mitigate infections
▪ Provides the guidelines for • Infection control prevents or stops the spread of
writing laboratory procedures and infections in healthcare settings
policies
o Healthcare facilities (laboratories) should develop
procedures to control and monitor infections
TYPES OF SAFETY HAZARDS occurring within the facilities
• The clinical laboratory contains a variety of safety
hazards and laboratory personnel are exposed daily to
Chain of Infection
this variety of real or potential hazards, hence the need
to develop an understanding of the risks associated with • Break the chain of infection to stop the spread of
these hazards and must be safety conscious at all times infectious disease.
• Requires a continuous link between:
o (1) Infectious agent
o (2) Reservoir
o (1) Biological o (3) Portal of exit
o (2) Sharp o (4) Means of transmission
o (3) Chemical o (5) Portal of entry
o (4) Radioactive o (6) Susceptible host
o (5) Electrical
o (6) Fire/ Explosive
o (7) Physical

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MORENO. RATILLA. ROSALINDA. TAMBA. TABUDLONG. PACANA BSMLS 3 1
 LABORATORY UNIT 1: LABORATORY SAFETY

• (1) Infectious agents: bacteria, fungi, parasites, and o Patient may become infected depending on such
viruses factors like during invasive procedures, visitors, and
healthcare personnel when exposed to infectious
o The harmful germ or pathogen that causes the
specimens
infection, illness, or disease (causative agent)
o Immunocompromised patients, newborns and
• (2) Reservoir: AKA source; location of potentially infants, and elderly (weak immune system =
harmful organisms vulnerable for infections)
o This could be in/on a person, or an animal
o Some replicate in environmental reservoir: water or
soil
• (3) Portal of exit: mucous membranes of nose, mouth,
and eyes, and in blood or other body fluids.
o How the pathogens leave the source
▪ Example: Pathogens that live in a respiratory
tract.
 The lungs or the throat can leave the body
through mouth or nose with saliva/mucus
through coughing or sneezing
• (4) Means of transmission:
o How the pathogen is passed from one person to
another
▪ Direct contact
 Common route of transmission of the
pathogens
 The unprotected host touches the patient
specimen or a contaminated object
▪ Airborne
 Inhalation of dried aerosol particles
circulating on air currents or attached to dust
particles • Each chain has different corrective actions and
practices to break the chain
 Pathogens such as influenza or flu virus stays
in the air for a long time and can be breathed o e.g., proper hand hygiene, correct disposal of
in by other people contaminated materials, and wearing personal
protective equipment (PPE).
▪ Droplet
 When the host inhales materials from • As a laboratory personnel, we should always practice all
reservoir (e.g., aerosol droplets from a of these preventions and corrective actions to stop the
patient, or uncapped body fluid samples such spread of infection.
as plural fluid after it was being centrifuged, or
when specimens are aliquoted or spilled) Universal Precautions (UP)

▪ Vehicle • Instituted by CDC in 1987

 Someone ingested a contaminated • Under this guideline, all patients are considered
substance (food, water, or specimens) possible carriers of bloodborne pathogens
• The guideline recommends:
▪ Vector
o Wearing gloves when collecting/handling
 From animal or insect bite
contaminated blood and body fluids
 E.g., Anopheles mosquito – vector of o Wearing of face shields when there is a danger of
dengue virus and malaria blood splashing on mucous membranes
• (5) Portal of entry: (same as portal of exit) mucous o Disposing of all needles and sharp objects in
membranes of the nose, mouth, and eyes, skin puncture-resistant containers
abrasions, and open wounds. • The CDC excluded urine and body fluids not visibly
o The way that the pathogen enters the body of the contaminated by blood from UP
potential host o However, many specimens can contain a
• (6) Susceptible host: considerable amount of blood before it becomes
visible.
o Person at risk o As a result, the CDC modifies the UP and develops
o Final leg of the chain of transmission body substance isolation.

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 LABORATORY UNIT 1: LABORATORY SAFETY
• Body Substance Isolation (BSI)
o Not limited to bloodborne pathogens but also
consider all body fluids and moist body substance to
be potentially infectious. Thus, lab personnel should
wear gloves at all times.
o Disadvantage: does not recommend hand washing
after removing gloves unless visual contamination is
present.
Standard Precautions
• In 1996, there are new guidelines by CDC and HICPAC
(Healthcare infection Control Practices Advisory
Committee), a combined features of UP and BSI
guidelines
• Standard precautions not just focus on patient contact
but also applies the principles in handling patient
samples.
• The following are under standard precaution guidelines:
o (1) Hand hygiene
▪ Includes both hand washing and using of
alcohol-based antiseptic cleaners
o (2) Gloves
▪ Wear gloves (clean, nonsterile gloves are
adequate) when touching blood, body fluids,
secretions, excretions, and contaminated items
OSHA Standard Requirements
o (3) Mouth, nose, and eye protection
▪ Wear a mask and eye protection or a face shield
to protect mucous membranes of the eyes, nose,
and mouth during procedures and patient care
activities that are likely to generate splashes or
sprays of blood, body fluids, secretions, or
excretions.
o (4) Gown
▪ Wear a gown (a clean, nonsterile gown is
adequate) to protect skin and to prevent soiling
of clothing during procedures and patient care
activities that are likely to generate splashes or
sprays of blood, body fluids, secretions, or
excretions.
o (5) Patient care equipment
▪ Handle used patient care equipment soiled with
blood, body fluids, secretions, and excretions in
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MORENO. RATILLA. ROSALINDA. TAMBA. TABUDLONG. PACANA
a manner that prevents skin and mucous
membrane exposure, clothing contamination,
and transfer of microorganisms to other patients
or environments
o (6) Environmental control
▪ Ensure that the hospital has adequate
procedures for the routine care, cleaning, and
disinfection of environmental surfaces, beds,
bedrails, bedside equipment, and other
frequently touched surfaces.
o (7) Linen
▪ Handle, transport, and process linen soiled with
blood, body fluids, secretions, and excretions in
a manner that prevents skin and mucous
membrane exposures and clothing
contamination and that avoids the transfer of
microorganisms to other patients and
environments.
o (8) Occupational health and blood-borne
pathogens
▪ Take care to prevent injuries when using
needles, scalpels, and other sharp instruments
or devices; when handling sharp instruments
after procedures; when cleaning used
instruments; and when disposing of used
needles.
o (9) Patient placement
▪ Place a patient in a private room who
contaminates the environment or who does not
(or cannot be expected to) assist in maintaining
appropriate hygiene or environment control.
o (10) Respiratory hygiene / cough etiquette
▪ Educate health-care personnel, patients, and
visitors to contain respiratory secretions to
prevent droplet and fomite transmission of
respiratory pathogens.
▪ E.g., wearing masks, face shields, washing of
hands and social distancing
• Employer mandates all employees to adhere with the
preventive controls.
• Engineering Controls
o (1) Providing sharps disposal containers and
needles with safety devices.
o (2) Requiring discarding of needles with the safety
device activated and the holder attached.
o (3) Labelling all biohazardous materials and
containers.
• Work Practice Controls
o (4) Requiring all employees to practice Standard
Precautions and documenting training on an annual
basis.
o (5) Prohibiting eating, drinking, smoking, and
applying cosmetics in the work area.
o (6) Establishing a daily work surface disinfection
protocol.
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 LABORATORY UNIT 1: LABORATORY SAFETY

• Personal Protective Equipment Hand Hygiene

o (7) Providing laboratory coats, gowns, face shields,
and gloves to employees and laundry facilities for • Laboratory personnel must always sanitize hands…
non-disposable protective clothing. o Before patient contact
• Medical o After gloves are removed
o Before leaving the work area
o (8) Providing immunization for the hepatitis B virus o When hands have been knowingly contaminated
free of charge. o Before going to designated break areas
o (9) Providing medical follow-up to employees who o Before and after using bathroom facilities
have been accidentally exposed to blood-borne
pathogens.
Biological Waste Disposal
• Documentation
o (10) Documenting annual training of employees in
safety standards.
o (11) Documenting evaluations and implementation
of safer needle devices.
o (12) Involving employees in the selection and
evaluation of new devices and maintaining a list of
those employees and the evaluations.
o (13) Maintaining a sharps injury log including the
type and brand of safety device, location and
description of the incident, and confidential
employee follow-up.
Personal Protective Equipment

• PPE used in the laboratory includes gloves, fluid-

resistant gowns, eye and face shields, and Plexiglas
countertop shields
• Gloves should be worn at all times when in contact with
patients, specimens (e.g., urine and stool samples),
handling machines, and laboratory equipment or
fixtures.
• Fluid-resistant laboratory coats with wrist cuffs are worn
to protect clothing and skin from exposure to patients’
body substances.
• Masks and goggles, full-face plastic shields that cover
the front and sides of the face, mask with attached
shield, and Plexiglas countertop shields.
o Eyes, nose, and mouth must be protected from
specimen splashes and aerosols

• All biologic waste, except urine, must be placed in

appropriate containers labelled with the biohazard
symbol.
• Urine may be discarded by pouring it into a laboratory
sink under a Plexiglas countertop shield directed to the
septic tank and not in the sewage. Otherwise, it may
pose as an environmental pollution.
• Decontaminated following institutional policy:
o (1) Incineration
o (2) Autoclaving
o (3) Pickup by a certified hazardous waste company

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• Disinfection of the sink using a 1:5 or 1:10 dilution of
sodium hypochlorite should be performed daily
• Empty urine containers can be discarded as
nonbiologically hazardous waste.
Sharp Hazards
• Needles, lancets, and broken glassware
o Should be handled and disposed
appropriately to prevent risk of
infection to the laboratory and
housekeeping staff.
• Must be disposed in puncture-resistant, leak-proof
container with the biohazard symbol
o Label with ‘sharps’
• Biohazard sharp containers should not be overfilled and
replace when the safe capacity mark is reached.
Chemical Hazards
• Exposure to toxic chemicals poses a
real threat to the health and safety of
laboratory staff. Thus, every
chemical in the workplace should be
presumed hazardous.
• Chemical Spills and Exposure
o When skin contact occurs, flush the area with large
amounts of water for at least 15 minutes, then seek
medical attention.
• Chemical Handling
o Chemicals should never be mixed together unless
specific instructions are followed.
▪ Chemicals must be added in the order that is
specified.
• Chemical Hygiene Plan (CHP)
o The OSHA requires all facilities to have a written
CHP for the employees. The purpose of this is to
detail the following:
▪ (1) Appropriate work practices
▪ (2) Standard operating procedures
▪ (3) PPE
▪ (4) Engineering controls
▪ (5) Employee training requirements
▪ (6) Medical consultation guidelines
• Chemical Labelling
o Hazardous chemicals should be labelled with a
description of their particular hazard (poisonous,
corrosive, flammable, explosive, teratogenic, or
carcinogenic)
▪ All chemicals (including solutions and chemicals
transferred from their original containers) should
be labelled with their common names,
concentrations, and hazards to prevent/reduce
incidents caused by exposure to toxic chemicals.
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o Chemical Hazard Symbols:
• Material Safety Data Sheets (MSDS)
o A major source of safety information for employees
who may use hazardous materials in the laboratory
o Information contained in MSDS:
▪ (1) Physical and chemical characteristics
▪ (2) Fire and explosion potential
▪ (3) Reactivity potential
▪ (4) Health hazards and emergency first aid
procedures
▪ (5) Methods for safe handling and disposal
▪ (6) Primary routes of entry
▪ (7) Exposure limits and carcinogenic potential
o The MSDS should be available to all employees
prior to use of hazardous materials and kept close to
where it is used and located.
Radioactive Hazards
• Radioactivity may be encountered in the
clinical laboratory when procedures
using radioisotopes are performed.
• Radioisotopes in clinical laboratory
o Only properly trained personnel will
work with radioisotopes
• “The amount of radioactivity present in the clinical
laboratory is very small and represents little danger;
however, the effects of radiation are cumulative related
to the amount of exposure”
Electrical Hazards
• Fully automated laboratories contain a
large amount of electrical equipment
with which workers have frequent
contact.
• In clinical microscopy, the danger of
water or fluid coming in contact with
equipment is greater in this laboratory
setting.
• Equipment should not be operated with wet hands.
• There should be a laboratory personnel that constantly
monitors electrical equipment closely such as frayed
cords and overload circuits.
• Equipment that has become wet should be unplugged
and allowed to dry completely before reusing.
• All electrical equipment must be grounded with three-
pronged plugs (recommended).
o The third prong provides an alternate pathway for
electricity in the event of a fault.
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 LABORATORY UNIT 1: LABORATORY SAFETY

Fire / Explosive Hazards Hazardous Materials Classification

• All laboratory personnel must be • NFPA (National Fire Protection Association)
alert for all conditions that might diamond
pose a risk of fires.
• When a fire is discovered, all o Provides a quick visual representation of the health
employees are expected to take the hazard, fire hazard (flammability), reactivity, and
actions in the acronym RACE: specific hazard that a chemical may pose during a
fire
o Rescue - rescue anyone in immediate danger o Also consists of four color-coded fields
o Alarm - activate the institutional fire alarm system
o Contain - close all doors to potentially affected ▪ Blue (health hazard), Red (fire hazard), Yellow
areas (reactivity), and White (specific hazard).
o Extinguish/Evacuate - attempt to extinguish the
fire, if possible or evacuate, closing the door.
• Flammable chemicals should be stored in safety
cabinets and explosion-proof refrigerators.
o Store corrosive, toxic and highly reactive chemicals
in a well-ventilated area.
o Store chemicals that can ignite at room temperature
in a flammable cabinet.
• Cylinders of compressed gas should be located away
from heat and securely fastened to a stationary device
to prevent accidental capsizing.
o Storage of compressed gases in the laboratory
requires precautions unique to the unusual
containers in which these materials are kept and the
high pressures they are subject to
o Cylinders are kept chained to the wall to avoid fall
over
o Safety cap must be secured over the valve of the
cylinder when moved or taken out of service.

Fire Extinguisher

Table 2. Types of Fires and Fire Extinguisher

Type /
Extinguishing
Fire Type Composition of Extinguisher
Material
Fire
Wood, paper, Water
Class A Class A
clothing
Flammable Dry chemicals,
Class B organic Class B carbon dioxide, Physical Hazards
chemicals foam, or halon
Dry chemicals, • Physical hazards are not unique to
Class C Electrical Class C carbon dioxide, or the laboratory, and routine
halon precautions observed outside the
Sand or dry
Combustible None workplace apply.
Class D powder
metals Class ABC • General precautions to consider are
Dry chemicals to:
Liquid designed to
Grease, oils,
Class K Class K prevent splashing o (1) Avoid running in rooms and
fats
and cool the fire. hallways
o (2) Watch for wet floors
o (3) Bend the knees when lifting heavy objects
• How to Operate Fire Extinguisher
o (4) Keep long hair pulled back
o The acronym PASS can be used to remember the o (5) Avoid dangling jewelry
steps in the operation: o (6) Maintain a clean and organize work area
o (7) Wear closed-toed shoes that provide maximum
▪ (1) Pull pin
support
▪ (2) Aim at the base of the fire
▪ (3) Squeeze handles ▪ Essential for safety and comfort
▪ (4) Sweep nozzle side to side

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[TRANS] LABORATORY UNIT 2: URINE SPECIMEN COLLECTION

URINE SPECIMEN COLLECTION o Computerized: input, print, and accompanied to the

urine specimen
• Important thing to consider prior to the actual urinalysis
testing because there are a lot of things that should be • Must accompany specimens delivered to the
prioritized and considered in order to achieve the main laboratory
goal o To make sure that the specimen is for the specific
o Main goal: obtain accurate and reliable lab results patient
o Information must coincide
Urine Containers
Specimen Rejection
• clean, dry, leak-proof containers
• Improperly labelled and collected
o Leakage may affect testing hence
must be avoided o Double check

• Disposable containers must be used • Unlabelled containers

o Single-use only
▪ Should not be washed, autoclaved, and reused
• Wide mouth and with wide flat bottom
o For easier access for patients in pooling of urine
• Clear material
o Except for its cap, the overall appearance should be
clear so the urine’s level of volume can be
immediately seen
o Contamination is noticeable
• Recommended capacity: 50 mL
o Specifically for routine urinalysis testing
Labels
• Must be attached to the container (not to the lid) and
should not detach if the container is refrigerated or
frozen
o There are some instances that the samples are
processed by batch (e.g. 50 samples) and there is a
possibility that the urine container lids are
interchanged → prone to error
• Patient’s name and identification number
• Date and time of collection
o Very crucial
o The integrity of the samples should be preserved
o Instruct the patients to take note of the time of
collection before they are going to submit the
sample to the lab
• Patient’s age and location (address)
• Healthcare provider’s name
o Hospital or laboratory
Requisitions
• May be manual or computerized
o Manual: handwritten
o Double check; if the patient is still there, ask them to
label their specimen
• Nonmatching labels and requisition forms
• Contaminated with feces or toilet paper
• Contaminated exteriors
• Insufficient quantity
o Recommended: 50 mL
o However, there are exemptions.
• Improperly transported
o Specifically on urine specimens
▪ 24hr urine: requires a specialized transportation,
in which it should be transported with ice prior to
laboratory delivery
Specimen Handling
Specimen Integrity
• Specimens should be delivered to the lab promptly
o After collection, the patients should take note of the
time and deliver the sample immediately
• Tested within 2 hours
o Within the 2hrs, the MT is already done performing
the physical aspect, the chemical test, and the
microscopic analysis
• If cannot be delivered and tested within 2 hours:
refrigerate or add chemical preservative
o Refrigeration – most common method of preserving
the urine
NOTE: If the urine sample can be processed within 2 hours,
there’s no need for refrigeration or chemical preservative
UNLESS the specimen is needed for further testing.
Specimen Preservation
• Refrigeration: 2 to 8 degrees C (return to RT before
testing)
o Most common method

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 LABORATORY UNIT 2: URINE SPECIMEN COLLECTION

o RT – room temperature Inhibits Prone to false-

chemical tests negative
▪ Allow the sample to return to RT before Good
Sodium for glucose, results
preservative for
performing tests for it. fluoride blood, and LE
drug analyses
(Leukocyte
• Urine for culture: refrigerated during transit and kept Esterase)
refrigerated until cultured up to 24 hours Convenient
when
Check tablet
refrigeration is
Urine Preservatives Commercial composition to
not possible;
preservative determine
have controlled
• If refrigeration is impossible, chemical preservatives tablets
concentration
possible effects
may be added on desired tests
to minimize
• Ideal preservative: interference
Contains
o bactericidal/bacteriostatic collection cup,
transfer straw,
▪ Bactericidal: ability to kill bacteria
Urine C&S (Culture &
▪ Bacteriostatic: prevents growth of the bacteria Collection Sensitivity)
Burns or
dismemberment
o inhibits urease Kits preservative
tube, or UA
▪ Bacteria that produce urease: Proteus, (Urine
Providencia, etc. Analysis) tube
Uses boric
o preserves formed elements in the sediment Stable at RT
acid, sodium
(Room
borate, and
▪ 3 Assessments in urinalysis: Physical, Chemical, A. Light gray
Temperature) Do not use if
sodium
and Microscopic for 48 hours; urine is below
and gray formate;
▪ In the microscopic examination, look if there are prevents the minimum fill
C&S tube keeps pH at
bacterial line
elements/sediments in the urine. about 6.0
growth and
▪ Chemical preservatives should preserve the metabolism
(ideal for
formed elements such as RBC, urinary cast, and sediments)
WBC crystals, not cause deterioration or Round or
Use on
B. Yellow UA Must refrigerate conical bottom,
disintegration of these elements. automated
Plus tube within 2 hours no
instruments
o should not interfere with chemical tests preservative
Must be filled to Preservative is
▪ In Chemical examination of urine, reagent strip minimum fill sodium
testing is performed with different parameters. line; bilirubin propionate,
▪ Chemical preservatives are added to preserve C. Cherry Stable for 72 and ethyl paraben,
the urine but should not interfere with chemical red/yellow hours at RT; urobilinogen and
tests because this may cause false-positive or Preservative instrument- may be chlorhexidine;
Plus tube compatible decreased if round or
false-negative results or false increase in
specimen is conical
reagent slip parameter exposed to light bottoms
and left at RT
Table 1. Urine Preservatives
NOTE:
• Urobilinogen will photo-oxidize if exposed to light
Additional which can lead to a false decrease
Preservative Advantage/s Disadvantage/s
Information
• Amorphous phosphates precipitates in alkaline urine
while urates precipitates in acidic urine.
Precipitates Prevents
Does not amorphous bacterial
Refrigeration interfere with phosphates and growth for 24
chemical tests Urine Collection Kit (BD
urates which are hours
urinary crystals Vacutainer)
Bacteriostatic: Keeps pH at
Interferes with
Prevents about 6.0, can
drug and
Boric acid bacterial be used for
hormone
growth and urine culture
analyses
metabolism transport

Acts as a Rinse
Excellent
reducing agent, specimen
sediment
interferes with containers with
preservative
chemical tests formalin to
that can
Formalin
preserve cells
for glucose, preserve cells • Left picture (start from the leftmost tube)
blood, and casts
such as RBC, o 3rd tube – Yellow UA Plus tube
Leukocyte
WBC, and
Esterase (LE), o 4th tube – Cherry red/Yellow Preservative Plus tube
urinary cast
and copper
reduction

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 LABORATORY UNIT 2: URINE SPECIMEN COLLECTION

Changes In Unpreserved Urine • RBC and WBC are very sensitive in terms of pH

Table 2. Changes in Unpreserved Urine o Will disintegrate in increased pH

• Trichomonas vaginalis is motile in a freshly voided
urine.
ANALYTE CHANGE CAUSE

Oxidation or reduction TYPES OF SPECIMENS

Color Modified/darkened of metabolites such as
ketones or bilirubin Random specimen
bacterial multiplication
Odor Increased causing breakdown of • Most common specimen; ideally collected midstream
urea to ammonia clean catch
Increased Breakdown of urea to o First portion of the urine stream should be voided in
pH
ammonia by urease- which the midstream portion of the urine should be
producing bacteria/loss collected
of CO2
• Actual time of voiding should be recorded
Increased Multiplication of nitrate-
Nitrite
reducing bacteria o Time after urinating and collection is recorded
Increased
Bacteria Multiplication First morning specimen
Bacterial growth and
Clarity or clearness Decreased precipitation of • Ideal screening specimen
amorphous material • Most concentrated specimen
• Assures detection of chemicals and formed elements
Decreased Glycolysis and
Glucose
bacterial use • Must be delivered to the lab within 2 hours or keep
refrigerated (to preserve the analytes present in the
Decreased Volatilization and urine)
Ketones
bacterial metabolism
• Essential to prevent false-negative urine pregnancy
Bilirubin
Decreased Photo-oxidation to tests, and to evaluate orthostatic proteinuria
biliverdin
o HCG is high during the morning
Urobilinogen Decreased Oxidation to urobilin o First morning urine is ideal for pregnancy testing

Decreased Disintegration in dilute 24-Hour or Timed Specimen

RBC and WBC
alkaline urine
casts
(increased pH) • To measure the exact amount of a urine chemical
Decreased o E.g., Glomerular filtration rate (GFR)
Trichomonas Loss of motility, death
vaginalis • Urine chemicals that that can be measured at 24-hour
period
NOTE:
• UNPRESERVED = not processed beyond 2 hours o E.g., Creatinine, Inulin, Cystatin C, Radioisotopes
• Increased Analytes: “PaBaON” • Urine chemicals
o Pa – pH • If a substance exhibits diurnal variation, a 24-hour
o Ba – Bacteria collection is required
o O – Odor • Example:
▪ Normal urine odor is faintly aromatic o Day 1: 7 AM: patient voids and discards specimen;
▪ Ammoniacal/strong ammonia-like odor for collects urine for 24 hours
unpreserved urine
▪ The patient's first pee will be discarded.
o N – Nitrite ▪ The next urine will be included once the previous
urine has been discarded.
▪ pH, Nitrite, and Bacteria are connected; so an
increase in either one will also increase the other o Day 2: 7 AM: patient voids and adds this urine to
two previously collected urine
• If certain bacteria are present, it can affect the pH and ▪ The moment the patient begins collecting urine
nitrite on Day 1 will determine when the last urine will
be added on day 2.
o Bacteria multiplication will affect the pH, reducing
nitrate to nitrite • All specimens must be refrigerated or kept on ice during
collection
• Ketones are volatile, thus precipitate/disintegrate when
exposed to air. o It will be rejected if the specimen is not adequately
kept or transported.
o will be the first to be unpreserved o Refrigeration helps in the preservation of analytes
• Bilirubin will be oxidized into Biliverdin when exposed that should be measured.
to light

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 LABORATORY UNIT 2: URINE SPECIMEN COLLECTION
• In the lab, sample must be thoroughly mixed before
aliquoting
o This allows proper segregation of analytes and
sediments
Midstream Clean-Catch Specimen
• Safer, less traumatic method for obtaining urine for
bacterial culture and routine urinalysis
o Can be used for Culture and Sensitivity in
Bacteriology and for Routine Urinalysis
• Less contamination (with proper instruction to patients)
• Patients are provided with appropriate cleansing
materials (mild antiseptic towelettes), a sterile container,
and instructions
o Patients must be adequately instructed for them to
comprehend the instructions given to them
Catheterized Specimen
• Collection of urine under sterile conditions by passing a
catheter through the urethra into the bladder
o A hollow tube will be inserted into the genitals and
advanced until it reaches the urinary bladder.
• Commonly requested for bacterial culture
• It is also done with bedridden patients
Suprapubic Aspiration
• External introduction of a needle
through the abdomen into the
bladder
• Ideal for culture and cytology
tests; urethra bypass
2-Hour Post-Prandial Urine
• For monitoring diabetes mellitus (DM)
• 2 hours after eating
Glucose Tolerance Specimen
• To correspond with the blood collection during GTT
o GTT – collects blood at various times to see if it
correlates with other results.
• Tests the patient’s ability to metabolize a measured
amount of glucose
o NOTE: Limitations: the body does not re-absorb all
of the glucose it consumes.
RAMOS, MANZANO, BAUZON, NUEVO, CRUDA, BOYOSE, VILLANUEVA, EVANGELISTA, TEVES, MARASIGAN. PACANA
Prostatitis Specimens
• Prostatitis is the swelling or inflammation of the
prostate gland.
o Commonly in males
3 Methods of Collecting Urine in Prostatitis
• Three-Glass Collection (3 specimens)
• Pre- and Post-Massage Test (2 specimens)
• Meares-Stamey Test (4 specimens)
Three-Glass Collection
• (1) Specimen 1: first urine/stream voided
• (2) Specimen 2: midstream portion
• (3) Prostate massage
o Prior to the third specimen, the prostates are being
massaged
• (4) Specimen 3: remaining urine (w/ prostate fluid)
o Does not only contain pure urine but it has also
prostate fluid
• Prostate infection: if the third specimen has a WBC
per high power field and bacterial count 10 times that
of the first specimen
o E.g., The first specimen has only 3 WBC or bacteria
while on third specimen is numerous to count, this
indicates a prostate infection.
o Read the first and third specimen under the high-
power field and look for WBC and bacteria.
o Comparison of the WBC and bacterial count
NOTE: If the 2nd specimen is positive, the results are
invalid (infected urine contamination)
o Control specimen – 2nd specimen
▪ WBC and bacteria should not be seen in this
specimen
▪ If there is, it indicates urine contamination
Pre- and Post-Massage Test
• Before and after the prostate massage
• (1) Specimen 1: clean-catch midstream prior to
massage
• (2) Prostate massage
• (3) Specimen 2: urine sample after massage
• Positive result: bacteriuria in the post-massage
specimen of greater than 10 times the pre-massage
count
o Comparison in the number of bacteria
o E.g., Indication of prostate infection is if the bacteria
are greater than specimen 2 with specimen 1
Meares-Stamey Test
• (1) Specimen 1/VB1: initial voided urine (test for
urethral infection or inflammation)
• (2) Specimen 2/VB2: midstream urine (test for bladder
infection)
• (3) Massage the prostate
• (4) Specimen 3/EPS: Expressed Prostatic Secretions
BSMLS 3 4
 LABORATORY UNIT 2: URINE SPECIMEN COLLECTION
o NOTE: Prostatic secretions are being cultured and
examined for white blood cells.
o Specimen that is being examined under the
microscope.
o Abnormal result: more than 10-20 WBCs per high
power field on EPS
▪ Indicates that the patient has prostatitis infection.
• (5) Specimen 4/VB3: post-prostatic massage urine
specimen
Pediatric Specimens
• On pediatric specimens, it is hard to collect their urine in
a container because we do not know when they are
going pee.
• Use: Soft, clear plastic bags with hypoallergenic skin
adhesive (check every 15 minutes)
o It will attach on the genitals of babies
o These urine bags are being checked every 15
minutes
• Routine Testing: ensure area is free of contamination,
attach bag firmly over the genital area avoiding the anus
o To avoid contamination, the bag should avoid
touching the anus of the baby.
o But this technique is very prone to contamination
because we cannot control the actions of the baby
• Microbiology: clean the area with soap and water, dry
the area, and firmly apply the sterile bag
o Apply soap and water to ensure that the site is
sterile because it is for microbiological studies
• Samples must be transferred to a sterile urine container
upon submission to the laboratory
o If the urine bag will be full, transfer it to a urine
container before submitting it to the laboratory.
1 2 3 (diuretics, water)
4 5
Based on the illustration:
• (1) Urine Bag: We will attach a hypoallergenic plastic
urine bag for babies
• (2) Clean catch: A Clean catch can also be applicable
if the baby is already peeing
• (3) Voiding stimulation
• (4) Catheter: If it is really needed
• (5) Suprapubic aspirate
o The catheter and suprapubic aspirate method are
traumatic for the baby.
RAMOS, MANZANO, BAUZON, NUEVO, CRUDA, BOYOSE, VILLANUEVA, EVANGELISTA, TEVES, MARASIGAN. PACANA
Drug Specimen Collection
NOTE: Drug specimen collection is used for drug testing. The
most common specimen used for testing drugs in humans is
the urine.
• Urine collection is the most vulnerable part of drug
testing
o The collection of urine is crucial because it is very
prone to tampering and adulteration.
• Chain of custody (COC): the process that documents
sample identification from time of collection to the
receipt of results
o To ensure that the collection is free from
interferences and any illegal actions
o From the time that you fill up the forms, collecting
the sample, up to the releasing of results.
• All personnel handling the specimen must be noted
• Collection may be ‘witnessed’ or ‘unwitnessed’
o Commonly there is a witness when collecting
Tampering of specimens
• Adulteration
o with a substance not normally present in the test
specimen
o Examples of the common ways specimens are
adulterated:
▪ Bleach (slows Ab-Ag reaction)
▪ Ammonia/liquid soap/table salt (increase pH of
urine)
▪ Hydrogen peroxide (destroys 50% of THC)
 THC – Tetrahydrocannabinol
 It is possible that the THC won’t be detected
▪ Vinegar (decreases pH)
• Dilution
o Less than normal physiological constituents
o Commonly it is water, but the comfort rooms used in
the collection of urine for drug testing, there are no
outlets for water.
• Substitution
o common
o E.g., Submitting “patis” instead of urine, using
someone else’s urine
Parameters for validity test
• Color
• Appearance: clear
o Clarity of the urine
• Odor: aromatic (or faintly aromatic)
• Volume: 30-60 mL
o Required for drug testing
o Drug testing requires a large volume of urine
• Temperature: 32-38 degrees C (Freshly voided)
• pH: 4.0-9.0 (adulterated if < 3 or >11)
o < 3 – too acidic
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 LABORATORY UNIT 2: URINE SPECIMEN COLLECTION

o >11 – too alkaline or diluted

• Specific Gravity: 1.003-1.030 (diluted: <1.003)
• Creatinine: if < 20 mg/dL, sample is diluted
o For us to know if the patient submitted undiluted
urine, you can test for creatinine.
▪ Creatine levels is detected in normal urine.

