ANALYSIS IN URINE AND BODY FLUIDS Laboratory

1st Semester | A.Y. 2024-2025

Lecturer: Ma’am Moira Patricia Soliman
By: Aimee G. Montes

MODULE 1
SAFETY AND QUALITY MANAGEMENT
SAFETY
 The clinical laboratory contains a variety of
safety hazards, many capable of producing
serious injury or life-threatening disease. To
work safely in this environment, laboratory
personnel must learn what hazards exist, the
basic safety precautions associated with them,
and how to apply the basic rules of common
sense required for everyday safety for patients,
coworkers, and themselves.
Terminologies
 CDC – Centers For disease Control and
prevention
 OSHA – Occupational Safety and Health
Administration
 CLSI – Clinical and Laboratory Standard
Institute
 PPE – Personal Protective Equipment
 UP – Universal Precautions
 BSI – Body Substance Isolation
 NFPA – National Fire Protection Association

Biologic Hazard
 Source: Infectious agent
 Possible Injury: Bacterial,
fungal, viral, prions,
parasitic infection
 Note: The symbol of Biologic
Hazard is fluorescent orange
in color

6 Components of Chain of Infection (IREMES)

Six Components Examples
Infectious agent Bacteria, fungi, viruses,
parasite
Reservoir Animals, humans,
fomites, insects, blood,
body fluids
Portal of Exit Nose, mouth, mucus
membrane
Mode of Transmission Droplets, airborne,
contact, vector, vehicle
Portal of Entry Nose, mouth, mucus
membrane, skin,
unsterile equipment
Susceptible Host Patients, elderly,
newborns, immune-
compromised,
healthcare workers
3 Links
 Infectious agents/source
 Mode of transmission
 Susceptible host

MODE OF TRANSMISSION
 Airborne/ aerosol
o Centrifugation of unstoppered
tubes
o Heating cultures of specimens too
rapidly
o Sterilization of inoculating loops in
the Bunsen burner flames
o Leakage from a container that holds
Note:
contaminated specimen
o Broken centrifuge and spills  Best way to break the link of infection is
through Hand washing/hygiene.
 Ingestion
o Failure to wash hand
BIOLOGIC WASTE DISPOSAL
o Eating
o Drinking  All biologic waste, except URINE, must be
o Smoking placed in appropriate containers labeled
o Applying cosmetics (ex.lipstick tester with biohazard symbol
in malls)  All biologic specimens, except urine, must
o Pipetting with mouth be sterilized or decontaminated before
 Direct inoculation disposal.
o Needlestick  Urine may be discarded by pouring it Into a
o Broken glass laboratory sink under a plexiglas
o Animal bites countertop shield. Care must be taken to
o Small scratches avoid splashing. And the sink should be
 Mucous membrane flushed with water after specimens are
o Infection may occur if the organism discarded
can directly enter through the  Disinfection of the sink using a bleach
mucous membrane such as through dilution of 1:10 sodium hypochlorite
the conjunctiva of the eye should be performed daily
 Arthropods/ vector  Empty urine containers can be discarded as
o Infectious sources include ticks, non-biologically hazardous waste.
fleas, and mosquitos, which may
harbor various microorganisms Sharp Hazard
Note:  Source: needle, syringe, lancet, broken
Head lice – direct contact glasswares
Pubic lice – sexual contact  Possible injury: cuts, puncture, blood-
borne pathogen exposure
 All sharp objects must be
disposed in puncture-resistant,
leak-proof container, with the
biohazard sharp symbol.
 Note: Biohazard sharp symbol
has syringe facing northwest
 The biohazard sharp containers
should not over-filled and must
always be replaced when the safe
capacity mark is reached.
 Chemical/Poison Hazard  Equipment should not be operated with wet
 Source: preservatives and reagents hands
 Possible injury: exposure to toxic,  Laboratory personnel should continually
carcinogenic, and caustic agents observe for any dangerous conditions, such
 Hazardous chemical should be labeled with as, frayed cords and overloaded circuit, and
description of their particular hazard, such report to the supervisor.
as poisonous, corrosive, flammable,  Equipment that has become wet should be
explosive, or carcinogenic. unplugged and allowed to dry completely
 In cases of spills, when skin before reusing
contact occurs, the best aid is  Equipment should also be unplugged before
to flush the area with large cleaning
amount of water at least 15  All electrical equipment must be grounded
minutes, then seek medical with three-pronged plugs.
attention.  When as accident involving electrical
 Note: The symbol for shocks occur:
Chemical/Poison hazard is a skull with o Turn off the circuit
two crossbones. breaker
o Unplug the equipment
MATERIAL SAFETY DATA SHEET o Move the equipment using
 contains the information about chemicals a nonconductive glass or
 includes the following information: wood object
o Physical and chemical  Note: The electric hazard symbol
characteristics has 2 lightning bolts
o Fire and explosion potential
o Reactivity potential Fire Hazard
o Health hazards and emergency first  Source: Open flames, organic chemicals
aid procedures  Possible injury: Burns or dismemberment
o Methods for safe handling and  When a fire is discovered, people are
disposal expected to:
o Primary route of entry o Rescue – anyone in immediate
o Exposure limits and carcinogenic danger
potential o Alarm – activate the institutional
fire alarm system
Radioactive Hazard o Contain – close all doors to
 Source: Equipment and radioisotopes potentially affected areas
 Possible injury: Radiation exposure o Extinguish/Evacuate – attempt to
 The amount of radiation is related to a extinguish the fire if possible or
combination of time, distance and evacuate, closing the door
shielding.  To operate the extinguisher
 Exposure to radiation during pregnancy o Pull the pin
presents a danger to the fetus, personnel o Aim at the base of the fire
who are pregnant or think they may be o Squeeze the handles
should avoid areas with o Sweep from side-to-side
this symbol. It can cause
possible delays or
congenital disease that
could affect the
developing fetus.
 In utero exposure to
ionizing radiation can be
teratogenic, carcinogenic,
or mutagenic.