RAMOS, MANZANO, BAUZON, NUEVO, CRUDA, BOYOSE, VILLANUEVA, EVANGELISTA, TEVES, MARASIGAN. PACANA BSMLS 3 6
 [TRANS] LABORATORY UNIT 03: PHYSICAL EXAMINATION OF URINE

URINALYSIS THREE IMPORTANT PARTS OF URINALYSIS:

• Subdivided into 3 important parts: Physical Examination
o (1) Physical examination • Color – pale yellow, yellow, dark yellow
o (2) Chemical examination
• Appearance/Transparency – clear, turbid, milky
o (3) Microscopic examination
(depends on the clarity)
• Reagent strip – test currently employed in determining
Chemical Examination
specific gravity
Steps in Urinalysis • The specific gravity is included in the chemical
examination but it should be part in the physical
• (1) Specimen Collection examination.
• (2) Sample Verification • Why is it part of the Chemical Examination?
o Proper verification gives the assurance that correct o One way of determining the chemical examination is
specimen is being processed through the reagent strips (most commonly done in
the laboratory)
• (3) Physical Examination of Urine o Parameters included in the reagent strip are:
• (4) Chemical Examination of Urine
• (5) Microscopic Examination of Urine ▪ (1) Blood
• (6) Release of Results ▪ (2) Bilirubin
▪ (3) Urobilinogen
Physical Examination of Urine ▪ (4) Ketone
▪ (5) Protein
• (1) Color ▪ (6) Nitrite
• (2) Clarity – transparency or turbidity of the sample ▪ (7) Glucose
• (3) Specific gravity ▪ (8) pH
• (4) Odor – not part of the routine physical examination, ▪ (9) Specific Gravity  (part of the parameters)
but it is a noticeable property that may give a clue of the ▪ (10) Leukocyte
possible disorder or diseases of patients. o Specific gravity was considered as one of the
o E.g., Case of Maple Syrup Urine Disease (MSUD) reagent strip

▪ Rare metabolic disorder, common in pediatric Microscopic Examination

patients
▪ Caused by a defect of an enzyme that breaks • Any parameter that has to be identified microscopically
down amino acid (sediments, cellular, etc.)
▪ Very sweet urine odor o (1) Cast
o (2) Crystals
• Based on observation or senses and judgment of the
medical technologists. o (3) Epithelial Cells
o (4) Parasites
• Specific parameters are followed to come up with
o (5) Bacteria
almost the same observation and to avoid
o (6) Spermatozoa
subjectiveness and differences in interpretation.
Urinalysis Result PARAMETERS:
Urine Color

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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

• Varies from almost colorless to black

• A noticeable change in urine color is often the reason
that a patient seeks medical advice
o E.g., Sudden change in color: from yellow to red
• Common urine colors
o (1) Pale yellow (AKA straw color) Milky/Precipitated/Clotted
o (2) Yellow
o (3) Dark yellow Examples of Different Appearances of Urine
o (4) Red
o NOTE: Do not put “-ish” (yellowish) as urine color
• Urochrome
o The pigment that causes the yellow coloration of
A. B. C.
urine
o Metabolism of Bilirubin:
▪ Bilirubin will undergo several processes from the
liver. It needs to be conjugated because bilirubin
is water insoluble.
▪ It is very toxic in the body. It must be converted
into water soluble to be easily eliminated into the
body • (A) Red, cloudy; (B) Red, clear; (C) Yellow, cloudy
o Product of endogenous metabolism
o Under a normal condition, the body produces it at a Urine Color and Clarity Procedure
constant rate – able to identify the yellow color of • (1) Evaluate an adequate volume of a specimen
the urine
o It would be difficult in identifying the color and clarity
Urine Clarity of the sample if you only have a portion less than an
mL
• General term that refers to the transparency or
turbidity of a urine specimen • (2) Use a well-mixed specimen
• Visual examination of a mixed specimen while holding it
o The specimen must be thoroughly mixed the
in front of a light source
specimen before identifying the color and clarity.
o As long as the specimen is clearly visible o Urine has sediments. If not properly mixed, the
sediment will settle down because of the gravity and
• May be reported as: clear, hazy/slightly cloudy, the sample becomes clear and may create a falsely
cloudy, turbid, and milky negative result
o Unlike the color, clarity has a parameter to identify
▪ This will create confusion to the result since the
what is clear, hazy, cloudy, turbid, and milky
physical examination shows a clear result. But in
the microscopic examination, a lot of different
Table No. 1 Urine Clarity sediment were seen.
▪ The physical examination must coincide with the
CLARITY Term microscopic examination.
• (3) View the urine through a clear container
Clear
No visible particulates, • (4) View the urine against a white background using
transparent adequate room lighting
Hazy
Few particulates, print easily • (5) Maintain adequate room lighting
seen through urine • (6) Evaluate a consistent volume of urine
Many particulates, print blurred • (7) Determine color and clarity
Cloudy
through urine
Print cannot be seen through Specific Gravity
Turbid
urine
• Evaluation of urine concentration
Milky May precipitate or be clotted o Measures the kidney’s ability to concentrate
glomerular filtrate
o Determines the adequacy of specimen
Newsprint concentration to ensure accuracy of chemical tests

• Place the urine sample at the back of newsprint
o Through time or experience, it is not necessary to
use this technique anymore. As long as you are
already confident in identifying which is clear or
cloudy.
• Measures the density of the dissolved chemicals (e.g.,
sediments, crystals) in the urine
o Solvent – Water
o Solute – dissolved chemicals.

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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE
• Defined as density of the solution compared to the
density of a similar volume of a distilled water with a
specific gravity of 1.000 at a similar temperature.
o Technically, urine is water but contains dissolved
chemicals that makes water different from urine
o The specific gravity of urine is a measure of the
density of dissolved chemicals in the specimen
▪ In the urine, it is referring to the solute (dissolved
chemicals) rather than solvent (water).
• Normal random specimens: 1.002-1.035
o Most random specimens: 1.015-1.030
o There is a value in specific gravity unlike in water –
1.000
▪ No numerical value since it does not contain any
dissolved materials or chemicals.
▪ Purely solvent.
• Q: Other than specific gravity, how can we confirm
that the sample submitted by the patient is water
and not urine?
o Microscopic examination
▪ No sediments, no cells
o Chemical exam
• SG of plasma filtrate leaving the glomerulus: 1.010
o Isosthenuric: 1.010
o Hyposthenuric: below 1.010
o Hypersthenuric: above 1.010
• Influenced not only by the number of particles present
but also by their size
o Influence the density of the sample.
o Presence of large molecules requires correction (in
some methods of SG determination)
DIFFERENT METHODOLOGIES OF SPECIFIC
GRAVITY
• (1) Urinometry and (2) Harmonic oscillation
densitometry
o Part of historical methodology of identifying specific
gravity of urine
• (3) Osmolality, (4) Refractometry and (5) Reagent strip
o Currently being employed in laboratories
o Reagent strip – most common
Urinometry
• Specific gravity measurement
• Uses a urinometer
• Principle: based on buoyancy or density
o Water – purely solvent, therefore the
urinometer will sink.
o Urine – solute with dissolved
chemicals and sediments, therefore
the urinometer will float due to
additional mass.
• A weighted float attached to a calibrated
scale
• Displaces a volume of liquid equal to its weight
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• Designed to automatically sink to a level of 1.000 in
distilled water
• Additional mass causes the float to displace a volume of
urine smaller than that of distilled water (will float higher
in urine)
• The level to which the urinometer sinks represents the
specimen’s specific gravity
Parts of Urinometer
• Stem
• Float
• Weight
Procedure
• Allow urine to reach room temperature.
• Fill the glass cylinder with urine up to
2/3rds (at least 15 ml of urine).
• Make sure that there are no
bubbles/foam in the glass cylinder.
• Gently drop the urinometer into the
container. Make sure the urinometer
does not touch the bottom or the sides of
the container and allow it to float freely.
• Impart a slight spin to the float as it is
released.
• Read the calibrated graduation at the
lower meniscus.
Disadvantages
• Large volume of urine is needed
o Minimum is 15 ml
o Less accurate than other methods
o Not recommended by CLSI
• Turbid specimens are difficult to read
o There are a lot of sediments and the urinometer will
be difficult to sink
• A lot of corrections are needed to be done
o Temperature correction
▪ Add 0.001 for every 3C above the calibration
temperature
3
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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

▪ Subtract 0.001 for every 3C below the Osmolality

calibration temperature
▪ Sample Problem:
 Calibration temperature: 20C
 Urine temperature: 23C
 Urinometer initial SG reading: 1.015
▪ Correction:
 Urine temperature is 3 degrees higher than
calibration temperature: add 0.001
 Corrected urinometer SG reading: 1.025 +
0.001 = 1.016
o Large substance correction
▪ Subtract 0.003 for each gram of protein present
▪ Subtract 0.004 for each gram of glucose present
▪ Sample Problem:
• Based on the number of particles of solute per
kilogram of solvent
• Different from osmolarity
o The unit of solvent is in liter
o Number of particles of solute per liter of solvent
• Measurement of the colligative properties of a
solution and comparing their value with the value of a
pure solvent (osmometer)
o Colligative property
▪ Property that is mathematically related to the
number of particles in a solution
o Specific gravity is based on the colligative property
▪ Compared with normal pure water point

 Urinometer initial SG reading: 1.035

Table No. 2 Particle Changes to Colligative Properties
 Glucose present: 2g/dL (subtract 0.004 per
gram=0.008) Normal Pure Water Effect of 1 Mole of
Property Solute
 Protein present: 1g/dL (subtract 0.003 per Point
gram) Lowered 1.86°C
Freezing Point 0°C
▪ Correction: Raised 0.52°C
Boiling Point 100°C
 1.035 - 0.003 (protein)= 1.032 - 0.008
Lowered 0.33
(glucose) = 1.024 (corrected SG) Vapor Pressure 2.38 mm/Hg at 25°C mm/Hg at 25°C
Harmonic Oscillation Densitometry Increased 1.7 x 109
Osmotic Pressure 0 mm/Hg mm/Hg
• Old test similar to urinometry
• Frequency of a sound wave entering a solution changes • Dissolving solute in solvent will cause changes in the
in proportion to the density of the solution colligative properties such as:
o Principle: Sound wave o Low freezing point
• Uses a U-shaped instrument with electromagnetic coil o Higher boiling point
in one end and motion detector in the other end o Increased osmotic pressure
• Electric current is applied and allows sound waves to o Lower vapor pressure
pass through the solution • In the urine sample:
• Amount of sound frequency reflected = density (specific
gravity) o Water is the solvent in the urine
o The number of particles present in a sample can be
Procedure determined by comparing the colligative property
value with that of the pure water
• Urine enters the U-tube (glass
tube) with an electromagnetic Osmometer
coil.
• Sonic oscillation is generated • Device that measures
when an electric current is the osmolality in the
applied to a coil. urinalysis laboratory
• When there is already an • Detects the changes
electric current, then there that occurs when the
would also be a sonic changes in the
oscillation colligative property is
• The oscillation will then be compared with the
detected which will then be normal pure water
proportional to the density of point (osmolality)
the urine • Basis for calculated osmolal gap
• A microprocessor will correct Refractometry
a sample temperature and the result will be valid up to a
specific gravity of 1.080 • Uses a refractometer
o Specific gravity is 1.080 (maximum result that can • Measures the refractive index (comparison of the
be read) velocity of light air with the velocity of light in a
solution)

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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

• Concentration of dissolved particles present in the on the refractive

solution determines the velocity and angle at which light index.
passes through a solution

Basic Parts of Refractometer

• Basic Parts:
o Daylight plate
▪ Where
urine Wipe the sample
sample is from the prism clean
placed STEP 6. with a tissue and
water.
o Calibration
screw
o Rubber grip
o Eyepiece
o Focus Adjustment Advantages

▪ The part that rotates to clearly check the

• Small volume of specimen
calibration in the eyepiece
o 1 to 2 drops of urine
o Unlike the 15 ml of urinometer
Procedure • No temperature corrections

PROCEDURE IN USING REFRACTOMETER

Disadvantages

• Correction for protein and glucose

Put one or two drops
STEP 1. of sample on the o The same with urinometer
prism o Must be calculated by calculating 0.003 for each
gram of protein present and 0.004 for each gram
of glucose present

Calibration
Close the daylight
STEP 2. plate gently. • Use distilled water that should read 1.000
• Use 5% NaCl that should read 1.022 + 0.001
• Use 9% sucrose that should read 1.034 + 0.001
• Urine control samples with low, medium, and high
concentration
• Determines if the refractometer is working and accurate

The same sample Calculation

STEP 3. spread all the prism
surface.
• The same with urinometer
• Must be calculated by calculating 0.003 for each gram
of protein present and 0.004 for each gram of glucose
present

Look at the scale Reagent Strip

STEP 4. through the
eyepiece. • Commonly done in the laboratory
• Very simple
• Included in the chemical examination
Read the scale
• Already includes different parameters of the chemical
where the boundary examination
line intercepts it. o 14 parameters
STEP 5. o 12 parameters
Read the specific
gravity in the
boundary line based

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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

 Not expected to a urine sample

 Must not be released
▪ For specific gravity, results start at 1.005, the
interval is by 5, so add by 5
 1.005, 1.010, 1.015, 1.020. 1.025, 1.030

Specific Gravity

• Strip/pad is impregnated with pre-treated polyelectrolyte

susceptible to pKa changes in relation to ionic
concentration which reads into specific gravity → ion
dissociation → pH change
• pH change will be reflected in the different colors of the
strip/pad
o deep blue – green – yellow green
o 10 parameters (commonly employed in the
laboratory)
▪ (1) Leukocyte
▪ (2) Nitrite Summary
▪ (3) Urobilinogen Table 3. Current Urine Specific Gravity Measurements
▪ (4) Protein
▪ (5) pH Method Principle
▪ (6) Blood Refractometry Refractive index
▪ (7) Specific Gravity Osmolality Changes in colligative
▪ (8) Ketone properties by particle number

▪ (9) Bilirubin Reagent strip pKa changes of polyelectrolyte

by ions present
▪ (10) Glucose
Table 4. Old Method
o Each of the pads has its own principle
Procedure Method Principle
Urinometry Density
• (1) Dip the reagent strip in urine
• (2) Wait for a certain time Harmonic oscillation Density
densitometry
• (3) Compare the color changes
o It becomes subjective since it depends on the color
seen by the lab personnel
o It is a lot easier because you are simply comparing DEMO VIDEO: URINOMETRY
colors CALIBRATION
• More convenient way STEP 1. • Urinometer must be
• Based on the change in pKa of a polyelectrolyte in calibrated in distilled
alkaline medium water
• Polyelectrolyte ionizes, releasing hydrogen ions in o SG of distilled
proportion to the number of ions in the solution water = 1
• More concentrated urine, more hydrogen ions released,
pH is decreased
• Indicator: bromothymol blue (measures the change in
pH) STEP 2. • Fill the urinometer vial
o Blue (1.000/alkaline) to green to yellow 2/3 full with room
temperature distilled
(1.030/acid)
water
▪ Yellow = higher specific gravity; acidic; more
concentrated
 More acidic = urine is more concentrated =
more hydrogen ions = higher specific gravity
o 0.005 intervals
▪ 1.000 is not included since it is the specific
gravity of distilled water

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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

o The calculation shows a correction of .005

STEP 3. • Carefully lower the
float into the vial o .005 should be added to the urinometer reading
since the water temperature was above 20°C
o Do not drop into o In this case. This correction factor must be added to
the vial as the all subsequent urine readings.
float has a
weighted end Distilled Water Colder than 20°C
▪ Can cause it
to break • Temperature of water = 14°C
STEP 4. • Impart a slight spin to 14°C − 20°C = −6°C
make sure it is
6°C/3°C = 2
floating in the middle
of the vial and is not 2 x .001 = .002
stuck to the wall
Specific gravity was 1.002
o Try not to wet the
stem as this will Subtract .002 to get 1.000
throw off the
reading • The calculation shows that the correction factor of .002
needed to be subtracted from the reading since the
STEP 5. • After the float temperature of distilled water was lower than 20°C
stabilizes, read the • In this case .002 would also need to be subtracted from
bottom of the all subsequent urine sample readings
meniscus
Measuring Specific Gravity of Urine Samples
o Eyes are level
with the meniscus • After calibration is
complete, we are ready to
STEP 6. • If the urinometer measure the specific
reads 1.000, then gravity of urine samples
calibration is complete
• Follow the same steps as
before
• Results:
o Specific gravity= 1.011
▪ Does not need correction because the
• If the observed SG for distilled water is not 1.000, experiment was conducted in a cool room and
record the temperature of the water the calibration reading for distilled water was
1.000 without having to use a correction factor
Temperature Adjusted Calibration
• After testing the urine sample, pour it back into its
• Adjustments you must make for samples not held at container
20°C • Rinse the vial thoroughly with water before testing
• + .001 for every 3°C above 20°C another urine sample
• - .001 for every 3°C below 20°C • Clean the urinometer float and vial with soap and water
NOTE:
DEMO VIDEO: REFRACTOMETRY
• Lab room is typically warmer than 20°C, so it is likely PROCEDURE
that you have to make some adjustment
STEP • Open the top
Distilled Water Warmer than 20°C 1.

• Temperature of water = 35°C

o Since the urinometer did
not read 1.000, the
temperature of distilled
water was taken and
found to be 35°C STEP • Add 2-3 drops of
2. urine using
𝑇𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 = 35°C
transfer pipette in
35°C − 20°C = 15°C the circle hole
15°C/3°C = 5 underneath the
glass of
5 x .001 = .005 refractometer
Specific gravity was .995 o The urine will
Add .005 to get 1.000 spread out
across the

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 7
PACANA
 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

piece of glass STEP • Clean off the

in the 7. device with
refractometer
alcohol swab and
wait until the
STEP • Pull it up in the alcohol is
3. eye (like a completely
telescope) and evaporated
walk underneath before storing the
a bright overhead device or using it
light again
o To shine STEP • Make sure to
through the 8. also clean the
liquid for
countertop in
easier
case urine got
visualization
spilled
of the specific
gravity

STEP • Lower left scale –

4. SG Specific Gravity
• Look for the • good for assessing hydration
black line across status
the scale and
• SG: 1.020 is considered as
read to find the
dehydrated
specific gravity of
• Two of the samples are
the urine
dehydrated and one is hydrated
• If there’s a
• Dehydrated samples had higher
distinctive black
sodium levels
bar:
• Normal hydration or hyper hydration had lower sodium
o Open the levels
refractometer, • Sodium and other electrolytes in urine is what makes up
clean it off, the urine, and affects the specific gravity
and start over
again Table 5. Specific Gravity Samples
o Might be due
to air bubbles SG (g/cm3) Na+ K+ Cl-
or the urine
did not Electrolytes Electrolytes Electrolytes
completely (mmol/L) (mmol/L) (mmol/L)
cover the
1.004 46.4 16.1 56.0
glass
1.028 91.0 90.3 168.5
STEP • Write down the 1.029 103.5 121.8 141.3
5. value

DEMO VIDEO: URINALYSIS

PROCEDURE
STEP 1. • Gather equipment
o Hand wash
STEP • Open the top of
6. o Gloves
the refractometer
o Apron
and clean with
o Urine dipsticks
tissue
o Urine sample
o Make sure it o Paper towels
will not affect
the next STEP 2. • Wash hands
measurement
and soak all
the urine in
the device

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 8
PACANA
 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

STEP 3. • Wear apron STEP • Remove testing

10. strip from the
container

STEP • Place test strip into

11. the urine sample
STEP 4. • Wear gloves
o Ensuring all
zones are
immersed

STEP • Remove test strip

12.
o Removing
excess urine
STEP 5. • Confirm patient
details
o Name
o Date of birth
o Hospital
number
STEP • Ensure test strip
13. remains in a
horizontal
STEP 6. • Inspect urine color orientation
o Straw colored = o To avoid cross-
normal contamination
o Dark of zones
concentrated =
dehydration STEP • Interpret each test
o Red/pink = 14. at the appropriate
hematuria time interval using
o Brown = bile the dipstick
pigments/myogl analysis chart
obin o Test require
differing
STEP 7. • Inspect urine clarity amounts of
o Clear = normal time to
o Cloudy/debris = complete
UTI
o Frothy = STEP • Discard clinical
nephrotic 15. waste into the
syndrome appropriate bin

STEP 8. • Assess urine odor

o Offensive = UTI
o Sweet =
diabetes STEP • Wash hands
16.

STEP 9. • Check urine

dipstick expiry date

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 9
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 LABORATORY UNIT 02: PHYSICAL EXAMINATION OF URINE

STEP • To complete the

17. procedure:
o Document
findings
o Perform further
investigation if
appropriate
(e.g. MSU)

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 10
PACANA
 [TRANS] LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE

INTRODUCTION 4 Versus 10 Parameters

Reagent Strips • In the Philippines (Davao), the laboratories make use of

either 4 parameters or 10 parameters reagent strip.
4 parameters

• Glucose
• pH
• Specific gravity
• Protein

• “Dipstick” NOTE: Specific gravity is still a physical parameter.

• 2 major brands: However, it is tested under chemical examination because
reagent strip provides or offers a more reliable and more
o Multistix
convenient way of checking for specific gravity than the
o Chemstrip
manual methods.
o There are other brands of reagent strips aside from
these two Figure 1. 4 parameters
• Chemical-impregnated absorbent pads attached to a
plastic strip
• Anatomy of reagent strip:
o Plastic strip
o Pads – attached to the strip
▪ Chemically impregnated
▪ Each pad contains reagents,
chemicals, and enzymes that
allow us to test for chemical
constituents of urine.
▪ Looks simple but is actually
modern state of art
• A color-producing chemical reaction takes place
when the strip comes in contact with urine
o Color change: end result in the reagent strip test for
chemical examination of urine
10 Parameters of Reagent Strip
• Reactions are interpreted by comparing the color
produced on the pad with the chart provided by the
manufacturer (Semiquantitative)
o The chart may be attached to the reagent strip
bottle
o Results are Semiquantitative – provide
approximate concentrations, not exact
concentrations
▪ Even though the results provided are
semiquantitative, the reagent strips still provide
good value as to urine testing because they are
very sensitive and very specific
• Procedure:
o (1) Dip the strip in the urine
o (2) Allow for certain time to elapse
o (3) The color reactions develop
o (4) Compare to the comparator (chart)
Figure 2. 10 parameters

BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3 1
VILLANUEVA. PACANA
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE

• pH ▪ The last parameter must come into contact with

• Proteins the urine sample so reaction can occur
• Glucose o Dip briefly (1-2 seconds)
• Specific Gravity
• Ketones ▪ Do not allow it to be immersed into the urine for
• Blood a long period of time
• Bilirubin Step 2
• Urobilinogen
• Nitrite • Remove the excess urine by
• Leukocytes (leukocyte esterase) running the edge of the strip on
the container/test tube when
NOTE: Most labs in Davao use the 4 parameters more due to withdrawing it from the
its cheaper price specimen, and blotting its back
o 4 parameters - approximately 700-800 Php / bottle portion horizontally on any
o 10 parameters - approximately 1,500 – 3,000 Php / absorbent medium (e.g. tissue)
bottle o Upon extracting out/pulling
out the reagent strip, make
When to perform? sure that the back (plastic)
portion must touch the lip
• After physical exam and before microscopic exam
of the test tube while removing it.
• Perform chemical examination of urine in a sample that
has not been centrifugated yet. ▪ Pad portion should not be touched
• Urinalysis is 3-step process. After receiving the sample ▪ To drain the excess fluid present in the strip
and assessing its validity: ▪ Reagent strip should not be that drenched
o (1) Physical examination o Blotting facilitates further drainage of excess fluid
▪ First process in urinalysis Step 3
▪ Assess physical traits and characteristics of
urine samples (Color, Clarity, Specific Gravity, • Wait for the specified length of time
Odor) for the reactions to take place, and
o (2) Chemical examination compare colored results to the
manufacturer’s chart under a good
▪ Use of reagent strip source of light
▪ Interpret results and centrifuge sample
▪ Yield results for chemical analysis o Timing is very crucial in the
interpretation of the reaction of
o (3) Microscopic examination results in the reagent strip
▪ Last part of urinalysis process ▪ There is specified time in reading the results per
Process in Laboratory pad:
 For leukocyte, read after 120 seconds
• (1) Receive a sample  For specific gravity, wait for 45 seconds
• (2) Assess if the sample is sufficient, valid, or integral
• (3) Transfer the sample to a clean test tube o After achieving the specific time, compare the strip
to the chart in the bottle
o Assess the physical parameters in the test tube
▪ Good light source to properly assess the color
• (4) Make use directly of the reagent strip because some shades are close to each other.
o Dip the reagent strip in the urine Summary of Procedure
• (5) Interpret the results
• (6) Centrifugate the urine sample in the test tube
• (7) Do the microscopic exam
o After chemical exam, that’s the only time to
centrifugate the sample → Microscopic exam

Procedure in Using Reagent Strip

Step 1

• Dip the reagent strip completely but

briefly into a well-mixed specimen • Mix specimen properly→ Dip strip briefly → Remove
o Make sure that all the parameters excess → Blot horizontally the back portion→ Time
are completely dipped into the → Compare with the chart
urine sample

BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3 2
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE
• In the academe, it is encouraged to transfer the urine
sample immediately in the test tube after receiving it,
rather than letting it stay in the container
o Containers are plastic and test tubes are glass
o Test tube (glass) allows to properly assess physical
characteristics such as color and clarity
▪ Permits more light to enter the specimen. This
will give better interpretation compared to
plastics (opaque).
• Advantage in the process of Urinalysis: Short/Fast
and easy procedure
• Without reagent strips, chemical testing of analysis of
urine makes use of conventional method (e.g. manual
Benedict’s test and Gerhardt's Test) which takes time
Errors Caused by Improper Technique
• (1) Formed elements such as red and white cells sink to
the bottom of the specimen and will be undetected in an
unmixed specimen
o Mix the urine sample before processing it so that
cellular sediments will not settle at the bottom
o If RBCs, WBCs, and any other cellular sediment
settle at the bottom, they will not come in contact
with the reagent pad (blood/leukocyte esterase).
▪ No contact = no reaction
 False negative reaction even if RBCs and
WBCs are present
o Resuspend the sample to redistribute the cellular
sediment in the urine sample so that they become
available for reaction for their respective reagent
strip pad
• (2) Allowing the strip to remain in the urine for an
extended period may cause leaching of reagents from
the pad
o Dip the strip completely and briefly
o Prolonged exposure of reagent strip to urine cause
chemicals in reagent pad to leak out
▪ No reactions will occur due to loss of chemicals
and reagents
• (3) Excess urine remaining on the strip after its
removal from the specimen can produce a run-over
between chemicals on adjacent pads, producing
distortion of color.
• To ensure against run-over, blotting the edge of the
strip on absorbent paper and holding the strip
horizontally while comparing it with the chart
o Drenched strip = tendency of mixing or run-over
between pads
• (4) The timing for the reactions to take place varies
among manufacturers and ranges from immediate
reaction for pH to 120 seconds for the leukocyte
esterase. (60-120seconds)
• (5) A good light source is essential for accurate
interpretation of color reactions, aside from good vision
o Room must be well-lit to facilitate proper
assessment
BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3
• (6) The strip must be held close to the color chart
without actually placing it on the chart
o Close but not in direct contact due to hygienic
reasons
• (7) Reagent strips and color charts from different
manufacturers are not interchangeable
o Be brand-specific
• (8) Specimens that have been refrigerated must be
allowed to return to room temperature prior to
reagent strip testing as the enzymatic reactions are
temperature-dependent
o Enzymes have optimum temperature at which they
function best
Handling and Storing of Reagent Strips
• Reagent strips must be protected from moisture, volatile
chemicals, heat and light
• Packaged into opaque containers with desiccant
o Opaque so there is no entry of light; and desiccant
to get rid of moisture.
• Strips are removed just prior to testing and bottle is
resealed immediately
o When we do chemical testing analysis of urine, we
need reagent strip.
o We do this from the time we use it: After Physical
examination and before microscopic examination
o Reseal immediately to avoid exposure to light since
pads are very sensitive
• Stored at room temperature below 30° Celsius (but
never refrigerated)
o Reagent strips are usually put into the shelves
o Airconditioned lab: Cold but not refrigerator-temp
• Must not be used beyond expiration date
• Care must be taken not to touch the chemical pads
when removing the strips from the container
o Reagent pads are very sensitive
o Chemicals will leak out from the pads
o When we get from the bottle, make sure that the
plastic strip is the only thing that we are holding
o Plastic strips have a long empty portion
▪ Position: Pads are positioned inferior
▪ Near at the storage of the cap is the empty
portion
• Visual inspection should be done every use to detect
deterioration. Do not use if pads are discolored
o Before using reagent strip for analysis, look briefly
for discoloration (change in color)
o Assessment will not be factual due to change in
colorimetric reaction.
3
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE
Quality Control of Reagent Strips
• Reagent strips must be tested with positive and
negative controls
o minimum of once every 24 hours
▪ As to which shift to perform it, that would depend
on the protocols of the lab
 Most of the time, it is performed during the AM
shift, 7-3
o every after new bottle is opened
o when questionable results are obtained
o when there is concern about the integrity of the strip
• Record all control results
o For referencing purposes
• Distilled water should not be used as negative control.
The manufacturers will provide the controls.
o Reagent strips/pads are designed for Ionic
concentrations
▪ Because distilled water has undergone the
process of distillation, it is already deionized.
Error Sources in Reading the Reagent Strip
• Interfering substances in the urine
o e.g., metabolite of drug Phenazopyridine which
has an orange to yellow shade
▪ May obscure the color changes
• Technical carelessness
o Proficiency
• Color-blindness
o There might be havoc in the interpretation
Value and Application
• “Results should coincide with the physical examination
results and must be suggestive of the probable
sediments that can be seen during microscopic exam.”
o Chemical and Physical examination should coincide
most of the time, but not all the time
o Ex: Red and cloudy observation during physical
exam
▪ Positive with the blood reagent strip (Chemical
exam)
▪ Suggestive for microscopic exam: expect for
RBC
 Potential probable sediments
Reporting of Results
• Depending on the test performed, the results are
reported in the following manner:
o in concentration (mg/dL)
o as small, moderate, or large
o using the plus system (1+,2+,3+,4+)
o as positive, negative or normal
o specific gravity and pH
▪ results are estimated in their respective units
BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3
▪ Expressed in number
• The units can be seen in the chart, we just have to
compare
Individual Parameters of the Strip Test
pH
• Ideally read at 60 seconds
• The average adult on a normal diet excretes about 50–
100 mEq of hydrogen ions in 24 hours to produce urine
of about pH 6.
o In healthy individuals, normal urine pH may vary
from 4.5–8
o If the urine sample pH is above 8, it usually
indicates that the sample is old and not suitable for
testing → should be rejected
• Reagent Strip Reaction Principle:
o Double Indicator System
▪ Methyl Red (pH range 4 to 6) – detects for
acidity
 Acidic = red
 Alkaline = yellow
▪ Bromthymol Blue (pH range of 6 to 9) – detects
for alkalinity
 Acidic or near neutral = yellow
 Alkaline = bluish-green
𝑀𝑒𝑡ℎ𝑦𝑙 𝑟𝑒𝑑 + 𝐻 𝑖𝑜𝑛 → 𝐵𝑟𝑜𝑚𝑡ℎ𝑦𝑚𝑜𝑙 𝑏𝑙𝑢𝑒 − 𝐻 𝑖𝑜𝑛
(𝑅𝑒𝑑 → 𝑜𝑟𝑎𝑛𝑔𝑒 → 𝑌𝑒𝑙𝑙𝑜𝑤) (𝐺𝑟𝑒𝑒𝑛 → 𝑏𝑙𝑢𝑒)
• Measurement of urine pH and acidity must always be
made on freshly voided specimens.
• The container should be filled to minimize the amount of
dead space, and the urine covered tightly.
o Covered tightly because air can change pH
• The container should be kept cold, preferably on ice,
but not frozen.
Protein
• Read at 60 seconds
• The presence of increased amounts of protein in the
urine can be an important and primary indicator of renal
disease.
• Reagent Strip Reaction Principle:
o Protein Error of Indicator
▪ Certain indicators can change color in the
presence of proteins even though the pH of the
medium remains constant (due to the PLUS
Buffer)
 No pH change in the reagent pad of protein
 A deviance in the rule that indicators change
color together with the change in pH
4
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE
▪ Proteins (amino group) accepts hydrogen ions
from the indicator.
▪ Albumin is the major protein detected by the
reagent strip
 Albumin has a lot of amino groups in its
structure so it accepts more hydrogen ions.
▪ Explanation of the Protein Error of Indicator:
CASE 1: Urine sample contains albumin
• How does the protein pad change color?
o since albumin has a lot of amino groups, the
indicators will donate its hydrogen ions to albumin
o The more albumin, the more the indicators will lose
hydrogen, the more it will become alkaline (bluish-
green)
o Hence, if urine sample has a lot of proteins (4+), the
strip will be bluish-green, because the indicator has
lost hydrogen, as it has migrated to albumin
CASE 2: Urine sample containing no albumin
• Indicators did not give off their hydrogen
• Hydrogen ions remain within the indicators
o Internal pH of the indicators will just be acid
o Hence, yellow color = acid pH
o Error: Buffer
▪ Maintaining the pH of the reagent pad
▪ Working condition is maintained at pH 3
• Protein error of indicators: There will still be changes
in color because indicators will lose hydrogen in
response to the presence of the albumin or protein
present in the urine sample
• Multistix indicator:
o Tetrabromophenol blue
• Chemstrip indicator:
o 3’,3”,5’,5”-tetrachlorophenol-3,4,5,6-
tetrabromosulfonphthalein
• Multistix and Chemstrip may have different indicators
but they have the same behavior
o Acid pH = yellow
o Alkaline pH = bluish-green
• The reagent strip pad for protein contains PLUS
BUFFER
o Buffer maintains the pH of the pad at constant level
which is pH 3 (acidic)
▪ the default negative color is yellow
• Very sensitive to albumin
o Albumin: protein primarily detected by reagent strip
• Other urine proteins such as gamma globulin,
glycoprotein, ribonuclease, lysozyme, hemoglobin,
Tamm–Horsfall mucoprotein, and Bence-Jones protein
are much less readily detected than albumin.
BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3
o Albumin is the major protein a man excretes; major
protein in the blood
• Implication: Therefore, a negative urinary dipstick
result does not necessarily rule out the presence of
these proteins.
o Other proteins can still be present even without the
presence of albumin
Glucose
• Read at 30 seconds after testing
• When the blood glucose exceeds the renal threshold,
the tubules cannot reabsorb all of the filtered glucose,
and so glycosuria occurs
o Glycosuria: presence of glucose in urine
▪ It can be detected in reagent strip
• Reagent Strip Reaction Principle:
o Double Sequential Enzymatic Reaction
▪ Make use of the two enzymes
▪ 1st reaction and 2nd reaction
 The product of the first reaction sequentially
becomes the substrate of the second reaction
 If there is no glucose in urine, then glucose
oxidase will not have its substrate → no
oxygen peroxide → nothing will be used in the
2nd reaction → no oxidation of chromogen →
chromogen remains uncolored = negative
reaction
1st reaction: glucose oxidase (enzyme)
𝐺𝑂𝑋
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 → 𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝐴𝑐𝑖𝑑 + 𝐻2 𝑂2
o If there is glucose in urine, plus the oxygen in air,
will be acted upon by the first enzyme present in the
reagent strip which is glucose oxidase.
▪ Glucose oxidase will act on the glucose in urine,
forming gluconic acid and hydrogen peroxide
 The hydrogen peroxide from this first reaction
becomes the substrate of the second reaction;
hence “sequential”
o It can only have hydrogen peroxide if glucose was
hydrolyzed by glucose oxidase
2nd reaction: peroxidase (enzyme)
𝐻2 𝑂2 + 𝑅𝑒𝑑𝑢𝑐𝑒𝑑 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → 𝐻2 𝑂 + 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛
(Uncolored) (Colored)
o (1) Peroxidase enzyme will hydrolyze hydrogen
peroxide, forming water and oxygen
o (2) Oxygen produced from the breakdown of
hydrogen peroxide will oxidize the reduced
chromogen
o (3) When chromogen is already oxidized, it will now
achieve color
▪ Chromogen will only change in color if it’s
oxidized
5
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE

▪ It can only have oxygen in the secondary action ▪ Ketone bodies - by product to fatty acid
if there is hydrogen peroxide hydrolyzed by metabolism to fat metabolism
peroxidase enzyme
• 3 major ketone bodies that can form in the body:
• Chromogens used are the following;
o Acetoacetic acid (diacetic acid) – parent
o Chromogens - Indicate that there is a reaction; compound; the other ketone bodies can only arise if
hence, there will be color changes it’s present
▪ Multistix: Potassium iodide (green to brown)
▪ Most of the reagent strips only detect
 Green – at negative acetoacetic acid.
 Brown – when it detects glucose o Beta-hydroxybutyric acid – produced
▪ Chemstrip: Tetramethylbenzidine (yellow to spontaneously from acetoacetic acid
green)
▪ No method can detect it
 Yellow – at negative
o Acetone – from decarboxylation of acetoacetic acid
 Brown – at positive
▪ cannot be detected if there is no glycine present.
Other Reagent Strip Brands with their Chromogens
Table 1. Reagent Strip Brands with their Chromogens

Brand and Sensitivity Chromogen

AimSticktick9 (50 mg/dL) Potassium iodide

Chemistrip5 (40mg/dL) Tetramethylbenzine

Glucose oxidase
o There is no problem even if the reagent strip can
Combi-Screen PLUS10 (40mg/dL) Peroxidase only detect acetoacetic acid; its presence safely
O-tolidine-hydrochloride assumes the presence of the other two
DiaScreen11 (50mg/dL) Potassium iodide • Reagent Strip Reaction:
Glucose oxidase o Sodium nitroprusside reaction
Dirui H-series12 (2.8-5.5 mmol/L) Peroxidase
Potassium iodide ▪ Used to detect ketone bodies
Glucose oxidase ▪ Acetoacetic acid in alkaline medium reacts with
Mission13 (25-50 mg/dL) Peroxidase sodium nitroprusside to yield a purple color =
O-tolidine positive reaction.
Multistix2 (75 mg/dL) Potassium iodide Other Reagent Strip Brands with their Sensitivities
Glucose oxidase
Self-Stik14 (50-100 mg/dL) Peroxidase Table 2. Reagent Strip Brands with their Sensitivities
Potassium iodide
Glucose oxidase
URiSCAN15 (50mg/dL) Peroxidase Brand and Sensitivity Reagent
Potassium iodide
Glucose oxidase AimSticktick9 (5 mg/dL diacetic acid; 48
Uritest 13G16 (2.2-2.8 mmol/L) Peroxidase Sodium nitroprusside
mg/dL acetone)
4-Aminoantipyrine
Chemistrip5 (9 mg/dL diacetic acid; 70
Glucose oxidase Sodium nitroprusside
mg/dL acetone)
Uro-dip 10C17 (100-150 mg/dL) Peroxidase 10
Potassium iodide Combi-Screen PLUS (5 mg/dL
Sodium nitroprusside
Glucose oxidase acetoacetic acid; 50 mg/dL acetone)
18 Sodium nitroprusside
URS (100 mg/dL) Peroxidase
Potassium iodide DiaScreen11 (5 mg/dL diacetic acid)
Sodium nitroprusside
Ketones Dirui H-series12 (0.5-0.1 mmol/L)
Sodium nitroprusside
Mission13 (2.5-5 mg/dL)
Sodium nitroprusside
Multistix2 (5 mg/dL diacetic acid)

• Should be read at 40 seconds Self-Stik14 (5 mg acetoacetic acid per Sodium nitroprusside

• Ketones, or ketone bodies are formed during the 100 mL of urine) Magnesium sulfate
catabolism of fatty acids. URiSCAN15 (5 mg/dL acetoacetic acid; Sodium nitroprusside
70 mg/dL acetone)
o NOTE: primary source of energy is carbohydrates,
Uritest 13G16 (0.5 – 1.0 mmol/L Sodium nitroprusside
but in cases of its depletion, the body turns to fat for
acetoacetic acid)
source of energy

BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3 6
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE

Uro-dip 10C17 (5 mg acetoacetic acid Sodium nitroprusside
per 100 mL of urine)
Sodium nitroprusside
URS18 (5-10 mg/dL acetoacetic acid)
Blood
• Should be read at 60 seconds
• Blood in the urine sample is an indication of a possible
bleeding episode in the renal tract.
o Red - Most abnormal color of urine
• 3 different conditions of red urine sample, may
mean:
o Hematuria – presence of intact red blood cells that
have not been destroyed
𝐻2 𝑂2 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 →
▪ Positive reaction
▪ speckling or speckled pattern in the reagent
strip, or pigmentation
 results from the remnants of intact RBCs
coming into contact with the reagent pad, and
undergoing lysis
Other Reagent Strip Brands with their Chromogens
▪ Color is homogenous, uniform all throughout the
boxes
• Reagent strips – can differentiate the redness of the
urine
• Reagent Strip Reaction Principle:
o Pseudoperoxidase Activity of Hemoglobin
o Other references: Pseudoperoxidase activity of
Hemoiety
▪ The reagent strip will become positive as long as
there is the presence of heme (creates all the
reaction).
▪ Hemoglobin has similar activity to peroxidase
 Peroxidase - enzyme that destroys hydrogen
peroxide forming water and oxygen
▪ Heme can hydrolyse hydrogen peroxide forming
water and oxygen, hence the name
pseudoperoxidase.
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛
𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 + 𝐻2 𝑂2
𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑒
• Heme → hydrolyse the hydrogen peroxide
o Product: water and oxygen
• Oxygen → oxidize the chromogen
o Result: color changes

Figure 3. Progression of results in Hematuria Table 3. Reagent Strip Brands with their Chromogens

o Hemoglobinuria - presence of hemoglobin in urine Brand and Sensitivity Oxidant; Chromogen

▪ Red blood cells have already been destroyed or 𝐀𝐢𝐦𝐒𝐭𝐢𝐜𝐤 𝟗 (5 RBCs; 0.3
lysed mg/dL Hb)
▪ Produce almost similar results with 𝐂𝐡𝐞𝐦𝐢𝐬𝐭𝐫𝐢𝐩𝟓 (5 RBCs; Hb ~
myoglobinuria in urine analysis 10 RBCs)
▪ Positive but do not have a speckled pattern 𝐂𝐨𝐦𝐛𝐢 − 𝐒𝐜𝐫𝐞𝐞𝐧 𝐏𝐋𝐔𝐒𝟓 (5
▪ Color is homogenous, uniform all throughout the Ery/uL)
boxes
𝐃𝐢𝐚𝐒𝐜𝐫𝐞𝐞𝐧𝟏𝟏 (5 RBCs; 0.02
mg/dL Hb)
𝐃𝐢𝐫𝐮𝐢 𝐇 − 𝐒𝐞𝐫𝐢𝐞𝐬𝟏𝟐 (5-15
Ery/uL)
Figure 4. Hemoglubinuria & Myoglobinuria
𝐌𝐢𝐬𝐬𝐢𝐨𝐧𝟏𝟏 (0.018-0.060
mg/dL)
o Myoglobinuria 𝐌𝐮𝐥𝐭𝐢𝐬𝐭𝐢𝐱 𝟐 (5 RBCs; 0.015
▪ Hematuria, hemoglobinuria and myoglobinuria mg/dL Hb)
have the same result in chemical exam 𝐒𝐞𝐥𝐟 − 𝐒𝐭𝐢𝐤 𝟏𝟒 (5-10
▪ Brownish–red urine RBCs/mL urine)
▪ Myoglobin pigment is present in the urine which 𝐔𝐑𝐢𝐒𝐂𝐀𝐍 𝟏𝟓 (5 RBC/uL or 3-
behaves like hemoglobin 5 RBC/ HPF; 0.015 mg/dL
hemoglobin)
 Located in muscles 𝐔𝐫𝐢𝐭𝐞𝐬𝐭 𝟏𝟑𝐆𝟏𝟔 (0.3-0.6 mg/L
 Oxygen-delivering substance in muscles hemoglobin)
▪ If there is a problem in the muscles, the 𝐔𝐫𝐨 − 𝐝𝐢𝐩 𝟏𝟎𝐂 𝟏𝟕 (0.05
mg/dL hemoglobin)
myoglobin released will be delivered in the urine
causing the color change in urine. 𝐔𝐑𝐒𝟏𝟖 (0.015 mg/dL Hb or
▪ Produce almost similar results with 5-10 intact RBCs/uL)
hemoglobinuria in urine analysis • Teramethylbenzidines
▪ Positive but do not have a speckled pattern
o Chromogens for blood
Diisoproplbenzene
Dihydroperoxide; teramethylbenzidine
2,5-Dimethylhexane-2,5-
dihydroperoxide; teramethylbenzidine
Teramethylbenzidine-dihydrochloride
Isopropylbenzol-hydroperoxide
2,5-Dimethylhexane-2,5-
dihydroperoxide; teramethylbenzidine
Diisoproplbenzene
Dihydroperoxide; teramethylbenzidine
Diisoproplbenzene
Dihydroperoxide; teramethylbenzidine
Diisoproplbenzene
Dihydroperoxide; teramethylbenzidine
Cumene hydroperoxide
O-Tolidine
Cumene hydroperoxide
Tetramethylbenzidine
Cumene hydroperoxide
3,3’, 5, 5’-Tetramethylbenzidine
Cumene hydroperoxide
Tetramethylbenzidine
Cumene hydroperoxide
Tetramethylbenzidine

BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3 7
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE
Bilirubin
• Read at 30 seconds
• It is a breakdown product of hemoglobin that is formed
in the reticuloendothelial cells of the spleen, liver, and
bone marrow
o At the end of the lifespan of red blood cells,
haemoglobin is degraded, and one of the products is
bilirubin.
• Check the screen for urine bilirubin to reflect liver
diseases
• Two types of bilirubin after heme degredation B1
versus B2
o B1
▪ Unconjugated
▪ Water insoluble
o B2
▪ Conjugated
▪ Water soluble
 Hence, the only type of bilirubin that reaches
urine
▪ Only one detected in reagent strip
• Reagent Strip Reaction Principle:
o Diazo Reaction/Azo-coupling Reaction
▪ direct reaction
o Bilirubin reacts with 2,4-dichloroaniline-diazonium
salt (Multistix) or 2,6-dichlorobenzene-diazonium-
tetrafluoroborate (Chemstrip) in an acidic medium to
produce an azo dye (tan/pink-violet)
▪ Can only form a product if bilirubin is present
• Precautions in bilirubin testing:
o Make use of fresh urine
▪ B2 will be converted into free bilirubin if the
sample is not fresh
 Free bilirubin is not detected in reagent strip
o Avoid exposure to sunlight
▪ To procure accurate results for urinary bilirubin
▪ Sunlight will convert bilirubin back to biliverdin
▪ Can cause false negative reaction
• Foam test
o Yellow-brown to greenish-brown
urine that may have a yellow
foam
▪ Can suspect the presence of
bilirubin
o Shake to achieve foam testing
▪ If the yellow foam appears and does not
integrate or a yellow foam that is stable, then
bilirubin is present.
BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3
o Major interference factor: drug Phenazopyridine
(can also produce the same color and foam)
▪ History taking is important
Urobilinogen
• Read at 60 seconds
• It represents a group of closely related tetrapyrrole
compounds formed from reduction of bilirubin
o Not a stand-alone, solitary substance
o After heme degredation in the body, bilirubin will be
delivered to the small intestine and converted to
urobilinogen.
• Reagent Strip Reaction Principles:
o Ehrlich Aldehyde Reaction
▪ For Multistix brand
▪ Reagent: para dimethylamino benzaldehyde
▪ Urobilinogen react directly with Ehrlich reagent
giving a positive result
o Diazo Reaction/Azo-coupling
▪ For Chemstrip
o Both produce pink to red colors if positive
• Urobilinogen is normally present in the urine but in
concentrations of 1 Ehrlich unit or less per 100 mL of
urine
o Strip can still detect this level, lower than this, not
anymore
o Urine normally contains urobilinogen
o Minimum reporting is normal, not negative
• Urobilinogen instability is a problem
o Make use of fresh specimen
▪ In cases of not fresh specimens, and the
urobilinogen is exposed to air, oxygen will help
the conversion of urobilinogen to urobilin.
 Urobilin will not be detected by reagent strip, it
can only detect urobilinogen
▪ Used to investigate liver disorders
• Best time of specimen collection - between 2pm to 4pm
o In this timeframe, it is the peak of urobilinogen
excretion in urine
Nitrite
• Read at 60 seconds
• The nitrite test is a rapid, indirect method for the early
detection of significant and asymptomatic bacteriuria
o Indirect because it does not directly isolate or
cultivate the organism/bacteria
▪ Direct – means that the bacteria can be
seen/isolated
8
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE
o It can only tell if it is positive. A positive reaction can
be an assumption that bacteria are present.
• The primary condition that is being detected/diagnosed
when we perform nitrite testing is urinary tract
infection.
o Common cause of UTI: Enterobacteriaceae (has
the enzyme nitrate reductase)
▪ Our urine contains nitrate (comes from diet). If a
person has UTI and the causative agent of UTI
has the enzyme reductase, these organisms can
convert nitrate to nitrite.
▪ When there is nitrite, that’s the time where
assumptions of organism (which have reductase)
are present
 there will be indirect method to detect
bacteriuria
o However, if the causative agent of UTI are
organisms that do not possess nitrate reductase
such as Staphylococcus/Streptococcus, do not
expect for a nitrite positive result.
• Reagent Strip Reaction Principle:
o Greiss reaction (other references: Griess)
o Nitrite at an acidic pH reacts with an aromatic
amine (para-arsanilic acid or sulfanilamide) to form
a diazonium compound that then reacts with
tetrahydrobenzoquinolin compounds to produce a
pink-colored azodye
▪ Any degree of uniform/homogenous pink color
should be interpreted as a positive nitrite test
 Suggesting the presence of 105 or more
organisms per milliliter.
▪ If it is not homogenously pink → Negative
• First morning urine is the specimen of choice
o The type of specimen that is collected after waking
up.
o Very concentrated urine as it has stayed in the
bladder for a long period of time
o It will take organisms 4 hours to convert enough
nitrate to nitrite.
Leukocyte Esterase
• Read at 120 seconds or 2 minutes
• Detects the presence of esterase in the granulocytic
white blood cells (e.g. Neutrophil, Basophil, and
Eosinophil
o It will not detect WBC as a whole, only the esterase
which is a part of the granules in the WBC
o Monocyte also has esterase
▪ If monocyte is present in the urine, the reagent
strip can still detect this.
o Advantage: Even if the WBCs are already lysed,
the esterase can still be detected.
BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3
• Application: Used to help in establishing that the
patient is suffering from Urinary Tract Infection (UTI)
o Can be used together with nitrite
o If a person has UTI, there is a presence of an
offending agent/ bacteria, so expect that there will
be migration of WBC, especially neutrophils.
▪ If neutrophils are present, the esterase enzyme
will be detected.
• Esterases also are present in Trichomonas and
histiocytes
o Trichomonas and histiocytes can be interferences.
The MedTech should rule out the presence of these
two.
o If these two are present, the patient can be positive
for esterase even without the presence of WBC.
o interferences → false positive
• Reagent Strip Reaction Principle:
o Action of LE to catalyze the hydrolysis of an acid
ester
Reaction A:
𝑔𝑟𝑎𝑛𝑢𝑙𝑜𝑐𝑦𝑡𝑖𝑐
𝐼𝑛𝑑𝑜𝑥𝑦𝑙 𝑜𝑟 𝑝𝑦𝑟𝑜𝑙𝑒 𝐶𝑎𝑟𝑏𝑜𝑛𝑖𝑐 𝐴𝑐𝑖𝑑 𝐸𝑠𝑡𝑒𝑟 → 𝐼𝑛𝑑𝑜𝑥𝑦𝑙 𝑜𝑟 𝑝𝑦𝑟𝑜𝑙𝑒
𝑒𝑠𝑡𝑒𝑟𝑎𝑠𝑒
o If esterase enzyme is present, it will hydrolyze
indoxyl or pyrole carbonic acid ester forming indoxyl
or pyrole
Reaction B:
𝐼𝑛𝑑𝑜𝑥𝑦𝑙 𝑜𝑟 𝑝𝑦𝑟𝑜𝑙𝑒 + 𝑑𝑖𝑎𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡 = 𝒑𝒖𝒓𝒑𝒍𝒆
o The indoxyl or pyrole will react with the Diazonium
salt forming the purple color
o Postive result: Purple
• A positive LE test result is most frequently accompanied
by the presence of bacteria
o If offending agents are present in the urinary tract,
the normal response of the body is to send WBCs
(neutrophil) which contains esterase.
o NOTE: Which may or may not produce a positive
nitrite reaction.
▪ When diagnosing for UTI, it is important to study
the calisthenics of the three:
 Can see bacteria
 Positive result for LE
 Positive for nitrite reaction
Specific Gravity
• Read at 45 seconds
• Simple compared to urinometer and refractometer
• The addition of a specific gravity testing area to reagent
strips has eliminated a time-consuming step in
9
VILLANUEVA. PACANA.
 LABORATORY UNIT IV: CHEMICAL EXAMINATION OF URINE

routine urinalysis and has provided a convenient
method for routine screening.
• Reagent Strip Reaction Principle:
o Change in pKa (dissociation constant) of a
polyelectrolyte in an alkaline medium
▪ More solutes in the urine sample → more
hydrogen released → acid (yellow urine)
▪ Less solute in the urine sample → less hydrogen
will be released → alkaline
o Presence of protein is a sign for renal disease
• It is a cold precipitation test
• Add 3mL of 3% SSA to 3mL of centrifuged urine
• Mix by inversion and observe for cloudiness
o End result: Cloudiness
• Grade the turbidity
Table 4. Interpretation of Results

Ascorbic Acid (Additional Reagent Strip Parameter) Protein Range

Grade Turbidity
(mg/dL)
• Read at 10 seconds
• Considered to be the 11th reagent strip parameter Negative No increase in turbidity Less than 6
• Presence of Ascorbic acid (Vit. C) can be detected in
the urine if it is taken in excess amount Trace Noticeable turbidity 6-30

o The body cannot use the excess Vitamin C, thus will Distinct turbidity; no
1+ 30-100
be excreted via urination. granulation (or pigments)
Turbidity, granulation, no
• Ascorbic acid in urine can be problematic specially 2+
flocculation
100-200
when testing chemical constituents because, it may
Turbidity, granulation,
inhibit several reagent strip reactions 3+ 200-400
flocculation
o Since Vit. C is a reducing agent, it can neutralize the
reagent strip reactions which are oxidative by 4+ Clumps of proteins Greater than 400
nature.
o It may also neutralize the activity of the Azo dye
o Such as glucose, blood, bilirubin, nitrite, and
leukocyte esterase (MNEUMONICS: BBLNG)
▪ Blood and glucose – use oxidation
▪ Bilirubin, nitrite and leukocyte esterase - Azo
dyes
o Presence of ascorbic acid will result in false
negative
• Reagent Strip Reaction Principle:

Vitamin C (≥ 5 mg/dL) + Phosphomolybdate → Molybdenum
Blue
• Positive: Molybdenum blue
• Other reagent strip brands include:
o Calcium, creatinine, and microalbumin
Confirmatory Testing
• Can be the conventional test such as Benedict’s test or
newer tests
• Using different reagents or methodologies to detect the
same substance as detected by the reagent strip with
some or greater sensitivity and specificity
• Tablets and liquid chemicals
• But not often used today due to increased
sensitivity and specificity of reagent test strips
• Usually, it is to verify if the reagent strip results are true
or not
• Some confirmatory tests are better than the reagent
strip BUT most of the time, reagent strip are better than
the manual test
Sulfosalicylic Acid Test for Protein
• Sulfosalicylic acid testing
o If the reagent strip result to 3+ → perform SSA to
verify
Other conventional chemical tests (Liquid Reagent-based)
• Conventional: Archaic test
o Some are obsolete or not used anymore
o Heller’s Ring Test for Protein
o Benedict’s Test for Glucose
o Rothera’s Test for Ketone bodies
o Gerhardt’s Test for Ketone Bodies
o Gmelin’s Test for Bile Pigments
o Smith Iodine Test for Bile Pigments
▪ Bile pigments: include bilirubin
Tablet-based Tests
• More sophisticated than liquid based-test
• Clinitest for Glucose
o Reagent strip is better than Clinitest
• Acetest for ketone bodies
o Advantage over reagent strip: can use other body
fluids (e.g. blood or serum)
• Ictotest for bilirubin
o Better test than reagent strip for bilirubin
o More sensitive test which can detect lower
concentration of bilirubin in urine

BAUZON. BAGAL. BOYOSE. CRUDA. EVANGELISTA. MANZANO. MATABALAO. MARASIGAN. NUEVO. RAMOS. TEVES. BSMLS 3 10
VILLANUEVA. PACANA.
 [TRANS] LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

EXAMINATION OF URINE SEDIMENT o Sometimes, discrepancies really occur within the

• Microscopic examination is the third step and is
performed after performing physical and chemical
examination.
• In chemical examination, reagent strip is dipped into
the urine sample and its results are interpreted.
o (1) Centrifugate the urine
o (2) Decant the supernatant
o (3) Using a glass slide, make a smear using the
sediment
o (4) View and examine it in the microscope
(Microscopic examination)
• Theoretically, some laboratories do not perform
microscopic examination anymore.
results. It still depends on the pathophysiology of the
patient’s disease.
• Results of Specimen that do not correlate must be
rechecked for both technical and clerical errors
o Consideration: if results are not consistent, there
might be technical or clerical errors in the procedure.
o Hence, investigations are done again to solve the
problem.
Table No. 1. Macroscopic Screening and Microscopic
Correlations
Screening Test Significance

o This decision is based on the results acquired from

Color Blood
physical and chemical examination.
o Normal results in both physical and chemical Clarity Hematuria versus
examination = no need for microscopic examination, hemoglobinuria/myoglobinuria
or it becomes optional Confirm pathologic or
nonpathologic cause of
• Most of the laboratories in the Philippines, particularly in turbidity
Davao City still perform microscopic examination even if Blood RBCs, RBC casts
physical and chemical results are normal. Protein Casts, cells
Nitrite Bacteria. WBCs
o Because there are instances that physical and
Leukocyte esterase WBCs,WBC casts, bacteria
chemical examination are seemingly normal but
when microscopic examination is performed, a lot of Glucose Yeast
sediments and clinically significant objects can still
be seen contained in the urine.
Table No. 2. Routine Urinalysis Correlations
Macroscopic Screening
Microscopic Physical Chemical Exceptions
• Abnormalities in the physical and chemical portion of Elements
urinalysis play a particular role in the decision to
perform a microscopic examination
RBCs Turbidity + Blood Number
Red Color + Protein Hemolysis
MICROSCOPIC EXAMINATION WBCs Turbidity + Protein Number
• Importance of Microscopic Examination + Nitrite Lysis
+ LE
o Microscopic examination of urine sediment will Epithelial Turbidity Number
ultimately confirm the results acquired from physical cells
and chemical examination Casts + Protein Number
o Enhances proficiency of MedTech since performing Bacteria Turbidity ↑ pH Number and
proper urine sediment examination can be used to + Nitrite type
build on their expertise. +Leukocytes
Correlation of Results Crystals Turbidity pH Number and
Color + Bilirubin type
• Microscopic results should be correlated with the
physical and chemical findings
Procedure
o Give clues and support each other, most of the time,
not always Prepare Materials
o Especially, physical exam should give clue to
microscopic exam • Tubes, pipets, and slides used for standardizing urine
sediments

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 1
PACANA
 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE
o Test tubes used are capped,
calibrated with graduations so
urine volume can be accurately
measured
o Pipets should not be made of
glass
o Pasteur pipets (plastic) with
graduations are used
o Slides where smears are made so that it can be
viewed under the microscope
Procedure
• (1) A sample of well-mixed urine (usually 10-15 mL) is
centrifuged in a test tube at relatively low speed for 5
minutes until a moderately cohesive button is produced
at the bottom of the tube
o Volume used for microscopic exam: 10-15 mL
o Done after reagent strip testing
o Urine is mixed
o Centrifugate
o After centrifugation, there will be 2 parts:
▪ Button – sediment
▪ Remaining liquid – supernatant (decanted)
o Supernatant should be discarded
• (2) The supernatant is decanted and a volume of 0.5 to
1.0 mL is left inside the tube
o Left in the test tube will be the sediment with little
remaining fluid from the supernatant
o Remnants measures 0.5 to 1 ml.
▪ Composed of the sediment (button) and little
remaining fluid (supernatant)
o Two parts are separated
o When decanting, fluid should not be completely
evacuated because ideally, small amount of fluid
must remain
▪ That fluid might be used in creating a slide/
smear later
• (3) The sediment is resuspended in the remaining
supernatant by flicking the bottom of the tube several
times
o Resuspension: shake the specimen to redistribute
the button (sediment) in the bottom portion of the
test tube in the remaining fluid
o Ensures that sediment at the bottom is well
distributed in the remaining fluid
• (4) A drop of resuspended sediment is poured onto a
glass slide and coverslipped
o Resuspended liquid must be transferred from test
tube to a slide
o Transfer of sediment (e.g., direct transfer, Pasteur
pipet)
▪ In the Philippines, direct transfer from test tube
to the slide is commonly performed
▪ Pasteur pipet - preferred because the sediment
that will be transferred can be controlled
o Make a smear
o Coverslip – allow it to occupy the area of the
coverslip
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3
• (5) Examine
• (6) Report
o Includes physical, chemical, and microscopic exam
• Dedicated 4-5 laboratory sessions to do microscopy of
the urine
o COMPETENCIES IN EXAMINATION:
▪ (1) To enhance the microscopy skills, and
identify the different urine sediments
 E.g., of objects or sediments: RBCs, WBCs,
different types of epithelial cells, casts, and
crystals, microorganisms present
▪ (2) To be able to count the identified objects
contained in the urine, and how to properly make
a result out of them.
 These two competencies should be learned at
the same time
General Aspects
1. Specimen Preparation
• Fresh specimen preferred
o If the urine sample is not anymore fresh, then
there’s a tendency for the cellular materials and for
other sediments in the urine to disintegrate
o RBC, WBC, and hyaline casts begin to disintegrate
in 1-3 hour at room temperature
▪ Can no longer be counted in the microscopic
exam
▪ Positive in chemical exam, but cannot give out
an accurate count in microscopic exam
o Refrigeration for up to 48 h (little loss of cells)
▪ If the urine sample cannot be processed
immediately
▪ Read immediately, if possible, to prevent losing
cells
 Process and analyze the urine sample
immediately to produce a result that has
integrity
2.Specimen Volume
• Ensure that the volume of the urine sample that will be
used for the urinalysis process especially for
microscopic exam is within 10-15 mL, with an average
of 12 mL
o To have a greater chance to isolate or catch urine
sediments if present
o 12 mL → usual volume used for the whole urinalysis
process especially during microscopic exam
3.Specimen Concentration
• Centrifugation can have different speed and duration
depending on the sample being centrifuged.
• Speed of the centrifuge and the length of time should be
consistent at 5 min., RCF of 400
o Enough to separate the sediment from the
supernatant
o Allowance = 450 (if the RCF is not 400)
2
PACANA
 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE
o RCF Range = 400 to 450
o RCF → Relative Centrifugal Force or G-Force
• Ten to twenty-fold concentration by centrifugation
Centrifugation
• Before centrifugations, the
sediments are distributed
to the urine sample
o Collect a drop of urine
sample from the first
tube, then transfer it to
the slide; only a few
sediments will be
collected because it is
still distributed in a
large amount of fluid
▪ When conducting a microscopy exam, there is a
little probability of seeing the sediment.
• The scattered sediments cause the cloudiness of the
sample, and after centrifugation, because of the effect
of the centrifuge, the sediments will gather at the bottom
of the test tube; they will be concentrated
• By doing centrifugation, you are concentrating the
dispersed sediment into one button or one packet of
sediment
o To ensure that every content present in the urine
was already obtained.
• Able to concentrate the urine sample, the higher the
chance of being able to see objects or sediments in the
urine when doing microscopic exam
o The supernatant fluid will be decanted, and a
minimal amount of fluid will remain
▪ Remnant fluid + Sediment = 0.5 to 1mL after
decanting
• Re-suspend the sediment to mix it with the remaining
fluid by shaking it.
• After shaking, take a sample, make a smear, and
examine it under the microscope
Review:
• The RBCs in the sample are still scattered in the
abundant fluid
o (1) Centrifugate the urine sample to accurately count
the number of RBC
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3
o (2) After centrifugating it for 5 mins at 400 RCF, then
the scattered RBC will be concentrated to the
bottom
▪ The supernatant will be left with minimal amount
of fluid
o (3) Re-suspend and make a smear out of it
• If centrifuge is not available in the laboratory
o Use an alternative technique → Natural
sedimentation
▪ Natural sedimentation allows the urine sample
to sit undisturbed for how many hours
▪ The sediments are heavier than the fluid, then it
has a tendency to settle at the bottom
 Disadvantage: It takes time; does not allow
us to keep up with a short turnaround time
4. Sediment Preparation
• Sediments – contains the object that can
be seen under the microscope such as
RBCs
• Uniform amount should remain in the
tube after decantation (0.5 and 1.0mL
frequently used).
o The remaining fluid in the tube should
not be totally emptied out after
decanting
• Concentration Factor – relates to the probability of
detecting elements present in low quantities:
o It is most of the time used when the microscopic
portion of analysis of the urine is automated
o Not applicable in the Philippines (manually)
o Formula: Volume of urine centrifuged/Sediment
volume
▪ E.g., 12/1 = 12
• Resuspension is essential to provide equal distribution
of elements in the microscopic fields
Process
• After centrifugation, remove the
supernatant fluid with a polyethylene,
transfer pipet leaving 0.5 to 1.0mL urine
on the sediment
o Two techniques for decantation:
▪ (1) Pour the urine sample
directly; one swift motion.
▪ (2) Make use of the pipette for
urinalysis to remove the excess
supernatant
 Polyethylene transfer pipette – ideal pipette
for urinalysis
 Advantage: Regulates the amount of fluid
that will remain
• Resuspend the sediment in the remaining urine by
flicking the bottom of the tube or by running the tube
across a test tube rack
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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE
o Flick or shake the sediment with the little amount of
fluid
• Using a plastic or polyethylene or
polypropylene transfer pipet,
mount a drop of the urine in the
counting chamber of the prepared
commercial plastic slides
• Avoid using glass pipette when
mounting the urine sediment as
elements like casts tend to cling to
the glass surface.
o Cannot be read under the microscope because
casts have tendency to remain within the walls of
the glass pipette
o The cast will have false decreased values
▪ Casts, are most of the time clinically significant
5. Volume of Sediment Examined
• 20μL (0.02 mL) covered by a 22 x 22 mm glass cover
slip
o Get 20uL only and put it in the slide, then cover it
with the cover slip
• How to transfer sediments to slide?
o Conventional/ Manual (tak-tak method)
▪ From the test tube, the MedTech directly or
manually “tak-tak” the sediment to the glass
slide. Spread then cover with cover slip
o By using pipette
▪ Using a pipette, get 0.02 mL of sediments and
transfer it to the glass slide. Spread then cover
with cover slip
▪ Better to use a graduated pipette for an accurate
amount of sediment
6. Commercial Systems (machines)
• KOVA
• Urisystem
• Count-10
• Quick-Prep
• CenSlide 2000
• R/S Workstations
Advantages
• It makes use of cup, calibrated test tubes and slides,
and pipettes
o Pipettes for exact and accurate decanting of
supernatant.
• It forms a monolayer sediment
o It is important that the sediment is in a monolayer to
make sure there is no overlapping and for better
microscopic analysis
• To some MedTechs, manual is still better than
machines
o Sometimes, the machines will not identify and
instead may misidentify certain sediments
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3
▪ As commercial systems/ machines run on certain
principles while manual procedures are ru0n with
our eyes and brain, which is better
7. Examination of the Sediments
• LPO – 10 fields; detects casts and ascertain general
composition of the sediment
o scanner → LPO → scan 10 continuous fields
o In LPO, you can already detect casts (as they are
large) and have the impression that urine contains
RBCs, WBCs, or bacteria
o Note: Those sediments are still not confirmed, those
are impressions only
• HPO – 10 fields; confirms type of sediments found
o Can confirm types of sediments found in LPO as
HPO
provides higher magnification than LPO
QUESTION: Why don’t we use OIO?
• Because in urinalysis, we use wet mount preparation and we
can’t use oil for a wet smear.
• Highest magnification for urinalysis lab = HPO
• Always start with LPO then proceed to HPO.
• Larger sediment= LPO
• Smaller sediment = HPO
8. Reporting the microscopic examination
• Reported in average number per low-power field (lpf):
casts
o Count how many casts are seen within the 10 fields
of LPO the get the average number.
o (e.g.) 3-10 per LPF
• Reported in average number per high-power field
(hpf): RBCs and WBCs
• Reported in semi-quantitative terms per low power
field: epithelial cells and crystals
Sediment Examination Techniques
9. Sediment stains
QUESTION: Do we stain our slide smear for urine sample?
• Generally, NOT. In the Philippines, we don’t stain urine
samples on a routine laboratory test.
o Because it will require extra preparation and since we are in
a clinical laboratory, we have to follow a turn-around-time
o So, we enhance our expertise as we try to look at unstained
sample that doesn’t have color.
• But there are some instances that requires to make use of
stain
o Because, staining is advantageous compared to reading
unstained urine samples.
o E.g., when viewing under microscope, a stained smear will
be easier to identify, count, and look at.
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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

Advantages of Stain ▪ Acetic acid is used as a diluent to destroy RBCs

for WBC identification and counting
• Increases overall visibility of sediment/elements being ▪ (e.g.) If urine sample contains a lot of RBCs + 1-
examined 2 drops of acetic acid = RBCs will disintegrate
• Imparts identifying characteristics to cellular structures
• Makes objects more identifiable QUESTION: Do we really have to intentionally lyse/destroy
Urine Sediments Stain RBCs?
• YES. There are times that they need to be destroyed
Table No 3. Urine Sediments Stain Characteristics especially when we are after the WBCs.
o Sometimes, RBCs are too overwhelming or are too
numerous to count that they cover up the WBCs, hence we
STAIN ACTION FUNCTION cannot count them anymore. So, we lyse RBCs with acetic
acid.
Delineates o So, we enhance our expertise as we try to look at unstained
structure and Identifies WBCs, sample that doesn’t have color.
Stemheimer- o This also helps us distinguish RBCs from: yeast, oil,
contrasting colors epithelial cells,
Malbin droplets and crystals as they are sometimes confused with
of the nucleus and and cast RBCs, but cannot be destroyed by acetic acid.
cytoplasm
Differentiates
Enhances nuclear
WBCs and renal • Lipid stains: Oil Red O and Sudan III
Toluidine blue detail (i.e.,
tubular epithelia o Sometimes cells and casts can take up the lipids
segmentations)
(RTE) cells present in the sample, thus, this stain is used for
Distinguish RBCs identification of the lipids
Lyses RBCs and
from WBCs, yeast, o Only stains neutral fat (triglycerides) with the color
2% acetic acid enhance nuclei of
oil droplets, and orange-red
WBCs
crystal o Does NOT stain cholesterol
Stain triglycerides
and neutral fats Identify free fat • Gram stain
Lipid stains;
orange-red droplets and lipid-
Oil Red O and o To narrow down the choices as of the causative
containing cells
Sudan III agent of UTI
Do not stain and casts
o Casts originally do not have bacteria, but sometimes
cholesterol
bacteria can go inside the cast turning it into a
Differentiates gram-
Identifies bacterial cast
Gram stain positive and gram-
bacterial casts o Identifies bacterial cast
negative bacteria
Methylene blue and ▪ Bacterial cast may be hard to differentiate as it
eosin Y stains Identifies urinary resembles other sediments of urine. So, do gram
Hansel stain
eosinophilic eosinophil stain.
granules
• Hansel stain
Identifies yellow-
Prussian blue Stains structures brown granules of o Composed of methylene blue and eosin Y
stain containing iron hemosiderin in o Stains: eosinophilic granules
cells and casts o Note: just like any other fluids, the major WBC that
reaches in urine is neutrophil.
• Stemheimer-Malbin
o major stain ▪ But during certain conditions (allergic
conditions), eosinophil migrate to urine samples
o composed of crystal violet and safranin O
o can highlight the nucleus and cytoplasm of WBCs • Prussian blue stain
and epithelial cells which are better seen under the
o Stains: structures containing iron
microscope
o Sometimes, during cases of hemoglobinuria, iron is
• Toluidine blue deposited in renal structures
o Good to use when visualizing structures with ▪ Best way to identify them is by using Prussian
nucleus like WBCS and RTE cells blue stains
▪ RTE cells is a type of epithelial cells which has 1 Cytodiagnostic Urine Testing
nucleus
• Most of the time, what we do to our urine sample in
 So, when we use toluidine blue it can highlight
routine urinalysis is just routine urinalysis.
the nucleus so cells are more identifiable and
more differentiable o Routine urinalysis: physical, chemical, and
microscopic exam
• 2% acetic acid
▪ Sometimes, when performing routine urine
o Vinegar
examination, we could notice very abnormal or
o Must know: It can lyse RBCs
peculiar results. These results are unusual that it

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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

needs further testing like cytodiagnostic urine • Polarizing microscope

testing
 Examples of unusual results: too much cells
(epithelial, RTE cells, transitional cells) in
urine samples
• Provides additional method for detecting and
monitoring renal disease through cytocentrifugation
and Pap’s stain; endorsed and performed in the
Histopathology laboratory
• Detects malignancies of the lower urinary tract
• Recommends first morning urine which will be
performed by cytologists and pathologists
o Allows the visualization or demonstration of
birefringent objects
o Birefringent: objects that are capable of refracting
light at two directions. (E.g., fatty substances).
• Dark-field microscope
o Opposite with Bright-field microscope
o Object at focus is illuminated in contrast to the dark
view or field surrounding the object.
o Provide a better view of unstained specimens (i.e.,
Treponema pallidum) using Dark-field microscope.

o Only endorse if there’s too many abnormal cells • Fluorescence microscopy

present in urine analysis. o Rarely used in urinalysis, common in parasitology or
• Not anymore part of AUBF LAB. immunology.
• Interference microscope
Microscopy o Modified bright-field microscope
o Two types:
• Always use microscope in urine examination whether it
was stained or not. ▪ (1) Modulation contrast microscope: Hoffman
microscope
o Chemical examination = Reagent strip ▪ (2) Differential interference contrast microscope:
o Microscopic examination = Microscope Nomarski microscope
RECAP: Procedure
Table no 4. Urinalysis Microscopic Techniques
• (1) A sample of well-mixed urine (usually 10-15 mL) is
Technique Function centrifuged in a test tube at relatively low speed (400
RCF) for 5 minutes until a moderately cohesive button
Bright-field microscopy Used for routine urinalysis is produced at the bottom of the tube
• (2) The supernatant is decanted and a volume of 0.5 to
Enhances visualization of
elements with low refractive 1.0 mL is left inside the tube
Phase-contrast microscopy indices, such as hyaline casts, • (3) The sediment is resuspended in the remaining
mixed cellular casts, mucous supernatant by flicking the bottom of the tube several
threads, and Trichomonas times
Aids in identification of • (4) A drop of resuspended sediment is poured onto a
Polarizing microscopy cholesterol in oval fat bodies, glass slide and cover slipped
fatty casts, and crystals • (5) Examine
Aids in identification of • (6) Report
Dark-field microscopy
Treponema pallidum
Allows visualization of EXAMINATION OF URINE SEDIMENT
naturally fluorescent
microorganisms or those • At Low Power
Fluorescence microscopy
stained by a fluorescent dye o (1) To observe most
including labelled antigens and
antibodies ▪ crystals
Produces a three-dimensional ▪ casts
microscopy image and layer- ▪ squamous cells
Interference-contrast ▪ other large objects
by-layer imaging of a
specimen  LPO are sufficient for larger urine sediments
• Bright-field microscope
o (2) The numbers of casts seen are usually reported
o Similar to the ones in parasitology lab as number of each type found per low power field
o Object is dark and not receiving light while the field (LPF)
(background) surrounding the object is
bright/illuminated. ▪ E.g., Bacterial casts, WBC casts, Red Blood Cell
o It creates contrast to allow visualization under the casts, and Crystals
microscope. Contrast is key to readability. ▪ Quantified since they are clinically significant
o Objects that have low refractive indices are hard to ▪ Different types of casts are reported separately
visualize in bright-field microscope. o (3) Example: 5-10 hyaline casts/L casts/LPF
o Casts are commonly misidentified as hyaline casts
and mucous threads. ▪ Examine 10 fields for both LPF or LPO

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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

o (4) Since the number of elements found in each field o Bleeding in catheterization is not considered
may vary considerably from one field to another, pathologic since there’s no underlying disease and
several fields are averaged (range manner) rather caused by incorrect procedure or insertion.
• At High Power • (3) Theoretically, no red cells should be found, but
some find their way into the urine even in very healthy
o (1) To identify crystals, cells, bacteria and other
individuals
microorganisms
o The urine normally contains 0-1 range of red blood
▪ Abnormal pathologic crystals cells which are considered negative.
▪ Red Blood cells, White Blood cells and other o It only becomes clinically significant if the result is
significant and small cells that require higher increased.
magnification.
o (2) The various types of cells are usually described • (4) If one or more red cells can be found in every high -
as the number of each type found per average high- power field, and if contamination can be ruled out, the
power field (HPF). Example: 1-5 WBC/HPF specimen is probably abnormal.
o May mean that there is already a pathologic process
underneath, which the patient is experiencing
Components of Urine Sediment especially if you can rule out contamination.
o No contamination and yet there are abundant red
Organized components blood cells then it would most likely be a pathologic
hematuria.
• (1) Red blood cells
• (2) White blood cells • (5) RBCs may appear normally shaped, swollen by
• (3) Epithelial cells dilute urine (in fact, only ghost cells and free
• (4) Casts hemoglobin may remain), or crenated by concentrated
• (5) Bacteria urine
• (6) Yeast o Red blood cells in the urine may assume different
• (7) Parasites forms and morphology depending on the
• (8) Spermatozoa characteristic of the urine sample.
o Ghost cells are swollen but empty, these are
Unorganized components usually formed if RBCs are put on a hypotonic or
dilute urine sample.
• (1) Crystals of various salts
o If the urine sample are hypertonic or very
o (1) Oxalate concentrated the red blood cells may shrink, this is
o (2) Phosphate called crenated red blood cells.
o (3) Urates
• (6) Swollen, partly hemolyzed RBC’s and crenated
• (2) Chemical elements (E.g., Calcium) RBCs are sometimes difficult to distinguish from WBC’s
granules in the urine
Red Blood Cells
• (7) In addition, ghost cells may simulate yeast. The
• (1) Hematuria is the presence of abnormal numbers of presence of dysmorphic RBCs in urine suggests a
red cells in urine due to conditions that damage the glomerular disease such as a
renal tract: glomerulonephritis/glomerular damage
o (1) glomerular damage,
o (2) tumors which erode the urinary tract anywhere • (8) Dysmorphic RBCs have odd shapes as a
along its length, consequence of being distorted via passage through the
o (3) kidney trauma, abnormal glomerular structure
o (4) urinary tract stones, o Dysmorphic = distorted or very folded
o (5) renal infarcts, o Bleeding originates in the glomerulus
o (6) acute tubular necrosis (ATN),
o (7) severe upper and lower urinary tract infections,
o (8) nephrotoxins or drugs that act as nephrotoxins QUESTION: Why will glomerular bleeding produce dysmorphic
(e.g., antifungal drugs), RBCs?
o (9) and physical stress (e.g., strenuous exercise or • As the RBCs pass through the abnormal glomerular structure
sports training) and as it passes through the long renal tubules, the RBCs
tend to get distorted along the way.
▪ Hematuria conditions - there is wound damage
or bleeding and blood is found in urine.
• (9) Dysmorphic RBCs Irregular shape, size, density
• (2) Red cells may also contaminate the urine from the Indicate glomerular bleeding
vagina in menstruating women or from trauma produced
by bladder catheterization
o Menstrual contamination can be misdiagnosed for
pathological disease if the MedTech was not
informed.

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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

RBCs Summary of RBC

• Smooth, non-nucleated • Most difficult for students to identify

biconcave disks;
• 7um diameter Table no 5. Summary of RBC
• Note the central pallor of most
of the cells especially the one SUMMARY 6-1 Microscopic RBCs
labelled with a “C”
• Donut shape or biconcave disc • Non-nucleated biconcave disks
form of the cell • Crenated in hypertonic urine
• “A” points to a red cell on its Appearance • Ghost cells in hypotonic urine
side • Dysmorphic with glomerular
membrane damage
Crenated RBCs
• Yeast cells
• This is another field of RBCs
o Primary source of error
• Note that some of them look Sources of
granular Identification error: • Oil droplets
o That is because they are o If samples are contaminated
crenated or puckered with oil, especially when
▪ Colloquially, crenated using OIO
red blood cells are • Air bubbles
called Durian cells
because they resemble o Results from improper
durian. technique in smear
preparation
• The spicules make the cell look granular o Use acetic acid to
• “A” can be a ghost cell differentiate, it if lysed then it
Dysmorphic RBCs would most likely be RBC,
o But if it remains then, it might
be one of the three
• Note the irregular outlines of many of these RBCs,
compared to two relatively normal RBCs at the center Reporting:
left of the right panel. These abnormal RBCs are • Average number per 10 hpfs
dysmorphic RBCs. Complete urinalysis • Color
• Dysmorphic RBCs have odd shapes as a consequence correlations: • Reagent strip blood reaction
of being distorted via passage through the abnormal
glomerular structure • NOTE: Expect to see RBCs in Hematuria. But RBCs
are absent in both Hemoglobinuria and
Photo: Actual Appearance of RBCs on Urine Sample Myoglobinuria

WBCs

• (1) Pyuria refers to the presence of abnormal numbers

of leukocytes that may appear with infection in either
the upper or lower urinary tract:
o WBCs usually appear in the urine as a result of
bacterial infection.
▪ (1) Pyelonephritis – infection of the kidneys
▪ (2) Cystitis – infection of the bladder, UTI of the
bladder
▪ (3) Prostatitis – infection of the prostate gland
▪ (4) Urethritis – infection of the urethra
o These are all inflammatory reactions that are
commonly bacterial by nature
o Since they are bacterial by nature, they allow
Normal RBCs migration of RBCs which gives the condition Pyuria.

QUESTION: Is pyuria only associated with bacterial infections?

• NO, pyuria can also be associated with non-bacterial
conditions such as Glomerulonephritis, especially if it is
already severe. Other causes are: SLE, interstitial nephritis,
and tumors.

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• (2) Non-bacterial cause: Glomerulonephritis, SLE
interstitial nephritis, and tumors
• (3) Usually, the WBCs are granulocytes especially
Neutrophil
• (4) White cells from the vagina, especially in the
presence of vaginal and cervical infections, or the
external urethral meatus in men and women may
contaminate the urine
o There can be cases of contamination wherein there
are visible WBC in the urine, if the WBC is a result
of contamination, then it would not be clinically
significant.
• (5) If two or more leukocytes per each high power field
appear in non-contaminated urine, the specimen is
probably abnormal
o In order to identify the specimen as abnormal or
pathologic, contamination must be ruled out.
o If contamination is ruled out and there is increased
population of cells (either RBC or WBC), then you
can say that the patient is suffering from a
pathologic condition.
• (6) Leukocytes have lobed nuclei and granular
cytoplasm
o Segments and granules are not seen in RBCs.
• If exposed in hypotonic urine – WBCs become Glitter
cells
o In a hypotonic urine sample, the water will enter the
cytoplasm/WBC. When that happens, it will create
Brownian movement.
o When there is Brownian movement inside the WBC,
it will give a glittering appearance.
• Like erythrocytes, WBCs may lyse in very dilute or
highly alkaline urine
o When they disintegrate, WBC cytoplasmic granules
are released into the urine that often resembles
bacteria that are cocci in shape.
o This eventually creates confusion in the
identification of the cocci organisms in bacteria
(rarely happens but CAN happen)
Sources of Confusion in the Identification of WBCs
• Common confusers:
o Mononuclear cells
o Renal Tubular
Epithelial cells (RTE)

o Neutrophil • "C" points to a white

blood cell (WBC)
▪ Most common leukocyte seen in urine of persons
with certain conditions o Most likely a
▪ Easiest to identify in all leukocytes because of its neutrophil
number of segmentation (3-5) o Also note the granular appearance

o There are also other leukocytes seen in the urine • "B" points to two transitional epithelial cells
although it is very rare o An example of mononuclear cell
• (7) Eosinophil
o Associated with drug-induced interstitial nephritis, Table 6. Points of Differentiation between WBCs and
UTI, and renal transplant rejections Confusers
o Stained using Hansel Stain or Wright’s Stain
Mononuclear
• (8) Mononuclear Cells WBCs
Cells and RTE
o Cells having only one nucleus
Size Smaller Larger
▪ E.g., Lymphocytes, monocytes, macrophages,
Multi-lobed (e.g.,
and histocytes Nucleus Single-lobed
neutrophils)
o Very rare HEAVY granules
(e.g., neutrophils,
▪ If present in the urine, the indication most of the Granules
eosinophils, and
NO granules
time is for cytodiagnostic urine test because
basophils)
these cells are associated with malignancies
o In routine urinalysis, diagnosis of malignancies is not
Summary of WBCs
part of the scope.
▪ If there is suspicion of malignancies associated Table 7. Summary of Microscopic WBCs
with mononuclear cells, then it would be
endorsed with cytodiagnostic urine testing under
histopathology. Microscopic WBCs

Larger than RBCs

Appearance Granulated, multilobed neutrophils
Glitter cells in hypotonic urine
Mononuclear cells with abundant
Sources of
cytoplasm
▪ The appearance of WBC in the urine has lobes in its Identification
structure as well as granules. Error Renal tubular epithelial cells

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 LABORATORY UNIT 05: MICROSCOPIC EXAMINATION OF URINE

Average number per 10 hpf (high power ▪ Clue cell: A squamous epithelial cell with
Reporting cytoplasm studded with Gardnerella vaginalis
field)
Leukocyte esterase  Named as such because it serves as a clue
Complete for bacterial vaginosis with Gardnerella
Urinalysis Nitrite vaginalis as the causative agent
Correlation (for  In routine urinalysis, if clue cells are present,
the establishment Specific gravity
indicate it in the report so the female can be
of UTI diagnosis) diagnosed with vaginosis and eventually can
pH
be treated

Epithelial Cells Different Actual Appearance of SEC in Urine

• Three epithelial cells that can be seen in urine sample: • Image 1: Notice that the squamous epithelial cell is
o Squamous Epithelial Cells indeed the largest of all the sediments seen on the field
o Transitional Epithelial Cells
o Renal Tubular Epithelial Cells
Squamous Epithelial Cells (SEC)

• Most common epithelial cell

• Largest urine sediment – point of focus/point of
reference
o It’s the easiest to identify
o Serves as the point of focus when doing microscopy
▪ For beginners, make use of squamous epithelial
cell as a point of focus (look for it first)
▪ If you can focus at these structures, then you are • Image 2: Actual Urine Sample
on the right track
o If urine sample contains a lot of SEC, this is how it
• Originates from the linings of the vagina and female looks like
urethra, and from the lower portion of the male urethra o Most are squamous epithelial cells and some are
o Normal in the urine, especially in females folded
o Expected of female patients especially if the
For SEC identification: collection is not midstream

• Note the size of the cell, its nucleus, and its irregular
shape
o Large cell size
o Solitary nucleus and
most of the time
centrally located
o Irregular shape
cytoplasm (not circular
unlike RTE and TEC);
appears flat
• SEC may appear granular
o Urine smear dried up • Image 3: It contains 3-4
producing SECs
pseudogranules
o Can be solitary but if
▪ Pseudogranules it’s abundant, they can
are not actual be clumped together
granules but
artifacts of
dehydration
• Image 4: Look at the
o Sometimes the granules can actually be bacteria nucleus and irregular
shape of cytoplasm (SEC)
▪ If you have SEC with granules inside and the
smears are not dehydrated/dried up, those
granules may actually be a bacterium
▪ Bacteria: Gardnerella vaginalis (coccobacilli)

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Table 8. Summary of Squamous Epithelial Cells
Microscopic Squamous Epithelial Cells
Largest cells in the sediment with
Appearance abundant, irregular cytoplasm, and
prominent nuclei
Sources of Error
Rarely encountered, folded cells may
Sources of Error
resemble casts
Rare (1+), few (2+), moderate (3+), or
Reporting
many (4+) per lpf [low power field]
Complete
Clarity (SEC is most of the time harmless
Urinalysis
or innocuous)
Correlations
• You don’t count squamous epithelial cells unlike RBCs
and WBCs
• Clue
o If the sediment is not that much clinically significant,
the reporting would be rare, few, moderate, or many
per LPF
o But if the sediment is clinically significant such as
RBC and WBC, usually they are average 1-5 per
HPF etc.
Renal Tubular Epithelial Cells (RTE)
• More SEC = Impaired clarity
Transitional Epithelial Cells (TEC)
• Also known as Urothelial cells
• Smaller than SEC
• Can assume a lot of shapes
o Spherical, polyhedral, caudate (elongated and with
tail)
• Spherical TEC vs RTE – differentiated by the location
of nucleus
o TEC have centrally located nucleus
o RTE have eccentric nucleus
• They originate from the renal pelvis, ureter, calyces, or
bladder
o The normal lining of these structures, especially the
bladder, is a transitional epithelial cell
• Single, Pairs, Groups
o Referred to as syncytia if epithelial cells are in
groups which usually forms after catheterization
• “B” shows a transitional epithelial cell
• The three cells just above “B” is also transitional
epithelial
• “A” point to a squamous epithelial cell
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Table 9. Summary of Transitional Epithelial Cells
Microscopic Transitional Epithelial Cells
Spherical, polyhedral, or caudate with
Appearance
centrally located nucleus
Spherical forms resemble RTE cells
Rare, few, moderate, or many per hpf
Reporting (high power field) depending on the
protocol of the laboratory
Complete Clarity (more TEC = impaired clarity)
Urinalysis
Correlations Blood, if malignancy-associated
• If there is increased number of transitional cells found in
the urine plus positive in blood, malignancy may be
suspected
• If transitional epithelial cells look abnormal, malignancy
may be possible
o Pyknotic nucleus
o Vacuolated cytoplasm
• Cells originating from the renal tubules
o PCT, DCT and even the collecting ducts (CD) are
lined with renal tubular epithelial cells
• RTE cells reflect the status of renal tubules
• Usually larger than granulocytes (especially
neutrophils), contain a large round or oval nucleus
(eccentric) and normally slough into the urine in small
numbers (or none at all)
o In normal conditions, renal tubular cells may be
seen but in very small amounts or none at all
o Don’t expect to see RTE cells in a normal urine
because there are many cases that RTE cells are
not present
o If present in urine in small numbers (1-2), that’s still
normal because it just represents the normal
sloughing off process or normal turnover of cell
• RTE can come from different parts of renal tubule and
looks differently depending on the origin
o Proximal Convoluted Tubule
▪ RTE appears larger
▪ More rectangular (may resemble cast)
o Distal Convoluted Tubule
▪ RTE appears to be smaller and more oval
which can be confused with WBC and even with
spherical TEC
o Collecting Ducts
▪ RTEs appears to be cuboidal
▪ Cuboidal cells can form groups of 3 or more RTE
which can be referred to as renal fragments
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Clinical Significance o When RTE is filled/ingested with numerous fat

droplets, such cells are called oval fat bodies
• RTE cells are the most clinically significant (most
pathologic) epithelial cells WBC
• Often associated with nephrotic syndrome and in
conditions leading to tubular degeneration and RTE
necrosis, the number sloughed is increased
• RTEs are seen in the following conditions:
o Exposure to heavy metals
o Drug-induced toxicity
o Hemoglobin and myoglobin toxicity, viral infections
(Hepatitis B) TEC
o Pyelonephritis
o Allergic reactions
o Malignant infiltrations Oval Fat Bodies
o Acute allogeneic transplant rejection
• All these conditions, if not managed properly can lead to • Oval fat bodies consist of
tubular necrosis which promotes the appearance of degenerated renal tubular
RTE in the urine cells containing abundant
lipid, which appears
Appearance of RTE Cells refractile
• Use Sudan III and Oil Red
• Circle, generally smaller than the other epithelial cells O
• Nucleus - can be off centric (eccentric)
o Fat present: Triglyceride and Neutral fat
o Differentiates from TEC o Color = orange red
o Cholesterol is not stainable
• Most clinically significant in all the EC
• This frame compares a • Polarizing Microscope
WBC ("B") and the renal
o Fat present: Cholesterol
tubular cells ("A")
• Note the size • Oval fat bodies are clinically significant in as much as
comparison RTE cells
o RTE is larger than B o Essentially, oval fat bodies are still RTE cells but
WBC with absorbed lipids
• Associated with Nephrotic Syndrome, Severe Tubular
A Necrosis, and Diabetes Mellitus
o DM
• Also note how prominent the nucleus is in the renal
tubular cells ▪ There can be lipiduria
• It is much easier to see than the nucleus of the WBC. ▪ If there is lipiduria, formation of lipid fat body is
enhanced.
• When lipiduria occurs, these cells contain endogenous
fats. • Conditions to form Oval Fat Bodies:
o Lipiduria – excretion or appearance of fats/lipids in o (1) RTE cells must be present in urine
the urine; associated with nephrotic syndrome o (2) Patient must be exhibiting lipiduria or excreting
o Lipids in urine can be absorbed by the RTE fats in urine
▪ Since RTE line the renal tubule and one of the • NOTE: If no lipiduria and there is only RTE, oval fat
functions of renal tubule is tubular reabsorption, bodies can’t be formed.
so these cells can reabsorb and are active at
reabsorption o If there are no RTE, oval fat bodies are still not
formed
o If there is lipiduria, the tendency is that the RTE can
absorb the lipids present in urine. • Reported as average number per field using HPF
• May be seen with Bubble Cell
o If there are oval fat
bodies, expect to see
bubble cell
o Bubble cell -
vacuolated RTE which
resemble bubbles

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o Vacuole in bubble cells – dilated endoplasmic o Sources of error:

reticulum
▪ Distinction: look side by side and look for the
location of nucleus because most of the time
QUESTION: Why will Endoplasmic Reticulum dilate?
transitional cells are centrally located
• Endoplasmic reticulum usually dilates prior to cell death ▪ Granular casts: can be easily differentiated
• If RTE’s endoplasmic reticulum has been dilated, it can now o Reporting:
become a bubble cell
▪ RTE must be counted since RTE is the most
clinically significant cells out of the epithelial cells
• Under polarized light, oval fat bodies demonstrate the
"Maltese cross" appearance
Table 10. Summary of Oval Fat Bodies
o If you want to demonstrate cholesterol content of
oval fat bodies, use polarizing microscope.
Microscopic Oval Fat Bodies

Appearance Highly refractile RTE cells

Confirm with fat stains and polarized
Sources of Error
microscopy
Reporting Average number per hpf
Complete
Clarity, Blood, Protein, Free fat droplets/
Urinalysis
Fatty casts
Correlations

• Oval Fat Bodies

o Appearance:
▪ Since RTE cells absorbed fats, they become
highly refractile
o Sources of Error:
▪ Mostly the source of error in OFB is not being
Table 10. Summary of RTE Cells
able to identify an OFB
 This can be a problem since it is under the
Microscopic Renal Tubular Epithelial Cells diagnosis

Rectangular, columnar, round, ova, or Bacteria

cuboidal with an eccentric nucleus
Appearance
possibly bilirubin-stained or hemosiderin- • Common in urine specimens because of the abundant
laden normal microbial flora of the vagina or external urethral
meatus and because of their ability to rapidly multiply in
Spherical transitional cells urine standing at room temperature
Sources of Error
Granular casts o Urine will contain bacteria as a result of
contamination from all of these sites since the sites
Reporting Average number per 10 hpf mentioned contain normal flora
o When the person urinates, normal flora of the sites
Leukocyte esterase and nitrite can be brought to the urine → when in urine,
Complete bacteria multiply aggressively
(pyelonephritis)
Urinalysis
Color, Clarity, Protein, Bilirubin
Correlations • Significant if: Associated with WBC
(hepatitis), Blood
o If no WBC, only bacteria → Normal flora; purely
• RTE Cells contaminant
o Appearance: o If there is a bacteria and high WBC → True actual
infection
▪ Depends on which part of the renal tract or renal o WBC is the clue to know if the bacterium in urine is
tubule the RTE originates clinically significant or not.
▪ Absorbent by nature – can absorb fats, bilirubin,
hemoglobin • Diagnosis of bacteriuria in a case of suspected urinary
▪ Bilirubin stained – RTE becomes yellow since tract infection requires culture
bilirubin is yellow o Although urinalysis is important to screen for UTI,
▪ If there is hemoglobinuria – RTE cells can the gold standard test for the diagnosis of UTI is
absorb hemoglobin and hemoglobin can be bacteriology by doing culture and sensitivity
converted to hemosiderin

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• A colony count may also be done to see if significant Complete

numbers of bacteria are present Urinalysis pH, Nitrite, Leukocyte Esterase, WBCs
• More than 100,000/ml or CFU of one organism reflects Correlations
significant bacteriuria
• UTI – can develop at an alkaline pH
• Multiple organisms reflect contamination. However, the
presence of any organism in catheterized or suprapubic Yeast
tap specimens should be considered significant
o Since urine collected in catheterization or • Yeast cells may be contaminants or represent a true
suprapubic aspiration is expected to be sterile. yeast infection

▪ Do not expect to see bacteria

QUESTION: When do you consider it as a true yeast infection?
o If you see bacteria from a catheterized or
suprapubic sample, most of the time it is clinically • Same with the bacteria, consider it if it is accompanied by
significant the WBCs.
• HINT: Presence of WBCs (neutrophils)
• Presence of bacteria in urine is significant in diagnosing
UTI: Gram negative bacilli, Staph and Enterococcus
• They are often difficult to distinguish from red cells and
o E. coli - most common cause of UTI amorphous crystals but are distinguished by their
tendency to bud
• May be present as cocci or bacilli and reported per
HPF. o Major problem: RBC vs. Yeast
o The reporting is just like squamous epithelial cell- ▪ Since they are identical especially if RBCs are
rare, few, moderate, and many regular
o There is no need to indicate in the results if it is ▪ Point of differentiation lies on the tendency on
cocci or bacilli. Just put “bacteria are present” the ability of the yeast to bud.
o The one that will denote if it is cocci or bacilli is the ▪ Red blood cells – do not bud; circular with no
bacteriology department (culture and gram stain) buds
▪ Yeast cells – has buds; circular with budding
• This is an excellent
example of a mixed field • Most often the common yeast is Candida, which may
of red blood cells and colonize bladder, urethra, or vagina
bacteria • Small, refractile oval structure that may or may not bud
• "C" is pointing to the
o Characterization of Candida
bacteria
• “B” is pointing to RBC • In severe infections, may appear as branched, mycelial
• “A” is pointing to forms (rare)
crenated RBC • Seen in immunocompromised patients and in women
• Note how small they are with vaginal moniliasis.
compared to the RBC
o Moniliasis – other term of Candidiasis
• Bacteria can be confused
with amorphous crystals.
o Amorphous crystals – crystals which do not have • “B” is Budding Yeast
a definite form; resembles bacteria, especially cocci Cell
organisms o Note the budding
and the more oval
QUESTION: How to differentiate bacteria with amorphous appearance of
crystals? each cell

• Look at the motility • “A” is crenated red

• Bacteria will have the tendency to move blood cell
• Amorphous crystals are non-living things thus they do not • “C” is normal red
move blood cell

Table 11. Summary of Bacteria

Microscopic Bacteria

Small spherical and rod-shaped

Appearance
structures
Sources of Error Amorphous phosphates and urates
Few, moderate, or many per hpf
Reporting • Bacteria
The presence of WBCs may be required

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o Rod or cocci o T. vaginalis do not move rarely

• Yeast
o Oval and budding
• Mycelial form
o Can only be present in severe conditions

Table 12. Summary of Yeast

Microscopic Yeast Table 13. Summary of Trichomonas vaginalis

Small, oval, refractile structure with buds

Appearance Microscopic Trichomonas vaginalis
and/or mycelia
Sources of Error RBCs Appearance Pear-shaped, motile, flagellated
Rare, few, moderate, or many per hpf
Reporting Sources of Error WBCs, renal tubular epithelial cells
The presence of WBCs may be required
Complete Rare, few, moderate, or many per hpf
Urinalysis Glucose, Leukocyte Esterase, WBCs Reporting (sometimes reported as “presence of T.
Correlations vaginalis)
Complete
Leukocyte Esterase, WBCs (if actual
• Considered as true yeast infection and reportable if Urinalysis
parasitic infection)
WBC is present Correlations

QUESTION: Why correlate yeast with glucose?

Other Parasites in Urine

• Yeast infections are common in persons with Diabetes • Schistosoma haematobium

Mellitus; there will be glucosuria
• Yeast cells love glucose, that’s why glucose favors yeast o Ova can be found in urine samples
growth o Can reside in the plexuses of the blood vessels that
• If a patient has DM, expect to see yeast (pathogen) in the supply the urinary bladder
urine. o If a person is positive for S. haematobium, it can
associate with urinary bladder cancer
• Enterobius vermicularis
Trichomonas vaginalis
o Fecally contaminated
o Most common fecal contaminant
• Trichomonas vaginalis is
o Resides in the intestine. At certain intervals, it goes
the most common parasite
in urine in USA and is found out of the perianal folds and deposits their eggs
in approximately 25% of there.
women in casual vaginal ▪ There is a tendency that the ova in perianal folds
cervical examination. will go to the urine when urinating
o Females have clinical ▪ Mostly happens in women
manifestation if infected with T. vaginalis, whereas o Not an actual pathogen in urinary tract
males are asymptomatic and carrier of T. vaginalis
Spermatozoa
• A pear-shaped flagellate with an undulating
membrane • Easily identified in the urine by their oval, slightly
• The presence of this organism in urine specimens of tapered heads and long-flagella-like tails
females is due to the contamination of the urine with
vaginal secretions o Urine is toxic to
sperm cells and it will
o Can still be clinically significant die
• T. vaginalis is the most common parasites seen in the ▪ Dead and
urine since it can be an actual pathogen in the urinary immotile in urine
structure especially in girls ▪ Whole anatomy
• May resemble a WBC, transitional, or RTE cell when it’s can be seen but
not moving already dead
o Especially if the WBC is large in size • Sperm cell appearance in
o Most of the time T. vaginalis will move (jerk) semen is the same appearance in urine
• Associated with positive protein reagent strip

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o Too much sperm cells in urine rises protein level Cytomegalovirus

• Some laboratories are not reporting its presence:
o Cite lack of clinical significance
o Possible legal consequences – rape cases

Table 14. Summary of Spermatozoa

• Can be present in urine but very rare to be found or
seen
Microscopic Spermatozoa • CMV is a common virus that infects people of all ages
and once CMV is in a person’s body, it stays there for
Appearance Tapered oval head with long, thin tail life

Sources of Error None o Cannot see the virus in itself; too small to be directly
seen by microscopy
Reporting Present, based on laboratory protocol o Presence of CMV in the urine can be identified if
cells like the photo above are seen
Complete Urinalysis
Correlations
Protein • Most infections with CMV are “silent,” no signs or
symptoms
• It rarely causes serious consequences except in people
Mucus Threads with suppressed or impaired immune systems
• It is found in saliva, urine, and other bodily fluids
• Protein material produced by the glands and epithelial • Because it is often found in semen as well as in cervical
cells of the lower genitourinary tract and the RTE cells. secretions, the virus can be spread by sexual contact
o DCT and collecting duct (lower genitourinary tract) • Cells which are infected with CMV:
o Enlarged cells
▪ These cells produce proteins that make up
o There is an inclusion body or cytopathic effect (halo
mucous threads
is formed inside the cell)
• Tamm Horsfall protein or Uromodulin is its major
constituent. • CMV can only be appreciated/visible if it is stained

o Protein, raw material of mucous threads

• Thread-like structures with low refractive index often
confused with cast
o Resolution: Make use of subdued light in low light
to appreciate cast and mucous threads
• Clinical significance: No clinical significance but is
sometimes associated with Diabetes Mellitus

Table 15. Summary of Mucus Threads

Microscopic Mucus Threads

Single or clumped threads with a low

Appearance
refractive index
Sources of Error Hyaline casts

Reporting Rare, few, moderate, or many per lpf

Complete Urinalysis
None
Correlations

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MICROSCOPIC EXAMINATION OF URINE o The raw ingredients to make casts are Tamm-
Horsfall proteins or uromodulin
Significance of Cellular Casts
• (2) Interweaving of protein fibrils to form a loose fibrillary
• Individual network (urinary constituents may become enmeshed in
erythrocytes the network at this time)
may mean o “Interweaving” – weave to form a cast in urine
bleeding in o “Enmeshed” - When the urine inside the tubules
any portion of contains other urine sediments, those urine
the sediments may be trapped in the cast that are being
genitourinary interweaved in the renal tubule so they become part
tract of the cast
o Can be in
• (3) Further protein fibril interweaving to form a solid
the kidneys, ureter, urinary bladder, or even in the
structure
urethra
o Weave further to form a solid structure (continuation
• Individual leukocytes may represent infections or of step 2)
inflammation in any part of the urinary tract
▪ The solid structure will be flushed in the urine
o From kidney down to the lower structures
• (4) Possible attachment of urinary constituents to the
• Individual bacteria may mean infection in any part solid matrix
and in any length of urinary system/urinary tract
o The RBC, WBC, and bacteria may attach to the
Cast solid matrix of the cast so they become part of it

• Cast – problem lies only in the nephron
o Sediments which are exclusively formed in the
kidneys, especially in the renal tubules.
• Examples:
o Erythrocyte or RBC cast
▪ The bleeding is originating from the kidney itself
and not in any other part of the urinary tract
• (5) Detachment of protein fibrils (cast) from the
epithelial cells
o Detachment of the cast from the epithelial cells that
sponsored its growth from the RTE cells that used to
hold it
▪ Once detached from RTE cells, the cast can be
excreted via urination
▪ The cast is now visible in the urine samples

o Leukocyte cast • (6) Excretion of the cast

▪ The infection is solely confined in the nephrons, Figure 1. Cast Formation and Degradation
in the kidneys themselves and not in any part of
urinary tract
o Bacterial cast
▪ The infection or the colonization of the pathogen
is only happening in the nephron
NOTE:
• Significance of cellular cast: To narrow down possible
choices of pathologic conditions in case studies
o Cast – the problem is solely on kidneys

Cast Formation

• Pneumonic: AIFADE
• (1) Aggregation of Tamm-Horsfall protein into individual
protein fibrils attached to the RTE (Renal Tubular
Epithelial) cells
o The cells that allow or facilitate cast formation are
the RTE cells (thus, in the renal tubules)

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• There are many types of cast but the prototype cast o represent the end portion of the cast degradation
will always be the hyaline cast process
o Prototype cast – first cast that was formed; core or Figure 2. Cast Formation and Degradation
basic cast
o Hyaline cast – empty cast
▪ The other urinary constituents may be added
here

REMEMBER:
• In the process of cast formation, urinary constituents
may be attached in the matrix of the cast
• If RBC, WBC, bacteria, or epithelial cells are present in
the urine where casts are also present, those urinary
sediments can be incorporated in the cast
• After hyaline cast, cellular casts are usually formed.
o Cellular cast – general term
▪ Can be RBC cast or WBC cast, depending on EXAMINATION OF URINE SEDIMENT
which cell is incorporated in the hyaline cast
• The cellular sediments that are incorporated in the cast
can be degraded or dissolved
• From being cellular cast, once the cellular components
of those cast in the matrix begin to dissolve, they
become granular cast

Cast Degradation

• (1) The cellular cast eventually degrade

o Degradation of RBC, WBC, and epithelial cells
o Cellular elements become granular casts
▪ 2 types of granular cast:
 Coarse granular cast (bigger)
 Fine granular cast (smaller)
• (2) If the casts are degraded, they will first become the
coarse granular cast.
• (3) They will continue to dissolve and become the fine
granular casts.
• (4) The fine granules will eventually be dissolved totally
that they become a wax-like material
o Hence, the next casts formed are the waxy casts.
Summary
• Hyaline cast → Cellular cast → Coarse granular cast →
Fine granular casts → Wax-like material
• Hyaline cast – first cast that is formed
o Empty; cellular material can be incorporated in its
matrix
• Cellular cast – second formed cast
o The name depends on what cell is incorporated in
the matrix
• Coarse granular cast
• Fine granular cast
• Waxy cast
o Formed after the further dissolution, followed by
homogenization, of the fine granular cast
Casts
• Casts are cylinders of Tamm-Horsfall protein that
solidified in distal tubules and are unique to the kidneys
o Raw ingredient: Tamm-Horsfall protein or
uromodulin
• Urinary casts are formed only in the lumen of the
distal convoluted tubule (DCT) or the collecting duct
(distal nephron)
o Casts are unique to the kidneys
o It is only in the kidneys where casts are formed.
o Shape of cast: cylindrical (box type)
▪ Represents the lumen of DCT and collecting
duct where they are formed
• Uromodulin excretion increases: Stress and Post-
Exercise
o After exercise, expect casts in urine (especially
hyaline cast) which is normal
▪ Does not necessarily mean that the person is
sick
▪ Cast excretion will eventually subside after
relaxation
• Urine-flow stasis, acidity and calcium and sodium
o Urine-flow stasis - No movement of urine
▪ The person is rarely urinating or not urinating at
all

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▪ No urine flow = Urine will stay in the tubules
 If urine is stagnant in renal tubules, this
facilitates cast formation.
 High chance of Tamm-Horsfall protein
aggregation leading to formation of cast
o Acidity
▪ Low pH will favor cast formation
o Ca and Na
RBC Cast
▪ will also favor cast formation
• Although albumin and some globulins are also
incorporated in cast matrix.
▪ Congestive Heart Failure (CHF)
• Greater numbers of hyaline casts may be seen
associated with proteinuria of renal glomerular disease
or extrarenal (overflow proteinuria as in multiple
myeloma)
• Stain with Sternheimer-Malbin (SM) to highlight
hyaline cast
o Pink when stained
Figure 3. Histologic Preparations (smears) of RBC Casts

Factors Favoring Protein Cast Formation

• (1) Low flow rate (urinary stasis)

o Favors cast formation; no urine movement will allow
for aggregation of uromodulin proteins
• (2) High salt concentration (Ca and Na)
• (3) Low pH
• All of these will favour protein denaturation and
precipitation, particularly that of the Tamm-Horsfall
protein
• Cylindroids - protein casts with long, thin tails formed
at the junction of Henle's loop and the distal convoluted
tubule
o Sometimes casts are formed in the junction of
Ascending loops of Henle and the DCT • The presence of this red blood cell cast on urine
microscopic analysis suggests a glomerular bleeding,
▪ Ascending loop – thick walls; water-impermeable renal tubular injury,or bleeding in any part of the
 The casts formed here become tapered and nephron
will achieve long thin tails • Observation of Individual RBC
• Cylindruria – presence of cast in urine o bleeding in the GenitoUrinary Tract (GUT)
o Has the same clinical significance with Cylindroids ▪ Clinical difference between finding a solitary
RBC and RBCs trapped in a cast
▪ Difference is location where they are formed.
• Glomerular damage: RBC cast, proteinuria, and
• Hyaline casts can be seen even in healthy patients dysmorphic RBC

Type of Casts Figure 4. Histologic Section stained with Trichrome Stain;

at medium power
Hyaline Cast

• Hyaline casts are

common findings in urine
o appear colorless and
slightly refractile
• Difficult to identify
o Hallmark: empty
matrix with borders
• 0-2/lpf is normal
• Seen after stresses (SE), dehydration, heat exposure,
and emotional breakdowns • The photo highlights red blood cells grouping together
• Most of the time, it is physiologic; however: in tubules to form casts
• RBC Cast: Orange-red at LPO
o Observed in Pathologic Conditions such as:
o Due to presence of hemoglobin
▪ Acute Glomerulonephritis (AGN),
▪ Pyelonephritis (Pye) • Red-brown casts
▪ Chronic Renal Disease (RD), o Severe hemoglobinuria or myoglobinuria

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• Granular, Dirty brown casts
o Represents hemoglobin degradation products such
as methemoglobin
o Acute Tubular Necrosis (ATN) – due to massive
and persistent hemoglobinuria, which is toxic to
renal tubules, leading to renal failure
• RBC casts are more fragile than the other casts
• Tendency of RBC cast:
o Once the RBC cast has aged, the RBCs inside will
begin to lyse
▪ The cast will develop a more homogenous
appearance, but still orange-red in color Bacterial Casts
▪ Will now be called as blood cast, which means
greater urinary stasis
 Stagnant urine in tubule
 Patient is not urinating anymore
 Potentially indicate renal failure
REMEMBER:
• The content of the cast’s matrix determines the identity
of the cast
• Sometimes, the many RBCs may clump or consolidate
together resembling an RBC cast
o An RBC cast is differentiated by looking for the Epithelial Casts – RTE Cast
outline of a cast, and the cast matrix
▪ Most of the time, the matrix is not fully occupied
by RBCs
WBC Cast
• Most typical for acute pyelonephritis (differentiates
from lower UTI)
o UTI – generic term for infection in any part of the
urinary tract
▪ Pyelonephritis – when the UTI is in the kidney;
aka upper UTI
▪ Cystitis – UTI is in the urinary bladder; aka lower
UTI
• May also be present in severe cases of
glomerulonephritis and acute interstitial nephritis (AIN)
o However, glomerulonephritis is more associated
with RBC cast than WBC cast
• Their presence indicates inflammation or infection of the
kidney, because such casts will not form except in the
kidney
• Granular
o WBCs, especially neutrophils which most of the time
are abundant in the cast, are innately granular
• If disintegrated, multilobed
nuclei will be present
(stained supravitally)
• To establish the presence of
WBC cast, also observe for
free WBCs
o Absence may potentially
mean that there is also
no WBC cast
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• WBCs have the tendency to clump (more common than
RBC clumping), resembling a cast
o A WBC cast is differentiated by looking for the
border of a cast, and the cast matrix
o WBC clump and WBC cast have different clinical
significance
▪ WBC clump with no casts = infection can be in
any other part of the urinary tract (especially the
lower UT)
▪ WBC casts = infection is localized in the
nephrons
• Casts in which the matrix is studded with bacteria
• Clinical Significance: Pyelonephritis
(inflammation/infection of the kidney)
• May be mixed with WBC
• May mimic granular casts, especially the fine granular
casts
o To differentiate bacterial and granular cast → gram
stain
▪ matrix gives out color
▪ Gram stain will not color granular casts
• RTE cells get incorporated in casts
• Renal Tubular Cells seen are small, round, and oval
• These cells are slightly larger than leukocytes (10-14
um) with lightly granular cytoplasm
o Formation of casts is
in the DCT that’s why
the visible cells in the
cast matrix are the
smaller versions of
cells (round and oval)
which can be confused
with WBC
▪ Leukocytes has
heavier
granulations than
RTE casts
o Use stains and phase contrast microscopy to
differentiate RTE from WBC
• Represents advanced tubular destruction; Bilirubin-
stained RTE cells seen in Hepatitis
o Production of urinary stasis along with destruction of
tubular linings
o Clinical implications of RTE casts are almost the
same with RTE cells
o RTE cells or casts are absorbent and can absorb
bilirubin, turning them yellow → suspect for
hepatitis
• May accompany WBC Casts in Pyelonephritis
4
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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

• Observations for figure 4:

o Notice the WBCs and
RBCs above
o It is a squamous
epithelial cell

• This renal tubular cell cast suggests injury to the tubular

epithelium Granular Cast
Fatty Cast • Fine granular casts – “pinong-pino”, powdered
• Coarse granular casts – larger granules
• They are identified by the
presence of refractile lipid
droplets
• They are hyaline casts
that contain lipids such as
cholesterol, triglycerides,
and neutral fats
• The background matrix of
the cast may be hyaline
or granular
o Hyaline only – clear, no content
o Granular – matrix has fatty contents
• Seen in conjunction with Oval Fat Bodies (OFB) and
free-fat droplets in disorders that cause lipiduria
• Conditions that manifest with lipiduria: Fine Granular Coarse Granular
Cast Cast
o Nephrotic Syndrome (NS) • Can be Pathologic or Nonpathologic
o Toxic tubular necrosis
o Diabetes mellitus o Nonpathologic – the origin of the granules may
come from the lysosomes excreted by RTE cells
o Crush injuries
excreted during normal metabolism
• Use Polarizing Microscopy or fat stains (e.g. Sudan) for o Pathologic – may represent disintegration of
better recognition cellular casts and tubule cells or protein aggregates
filtered by the glomerulus

Mixed Cellular Casts ▪ Granular casts may form from direct aggregation
of plasma proteins or immunoglobulin light
• Most commonly mixed up cells are RBC and WBC chains which can also be a pathologic process
casts, but in: • When cellular (hyaline) casts remain in the nephron for
o Glomerulonephritis some time before they are flushed into the bladder
urine, the cells may degenerate to become a coarsely
▪ Matrix is predominantly RBC casts since granular cast
glomerulonephritis is associated with bleeding
o Casts which persist may break down, so that the
o Pyelonephritis cells forming it are degenerated into granular debris
▪ WBC and RTE Casts, or; until it becomes fine
▪ WBC and Bacterial Casts • Clumps of small crystals and fecal debris may look like
▪ Predominant cast should be WBC casts granular casts
o Differentiate by looking at the borders and matrix
Figure 4. Stained Mixed Cellular Cast (their presence indicates that it is a granular cast)
• Distinguish from columnar RTE cells by staining to
Squamous epithelial cell enhance nuclear details
o If nucleus is seen after staining, then it is an RTE
cell
• Granular casts have a textured appearance which
ranges from fine to coarse
RBCs
• Since they usually form as a stage in the degeneration
of cellular casts, the interpretation is similar to that for
cellular casts
WBCs

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

• Clinical significance of • Represents extreme

finding granular cast is the urinary stasis
same with cellular casts
o Similar to waxy cast
o Ex. RTE or RBC casts
disintegrated → • Indicates widening of
becomes granular DCT = broad
casts → clinical o DCT lumen widens
significance will be the during renal failure
same with RTE or
RBC casts ▪ All types of casts may occur in broad form
o The added implication
Coarse Granular Cast ▪ RBC/WBC cast will also broaden
(short) beside a waxy
of finding granular casts cast (long) • Granular and Waxy broad cast - more common
is urine stasis. If there
is no urine flow, cellular casts will favor degradation o Extreme urinary stasis = prequel to urinary failure

• Granular casts disintegrates further to become • May be Bile-stained waxy casts – tubular necrosis by
waxy cast viral hepatitis

o Fine granules are completely dissolved, REMEMBER:

homogenizing the content which appears to be waxy
o Waxy casts assume the shape of tubules • Casts assume the shape of DCT and the collecting duct

Waxy Cast
• Have a smooth
consistency, but are more
refractile and easier to see
compared to hyaline casts
o Hyaline and waxy casts
are the same in the
sense that they
seemingly don’t
contain anything
o Waxy casts have
dissolved granules that make them more refractile
and easier to see than hyaline casts
• Represents extreme urine stasis
o Patient does not urinate often when there is renal
failure (no urinary movement)
o Urine stasis will favor degradation
• They are found in conjunction with other types of urinary
casts that are associated with conditions that cause
renal failure
o Renal failure due to severe, unresolved UTI – you
will see WBC casts along with waxy casts
o Renal failure due to massive bleeding in the
glomerulus – you will see RBC casts along with
waxy casts
• They commonly have squared off, jagged and
notched ends, as if brittle and easily broken
• They are found especially in chronic renal diseases
o Diabetic nephropathy
o Malignant hypertension
o Glomerulonephritis
o Other conditions promoting renal failure
• Supravital Stains – used to enhance visualization;
produce a homogenous dark pink
o enhance visualization by addition of supravital stains
Urinary Crystals
• Detects the presence of the abnormal types (abnormal
crystals) in:
o Liver diseases
o Inborn error of metabolism
▪ Genetic defect, usually a missing enzyme
▪ Manifest as an amino acid disorder
o Renal tract damage caused by crystallization of
medications
▪ e.g. ampicillin, sulfonamide
• Normal crystals reported as rare (1+), few (2+),
moderate (3+), and many (4+) per low power field (lpf)
o Abnormal crystals averaged per lpf
o Ideal reporting in normal and abnormal crystals
o But it depends on laboratory protocols.
Crystal Formation
• Formed by the precipitation of urinary solutes (raw
materials)
o e.g. inorganic salts, organic compounds,
medications
▪ When they precipitate that’s the time they
become crystals.
NOTE:
• Soluble substances: will not precipitate → will not
crystalize; Insoluble substances: will precipitate → will
crystalize
• Precipitation subject to the following factors:
o Temperature
▪ Low temperature = favors crystallization
▪ Refrigerated samples after extracting out from
the refrigerator, it is expected to have crystals

Broad Cast o Solute concentration

▪ High: more precipitation (it will crystallize)
• a.k.a. Renal Failure casts

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

o pH • Most of the time the crystal is alkaline, if a crystal ends

a phosphate or carbonate.
▪ pH is a good determinant as to precipitation of
different crystals
▪ Some crystals are formed acidic, alkaline or Normal Acid: Amorphous Urates Crystals
neutral pH
▪ Knowing the pH will narrow down the choices of • No definite shape; like
the possible crystals present bacteria
▪ Affects solubility • Yellow-Brown granules
 Acidic crystals will dissolve when added with • Occur in clumps resembling
alkaline substance granular casts
 Alkaline crystals will dissolve when added with o Differentiate: cast has
acidic substance borders look and matrix
 Although different crystals have different • Appear after refrigeration producing characteristic pink
solubility characteristics sediment at pH greater than 5.5, whereas uric acid
crystals appear when pH is lower
General Identification Techniques
o Uroerythrin (pink color) present in the urine will also
• Have characteristic shapes and colors precipitate together with amorphous urates; hence
o Some are in geometric shapes will impart pinkish or pinkish red color
Normal Acid: Calcium Oxalate Dihydrate Crystals
• Consider pH
o To know either crystals formed in acid or alkaline. • 2 forms of Calcium
• Aids in the identification of crystals: Oxalate (CaOx):
dihydrate and
o Use of solubility characteristics (acid or alkaline) monohydrate
o Use of polarized microscopy (through birefringence
or its ability to polarize light)
▪ Birefringence – refract light
▪ Crystals are birefringent
• (1) Dihydrate form
Type of Crystals o most common
o aka “Weddellite”
• Normal Acid – normal crystals formed in acidic pH
o look like little envelopes
o Amorphous Urates (or tetrahedrons,
o Calcium Oxalate depending upon your
o Uric Acid point of view), octahedral
o Acid Urates and Sodium Urates
▪ Two pyramids joined
• Normal Alkaline – normal crystals formed in alkaline at their bases
pH ▪ or like a box with “X”
o Amorphous Phosphates o Oxalate crystals are
common in acidic urine
▪ whitish
▪ Also found in neutral
o Triple Phosphates
urine and rarely in
o Calcium Phosphates and Carbonates
alkaline urine
o Ammonium Biurates
o Increased Oxalic Acid
• Abnormal – most of the time formed at an acidic pH
▪ Favors the formation of calcium oxalate
o Cystine Crystals ▪ Associated with Tomatoes, Asparagus, and
o Cholesterol Crystals Vitamin C
o Radiographic Dye
o Tyrosine, Leucine and Bilirubin  Vitamin C will be converted to oxalic acid,
then the oxalic acid will precipitate becoming
▪ Liver Disorder Crystals calcium oxalate.
o Sulfonamide and Ampicillin • (2) Monohydrate form
▪ Out of crystallized medications because it can be o “Whewellite”
used as antibacterial o Rarely found, vary in size
NOTE: and may have a spindle,
oval, or dumbbell shape
• Urates crystals are most of the time acid except for o Appear as flat, elongated,
ammonium biurate (alkaline) six-sided crystals (“fence
pickets”)

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

o Ethylene glycol poisoning Figure 4. Polarized fields of Uric Acid (birefringent)

▪ a substance found in antifreeze agents
o Most of the time pathologic
▪ Presence in urine indicates ethylene glycol
poisoning
 Ethylene glycol – a substance that can be
found in anti-freeze agents
 Ingestion of anti-freeze agents that has
Partially-polarized Fully-polarized
ethylene glycol will precipitate to calcium
oxalate monohydrate crystals Normal Alkaline: Amorphous Phosphates
Normal Acid: Uric Acid
• Counterpart of amorphous urates
• Very common • Found in alkaline pH
• Lemon-shaped • Amorphous appearance – no definite shape
• Normally found in the urine o Granular in appearance and similar to amorphous
• Can be ambiguous because it may appear in a lot of urates and differentiated through color and pH
shapes
• White precipitates that don’t dissolve on warming
o Uric acid crystals may appear as: • Common between amorphous urates and phosphates
▪ rhombic (4 sided), o Refrigeration will enhance the formation of the two
▪ whetstones o Both do not have clinical significance
(mahahabang bato),
▪ wedges,
▪ rosettes (rose
shape)
▪ hexagonal
o Most common based in experience (pic above)

Normal Alkaline: Calcium Carbonate

• Small and colorless, Dumbbell or Spherical

• No clinical significance
• May resemble amorphous crystals but distinguished by
evaporation of gas after addition of acetic acid
o Calcium carbonate is soluble to acetic acid
• Usually yellow-brown and have a six-sided shape • Birefringent, differentiating it from bacteria
(common shaped according to the book)
• May be normal, but excessive amount in urine is
associated with increased Purine and Nucleic Acid
metabolism conditions of:
o Patients with leukemia, Lesch-Nyhan Syndrome,
and gout
o Increased metabolism of purine and nucleic acid =
uric acid
▪ Since there is many uric acid (raw material), it
will precipitate to form uric acid crystals Normal Alkaline: Calcium Phosphate
• After centrifugation, if the sediments are orange,
expect to see a lot of uric acid crystals
• Cystine crystal can be confused with uric acid crystals
because of its six-sided shape
o Pathologic compared to uric acid crystals
• May resemble ova of Trichuris trichiuria

• Not frequently encountered

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

• Colorless, flat rectangular plates or thin prisms in
rosette formations
• Confused with sulfonamide crystals.
o Calcium phosphate dissolves in dilute acetic acid,
sulfonamides do not
• Common constituent of renal calculi (renal stones)
Normal Alkaline: Ammonium Biurate Crystals
o Failure in the reabsorption of the amino acid
cysteine
o Too much cysteine in the urine = increased
concentration that will lead to precipitation
• Cystine crystals are seen as flat, colorless, hexagonal
plates
o shaped like stop signs

• These crystals generally appear

as yellow-brown, radially-
striated spheres with irregular
"thorn-apple" or "ox-horn"
projections
o Thorny-apple description will
help to identify the • Confirmed using Cyanide-Nitroprusside Test
ammonium biurate crystals o Cystine crystals = positive
o Uric acid crystals = negative
• May be seen in acid urine (rare), their formation is
favored in neutral to alkaline urine Abnormal: Cholesterol Crystals
• Dissolves at 60°C
• Converts to uric acid crystals in the addition of acetic • Rare except when
acid refrigerated cause lipids
• Frequently encountered in old specimens and usually are free floating
associated with presence of ammonia produced by • Requirements to satisfy
urea-splitting bacteria formation of cholesterol
o Ammonium biurate crystal = old sample (pH crystals:
exceeds 8) o Lipiduria
▪ Too alkaline = too much ammonia (too many o Refrigerated urine
molecules of urea has broken down) sample
▪ Refrigeration enhances lipid precipitation, thus
forming the crystals
• They are actually rectangles in cross-section with
notched ends
o Notched ends resemble a staircase (Staircase
crystals)
• The cholesterol crystals appear to be needle-shaped
(sideview)
• Associated with lipiduria together with fatty casts and
Normal Alkaline: Triple Phosphate (Ammonium oval fat bodies (OFB)
Magnesium Phosphate) o Nephrotic syndrome will exhibit lipiduria and
proteinuria
• Usually appear as colorless,
prism-like "coffin lids" • Highly birefringent
• Often seen in urine from
normal individuals.
• A.k.a. Struvite or Staghorn
• Fern leaf and feathery when
it disintegrates
o 3Fs – fern leaf, feathery,
coffin lids
• Urinary tract infection with urease producing bacteria
can promote its formation (and urolithiasis) by raising
urine pH and increasing free ammonia Side view: Front view:
needle-shaped rectangle w/
o Urolithiasis – renal stone formation notched ends
Abnormal: Cystine Crystals
• Looks similar with radiographic dye crystals

• Seen in Cystinuria o radiographic dye crystals are formed only if injected

with radiographic contrast material (e.g. CT scan, MRI)

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

o Radiographic contrast materials are oily in nature • Appear as clumped needles

or granules with the
▪ May crystallized when excreted in urine
characteristic yellow color of
o Also highly birefringent bilirubin
o How to differentiate Cholesterol crystals and • In disorders that produce renal
Radiographic dye crystals? tubular damage, such as viral
hepatitis, bilirubin crystals may
▪ Take note of other urinalysis results and patient
be found incorporated into the
history
matrix of casts.
 Rectangular with notched edge with fatty
casts and OFB → Cholesterol crystals o Crystals can also be incorporated in casts (Crystal
casts)
 Rectangular with notched edge without fatty o Bilirubin Crystal casts is the very probable crystal
casts and OFB → Radiographic dye crystals cast that is formed
▪ Check specific gravity Abnormal: Sulfonamide Crystals
 If radiographic dye crystals → very high SG
(>1.035) • Sulfonamide crystals are synthetic drugs that are used
 Make use of refractometer to treat bacterial infection.
Abnormal: Tyrosine Crystals o Very insoluble
▪ If insoluble, they have a high chance to
• Raw ingredient: Tyrosine amino acid precipitate
• Happens if there is failure in the metabolism of the
amino acid tyrosine. o When taken can precipitate into sulfonamide
crystals
• If it is not metabolized, it will precipitate forming tyrosine
o Sulfonamide in the present time is more soluble than
crystals.
the old sulfonamides.
• Fine, colorless to yellow needles in clumps or rosettes
• Usually seen in conjunction with Leucine Crystals with a • Sulfonamide crystals are typically yellow to brown in
positive bilirubin chemical test color and often resemble uric acid crystals (due to
o It is associated with liver disorder whetstone appearance)
o However, sulfa crystals are easily distinguished from
• May signify also amino acid disorder uric acid by confirmatory tests.
• Confirmatory tests:
o Acetone Test - Sulfa crystals are readily soluble in
acetone
▪ If suspected crystal did not dissolve, most likely
uric acid crystal
needle fine needle rosette
o Dextrine/Sulfuric acid test
Abnormal: Leucine Crystals
▪ positive in dextrin test (a.k.a "old yellow
newspaper" test
• Yellow-brown spheres that demonstrate concentric ▪ negative sulfuric acid test
circles and radial striations
• Needles, Rhombics, Whetstones, Sheaves of Wheat,
o Resembles the ova of Taenia spp, and and Rosettes
Echinococcus
o Sulfonamide crystals are polymorphic
• Seen less frequently than tyrosine crystals and, when
present, should be accompanied by tyrosine crystals • Problem: May damage the urinary tract since crystals
are sharp, resulting to hematuria
o Tyrosine may not be accompanied by leucine
crystals, but it is very rare for leucine crystals to be
seen alone

Sheaves of wheat Rosette

Abnormal: Ampicillin Crystals

Abnormal: Bilirubin Crystals • Ampicillin is a penicillin drug

• Associated with the intake of drugs
• Present in hepatic disorders producing large amounts • Formed after massive dose of penicillin compounds
of bilirubin in the urine without proper hydration

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

• Colorless needles that tend to bundle after refrigeration o When they are small,
• Obtain patient ’s history to differentiate from other they can be confused
crystals as RBC
• Oil droplets resulting from
contamination by
immersion oil or lotions
and creams
• Air bubbles occur when
using cover slip

Pollen Grains

• Seasonal contaminants
Artifacts and are very rare
appearing as spheres
• May be found in improperly collected urine or dirty
with a thick cell wall and
containers
occasional concentric
• Frequently encountered:
circles as well as they are
o starch, large
o oil droplets,
o air bubbles, Hair and Fibers
o pollen grains,
o fibers, • From clothing and diapers
o fecal contamination may be occasionally
mistaken for casts, but
• Highly refractile larger and longer and
o Helpful in differentiation from the true urinary more refractile
constituent o Fibers – common
artifacts of the urine
• Are confusers and miscellaneous, thus are not
reported ▪ Confused with casts, but latter is shorter

Starch Granules • Examine under polarized light since they exhibit such
o Fibers will polarize light whereas cast don’t with the
• seen in powdered gloves exception of fatty cast.
with highly refractile
spheres usually with PHOTO IDENTIFICATION
dimpled center
o Dimpled center -
invagination in the
center
• Resemble fat droplets when polarized producing
maltese cross formation
o Should be noted because they can obscure vision A. B.
and can be confused as fat droplets wherein most of
the time it is clinically significant

C. D.

• Occasionally confused with RBCs

• Consider reagent strip parameter results • A: Mucus threads
• B: Waxy Cast
o Fat droplet: there is OFB, cholesterol crystals, heavy
proteinuria o It contains a homogenous substance, thus it is not a
o RBC: positive for blood reagent strip hyaline cast
• C: Squamous epithelial cell (pinkish-stained with
Oil Droplets and Air Bubbles
centrally-located nucleus)
• Highly refractile and resembles RBC

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 LABORATORY UNIT VI: MICROSCOPIC EXAMINATION OF URINE PART II

o Small circles with biconcave and central pallor are

RBCs
• D: Uric Acid
o Resembles T. trichiura ova

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 [TRANS] LABORATORY UNIT 07: URINE SMEAR PREPARATION, READING, AND REPORTING
STEPS IN URINALYSIS
• (1) Specimen Collection
• (2) Sample Verification
• (3) Physical Examination of Urine
• (4) Chemical Examination of Urine
• (5) Microscopic Examination of Urine
o Reading and reporting cannot be accomplished
without performing the microscopic examination
o When accurate results are made, releasing of
results can now commence.
• (6) Release of Results
o Before releasing the results, reading and reporting
of urine sediments must be done correctly.
Urinalysis Result
Figure 1. Example of Urinalysis Report
• Different urinary sediment has different ways of
reporting it.
URINALYSIS
(1) Specimen Preparation
• Specimens should be examined while fresh or
adequately preserved.
o Read the result as soon as possible
o Fresh is preferred over refrigerating the sample
because refrigerating the sample can cause
precipitation of different urinary sediments or
crystals such as amorphous urates and
amorphous phosphates
▪ There would be precipitation of these crystals
that could obscure the different urinary
sediments → difficulty in reading the results
o Warming the sample at 37oCmay dissolve other
crystal and could lead to false negative result.
• The midstream clean-catch specimen minimizes
external contamination of the sediment.
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG.
(2) Specimen Volume
• A standard amount of urine, usually between 10 and
15mL, is centrifuged in a conical tube (test tube)
o 10-15mL sample is used because it will provide an
adequate volume from which to obtain a
representative sample of the elements present in the
specimen
o Average: around 12mL
▪ Frequently used because multi-parameter
reagent strips are easily immersed in this volume
▪ It will be easier to dip the entire reagent strip
o If the patient cannot provide a 12mL sample (e.g.,
pediatric patient), the volume must be noted in the
report form
▪ This guideline is based on the book. However, it
will also depend on the hospital protocol if there
is a need to report it
▪ Ideally, do repeat collection. But it will still
depend on the patient.
 If it is a pediatric patient, check the result then
release it if there is somehow a correlation
with the diagnosis of the patient (still depends
on the laboratory protocol)
(3) Centrifugation
• 5 minutes at a relative centrifugal force (RCF) of 400
o This setting will produce an optimum amount of
sediment with least chance of damaging the
elements
• The speed of the centrifuge and the length of time of the
specimen should be consistent
(4) 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.
o (1) Centrifugate the sample
o (2) Decant by throwing away the supernatant in the
sink, leaving out the remaining sediments with a bit
of supernatant
▪ When decanting, it will automatically remove the
supernatant, and there will always be remaining
sediment.
o (3) Flicker the remaining sample, mix, or resuspend
▪ Can be done by:
 Using a commercial system
 By the tips of our fingers,
 Tapping the tip of the tube or flicker
BSMLS 3 1
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 LABORATORY UNIT 07: URINE SMEAR PREPARATION, READING AND REPORTING

▪ The sediment must be thoroughly resuspended (6) Examining the Sediment

by gentle agitation
 DO NOT vigorously do agitation as it will
disrupt other some cellular elements →
inaccurate result
▪ Thorough resuspension provides an equal
distribution of elements in the microscopic
examination
 Sediments must be thoroughly mixed with the
supernatant
o (4) Transfer it to the glass slide
Commercial Urine Sediment Preparation System
• Most ideal setup
o KOVA System
• Microscopic examination:
o Observation of a minimum of 10 fields under both
low (10×) and high (40×) power
• When the sediment is examined unstained, many
sediment constituents have a refractive index similar to
urine:
o Examined under reduced light when using bright-
field microscopy
• If conventional glass slide method is used:
o Casts will have the tendency to locate near the
edges of the cover slip
o RECOMMENDATION: Low power scanning of the
coverslip parameter.

▪ Not a test tube but a (7) Reporting the Microscopic Examination

specialized KOVA
with graduation • Most sediments:
▪ Consists of: o Assessed or
 1 – KOVA top enumerated using at
least minimum of 10 lpf
 2 – KOVA tube
or 10 hpf
 3 – KOVA petter
Pipette -specialized • Two steps in reporting:
pipette o (1) Counted as a range
• Procedure o (2) Descriptive (rare,
few, moderate, many)
o (1) The clear plastic centrifuge tube is filled to the
appropriate graduation mark with a well-mixed urine Quantitative Assessment
o (2) Cover with KOVA top
o (3) After centrifugation, the KOVA petter is gently • Casts, RBCs, WBCs: counted as range (e. g. 0-2, 2-5,
slid into the tube (at the end until to the base) 5-10)
o (4) The bulb-like structure will fit snuggly such that o RBCs and WBCs are counted in 10 hpf
all but 1mL of urine can easily be decanted
▪ Count from tip to tip
▪ The remaining supernatant will be mixed with the ▪ Changes are also from tip to tip
sediment in preparation for urine smear
o How to range RBC and WBC?
• To maintain uniform urinary sediment, the supernatant
should be aspirated-off rather than poured off ▪ Do not add the cells counted all together
• Some system provides a pipette  Make the range based on the separate
o Pipette – used for sediment resuspension and individual fields
transferring of the specimen to the glass slide ▪ Do not need to count the entire 10 fields but
make a range
• NOTE: We do not use this type of system in our
laboratory  The minimum and the maximum cells counted
• In hospitals, the content of the test tube is decanted and per field seen
the remains are resuspended thoroughly before placing • Urinalysis Result:
it into the glass slide.
• The test tube is simply centrifuged.
(5) Volume of Sediment Examined

• The most recommended/ideal volume is 20 uL (0.02

mL) covered by a 22 × 22 mm glass cover slip.
o Glass cover slip is not really preferred because if
there’s too much sediments placed on the glass
slide, the large sediments (e.g., casts) would spread
out to the borders of the cover slip.

o The 0-1 hpf range on the pus cells means that there
are fields with no WBC and that the highest counted
WBC among all the fields is only 1

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 LABORATORY UNIT 07: URINE SMEAR PREPARATION, READING AND REPORTING

Example in Reporting the Microscopic Examination • It depends on hospital protocol on what to be used
(Quantitative) either qualitative assessment in descriptive or numeric
terms
• View 10 fields and write down the number of sediments o Numeric terms are usually used in chemical testing
per field o For microscopic examination, qualitative
o Example: Assessment of pus cells assessment in descriptive is utilized

▪ Field 1: 2 • TECHNIQUE:
▪ Field 2: 7 o Divide the field into four
▪ Field 3: 5 quadrants (make a cross)
▪ Field 4: 6 o Imagine the scattered cells:
▪ Field 5: 6
▪ Field 6: 4 ▪ If cells can fit on one
▪ Field 7: 5 quadrant = 1+
▪ Field 8: 8 ▪ Cells can fit half of the
▪ Field 9: 4 entire field = 2+
▪ Field 10: 5 ▪ ¾ of the entire field = 3+
▪ Cells can fit on the entire fields = 4+
o Result: 2-8 /hpf
o Does not require a total, just take note of the Reporting the Microscopic Examination Cont.
minimum and maximum only
• Casts: count in at least 10 lpf, get the average, and
• In practice, the lower and upper limit of the range must
report number of casts per lpf
not exceed 10
o Example: 3-5/lpf, 6-10/lpf, 0-2/lpf
o For example:
o In range and read in lpf
▪ 5-10/hpf
▪ Use hpf to identify casts by type
▪ 1-7/hpf
▪ 1-10/hpf • RBCs, WBCs, RTE, oval fat bodies: count in at least
▪ 3-24/hpf (incorrect) 10 hpf, get the average, and report number of cells per
▪ 1-15/hpf (incorrect) hpf
o If it exceeds 10, there might be a problem in the o Example: 3-10/hpf, 8-10/hpf
suspension of urine sample o In range and in hpf
▪ CAUSE: Urine is not resuspended well after • Squamous cells, mucus threads: rare, few, moderate,
decantation or many per lpf
▪ Main purpose of resuspending and mixing
thoroughly is for even distribution of sediments in o Reported in descriptive manner per lpf
all fields • Bacteria: few, moderate, or many per hpf; may be
reported as ‘packed field’
Qualitative Assessment
o Descriptive and per hpf
• Some sediments (mucus, crystals, bacteria): reported in
qualitative assessment in descriptive (rare, few, • Transitional cells, Yeast, Trichomonas: rare, few,
moderate, many) or numeric terms (1+, 2+,3+,4+) per moderate, or many per hpf
field of view o Reported on descriptive manner per hpf
• Report all the cells seen immediately
Table 1. Qualitative Terms and Descriptions for Fields o It will take time if reported one by one, depending on
of View (FOVs) the cell type
o Read all cells that are seen in the field
Descriptive Numeric
Description ▪ As long as reporting is correct
Terms Terms
 10 fields – lpf / 10 fields – hpf
Rare 1+ Present but hard to find
• Sperm cells: report only if present
One (or more) present in almost
Few 1+ o Depends on hospital protocols
every field of view (FOV)
Easy to find; number present in • Crystals: reported as rare, few, moderate, or many
Moderate 2+ FOV varies; “more than few, (descriptive manner) per hpf
less than many”
Prominent; large number o Can also be reported as present or positive
Many 3+ depending on hospital protocols
present in all FOVs
FOV is crowded by or
Packed 4+
overwhelmed with the elements

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 LABORATORY UNIT 07: URINE SMEAR PREPARATION, READING AND REPORTING

TNTC (Too Numerous To Count)

• Also known as “packed field” (4+ in numeric terms)

• No standard rule
• In practice, if more than 30/hpf, it can be reported as
TNTC or if the field is packed with or overwhelmed with
the sediment
• Usually applied to RBCs, WBCs/pus cells
• In bacteria, report as ‘packed field’

SUMMARY

Table 2. Factors that require standardization in the

Microscopic Examination

Urine volume used 10mL, 12mL, 15mL

Speed of centrifugation 400 or 450 x g

Time of centrifugation 5 minutes

Concentration of sediment
prepared
Volume of sediment examined
Result reporting
10:1, 12:1, 15:1, 30:1
Determined by commercial slide
used and microscope optical
properties (i.e., ocular field
number)
Format, terminology, reference
intervals, magnification used for
assessment

• Concentration of sediment prepared – not usually done

o Commonly used in the commercial system

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 [TRANS] LABORATORY UNIT 08: SEMEN ANALYSIS
SEMEN ANALYSIS
Introduction
Spermatogenesis
• Formation of spermatozoa (sperm) in seminiferous
tubules
• Begins with spermatogonia and eventually undergoes
mitosis
o Some spermatogonia remains near the basement
membrane of seminiferous tubule in an
undifferentiated state to serve as a reservoir of
cells for future cell division and subsequent sperm
production.
o The rest of spermatogonia will lose contact with
the basement membrane and squeeze through the
tight junctions of blood-testis barrier, which
undergoes developmental changes and
differentiate into primary spermatocytes until they
become sperm cells.
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG.
Physiology
• Semen is composed of fractions made from:
o Testes (seminiferous tubules)
o Epididymis
o Ductus deferens (vas deferens)
o Seminal vesicle
o Prostate gland
o Bulbourethral glands
• Each of these glands contains the fraction of semen
and mixed together during ejaculation
Testes (seminiferous tubules)
• Paired glands in the scrotum that contains
seminiferous tubules
• Seminiferous tubules – responsible for the secretion of
sperm
Epididymis
• Where the sperm matures, develop flagella, and remain
stored until ejaculation
Ductus deferens (vas deferens)
• Sperm will propel through the ductus deferens (vas
deferens) to the ejaculatory ducts, and received by
seminal vesicles
Seminal vesicle
• Produces the major fractions of the semen containing a
high concentration of fructose and flavin.
Prostate gland
• Aids in propelling the sperm through the urethra by
contractions during ejaculation
A muscular gland that contains acid phosphatase,
citric acid, zinc and proteolytic enzymes which are
responsible for the coagulation and liquefaction of
semen.
Bulbourethral glands
• Located below prostate gland
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 LABORATORY UNIT 08: SEMEN ANALYSIS

• Contains a thick alkaline mucus that helps neutralize o Varicocelectomy – removal of varicoceles (swollen
semen from the acidity of prostate secretions and the veins inside the scrotum)
vagina. o Vasectomy – removal of vas deferens

Composition of Semen Sample Collection

Table 1. Summary of the Components of 4 Fractions making Patient Preparation

an Essential Fluid for the Production of a Normal Semen
• (1) Abstinence: 2-7 days (average: 5 days)
o Minimum of 2 days
FRACTION COMPONENTS
o Maximum of 7 days of sexual abstinence
o Abstinence is not limited only to sexual intercourse,
60-70% • Major fraction of semen but also in masturbation and the like
Seminal Fluid • Contains fructose and flavin o It is important for patients to follow the abstinence
period prior to collection to maintain the integrity of
the specimen which will affect the results of the test
• Contains acid phosphatase, o What if the patient is not able to follow the
20-30% citric acid, zinc, and
Prostate Fluid abstinence period before collection time?
proteolytic enzymes
▪ Specimens collected following a prolonged
5% abstinence tend to have a higher volume and
• Contains thick alkaline mucus decrease motility in sperm.
Bulbourethral Fluid
5% • (2) Warm sterile glass or plastic container
Spermatozoa o Provided by the laboratory
o Most of the laboratory use the sterile urine container
Chemical Components because it is more cost-efficient than sterile glass
containers.
• Helps to nourish the sperm cells
• Collected in a private room near the laboratory
• Spermine and Choline
• Acid phosphatase o In order to limit the exposure of semen to fluctuation
• Fructose in temperature and to control the time between
collection and analysis
o Serves as the energy of sperm cells needed for the
flagella to propel them through the female • Give a clear written and verbal instructions concerning
reproductive tract the collection of semen
o Without fructose, the sperm cannot swim and
o Should be emphasized that semen sample needs to
remains immotile
be complete or collect the entire semen after
• Flavin ejaculation; the patient should report any loss of any
fraction of the sample
o Responsible for the gray appearance of semen
• Deliver to the laboratory within 1 hour after collection
• Potassium, Citric acid, and Ascorbic acid • Record the patient’s name and birth date, personal
• Ergothionine code number/hospital number/laboratory number, the
• Phosphorylcholine period of sexual abstinence, the date and time of
• Proteolytic enzyme collection, the completeness of the sample, difficulties
NOTE: Acid phosphatase, citric acid, zinc, and proteolytic with collection, and the times of specimen collection
enzymes – responsible for both coagulation and (interval between the collection and the start of the
liquefaction of semen following ejaculation semen analysis) and specimen receipt.

Purpose Methods of Collection

• Evaluation of reproductive dysfunction in males – • (1) Masturbation

infertility testing o Most practical and easiest way to collect
o Main purpose of semen analysis o If the patient is married, collection from sex is better
o A test requested by a doctor when a couple is than masturbation (due to the degree of arousal)
having trouble getting pregnant o The first part of the ejaculate should be collected →
o Both men and women can have problems in fertility it is the mostly concentrated
o Issues with male fertility can play a part in as many
• (2) Coitus interruptus
as half of all infertility cases
• (3) Common condom collection
o Male infertility is often caused by low sperm
• (4) Silastic condom collection
production
• (5) Aspiration from the vaginal vault after coitus
• Select donors for therapeutic insemination
• Monitor success of surgical procedures such as:

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Preservation

• Awaiting analysis
o For routine semen analysis → semen is not usually
preserved
o Stored in incubator at normal body temperature
▪ When the sample will not be immediately
processed
• For artificial insemination
o Frozen and stored for 1 year at -85°C at sperm bank
▪ The sample is viable for 1 year
Semenalysis Parameters
Appearance
• Semen analysis for fertility testing or fertility evaluation
consists of both macroscopic and microscopic Table 3. Color/Appearance under Macroscopic Analysis
examinations
• Parameters reported includes:
COLOR
o Liquefaction
o Appearance Gray-white, “Pearly-
o Volume white”, Light Yellow; Normal
o Viscosity Opaque
o pH
Shades of yellow High flavin concentration
o Sperm Concentration and Count
o Motility Associated with certain drugs,
o Morphology Deep yellow over abstinence, jaundice, urine
contamination
Table 2. Reference Values of Semenalysis Parameters Brown or red Presence of red blood cells
Presence of white blood cells and
Reference Values for Semen Increased white
infection within the reproductive
Analysis turbidity
tract
Volume 2 to 5 mL • The normal liquified semen sample has a homogenous
gray opalescent appearance
Viscosity Pours in droplets
o It may appear less opaque if the sperm
concentration is very low
pH 7.2 to 8.0
Sperm • The color may also be different/varied
concentration
>20 million/mL • If required, specimen culturing is performed prior to
continuing with the semen analysis
Sperm count >40 million/ejaculate
Liquefaction
Motility >50% within 1 hour
• Immediately after ejaculation into the collection vessel,
the semen is typically a semi-solid coagulated mass.
Quality >2.0 or a, b, c in Table 10-3
• NORMAL: Liquefy within 30 to 60 minutes after
>14% normal forms (strict criteria) collection
Morphology
>30% normal forms (routine criteria) o Within a few minutes, at room temperature, the
Round cells <1.0 million/mL semen usually begins to liquefy or becomes thinner
o At time, a heterogenous mixture of clumps will be
seen in the fluid
Initial Macroscopic Examination o The complete sample usually liquifies within 15
minutes at room temperature, although rarely, it may
• Gross examination – first step in performing a semen take up to 60 minutes or more
analysis once the sample is received in the laboratory
• In liquefaction, semen becomes more homogenous and
o Includes the color, volume, liquefaction, viscosity, quite watery; small areas of coagulation remain in the
and the pH of the semen final stage
• ABNORMAL: Failure of liquefaction within 60 minutes
o Deficiency in prostatic enzymes and should be
reported

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 LABORATORY UNIT 08: SEMEN ANALYSIS
• NOTE:
o Normal liquefied semen samples may contain jelly-
like granules (gelatinous bodies) – no clinical
significance
o Presence of mucus strands may interfere with
semen analysis
• In special cases:
o If after 2 hours, the specimen has not liquefied:
▪ An equal volume of physiologic Dulbecco’s
phosphate-buffered saline or proteolytic
enzymes such as alpha-chymotrypsin or
bromelain may be added to induce the
liquefaction and allow the rest of the analysis to
perform
▪ These treatments may affect the biochemical
tests as well as the sperm motility or sperm
morphology so their use must be documented.
Semen Dilution with Physiologic Saline1
• Prepare an equal volume of diluent and semen (1 part
diluent and 1 part semen) using Dulbecco’s phosphate-
buffered saline. Repeated pipetting of the prepared
dilution will induce liquefaction.
• Preparation of Dulbecco’s Phosphate-Buffered Saline1
o (1) Using a 1-L volumetric flask, add the following:
▪ 750 mL of purified water
▪ 0.20 g of potassium chloride (KCL)
▪ 0.20 g of potassium dihydrogen phosphate
(KH2PO4)
▪ 0.10 g of magnesium chloride hexahydrate
(MgCl2.6H2O)
▪ 8.0 g of sodium chloride (NaCl)
▪ 2.16 g of disodium hydrogen phosphate
heptahydrate (Na2HPO4.7H2O)
▪ 1.00 g of D-glucose
o (2) In a 10-mL volumetric flask, dissolve 0.132 g of
calcium chloride dehydrate (CaCl2.2H2O) in 10 mL
of purified water
o (3) To prevent precipitation, add calcium chloride
dehydrated solution to the 1-L flask slowly, stirring
continuously.
o (4) Adjust the pH to 7.4 with 1 mol/L sodium
hydroxide (NaOH)
o (5) Make up to 1000 mL with purified water.
Digestion with Bromelain1
• (1) Prepare 10 IU/mL bromelain in Dulbecco’s
phosphate-buffered saline
o Into a 100-mL volumetric flask, add 1000 IU of
bromelain.
o Add 60 mL Dulbecco’s phosphate-buffered saline.
o Mix to dissolve. It takes about 15 to 20 minutes.
Bring volume to calibration mark using buffered
saline.
• (2) Dilute one part semen 1:2 with the 10 IU/mL
bromelain (1 part semen + 1 part bromelain solution).
• (3) Stir with a pipette tip.
• (4) Incubate at 37°C for 10 minutes.
• (5) Mix the sample well before analysis.
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3
Volume
• The volume of the ejaculate is contributed mainly by the
seminal vesicles and prostate gland with a small
amount from the bulbourethral glands and epididymis
• Precise measurement of volume is essential in any
evaluation of semen
o It allows the total number of spermatozoa and non-
sperm cells in the ejaculate to be calculated
• Normal volume: 2-5 mL
• It can be measured by pouring the specimen into a
clean graduated cylinder calibrated in 0.1-mL
increments
NOTE: Measuring volume by aspirating the sample from
the specimen container into a pipette or syringe is not
recommended because not all samples will be retrieved
and the volume will therefore be underestimated.
• The amount of volume will also indicate a clinical
significance to the specimen or to the patient.
Table 4. Clinical Significance of Volume
LOW VOLUME HIGH VOLUME
- Obstruction of the ejaculatory - Active exudation in
duct, congenital bilateral absence cases of active
of vas deferens, collection inflammation of the
problems (loss of fraction of the accessory organs
ejaculate), partial retrograde
ejaculation or androgen deficiency - Extended abstinence
Viscosity
• Consistency of the fluid
• After liquefaction, the viscosity of the sample can be
estimated by gently aspirating it into a wide-bore
(approximately 1.5 mm in diameter) plastic disposable
pipette
o Allowing the semen to drop by gravity and observing
the length of any thread
• Normal viscosity:
o Form small discrete droplets (do not appear clump
or stringy)
• Abnormal viscosity
o Drop will form a thread more than 2 cm long
(viscous)
• Ratings: 0 - 4 or Low-Normal-High
o 0 → watery
o 4 → gel-like
NOTE: High viscosity can interfere with determination of
sperm motility, sperm concentration, detection of antibody-
coated spermatozoa and measurement of biochemical
markers.
4
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 LABORATORY UNIT 08: SEMEN ANALYSIS
pH
• Reflects the balance between the pH values of different
accessory gland secretions
o Mainly the alkaline seminal vesicular secretion and
acidic prostate prostatic secretion
• Measured after liquefaction at a uniform time
o WHO
▪ Preferably after 30 minutes
o Strasinger
▪ Within 1 hour of ejaculation since it is influenced
by the loss of CO2 that occurs after production
• pH paper range 6.0 to 10.0 should be used
o Mix the semen well
o Spread a drop of semen evenly onto pH paper
o Read within 30 seconds
▪ Wait for the color of the impregnated zone to
become uniform for less than 30 seconds
o Compare the color with the calibration strip to read
pH
• Normal pH: 7.2-8.0
• Increased pH: infection
• Decreased pH: increased prostatic fluid, ejaculatory
duct obstruction, poorly develop seminal vesicle
Initial Microscopic Examination
• Phase-Contrast Microscope
o Recommended for all examinations of unstained
preparations from fresh semen
• An initial microscopic examination of the sample
involves scanning the preparation at a total
magnification of 100x (10x objective lens with 10x
ocular lens); under low power objective
• Provides overview
o Mucus strand formation
o Sperm aggregation or agglutination
o Presence of cells other than spermatozoa
▪ E.g., epithelial cells, round cells (leukocytes,
immature germ cells), and an isolated sperm
heads or tails
• Then be observed at 200x or 400x magnification
o Assessment of sperm motility
o Determination of the dilution required for accurate
assessment of sperm number
Making a Wet Preparation
• Mix the semen sample well.
o Thorough mixing of semen
▪ Before removing an aliquot of semen for
assessment, mix the sample well in the original
container, but not so vigorously that air bubbles
are created.
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▪ This can be achieved by aspirating the sample
10 times into a wide-bore (approximately 1.5 mm
diameter) disposable plastic pipette (sterile when
necessary).
▪ Do not mix with a vortex mixer at high speed as
this will damage spermatozoa.
• Remove an aliquot of semen immediately after mixing,
allowing no time for the spermatozoa to settle out of
suspension.
• Remix the semen sample before removing replicate
aliquots.
• Place a standard volume of semen, e.g., 10ul, onto a
clean glass slide
• Cover it with a coverslip, e.g., 22mm x 22m for 10ul, to
provide a chamber approximately 20um deep. The
weight of the coverslip spreads the sample.
• Take care to avoid the formation and trapping of air
bubbles between the coverslip and the slide.
• Assess the freshly made wet preparations as soon as
the contents are no longer drifting.
Aggregation of Spermatozoa
• Adherence either of immotile spermatozoa to each
other or of motile spermatozoa to mucus strands, non-
sperm cells or debris is non-specific aggregation and
should be recorded
• This observation is optional to the test. But, for training
purposes, aggregation of spermatozoa must be
evaluated since it will give a clinical significance of the
test
Photo. Views of spermatozoa aggregated with an epithelial cell
(a), debris (b), or spermatozoa (c,d).
Agglutination of Spermatozoa
• Motile spermatozoa sticking to each other, head-to-
head, tail-to-tail or in a mixed way
• Motility is often vigorous, with frantic shaking motion
o But sometimes the spermatozoa is so agglutinated
that their motion is limited
• Any motile spermatozoa that stick to each other by their
heads, tails or midpieces should be noted.
• Evaluation:
o Major type of agglutination (grades 1-4)
o Site of attachment (grades A-E)
• REMINDER: The type of agglutination reflecting the
degrees (grades 1-4) should be recorded.
5
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 LABORATORY UNIT 08: SEMEN ANALYSIS

Table 5. Evaluation of the Agglutination of Spermatozoa

GRADE DEGREE DESCRIPTION

Grade 1 Isolated <10 spermatozoa per agglutinate, many free spermatozoa

Grade 2 Moderate 10-50 spermatozoa per agglutinate, free spermatozoa

Grade 3 Large Agglutinates of >50 spermatozoa, some spermatozoa still free

Grade 4 Gross All spermatozoa agglutinated and agglutinates interconnected

Table 6. Schematic Diagram of Different Extents of Sperm Agglutination Based from the Parts Involved and the Degree of
Agglutination

DEGREE OF AGGLUTINATION

PARTS INVOLVED 4. Gross (all sperm

1. Isolated (<10 2. Moderate (10-50 3. Large (agglutinates
agglutinated, and
sperm/agglutinate, sperm/agglutinate, >50 sperm, some
agglutinates
many free sperm) free sperm) sperm still free)
interconnected)

A. Head-to-Head

B. Tail-to-tail (heads are

seen to be free and
move clear of
agglutinates)

C. Tail-tip-to-tail-tip

D. Mixed (clear head-to-

head and tail-to-tail
agglutinations)

E. Tangle (heads and

tails enmeshed. Heads
are not clear of
agglutinates as they are
in tail-to-tail
agglutination)

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Cellular Elements other than Spermatozoa 11 Degenerating Spermatid

• The ejaculate contains cells other than spermatozoa in
12 Spermatid
which some may be clinically relevant
o Epithelial cells in genitourinary tract 13 Degenerating Spermatid
o Leukocyte and immature germ cells (round cells)
• They can be identified by examining the stained smear 14 Dividing Spermatocyte
at 1000x magnification
• Can be more precisely identified and quantified by 15 Cytoplasm
detecting peroxidase activity or the antigen CD45
• Usually, macrophage and some WBCs were the 16 Degenerating Spermatid
commonly found cells in the semen
17 Dividing Spermatocyte
Table 7. Examples of Other Cellular Elements found in
Semen aside from Sperm Cells 18 Abnormal Spermatozoon

19 Cytoplasm

20 Abnormal Spermatozoon

21 Spermatid

22 Phagocytosing Macrophage

23 Spermatocyte

24 Cytoplasm

Table 8. Examples of Other Cellular Elements found in

Semen aside from Sperm Cells

CELL CELL TYPE

1 Macrophage

2 Abnormal Spermatozoon

3 Cytoplasm

4 Abnormal Spermatozoon

5 Spermatocyte

6 Abnormal Spermatozoon
Abnormal Spermatozoon? Loose head on
7
cytoplasm?
8 Cytoplasm
CELL CELL TYPE
9 Dividing Spermatid
1 Macrophage
10 Spermatocyte
2 Abnormal Spermatozoon

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Table 9. Categories of Sperm Movement

3 (Dividing) Spermatid

4 (Dividing) Spermatid CATEGORIES OF SPERM MOVEMENT

5 Cytoplasm Spermatozoa moving actively, either

Progressive
linearly or in a large circle, regardless of
motility (PR)
6 Not Classifiable speed
All other patterns of motility with an
7 Degenerating Spermatid absence of progression (e.g., swimming
Nonprogressive
in small circles, the flagellar force hardly
motility (NP)
8 Degenerating Spermatid? displacing the head, observance of only a
flagellar beat)
9 Degenerating Spermatid Immotility (IM) No movement

10 Degenerating Spermatid NOTE: When discussing sperm motility, it is important to

specify total motility (PR + NP) or progressive motility (PR)
11 Macrophage
• A simple system for grading sperm motility that
distinguishes progressive motility or nonprogressive
12 Degenerating Spermatid
motility from immotility is recommended.
13 Degenerating Spermatid
Table 10. Sperm Motility Grading
14 Degenerating Spermatid

15 Degenerating Spermatid SPERM MOTILITY GRADING

16 Macrophage WHO
GRADE Sperm Motility Action
Criteria
4.0 a Rapid, straight-line motility
Sperm Motility
3.0 b Slower speed, some lateral movement
• Assessed as soon as possible after liquefaction
(preferably at 30 minutes) or within 1 hour following Slow forward progression, noticeable
2.0 b
ejaculation lateral movement
• NORMAL: 60% or higher progressively motile sperm 1.0 c No forward progression
• PROCEDURE:
o (1) Mix the semen sample well 0 d No movement
o (2) Remove an aliquot of semen immediately after
mixing, allowing no time for the spermatozoa to • Today, most of the laboratories use the WHO criteria to
settle out of suspension standardize the grading of the sperm motility
o (3) Remix the semen sample before removing a
replicate aliquot
o (4) For each replicate, prepare a wet preparation Table 11. Alternative Sperm Motility Grading Criteria1
approximately 20um deep.
o (5) Wait for the sample to stop drifting (within 60
seconds) ALTERNATIVE SPERM MOTILITY GRADING CRITERIA
o (6) Examine the slide with phase-contrast optics at
x200 or x400 magnification Progressive
o (7) Assess approximately 200 spermatozoa per Sperm moving linearly or in a large circle
motility (PM)
replicate for the percentage of different motile Nonprogressive Sperm moving with an absence of
categories motility (NP) progression
o (8) Compare the replicate values to check if they are
acceptably close. If so, proceed with calculations; if Immotility (IM) No movement
not, prepare new samples

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Sperm Morphology
o (b) Pipette method
• Sperm that are for washed
morphologically incapable samples. A drop of
of fertilization results in the sperm
infertility. suspension (SS) is
• Structures that are spread over the
evaluated: surface of the slide
by pushing the
o Head, neckpiece,
horizontal
midpiece, tail
pipette(P).
• Abnormalities:
Common Abnormalities of Sperm Heads and Tails
o Head – poor ovum
penetration
o Neckpiece, Midpiece,
and Tail – affects
motility
• Normal Sperm:
o Head – oval-shaped; approximately 5um long and
3um wide
o Tail – approximately 45 um long
o Acrosomal cap – located at the tip of the head,
encompass half of the head; cover 2/3 of sperm
nucleus
▪ An enzyme containing acrosomal cap is critical
to ovum penetration
o Neckpiece – attaches head to the midpiece and tail.
o Midpiece – 7 um long; the thickest part of the tail
because it is surrounded by a mitochondrial
sheath that produces the energy required by the tail • Normal
for motility • Double head – having two heads with one tail
• Normal values • Giant head
• Amorphous head – a sperm without a clearly defined
o Depend on the evaluation method used and vary shape/form and no presence of pure acrosomal cap
from:
• Pinhead – a variation of the small head sperm
▪ Routine criteria: >30% normal • Tapered head – cigar-shaped sperm that may indicate
▪ Kruger’s Strict Criteria: > 14% normal; the presence of a varicocele.
measures head, neck and tail • Constricted head
 The Strict Criteria evaluation requires the use • Double tail – one head and two tails
of a stage micrometer or morphometry. • Coiled tail
• Spermatid – refers to an immature sperm cell
 Normally at least 70% of sperm cells
demonstrate normal morphology Abnormal Sperms Stained with a Wright Stain
Semen Smearing Methods for Sperm Morphology

• To determine the morphology of the sperm, the sperm

morphology is evaluated from the thinly smeared
stained slide under oil immersion.
• Smears are made by feathering method.
• METHODS:
o (a) “Feathering” method for undiluted semen. The
semen drop (S)
spreads along
the back edge
of the angled
slide and is
pulled forwards
over the slide
to form the
smear.

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Sperms with Normal Morphology Sperm Cells with Abnormal Forms

• The head of the sperm is roundish, and it stains purple

with the Wright Stain.

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Sperm Concentration Ways of Counting of Sperm Cells

• “Total Sperm Number” vs. “Sperm Concentration”
o These terms are not synonymous
o The total number of spermatozoa per ejaculate and
the sperm concentration are related to both time to
pregnancy and are predictors of conception.
• Sperm concentration
o Number of spermatozoa per unit volume of semen.
▪ It is a function of the number of spermatozoa
emitted and the volume of fluid diluting them.
• Total sperm number
o Total number of spermatozoa in the entire ejaculate
and is obtained by multiplying the sperm
concentration by the semen volume.
▪ The total sperm count for the ejaculate can be
calculated by multiplying the sperm • 5 RBC squares: Using the Neubauer Hemocytometer,
concentration by the specimen volume. the sperms are usually counted in the 4 corner and
• Normal values: >40 million per ejaculate center squares of the large center square (same to
manual RBC count)
Reference Values for Sperm Concentration o Computation: #sperm cells counted x 1,000,000
(mL)
• Normal values: 20-250 million sperm per milliliter
o Concentrations between 10 and 20 million per • 2 WBC squares: Only 2 corner large squares (same to
milliliter are considered borderline manual WBC count)
o Computation: #sperm cells counted x 100,000 (mL)
• Markler counting chamber – undiluted semen, heat
instead ▪ Counting sperm cells under WBC squares is
o The previous method of counting the sperms cells is much better and more convenient in the
the use of the Markler counting chamber, but this computation of sperm cells
▪ You can use both ways. Just take note of what
gives inaccurate results.
grid and area you are counting
o Today, most of the laboratories use the Improved
Neubauer Counting Chamber (INCC) Considerations While Determining the Sperm
▪ The sperm are counted in the same manner as Concentration
the cells in the Cerebrospinal fluid cell count.
• Addition of stain (crystal violet) to diluting fluid helps
 By diluting the specimen and counting the visualization using bright-field microscope
cells in the Neubauer Chamber. • NOTE: Only FULLY DEVELOPED sperm should be
• Improved Neubauer Counting Chamber counted
o 1:20 dilution o Immature sperm and WBCs (“round cells”) must not
be included
▪ Most commonly used dilution prepared using a
mechanical pipette • Also, take note of the number of WBCs or pus cells and
spermatids in the specimen
o Diluting fluids: cold water, formalin, sodium
• A count of more than (>) 1 million WBCs/mL
bicarbonate, 0.5% chlorazene, 1% formalin in 3%
trisodium citrate o Associated with inflammation or infection of the
reproductive organs that can lead to infertility
▪ The traditional diluting fluid contains sodium
bicarbonate and formalin which immobilizes and • A count of more than (>) 1 million spermatids/mL
preserves the cells.
o Indicated disruption of spermatogenesis caused by
▪ However, today, most of the laboratories use the
viral infections, exposure to toxic chemicals, and
cold water as the diluting fluid to immobilize the
genetic disorders
sperm cells

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Inclusion Criteria for Counting Cells o (2) After obtaining the concentration per volume,
compute for the total number of sperm cells per
• In determining your sperm cell, we have the so called ejaculate.
“inclusion criteria” of counting the sperm cells using the
hemocytometer grid. 60,000,000 sperm/mL x 4 mL = 240,000,000
sperm/ejaculate
Using RBC square
Using WBC square

• Count only the whole spermatozoa with its head and tail
• Whether or not the sperm cell is counted, that is
determined by the location of its head
o Orientation of its tail is not important
• A sperm cell is counted if its head lies in the boundary
o Head will be the basis for sperm counting
• To avoid counting the same sperm cell in adjacent
square, a sperm cell with its head on the line dividing
two adjacent squares should be counted only if that line
is one of two perpendicular boundary lines • LEFT WBC SQUARE:

o Example: Cells may be counted if most of the o Focus on the black line
sperm head lies on the lower or left center o The middle of the three lines defines the square’s
boundaries which forms an “L” shape, but not if it boundary
lies on the upper right center line o All spermatozoa within the central square are
counted, as well as those with their heads between
• Practice: Determine the number of sperm cells as seen the two inner lines (white circles), but DO NOT
in the picture. Consider the “L” shape pattern inclusion INCLUDE those whose heads lie between the outer
criteria. two lines (black circle)
• MIDDLE AND RIGHT WBC SQUARES:
o A sperm cell with most of its head lying on the
central line is counted only if that line is the lower or
left-hand line of the squares (white circle, middle
panel)
o If the sperm cell is on the upper or right-hand line of
the square, they are not counted (black circle, right
panel)
o In the middle of the three boundary lines, the ones
o Number of sperm cells: 8 sperm cells counted are:
o NOTE: The sperm cell is not counted if the one that
has touched the grid line is the midpiece because ▪ Middle line
the head will be the basis for counting the sperm ▪ “L shaped” inclusion criteria: Left line and lower
cell. width line
• NOTE: Difference using “L-shaped” Inclusion
Criteria
Computation Example under 5 RBC Squares
• Using a 1:20 dilution, an average of 60 sperm are
counted in the five RBC counting squares on both sides o Using WBC square
of the hemocytometer. Calculate the sperm
concentration per millimetre and the total sperm count ▪ Count the sperm
in a specimen with a volume of 4mL. touching the lower
left grid.
o (1) Compute using the formula of 5 RBC squares

60 sperm counted x 1,000,000= 60,000,000

sperm/mL

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 LABORATORY UNIT 08: SEMEN ANALYSIS

Staining Principle of Traditional Fixation and Sequential

Staining
o Using RBC square
Table 12. Traditional Fixation and Sequential Staining
▪ Count the sperm
touching the upper
and right grid DESCRIPTION
pattern
Ethanol To fix cells; it also dehydrates them
To rehydrate the fixed smears
Graded ethanol gradually to permit water-soluble
Computation Example under 2 WBC Squares haematoxylin staining
To rehydrate dried smears to permit
• In a 1:20 dilution, 600 sperm are counted in two WBC Purified water
water-soluble haematoxylin staining
counting squares. Calculate the sperm concentration
per millilitre and the total sperm count in a specimen Haematoxylin To stain the nucleus blue
with a volume of 2 mL.
To remove unbound nuclear
• NOTE: Only 2 WBC squares are used Tap water
haematoxylin
• Same manner of computation but only differ with the To remove non-specifically bound dye
dilution factor use Acidic ethanol
from the cytoplasm (destaining)
600 𝑠𝑝𝑒𝑟𝑚 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 20 (𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛) 𝑠𝑝𝑒𝑟𝑚 To reduce acidity and return blue
= 60, 000 (𝑣𝑜𝑙𝑢𝑚𝑒 𝑐𝑜𝑢𝑛𝑡𝑒𝑑) Tap water
2 𝑠𝑞 𝑚𝑚 𝑥 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 0.1 𝑚𝑚 (𝑑𝑒𝑝𝑡ℎ) 𝑢𝐿 color to the nucleus
To return blue color to the nucleus (if
𝑠𝑝𝑒𝑟𝑚 𝑠𝑝𝑒𝑟𝑚 Scott’s solution
60, 000 𝑥 1000 = 60,000,000 tap water is insufficient)
𝑢𝐿 𝑚𝐿 To dehydrate smears to permit
000 Ethanol ethanol-soluble Orange G / EA-50
60, 000, 𝑥 2 𝑚𝐿 = 120, 000, 000 𝑠𝑝𝑒𝑟𝑚/𝑒𝑗𝑎𝑐𝑢𝑙𝑎𝑡𝑒 staining
𝑚𝐿
Orange G To stain the cytoplasm pink
Sperm Concentration Related Terms and Sperm Count
EA-50 To stain the cytoplasm pink
• Aspermia – no ejaculate at all To rehydrate the stained smears
• Oligospermia – sperm cells less than 20 million per Graded Ethanol gradually to permit the use of ethanol-
milliliter soluble mountants
• Necrospermia – immotile/dead sperms To permit the use of ethanol-insoluble
• Azospermia – complete absence of sperm Xylene
mountants
Staining Methods
• After staining of the smears, the slides can be viewed
• Used for assessing the sperm morphology mounted or unmounted
• Once the semen smears have been air dried, they o Slides can be viewed unmounted or mounted
should be fixed and stained to highlight the details of (without or with a coverslip attached).
the spermatozoa o Mounting the slides permits long-term storage, so
• Different Stains Used: that they can be reassessed if necessary and used
o Papanicolaou Stain- recommended in an internal quality control programme.
o Shorr or Diff Quick Stain- recommended o The refractive index (RI) of mountants after drying
o Wright’s Stain (1.50-1.55) is similar to that of glass (1.50-1.58), and
o Giemsa stain the best optical quality comes with the use of
immersion oil with a similar RI (1.52).
• Head
o Acrosomal Region – stained pale blue
Papanicolaou Staining
o Post Acrosomal Region – stained dark blue
• Midpiece – stained red Table 13. Steps in Papanicolaou Staining
• Tail – stained blue or reddish
• Excess residual cytoplasm (usually located behind
the head and around the midpiece) – stained pink Sequentially immerse the slides in:
o Red – Papanicolaou stain
o Reddish orange – Shorr stain 1. Ethanol 80% (v/v) 30 seconds

2. Ethanol 50% (v/v) 30 seconds

3. Purified water 30 seconds

4. Harris’s haematoxylin 4 minutes

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 LABORATORY UNIT 08: SEMEN ANALYSIS

5. Purified water 30 seconds Actual Pictures of Stained Smears of Semen in Different

Stains
6. Acidic ethanol 4-8 dips*
• PAP stain for
7. Running cold tap water 5 minutes sperm morphology
showing normal
8. Ethanol 50% (v/v) 30 seconds (centre) and
abnormal
9. Ethanol 80% (v/v) 30 seconds spermatozoa with
one pin head
10. Ethanol 95% (v/v) At least 15 minutes spermatozoa.

11. G-6 orange stain 1 minute

12. Ethanol 95% (v/v) 30 seconds

13. Ethanol 95% (v/v) 30 seconds • Spermatozoa

stained with Shorr
14. Ethanol 95% (v/v) 30 seconds

15. EA-50 green stain 1 minute

16. Ethanol 95% (v/v) 30 seconds

17. Ethanol 95% (v/v) 30 seconds

18. Ethanol 100% 15 seconds • Spermatozoa

stained with H&E
19. Ethanol 100% 15 seconds

*One dip corresponds to an immersion of about 1 second.

Other Tests and Techniques

Shorr or Diff Quick Stain
Sperm Vitality
Table 14. Steps in Shorr or Diff Quick Stain
• Assessed within 1 hour of
ejaculation.
Sequentially immerse the slides in: • Evaluated by mixing the
specimen with an eosin-
1. Running tap water 12-15 dips* nigrosin stain.

2. Haematoxylin 1-2 minutes o Then preparing a

smear, and counting
3. Running tap water 12-15 dips* the number of dead
4. Ammoniacal ethanol 10 dips* cells in 100 sperm using a brightfield or phase-
contrast microscope.
5. Running tap water 12-15 dips*
• Living cells are not infiltrated by the dye and remain
6. Ethanol 50% (v/v) 5 minutes bluish white; whereas dead cells stain red against the
7. Shorr stain 3-5 minutes purple background
• Normal vitality requires 50% or more living cells
8. Ethanol 50% (v/v) 5 minutes • Clinical Significance:
9. Ethanol 75% (v/v) 5 minutes o Presence of a large proportion of vital but immobile
10. Ethanol 95% (v/v) 5 minutes cells may indicate a defective flagellum
o High number of immotile and nonviable cells may
*One dip corresponds to an immersion of about 1 second indicate epididymal pathology.
NOTE: The slides can be viewed unmounted or mounted

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 LABORATORY UNIT 08: SEMEN ANALYSIS
Seminal Fluid Fructose Determination
• Low fructose levels are caused by an abnormality in the
seminal vesicles, bilateral congenital absence of vas
deferens, obstruction of ejaculatory duct, partial
retrograde ejaculation and androgen deficiency
• Screened for the presence of fructose using the
Resorcinol test that produces an orange color when
fructose is present
• A normal quantitative level of fructose is equal to or
greater than 13 μmol per ejaculate.
o This can be determined using spectrophotometric
methods
• Specimens for fructose levels should be tested within 2
hours of collection or frozen to prevent fructolysis.
Seminal Fructose Screening Test
• (1) Prepare reagent (50 mg resorcinol in 33 mL Microbial Testing
concentrated HCl diluted to 100 mL with water)
• (2) Mix 1 mL of semen with 9 mL of reagent.
• (3) Boil
• (4) Observe for orange-red color
Antisperm Antibodies
• This can be present in both men and women
• Detected in semen, cervical mucosa, or serum.
o Considered possible cause of infertility
• It is not unusual for both partners to demonstrate Chemical Testing
antisperm antibodies
o Male antisperm antibodies are more frequently
encountered
• Blood-testes barrier separates sperm from the male
immune system.
o When disrupted in surgery, vasectomy, trauma,
infection – antigens on sperm produce an immune
response
• Damage sperm may cause the production of antibodies
in female.
• In male, clumps of sperm are observed in routine
semenalysis
• Sperm-agglutinating antibodies cause to stick in a head-
to-head, head-to-tail, or tail-to-tail pattern
• Agglutination grading: “few”, “moderate”, or “many”
• In female, it results in a normal semen analysis
accompanied by continued infertility
o To determine, mix the semen with female cervical
mucosa or serum and observe for agglutination
2 Frequently Used Tests to Detect Antisperm Antibodies:
Special Case
• (1) Mixed Agglutination Reaction (MAR test)
o Screening procedure to detect IgG antibodies
o Semen + IgG (AHG) + latex particles or treated RBC
coated with IgG
o Bivalent AHG binds to both antibody on the sperm
and antibody on latex particles or RBCs, forming
microscopically visible clumps of sperm and
particles or cells
o Normal: < 10% of motile sperm attach to particles
BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3
• (2) Immunobead Test
o More specific to detect IgG, IgM, and IgA antibodies
and demonstrates what area of sperm (head,
neckpiece, midpiece, or tail) the autoantibodies are
affecting
o Head-directed Antibodies: interfere penetration
into the cervical mucosa or ovum
o Tail-directed Antibodies: affect movement through
the cervical mucosa
o Sperm + polyacrylamide beads (coated with anti-
IgG, anti-IgM, anti-IgA)
o Microscopic examination
▪ Beads attach to sperm at particular areas
o Reported as “IgM tail antibodies”, “IgG head
antibodies”, and so forth
o Normal: presence of beads on <50% of the sperm
(as defined by WHO)
• Presence of more than (>) 1 million WBCs/mL indicates
infection within the reproductive system
o Frequently, the prostate
• Aerobic/anaerobic cultures and test for Chlamydia
trachomatis, Mycoplasma hominis, Ureaplasma
urealyticum
o More frequently performed
• Decreased neutral alpha-glucosidase,
glycerophosphocholine, and L-carnitine suggest a
disorder of epididymis
• Decreased zinc, citric acid, glutamyl transpeptidase,
and acid phosphatase indicate lack of prostatic fluid
NOTE: Spectrophotometric methods – quantitate citric
acid and zinc
Table 15. Reference Semen Chemical Values
REFERENCE SEMEN CHEMICAL VALUES
Neutral α-glucosidase ≥ 20 mU/ejaculate
Zinc ≥ 2.4 umol/ejaculate
Citric acid ≥ 52 umol/ejaculate
Acid phosphatase ≥ 200 Units/ejaculate
• In cases of alleged rape
o Microscopically examining the specimen for the
presence of sperm may be possible with the best
results being obtained by enhancing the specimen
with the xylene
▪ Examine under phase microscopy
15
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 LABORATORY UNIT 08: SEMEN ANALYSIS

• Motile sperm – detected for up to 24 hours after Sperm Function Test

intercourse
• Nonmotile sperm – can persist for 3 days
• As sperm die off, heads remain and present for 7 days
after intercourse
• Seminal fluid contains high concentration of prostatic
acid phosphatase
o Detecting this enzyme can aid in determining the
presence of semen in a specimen
• Seminal glycoprotein p30 (prostatic specific antigen
[PSA])
o More specific detection
o Present even in the absence of sperm
NOTE: Further information, perform ABO blood grouping
and DNA analysis
• Advances in assisted reproduction and in-vitro fertility
have resulted in a need for more sophisticated semen
analysis to assess not only the characteristic of sperm
but also the functional ability
• Performed in specialized andrology laboratories
o (1) Hamster egg penetration assay
o (2) Cervical mucus penetration assay
o (3) Hypo-osmotic swelling test
o (4) In-vitro acrosome reaction
Table 16. Sperm Function Tests
TEST DESCRIPTION

Medico-Legal Tests Sperm are incubated with species-

Hamster egg nonspecific hamster eggs and
• Microscopic Exam in suspected material – enhanced penetration penetration is observed
with xylene microscopically
• Florence Test Observation of sperm’s ability to
Cervical mucus
penetrate partner’s midcycle cervical
o Reagents: Iodine Crystal + Potassium Iodide penetration
mucus
▪ Choline = (+) brown rhombic crystals Sperm exposed to low-sodium
▪ Not specific Hypo-osmotic concentrations are evaluated for
swelling membrane integrity and sperm
• Barbiero’s Test viability
o Reagents: Saturated Picric Acid + Trichloroacetic Evaluation of the acrosome to
In-vitro acrosome
Acid (TCA) produce enzymes essential for ovum
reaction
penetration
▪ Spermine = (+) yellow leaf shape crystals
▪ Specific
• Acid Phosphatase
• Glycoprotein p30 = more specific method
• Hyperimmune sera Test or Precipitin Hektoen Test

Post Vasectomy Analysis

• Much less involved procedure when compared with

infertility analysis
o The only concern is the presence or absence of
spermatozoa
• The complete length of time for completer sterilization
can vary greatly among patients and depends on both
the time and number of ejaculations
o Finding a viable sperm in a post-vasectomy patient
is not uncommon
o Care should be taken not to overlook even a single
sperm
• Specimens tested at monthly intervals, beginning at 2
months post-vasectomy, and continuing until two
consecutive monthly specimens show no spermatozoa
• The recommended testing includes examining a wet
preparation using phase microscopy
o Check for the presence of motile and non-motile
sperm
• Negative wet preparation is followed by specimen
centrifugation for 10 minutes and examination of
sediment

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 [TRANS] LABORATORY UNIT IX: FECAL ANALYSIS

FECAL ANALYSIS/ FECALYSIS
• “Uripara” section in some laboratories
o Where stool, urine, and other body fluid specimens
are received
• Normal feces are made up of:
o Water
o Non-pathogenic bacteria
o Food residues
o Intestinal and digestive secretions
o Limited number of epithelial cells
o Leukocytes
▪ Stool contains leukocytes but in low number
▪ If it exceeds a specific number, it may mean that
there is an underlying condition
• Normal 24 hour-amount: 100 – 200 g/day
o There is a possibility of exceeding the normal range
• Bacteria (non-pathogenic) constitute about 1/3 of the
total dry weight
Large number of leukocytes (WBCs) may be found in:
• Bacillary dysentery
o gastrointestinal disease due to poor sanitation
(bacterial infection)
• Ulcerative colitis
o Long-term condition of Inflammation in the colon and
rectum resulting in bleeding which leads to large
amount of pus
▪ Inflammation → bleeding → Infection → PUS
• Other inflammatory and ulcerative conditions
o Can be used to rule out possible conditions of the
patient
Specimen Collection
• Use clean, wide-mouthed and dry (sterile) container
o Wide-mouthed for easy collection of the sample
▪ Small-mouthed can be a struggle for the patient
to collect sample
• Qualitative testing for blood and microscopic
examination for leukocytes, muscle fiber, and fecal fats
are usually collected in a plastic or glass containers with
screw-capped tops.
o Looking for the presence/ absence of blood, WBC,
etc.
• For quantitative testing, such as for fecal fats, timed
specimens are required.
o Count the muscle fibers, muscle fats, etc.
o If timed specimens are required, these specimens
are frequently collected in paint cans to
accommodate the specimen quantity and to facilitate
emulsification prior to testing
▪ Timed specimens last for approx. 24 – 72 hrs (3
days)
▪ Longer collection time = great amount of
specimen is produced by the patient
 Thus, one screw-capped container is not
enough to allow the amount of stool that can
be submitted
▪ Paint cans have wider mouth and wider area
that allows proper emulsification of the specimen
 Need to emulsify specimen prior to testing

Fecalysis Request Macroscopic Examination

• Macroscopic and Table 1. Clinical Significance of Fecal Color

microscopic examination
including search for parasitic
Color Clinical Significance
ova
o Most common Upper GI bleeding, Iron, Charcoal,
o To diagnose/ rule out Brown
Bismuth, Digested Blood
underlying conditions Lower GI bleeding, Beets, Food color,
Black
Rifampin
• Chemical examination
o Fecal Occult Blood Test (FOBT) to rule out certain Gray Bile Duct Obstruction, Barium sulfate
cancers
Green Biliverdin, Green Vegetables
▪ Upper/ lower gastrointestinal bleeding = blood is
not visible in the blood Violet/Purple Porphyria
▪ Occult “Hidden”
▪ FOBT aims to rule out hidden blood in the stool • Brown
sample of the patient
o patient might have undergone iron therapy
• Bacteriological and Virologic Examination

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 LABORATORY UNIT 09: FECAL ANALYSIS

o bleeding from the upper portion → digest blood → o NOTE: If watery, there is a constant defecation
color brown
• Mucus
• Black
o Colitis
o Rifampin for Tuberculosis o Dysentery
o Malignancy
• Green
• Rice Watery Stools
o High level of biliverdin
o Cholera infection
▪ May be due to an obstruction where bilirubin is
released ▪ Caused by Vibrio cholera
o Conversion of bilirubin to biliverdin → Green • Butter-like
pigment
o Cystic Fibrosis
o May be due to high consumption of green
vegetables ▪ Constant sweating
• Violet/Purple
Table 3. Bristol Stool Chart
o Inborn errors of metabolism such as porphyria

Table 2. Clinical Significance of Fecal Consistency Type Characteristic

SEVERE CONSTIPATION
Consistency Clinical Significance Type 1
Separate, hard lumps
MILD CONSTIPATION
Type 2
Well Formed Normal Lumpy and sausage like
Pale, Bulky, NORMAL
Poor fat digestion, Steatorrhea Type 3
Frothy A sausage-shape with cracks in the surface
NORMAL
Hard, scybalous Constipation Type 4
Like a smooth, soft sausage or snake
Flattened and Obstruction in the lower bowel, LACKING FIBER
Type 5
ribbon like intestinal constriction Soft blobs with clear-cut edges
Digestive upset, mild diarrhea or after MILD DIARRHEA
Semisolid Type 6
taking laxative Mushy consistency with ragged edges
Bacterial infection or after taking SEVERE DIARRHEA
Watery Type 7
purgative Liquid consistency with no solid pieces

Mucus Colitis, dysentery, malignancy

Rice watery
Cholera
stools
Butter-like Cystic Fibrosis

• Hard, Scybalous
o Constipation – caused by small amount of water
intake
▪ Seldom defecation
• Flattened and Ribbon-like
o Obstruction in the lower bowel
o Intestinal constriction
• Semisolid
o Digestive upset, mild diarrhea or after taking laxative
o Mixture of a solid and liquid
• Watery
o Bacterial infection
▪ Different types of diarrhea
 Secretory or osmotic
o After taking purgative

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 LABORATORY UNIT 09: FECAL ANALYSIS

Microscopic Examination Normal = 100 droplets (<4

um)
• Done after macroscopic examination > 60 droplets / hpf =
Slightly Increased = 100
• White Blood Cells STEATORRHEA (increased
droplets (1-8 um)
fecal fat)
o Determination of the cause of diarrhea Increased = 100 droplets
(6-75 um)
▪ Diarrhea - < 200g per day with increased
liquidity and frequency, more than 3x a day NOTE:
▪ 3 types of diarrhea: • Qualitative tests determine the presence of fecal fats
 Secretory – Increased secretion of water and • Split fat stain is based on the size viewed under the
electrolytes microscope
 Osmotic Table 2. Fecal Fat Determination

 Altered motility
o Neutrophils – seen in conditions affecting the Quantitative Test
intestinal wall (ulcerative colitis, infection with
invasive bacterial pathogens)
VAN DE KRAMER TITRATION
▪ Can often be seen in secretory diarrhea
 Caused by microorganism (bacteria, viral,
protozoa) • GOLD STANDARD
 Commonly bacterial infection thus, large for fecal fat Sample = 3-day stool (72
number of WBCs can be seen on secretory determination hours)
diarrhea • For definitive Normal Value = 1 – 6g
diagnosis of fats / day
• Fecal Fats
STEATORRHEA Steatorrhea = >6g fats /
o Lipids included in the microscopic examination of • Titration with NaOH day
feces are neutral fats, fatty acids and salts, fatty (sodium hydroxide)
acids and cholesterol

Fecal Fats NOTE:

• Timed specimen is used
• Steatorrhea
• Possibility of overlapping of the specimen if wide
o Increased presence of fats mouthed container is used
in the stool about >6g per
day Muscle Fiber
• Tests: • Creatorrhea – abnormal
o Screening Test: Microscopic examination of feces excretion of muscle fibers in
for fat globules feces
• Increased amounts of striated
▪ (1) Perform Direct Fecal Smear (DFS) using fibers seen in Pancreatic
NSS and iodine solution insufficiency (cystic fibrosis)
 Fats globules are seen = presence of fecal • Determination if muscle fiber
fats is undigested in microscopic
examination:
o Definitive Test: Fecal fat determination
o Preparation:
Table 4. Fecal Fat Determination ▪ (1) stool + 2 drops of distilled water on slide + 2
drops of aqueous Eosin. Cover with coverslip
▪ (2) Stand for 3 minutes
Qualitative Tests ▪ (3) Count the number of undigested fibers
(HPF)
1. Neutral Fat Stain 2. Split Fat Stain (Fatty • Abnormal: > 10 undigested muscle fibers
(Triglycerides) Acids)
• Types of muscle fibers:
Stool suspension + 95% Emulsified stool + 36%
Ethanol + Sudan III Acetic Acid + Sudan III o Completely digested (No striations)

Orange Droplets (neutral Orange droplets (fatty

fats/Triglycerides) acids)

NUEVO. CRUDA. BOYOSE. VILLANUEVA. EVANGELISTA. BAGAL. BAUZON. TEVES. MARASIGAN. MATABALAO. PACANA BSMLS 3 3
 LABORATORY UNIT 09: FECAL ANALYSIS

o Partially digested (Striation in one direction) • Principle

Hemoglobin
Guaiac + H2O2 “Oxidized” Guaiac
Colorless Blue Color

o Hemoglobin facilitates the action of the

pseudoperoxidase which can oxidize the guaiac

▪ The striations either go upwards, downwards, or ▪ Colorless guaiac will then be oxidized and turns
by the side (in one direction) blue
▪ One angle/ one direction = Partially digested ▪ Positive Reaction: Blue color
muscle fiber o Reaction will only be possible if there is presence of
o Undigested (Striation in both direction) hidden blood in the stool sample
• Reagents/ Chromogens:
o Benzidine:
▪ most sensitive
▪ no longer available for clinical use due to
carcinogenic properties
▪ Striations either go sideways or upwards and o Ortho-toluidine:
downwards but they meet in one area =
undigested ▪ too sensitive for routine testing
▪ Type of muscle fiber that is counted while ▪ less sensitive than benzidine
performing this test. ▪ possible: false-positive reactions
▪ Abnormal: > 10 undigested muscle fibers o Gum-guiac:
Fecal Leukocytes ▪ least sensitive;
▪ preferred for routine testing (cheap)
• > 3 neutrophils / hpf =
invasive condition
o Per high power field of
the sample
o Pathologic
o Associated with
possible bacterial
infection
• Determination:
o Wet preparation = stool + Loeffler’s Methylene Blue
o Dried preparation = Stool + Wright’s/Gram Stain
o Lactoferrin Latex Agglutination Test
• Diarrhea with WBCs:
o Possible: Salmonella, Shigella, Yersinia,
Enteroinvasive E. coli, Campylobacter
• Diarrhea without WBCs:
o A lot of bacteria in microscopy but no WBCs
o Possible: Toxin-producing organisms (S. aureus, V.
cholerae)

Fecal Occult Blood Test

• Occults means hidden

• Screening test for colorectal
cancer
• Significant = > 2.5 mL blood /
150 g stool
• Sample = center portion of the
stool
• Upper GI tract bleeding
produces black, tarry stool &
lower – overtly bloody stool

NUEVO. CRUDA. BOYOSE. VILLANUEVA. EVANGELISTA. BAGAL. BAUZON. TEVES. MARASIGAN. MATABALAO. PACANA BSMLS 3 4
 [TRANS] LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

HUMAN CHORIONIC GONADOTROPIN 2. Galli-Mainini

• A hormone produced by the cytotrophoblast and • Animal used (AU):
syncytiotrophoblast
• Peaks during 1st trimester of pregnancy. o Male Frog
• This hormone can be detected/identified in the following • Mode of injection (MOI):
body fluids:
o Subcutaneous = urine/serum
o Blood (plasma/serum) o Beneath the skin of the male frog
o Urine
o Amniotic fluid • Positive result (PR):

• Composed of 2 subunits o Spermatogenesis

o Alpha • Sensitivity (Sen):

▪ Similar to TSH, LH, FSH, HCG o Variable

▪ Cannot be used to identify HCG because it’s not o Reference values varies between laboratories
specific for HCG
3. Friedman/Hoffman
o BETA
• Animal used (AU):
▪ Unique for HCG
▪ Seen and detected in testing o Virgin Female Rabbit

Urine HCG (Pregnancy Testing) • Mode of injection (MOI):

o Marginal ear vein = urine
• Specimen: 1st morning specimen
o Big vein on rabbit’s ear
o More concentrated
o High chance of identification and isolation of beta • Positive result (PR):
HCG o Corpora lutea (corpus luteum)
o Corpora hemorrhagica
Bioassays
▪ Bleeding corpora lutea
• Bioassays – animals are used
• Not done in modern laboratory anymore • Sensitivity (Sen):
• Most likely done for research purposes today o 10-15 IU/mL
1. Hogben 4. Ascheim-Zondek
• Animal used (AU): • Animal used (AU):
o Female Frog o Immature Female Mice
• Mode of injection (MOI): • Mode of injection (MOI):
o Lymph Sac = urine o Subcutaneous = urine
o If a patient’s urine is suspected of having beta HCG,
the urine is to be injected in the lymph sac of the • Positive result (PR):
female frog o Formation of hemorrhagic follicles and corpora
o If HCG is present in the urine of sample = positive lutea
result (oogenesis should be happening/transpiring)
• Sensitivity (Sen):
• Positive result (PR):
o 1-6 IU/mL
o Oogenesis
• Sensitivity (Sen):
o 75-100 IU/mL

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 1
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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

5. Frank-Berman o The body produces thick and sticky mucous that can
clog the lungs
• Animal used (AU): o It can obstruct the pancreas and other organs
affected by this disease
o Immature Female Rats
• Associated with pancreatic insufficiency, respiratory
• Mode of injection (MOI): distress, and intestinal obstruction and other
o Subcutaneous = urine manifestations of the disease
o Beneath the skin • It is a disease with multi-system involvement making it
really problematic
• Positive result (PR):
o Multiple organs are affected in cystic fibrosis
o Ovarian hyperemia
o Hyperemia – accumulation of blood or increased • For diagnosis of cystic fibrosis, the accurate
blood flow in the ovaries of female rats measurement of the sweat chloride concentration is
essential for its diagnosis
▪ Hyper – high
▪ Emia – blood Gibson and Cooke Pilocarpine Iontophoresis
• Sensitivity (Sen):
• A sweat test procedure
o 1 IU/mL • Gibson and Cooke → person
• Pilocarpine Iontophoresis → used to stimulate the
6. Kupperman sweating of the patient
• Animal used (AU): o Administer the drug: Pilocarpine
o Female virgin rat • Pilocarpine + mild current = stimulates sweat glands
• Mode of injection (MOI): o To harbor sweat
o Intraperitoneal = urine • Sweat is tested for Cl
o Peritoneum – at the back of the female virgin rat
o Once the sweat sample is collected, test for Chloride
• Positive result (PR): level of the sweat together with the sweat Sodium
o Ovarian Hyperemia • Sodium (Na) is also tested → mainly used as quality
control
• Sensitivity (Sen):
o Since discordant values between both ions suggest
o 1 IU/mL
that there will be or there are problems related to the
7. Kelso collection or analysis
o Control only; no diagnostic value
• Animal used (AU): • Values:
o Female Virgin Rat o Normal: <60 mmol/L
• Mode of injection (MOI): o Infants younger than 6 months: <30 mmol/L
o Subcutaneous = urine ▪ Borderline for CF: 30-59 mmol/L

• Positive result (PR):

GASTRIC FLUID
o Ovarian hyperemia
• The fluid that can be found in the stomach which is
• Sensitivity (Sen): important for digestion
o 1 IU/mL • The best fluid to represent the status/ condition of the
stomach
• Based on the current trends in the laboratory practices,
SWEAT it is very rare to receive gastric fluid sample
o There are now more sophisticated techniques to
Sweat Test
diagnose diseases of the stomach
• Primarily used to diagnose CYSTIC FIBROSIS • Scrumptious foods are the primary reason why gastric
Cystic Fibrosis fluid is secreted because it is needed for digestion
• At the site of the food, when chewing it (as mechanical
• Autosomal recessive genetic disease caused by digestion begins), gastric fluid deep inside the stomach
mutations in one single gene located in the long arm of is also being prepared to be produced for digestion
chromosome 7
• Metabolic disease that affects the mucous secreting
glands of the body
o That’s why the pathology lies in the mucous
exocrine glands of the body

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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

How Do You Digest Food After Chewing?

• (2) Rehfuss
• The food will travel through the esophagus → will go to
the stomach (where the gastric fluid is present) → o (1) Passed through the mouth
gastric fluid will contain secretions, enzymes, acid that o (2) Drive it deeper to the stomach
will help to digest the food that was devoured. o (3) Collect or aspirate gastric fluid

Cells of the Stomach Type of Specimen & Duration of collection

1. Specialized G Cells 1. Basal Acid Output (BAO)

• Produce gastrin • 1 hour collection

• Gastrin
o Four 15 minute specimen but a single 1-hour can be
o Signals the parietal cells to produce HCl and used
Intrinsic factor
▪ Collect every 15 minutes
2. Parietal Cells • 2 hour collection
• Produce HCl and Intrinsic o Done when testing for insulin hypoglycemia test
factor
• (HCl) Hydrochloric acid • BAO

o Promotes acidity in the o Total gastric secretion during unstimulated, fasting

stomach state

▪ Acidity is needed for ▪ Empty stomach; no encounter of food

digestion in the 2. Maximal Acid Output (MAO)
stomach
• Intrinsic factor • 1 hour collection
o Needed for Vitamin B12 o 15 minutes interval when Pentagastrin and
absorption Histamine are used
o Pernicious anemia – ▪ Every 15 minutes times
problem in the production
of intrinsic factor ▪ 4 intervals = 1 hour

▪ Thus, it will result to o Pentagastrin and Histamine as gastric stimulant

one of the manifestations of pernicious anemia ▪ Stimulate gastric secretion
which is the depleted acidity in the stomach
among all others • 2 hour collection
o Done for insulin hypoglycemia test and when
3. Chief Cells
Histolog is used as gastric stimulant
• Produce pepsinogen • MAO
o Zymogen, inactive enzyme o Total gastric secretion after gastric stimulation
o Pepsinogen should be converted to pepsin
Reference Values for BAO and MAO
• Pepsin
o Active form of pepsinogen Table 1. Reference Values
o Enzyme needed to digest proteins
• How to convert pepsinogen to pepsin? BAO MAO
o The acidity of the stomach (because of the HCl) will
help convert pepsinogen to its active form (pepsin) Men 0 to 5 mmol/hour 5 to 26 mmol/hour

Specimen Collection Women 0 to 4 mmol/hour 7 to 15 mmol/hour

Method of Collection: ASPIRATION

• (1) Levin Tube Gastric Stimulants

o Passed through the nose • Stimulate gastric secretions
▪ (1) Insert it to nose • Food – best gastric stimulants
• Can be in a form of test meals
▪ (2) Drive it down until you reach the stomach • Obsolete
▪ (3) Aspirate gastric fluid

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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

Different Pattern of Test Meals Used Clinically to Stimulate • Duodenal Ulcer

Gastric Secretions / Gastric Fluid Production o Associated with hyperacidity
o BAO and MAO are acidic and increased without
• (A) Ewald’s = bread and water diet stimulation.
• (B) Boa’s = oatmeal
• (C) Riegel’s = beef steak and mashed potato • Zollinger-Ellison
• (D) Heckman’s = egg albumin, water, methylene blue o Formed pancreatic tumors that produces excessive
• (E) Stasis = rice, raisins gastrin which results to high HCl levels (acidic) prior
• (F) Fischer’s = Ewald’s meal + hamburg stock to stimulation.
• (G) Lavine’s = ethyl alcohol, methylene blue
• (H) Motor = spinach or raisins + water Macroscopic Examination
• (I) Salzer = beef, lambchop, milk, rice, egg
• (J) Dock’s = biscuit Color

Chemical Stimulants • Pale Grey/Mucus

o Normal
• Chemicals used to stimulate gastric fluid secretion o Stomach contains mucoid layer.
o Pentagastrin – most preferred
• Yellow-Green
o Insulin – assess successful vagotomy procedure
o Histalog (Betazole) o Large amount of bile contamination (greenish)
o Histamine
• Red
Sham Feeding o Small amounts of fresh blood
• Mimics food consumption but food is recovered before • Coffee ground/Tarry/Blackish
being altered by the digestive processes
o Large amount of blood that mixes with acid results
• Usually done on experiments or studies on gastric fluid
to black or brown color.
status involving psychological processes that affect
digestion. Volume
o Fictitious (Scam) Feeding – sandwich
• Few mL – 50 mL
o Normal fasting specimen
Representative Normal and Abnormal Gastric Analysis
Results • > 50 mL

Table 2. Comparison of BAO and MAO Ratios in relation to o Abnormal fasting specimen since the stomach
the Different Conditions Associated with each Value should be unstimulated
• 20-60 mL up to 120 mL
BAO MAO o Usually achieved after Ewald’s test meal
BAO/MAO
mEq/hr mEq/hr
• 45-150 mL
Normal 2.5 25.0 10%
o Achieved after alcohol test meal or histamine
Pernicious Anemia 0 0 0 stimulation

Terminologies
Duodenal Ulcer 5.0 30.0 17%
• Different terms that relate to the acidity and presence of
Zollinger-Ellison 18.0 25.0 72% HCl in the stomach and gastric fluid.
• (1) Euchlorhydria
• BAO and MAO o Normal free HCl
o Expressed in milliequivalent per hour (mEq/hr) ▪ The term ‘eu’ is true
• MAO • (2) Hyperchlorhydria
o Refers to secretion after gastric stimulation o Increased free HCl = usually observed in peptic
o Relatively higher than BAO. ulcer
o Typically known as hyperacidity
• Pernicious Anemia
o There’s problem in HCl resulting in inadequate or no
gastric secretion.

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 4
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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

• (3) Hypochlorhydria • Lactic Acid – indicative of advance gastric

cancer/gastric stagnation
o Gastric fluid pH is greater than (>) 3.5 originally but
falls after gastric stimulation (decreased free HCl)
Table 4. Different Tests for Lactic Acid
▪ When there is no stimulation, it is > 3.5
▪ When there is HCl, pH should be 1
Test Reagents Endpoint
• (4) Achlorhydria
o Gastric fluid is greater than (>) 3.5 and does not fall Modified
FeCl3 + phenol Yellow
even after gastric stimulation (absence of free HCl) Uffelmann’s
o Pernicious Anemia Strauss FeCl3 + ether Yellow
• (5) Anacidity
Kelling’s FeCl3 Yellow
o Failure to produce pH of less than (<) 6.0 following
gastric stimulation
o Can also be associated with pernicious anemia
SPUTUM
Qualitative Tests for Free HCl • Different from saliva
• The results are not expressed in numbers o Comes from a deep expectoration on the lungs
o Only expecting for colorimetric reactions which are • Combination from upper respiratory tract (URT) and
not expressed/converted to numerical values lower respiratory tract (LRT)
• Dimethylaminoazobenzol o A fluid mixture from both structures
o Mixture of plasma, electrolytes, mucin, and water
o Positive = cherry red
▪ Mucin = makes up the mucoid structure of
• Gunzberg sputum
o Reagents: Phloroglucin, vanillin, alcohol
o Positive = purple red color Table 5. Different Ways to Collect for Sputum
• Boas
o Reagents: Resorcinol, Cane Sugar, Alcohol Manner Indication
o Positive = rose red color
1st morning Most preferred (routine)
Quantitative Tests for Free HCl 24-hour For volume measurement
Table 3. Different Quantitative Tests for Free HCl, Total
Acidity, and Combined HCl Throat swab For pediatric patients

Combined Sputum induction For non-cooperative patients

HCl
Free HCl Total Acidity
(Bound to Tracheal aspiration Debilitated patient
Proteins)
Titrant NaOH NaOH NaOH
Sodium Sputum Microscopic Examination
pH Dimethylaminoazobenzol
(Topfer’s reagent) Phenolphthalein (Na)
Indicator Volume
alizarin
Endpoint Canary Yellow Faint Pink Violet Table 6. Sputum Volume
Normal
25-50 mEq/L 50-75 10-15
Value
Decreased Increased
This is a titration method. When it reaches an endpoint, it can
express numerical values from that endpoint.

Bronchiectasis
Bronchial Asthma Lung Abscess
Acute Bronchitis Edema
Early Pneumonia Gangrene
Stage of Healing Pulmonary Tuberculosis (PTB)
Pulmonary Hemorrhage

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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

Odor Consistency

Table 7. Sputum Odor Table 9. Sputum Consistency

Odor Disease Relation Consistency Disease Relation

Odorless Normal
Mucoid Asthma, bronchitis
Lung Gangrene, Advance
Foul or putrid
Necrotizing Tumors Serous or Frothy Lung edema
Bronchiectasis, Pulmonary Bronchiectasis (enlargement
Sweetish
Tuberculosis (PTB) Mucopurulent of the airway), PTB with
Necrosis, Tumors, cavities
Cheesy
Empyema
Liver Abscess, Enteric
Sputum Macroscopic Structures
Fecal Gram-Negative (GNB)
Infections • Structures that can be seen without microscope

Dittrich’s Plug
Color
• Yellow or gray material,
• The color can give a preliminary impression of the pinhead size
probable condition • Foul odor when crushed
• Associated with Bronchitis,
Bronchiectasis, Bronchial
Table 8. Sputum Color Asthma

Color Disease Relation

Colorless or Translucent
White or Yellow (Purulent)
Gray
Lung Stones (Pneumoliths or Bronchitis Stones)
Made up of mucus only
(normal) • Yellow or white calcified TB
↑ Pus structures or foreign materials
(Pulmonary Tb, Bronchitis, • Associated with chronic
Jaundice, Pneumonia) bronchitis
↑ Pus and Epithelial Cell

↑ Bile
Bright Green or P. aeruginosa (secretes
Bronchial Casts
Greenish green pigment) infection,
Lung abscess • Branching tree-like casts of the bronchi
Fresh blood or hemorrhage
Red or Bright Red Pulmonary TB, o It assumes the shape of the bronchus
Bronchiectasis • Found in lobar pneumonia caused by S. pneumonia
Anchovy Sauce Old blood, Pneumonia, • Found in bronchitis and diphtheria infections
or Rusty Brown Gangrene
o In diphtheria, there are many discharges that are
Pneumonia, Chronic lung being produced in the neck area and will drip down
Prune Juice
cancer the lungs
Olive Green or
Cancer
Grass Green
Inhalation of dust or dirt,
Black carbon,
Anthracosis, Smoking
Lobar pneumonia (S.
Rusty with Pus
pneumoniae)
Rusty without
Congestive heart failure
Pus
Klebsiella pneumoniae
infection (causes
Currant Jelly-like
Hemorrhagic and
Necrotizing pneumonia)

BALIZA. CARBAJOSA. CORPUZ. MANINGO. MALASAGA. MORENO. RATILLA. RAMOS. ROSALINDA. TAMBA. TABUDLONG. BSMLS 3 6
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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

Layer Formation – 3 Layers: bronchiectasis, Lung Curschmann Spirals

Abscess, Gangrene
• Coiled mucus strands
• Can be observed macroscopically • Both microscopic and
• The sputum collected will be put inside a container and macroscopic structures
can form layers • Associated with bronchial
• The layering will be found in bronchiectasis, lung asthma
abscess, and gangrene
• Composition of the layering:
o 1st or top – frothy mucus (“mabula”) Myelin Globules
o 2nd or middle – opaque, watery material
o 3rd or bottom – pus, purulence, bacteria, tissues • Colorless globules
occurring in a variety of
Foreign Bodies sizes and bizarre forms
• No clinical significance
• Bronchial calculi – made up of calcium carbonate and but be mistaken as
phosphates Blastomyces dermatitidis
• Asbestos bodies – deposited if there is too much
o In the sense that they
inhalation of asbestos
are both globular
• Silica particles – too much inhalation of silica will be
deposited in lungs and reflected in mucus Blastomyces dermatitidis
• Pneumoconiosis – lung condition wherein there are
things that were being deposited in the lungs because
of extreme exposure to a certain substance and Epithelial Cells
eventually reflected in the sputum (e.g., anthracosis,
black lung disease, too much cold) • Cluster of ciliated columnar
cells
o Due to lining of the
Sputum Microscopic Structures
respiratory tract
Elastic Fibers • Often referred as Creola
bodies
• Slender fibrils with double contour and curled ends • Found in bronchial asthma
• Seen in Pulmonary Tuberculosis (PTB)
o In asthma, there is
Charcot-Leyden Crystals increase sloughing in
ciliated columnar
• Colorless, hexagonal, epithelial cells.
double pyramid, often o Seen in sputum
needle-like
• If found in sputum,
associated with bronchial Different Microorganisms Expected to Grow/Isolated in
asthma Sputum Samples
• Degraded products of
• Fungi
eosinophils
o C. albicans, C. neoformans, C. immitis, H.
o When eosinophils die and get
capsulatum, B. dermatitidis, A. fumigatus
degraded, there is a production
of Charcot-Leyden crystals • Parasites
o If eosinophil increases, expect
to see Charcot-Leyden crystals o Migrating larva:
in bronchial asthma ▪ Has heart lung phase migration
▪ Since bronchial asthma is an allergic condition, ▪ A. lumbricoides, S. stercoralis, Hookworm (ASH)
there will be eosinophilia  Three parasites known to exhibit heart lung
migration
Pigmented Cells
 If they have lung migration, they can be in the
• Heart failure cells: hemosiderin-laden macrophages sputum
o Observed in congestive heart failure o E. histolytica, E. gingivalis, T. tenax, P. westermani,
E. granulosus, T. canis
• Carbon-laden cells: cells studded with angular black
granules ▪ E. histolytica – invasive protozoa thus it is
possible to be seen in the lungs
o Observed if heavy smoker

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 LABORATORY UNIT 10: MISCELLANEOUS BODY FLUIDS

• Others: neoplastic cells, bacteria, leukocytes Other Structures Observed in BAL (Cytologic)
o Neoplastic cells – cancerous malignant cells
o Leukocyte – abundant sputum sample • Sulfur granules
o Associated with actinomycetes infection
▪ No leukocyte in saliva sample
• Hemosiderin-laden macrophages
Bronchoalveolar Lavage (BAL)
• Langerhans cells
• Washing of the lungs, especially in the bronchoalveolar • Cytomegalic cells
region • Fat droplets seen in fat embolism
• Lipid-laden alveolar macrophages.
o Lavage – washing
• A procedure in which a
bronchoscope is passed through
the mouth or nose into the lungs
and fluid is flushed into a small
part of the lung and then
recollected for examination.
o (1) Insert bronchoscope
o (2) Through the bronchoscope, flush the lung area
with saline
o (3) Wash it
o (4) Collect the washings from the lungs
• Body cells, lung cells, and microorganisms in the lungs
can be seen
• Important diagnostic test for P. jirovecii (P. carinii) in
immunocompromised patients
o P. jirovecii – innocuous/harmless pathogen; can only
cause infection in immunocompromised patients
▪ Causes Pneumonia in AIDS patients

Different Cells Observed in BAL and their Distribution

• Macrophage
o 56-80% - most predominant
o Specifically alveolar macrophage or dust cell
• Lymphocyte
o 1-15% - interstitial diseases, pulmonary lymphoma,
nonbacterial infections
• Neutrophil
o < 3% - cigarette smokers, bronchopneumonia, toxin
exposure
• Eosinophil
o <1-2% - hypersensitivity reactions
• Ciliated columnar bronchial epithelial cells
o 4-17%

Different Organisms Observed in BAL

• Pneumocystis jirovecii
• Toxoplasma gondii
• Strongyloides stercoralis
• Legionella pneumophila
• Cryptococcus neoformans
• Histoplasma capsulatum
• Mycobacterium tuberculosis
• Mycoplasma pneumoniae
• influenza A and B viruses
• Respiratory syncytial virus

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 [TRANS] LABORATORY UNIT XI: AUTOMATION IN URINALYSIS
URINALYSIS AUTOMATION
• Several instruments have been developed to partially or
completely automate routine urinalysis
• In addition to enhancing the workflow, automation can
standardize some aspects of manual urinalysis
• Most of these instruments can be interfaced with
laboratory information system (LIS), facilitating reports,
and results retrieval.
• In general, equipment in the laboratory (not just in the
clinical microscopy) is necessary to ensure an accurate,
reliable, and timely testing.
o Helps to maintain a high level of laboratory
performance
o Reduces the variations in test results and improves
the MT’s confidence in the accuracy of testing
• Goal: Improved reproducibility and color discrimination
while increasing productivity and standardization for
reporting urinalysis results.
o Color discrimination – color of reaction pads in the
urine strip (chemical analysis)
• Standardize:
o Sample processing
o Analyze test strips
o Perform urine sediment analysis
o Report results accurately (with consistent quality
and reduce hands-on time)
Features
• Automation brings a high level of performance and
greater confidence in the reliability of results. This may
be achieved by equipment with the following features
installed:
o Online computer capability
▪ With the laboratory information system (LIS)
▪ Interface that manages the inputting, processing,
and storing of data operations
o Bar coding
▪ For identification of samples
o Manual entry of color
o Clarity
o Microscopic results
o Flagging of abnormal results
▪ To know the abnormal findings in the patient’s
urine sample
o Storing of patients and control results
▪ Through LIS, patient and control results can be
stored in an archive for easy retrievals
o Minimal calibration
o Cleaning
BAUZON. TEVES. MARASIGAN. MATABALAO. MANZANO. VILLANUEVA. NUEVO. RAMOS. BOYOSE. EVANGELISTA.
o Maintenance
NOTE:
• The color, clarity, and microscopic results are
oftentimes manually encoded in the printed report. But
fully automated systems automatically generate the
results and ready for printing
• Minimal calibration, cleaning, and maintenance are still
performed by the medical technologists
Classifications of Urinalysis Automation
• Semi-automated analyzers
o The operation still depends on the MTs for specimen
mixing, tests, the strip-dipping, and microscopic
results input
• Fully-automated chemistry analyzers
o The sample tubes of urine are placed on a rack or a
carousel and move automatically through the
instrument
▪ Still operated by the MT by pushing the start
button
• Automated urine cell analyzers
o Instruments can perform the mixing, aspiration,
dilution, and staining of urine to classify the urine
sediment particles
• Automated urine system
o It performs a complete urinalysis that includes the
physical, chemical, and microscopic parts of a
routine urinalysis
o A.k.a. “walk away machines” because the MT will
just feed the urine samples and let the machine
perform all the works or testing in the urine.
▪ The MT will just come back if the processing is
done
Table 1. Available machines used in urinalysis
Equipment Manufacturer
Semiautomated Chemistry Instruments
Clinitek Advantus Siemens Healthcare Diagnostic, Inc.
Clinitek Status Siemens Healthcare Diagnostic, Inc.
Urisys 1800
Roche Diagnostics
system
COBAS u411 Roche Diagnostics
DiaScreen 50 U.S. ARKRAY
BSMLS 3 1
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 LABORATORY UNIT XI: AUTOMATION IN URINALYSIS

o Bilirubin
iChem 100 Iris Diagnostics o Urobilinogen
o pH
Fully Automated Chemistry Instruments o Specific gravity
o Color
Clinitek Atlas Siemens Healthcare Diagnostic, Inc. o Creatinine
Urisys 2400 o Protein-to-creatinine ratio
Roche Diagnostics
system • Well-suited for small-medium volume laboratories and
Aution Max AX-
U.S. ARKRAY physician’s offices
4030 • Self-calibrating and some instruments perform
automatic checks (Auto-Checks) to identify strip type
IChem Velocity Iris Diagnostics
and humidity exposure
Automated Microscopy
UF-1000i Urine
Sysmex Corporation
Cell Analyzer
iQ 200 Automated
Iris Diagnostics
Urine Microscopy
Urine Analyzer
Iris Diagnostics
(iQ 200 Sprint)

Table 2. Automated Urinalysis Systems

Automated Urinalysis Systems

iRICELL Iris Diagnostics Procedure

Urinalysis (iRICELL 3000plus, iRICELL 2000plus,
Systems iRICELL 3000pro, iRICELL 2000pro, • (1) Reagent strips are manually
iRICELL 1500) dipped and placed on the strip
CLINITEK AUWi reader
Siemens Healthcare Diagnostics, Inc.
System • (2) Reaction pads are read at
Body Fluid Analyzers the correct time
• (3) Strip is moved to the waste
ADVIA2120i with container
Boddy Fluids Siemens Healthcare Diagnostics, Inc. • (4) Results are displayed,
Software printed, or transmitted to an LIS
Sysmex XE-5000 • Patient identification, specimen color, clarity —
using Body Sysmex Corporation entered manually or through barcode reader
Fluids mode • Positive results are flagged — requires confirmation
iQ 200 using testing or microscopic evaluation
Body Fluids Iris Diagnostics
Software o MTs will need to perform another test just to confirm
the abnormal results

• Most of the machines installed in the laboratory came
from these manufacturers like:
o Sysmex Corporation – provider of UF-1000i Urine
Cell Analyzer
Semi-automated Urine Chemistry Analyzers
• Functions to analyze the chemical tests of urine
• Test for chemical components of urine
• Read and interpret the reagent strip results consistently
based from its chemical reaction
• Eliminates bias in reagent pad color analysis and
reading discrepancies
• Can perform the 10-parameter urine strip test:
o Leukocyte
o Nitrite
o Protein
o Blood
o Glucose
o Ketone
• Minimal daily maintenance — cleaning reagent strip
platform and emptying reagent strip waste container
Actual Process Flow
• (1) Dip the reagent strip into a well-mixed urine sample
• (2) Blot the strip to remove excess urine
• (3) Place the strip onto the reagent strip platform
• (4) Press the analyzer (enter button) to start processing
Fully Automated Urine Chemistry Analyzers
• High-volume urinalysis
laboratory
• User “walk-away” capability
• Ability to load many samples
with the capability to insert a
STAT specimen during the
run
• Press the button to begin testing and samples move
automatically through the instruments

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• Improved TAT (turnaround time) and hands-on time will Sysmex UF-1000i
be reduced
Process Flow • Laser-based flow
cytometry
• Measures the
• (1) Specimen Identification via barcode reader
following to identify
• (2) Sample probe aspirates exact amount of urine
stained urine
sample and dispenses it directly onto the reagent strip
sediment particles:
• (3) Reagent strip advances and is analyzed by
reflectance photometry to measure color change of o forward light
each reagent value scatter,
• (4) Automatically disposes strip test to waste box o side scatter,
• Instrument analyzes the following: o fluorescence
staining
o Leukocytes, Ketones, Protein, Glucose, Nitrite, characteristics,
Blood, Urobilinogen, pH, Bilirubin, Color, Clarity, o adaptive cluster
Creatinine, and Protein analysis
▪ Color – measured by reflectance photometry • Only requires 4 mL of uncentrifuged urine
(diffused light illuminates reaction mixture and • Offers 2 separate channels:
reflected light is measured) or spectrophotometry
(at multiple wavelengths) o urine particle analysis
▪ Specific gravity – the concentration of solute in o bacteria staining and detection
an aqueous solution = refractive index
▪ diluent stabilizes the pH and lyses the non-
methodology
bacterial particles
▪ Clarity – transmitted or scattered light
• Instrument uses Integrated bar-coded sample
Identification
• Abnormal ranges to be selected – microscopic
examination should be done or confirmatory testing will
be identified and flagged.
• Patient results, quality control results, calibrations –
stored for visual display, print-out, or transmission to a
laboratory computer system.

• Contaminators, intonations, bacterial and mycotic

infections can be easily indicated
• Staining the internal components of the cells
• staining the internal components of the cells
o the stain is specific to RNA in a bacteria cell
eliminating the nonspecific staining of the debris
o each channel has a specific stain that targets the
internal components of the cells
• stained urine sample passes through the flow cell where
Automated Microscopy it is hydrodynamically focused and presented to a red
semiconductor laser that sends the wavelength at 635
• Provide standardize nm
results in less than 1 • Identified by measuring the height and width of the
minute compared to fluorescent and light scatter signals, which are
manual method presented in scattergrams and histograms
• Automation reduces
workload, increases
accuracy, reliability,
rate production and
turnaround time
• Cost-effective and improve turnaround time
• Sysmex UF-1000i and iQ 200 (Iris Diagnostics)

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• Principle in identifying sediments is based from the • Flagged particles include pathologic casts, crystals,
signal wave form of the cells small round cells (renal tubular epithelial cells or
transitional epithet lial cells), sperm, mucus, and yeast-
o Width of the fluorescent signal = measurement of
like cells, and must be confirmed by manual
cellular inclusions
microscopy.
o Width of forward light scatter = measurement of
length of cells • Important parameter is the bacterial orientation which
provides two (2) kinds of typical slope:
• Resulting values are presented in quantitative cells
per microliter and cells per high- or low-powered o Slope 1 - the slope of the
field bacteria cluster is made up
o Abnormal results automatically flag in the computer of data points distributed
screen densely and broadly
o Medical technologist will confirm the result through along with the diagonal line
microscopic reading
▪ The scattergram
suggest that the
bacterium is a coccus
in the urine.
o Slope 2 – the slope of the
bacterial cluster is small
and concentrated in a
narrow zone
▪ Possibly indicates that
the bacterium in the
urine is a rod
iQ 200 Autmated Urine Microscopy Analyzer

• Digital imaging and Auto-Particle Recognition

o shortened turnaround
time with standardized
• Main particles enumerated in the machine are red blood results by leveraging
cells (RBCs), white blood cells (WBCs), epithelial cells, the digital flow
hyaline casts, and bacteria. The results are displayed morphology technology
as scattergrams using an auto-particle
o The signal intensity, signal width, signal waveform recognition or APR
area, signal wave form of forward scattered light, software
side fluorescence, side scattered light, and the o the urine particles are isolated, identified, and
polarized side scattered light of each particle characterized on the screen to virtually eliminate the
obtained in the flow cytometry are analyzed and need for manual microscopy
classified by applying differentiation of algorithms • Preclassifies urine particle; features of individual images
that take this data into account such as the size, shape, contrast and texture are used
o Scattergrams are the results displayed in the by the neural network pattern recognition software for
analysis classification into 1-12 particle categories.
o RBCs,
o WBCs,
o WBC clumps,
o Hyaline casts,
o unclassified casts,
o SECs,
o non-SECs,
o bacteria,
o yeast,
o crystals,
o mucus,
o sperms
• Can be use also for body fluid cell counts - add body
fluids software module
• Mixes sample and aspirates approx. 1 mL of urine to a
planar flow cell
• 500 digital photomicroscopic images taken per
sample

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• (1) Strobe light passes through the collimator in a single • Advantage:

beam
o Zoom capability to view images
• (2) Specimen is surrounded by lamina - orients the o Interpretation of images is similar to manual
particles for microscopic viewing microscopic smears
• (3) Digital video camera takes 500 pictures as the
specimen passes through the flow cell Automated Urinalysis Systems
• (4) Digital images are sent to the computer
o results can be reported without operator review or
stored images in each category can be displayed on
the screen for verification and for manual editing
• Allows the user to sub lassify 27 additional categories
to indicate the specific types of crystals, casts, epithelial
cells, yeast with pseudohyphae, trichomonads, and oval
fat bodies.
o can add free text comments to the report as needed

• Combination of urine chemistry analyzers and

sedMAX (77Elektronika)
• e.g. URISED 3 PRO - professional automated urine
sediment analyzer with a revolutionary new optical
system combining the bright field and phase contrast
microscopy
• Machine offers a uniquely advanced method for
visualization and recognition of your form elements in
urine samples
• Automating the gold standard method of sediment CLINITEK AUWi System (Siemens)
analysis increases reliability, improves your workflow
and decreases the turnaround time
• Optical System
o Capable of capturing the images of both the bright-
field and phase contrast microscope to gain more
information about particles in a sample
• Performs automated microscopy with digital imaging
• Requires a minimum of 2 mL of urine centrifuged in a
special cuvette to produce a monolayer of urine
sediment
• Sediment is analyzed by a bright-field microscope and
digital camera
• Sediment is captured; categorize 15 particle images
based upon size and shape using image-processing
software
automated urine cell analyzers
• Improved TAT (turnaround time) and hands-on time will
be reduced
• It provides a complete walkaway capability with minimal
specimen handling from sampling through results
o The MedTech will just feed the urine sample in a
tube and press the start button letting the machine
perform its work.
• Sample identification
o Machine is designed with bar code system and
interface with LIS
o Bar-coded samples are automatically identified and
processed according to the requested test
• Can independently perform both physical and chemical
testing, microscopy analysis, and a combination of both.
• A complete urinalysis report can be sent directly to the
LIS or printed out
o Reducing clerical error
• Processing
o Using the similar sample racks and moving on a
conveyor system, the samples are easily transferred
from one instrument to the next and process the
testing from chemical analysis down to microscopic
analysis of the sample
• The Clinitek Atlas
(automated urine
chemistry analyzer)
+ Sysmex UF-1000i
(automated urine
cell analyzer) =
CLINITEK AUWi
System (Siemens)
• Offer complete integrated urinalysis for unattended
operation from start to end
• Minimum of 5 mL of urine in the automated mode
• (1) The barcoded tubes are placed on the rack and
placed on the system.
• (2) Rack advances to the ATLAS analyzer,

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o Specimens are identified, mixed, aspirated, and • (6) Put the strip in the
tested for physical and chemical components equipment and wait for it to be
processed. (About 8 seconds)
• (3) Sample travels across a connecting bridge to the
UF-1000i for microscopic analysis.
• (4) The instruments automatically reflect the samples
requiring the sediment analysis thereby reducing the
time associated with manual microscopy analysis
• (5) Automatic verified results are reported directly to the • (7) Visually examine the color of the urine, and select
LIS which minimizes the medical technologist follow up on the screen what best describes it.
iRICELL Automated Urinalysis Systems • (8) After the processing is done, dispose the strip in the
biohazard bag. Press done on the machine, before you
• Combines urine chemistry and microscopy in a fully remove gloves, and take the result of the test.
automated walk away work cell that is easy to use and iQ200 Automated Urine Microscopy Analyzer
maintain
• Optimizes advanced urinalysis and body fluid testing in • Designed for all volume workloads, producing
the laboratory with the digital flow morphology shortened turnaround times, and standardized results
technology that uses APR (Auto-Particle Recognition) • System aspirates approximately 1.3 ml urine
to deliver the standardized and accurate results specimens from an unspun sample of at least 3 ml
• Minimum of 4 mL of urine is required
• Patient identification • (1) It surrounds the sample
with iQ Lamina in a planar
o Bar-coded tubes are placed into the 10-position rack
flow cell which
and moved to the iChem VELOCITY (sample is
hydrodynamically orients
mixed and the urine is aspirated)
and constrains particles
• Upon completion of the physical and chemical analysis,
the rack moves across the connecting bridge to the iQ
200 for microscopy testing
• Samples can be reflexed to urine microscopy based
upon the urine chemistry results
• Results are reported directly to the LIS and minimized • (2) A high-definition camera
medical technologies follow up captures 500 images per
sample, isolationg
thousands of particles
DEMO VIDEO
o Individual particles are
Urinalysis Using Clinitek Status Analyzer isolated within each
image and then classified
• (1) Turn on the analyzer
• The Auto-Particle Recognition (APR) Software uses
size, shape, texture, contrast to classify 12 urine
sediment particle types to reduce subjectivity
o The operator can subclassify into 27 additional
classes
• (2) While waiting for the analyser ▪ Unclassified crystals
to warm up, check the patients’  Calcium oxalate
sample if it has name and date
of birth; check the multistix open  Triple phosphate
date and expiry date.  Calcium phosphate
 Leucine
• (3) Wet a gauze with distilled water and use it to clean  Amorphous
the equipment and the table as well. After, dispose it in  Uric Acid
a biohazard bag.
 Calcium carbonate
• (4) Select strip test on the screen, and enter the new
proprietor’s name followed by the patient’s name and  Cysteine
ID.  Tyrosine
• (5) Take a strip and dip it into
the urine sample. Blot it twice ▪ Unclassified Casts
on a paper towel.  Granular casts
 Cellular casts
 Waxy casts
 Broad casts
 RBC casts

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 WBC casts
 Epithelial casts
 Fatty casts
▪ Yeast
 Yeast with pseudohyphae
 Budding yeast
▪ Non-squamous epithelial cells
 Renal Epithelial cells
 Transitional Epithelial cells
▪ Artifacts
▪ Other
 Trichomonas
 Fat
 RBC clumps
 Oval fat bodies
▪ Unclassified
 Dysmorphic RBCs
• Edit-Free Release (EFR)
o Auto-release results based upon user-defined
parameters, creating a true walkaway analyser
• Color-coded indicators
o Facilitate the efficient review of categories flagged in
yellow
• On-screen Verification
o Operator can release the urine report
• iWARE Integrated Urinalysis Software
o provides onboard clinical validation and result
verification into a single step, optimizing workflow
• Urine Culture Candidate Report
o Combines urine chemistry and urine microscopy
results to help screen out samples for culture
• iQ200 Body Fluids Module
o provides a standardized fully automated method for
the analysis of cerebrospinal, synovial, and serous
fluids
• Accurate and reproducible body fluids result can be
verified on the screen
o Proprietary technology isolates and counts RBCs
and nucleated cells with linearity down to zero

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