Electrical Hazard
 Source: Ungrounded or wet equipment,
frayed cords
 Possible injury: Burns or shock
Note: Halotron is the best extinguisher for computer
(Class C)

Physical/Ergonomic Hazard
 Source: Wet floors, heavy boxes, patient
 Possible injury: Falls, sprains, strains
 General Precautions:
o Avoid running in room or hallways
o Watch out for wet floors
o Bend knees when lifting heavy
objects
o Keep long hair pulled
back
o Avoid dangling
jewelries
o Maintain a clean, NSHED
organized areas
o Use a closed-toed
shoes

HAND HYGIENE
 Hand contact is the primary method of
infection transmission.
 Dr. Ignaz Semmelweise is the Father of
Handwashing
 Laboratory personnel must always sanitize
hands:
o Before patient contact
o After gloves are removed
o Before leaving the work area
o Anytime when hands has been
knowingly contaminated
o Before going to designated break
areas
o Before and after using bathroom
facilities
 Alcohol-based cleanser – used when
hands are not visibly soiled
 Hand washing – used when hands are
SUVSM
visibly soiled
 Hand washing songs: Degrees of Hazard (NSMSEx)
o Happy birthday 0 No hazard
o Twinkle, twinkle little star 1+ Slight hazard
o Alphabet song 2+ Moderate hazard
3+ Serious hazard
4+ Extreme hazard
 MODULE 2 substances are present in much higher concentrations in
urine than in other body fluids, a fluid that is high in
INTRODUCTION TO URINALYSIS urea and creatinine content can be identified as urine.
HISTORY AND IMPORTANCE
 References to the study of urine can be found
in caveman’s drawings and Egyptian
hieroglyphics, such as the Edwin Smith
surgical papyrus.
 Urine is a fluid biopsy of the kidney and
provides a “fountain” kind of information.
 Hippocrates – wrote the book “uroscopy”
 Frederick Dekker – discovered albuminuria by
boiling urine
 Thomas Bryant – published a book about
“pisse prophet” - inspired the passing of the
medical licensure law in England
 Thomas Addis – Addis count – method for
quantitating microscopic sediments
 Richard Bright – introduce the concept of
urinalysis as part of a doctor’s routine patient
examination.
 Thudicum – discovered Urochrome – the
pigment that cause yellow color urine.

Reasons for Performing Urinalysis (CLSI)

 Diagnosis of disease
 Screening asymptomatic populations for Urine Volume
undetected disorder  Urine volume depends on the amount of water
 Monitoring the progress of the diseases that the kidneys excrete.
 Monitoring the effectiveness of therapy  Factors that influence the urine volume include
fluid intake, fluid loss from non-renal
Urine Composition sources, variations in the secretion of ADH
 Urine consists of urea and other organic and and the need to excrete increased amounts of
inorganic chemicals dissolved in water dissolved solids, such as glucose or salts.
 Urine is normally 95% water and 5% solutes. o ADH – antidiuretic hormone – controls the
However, considerable variations in the body water excretion
concentration of these solutes can occur owing  Normal daily urine input is usually 1200-1500
to the influence of factors such as dietary mL, a range of 600 – 2000 mL is considered
intake, physical activity, body metabolism, and normal.
endocrine functions.  The kidneys excrete 2-3 times more urine
 Nitrate – a normal urine constituent; Nitrite is during the day than during the night.
not normal  Oliguria – decreased urine output
 Others – carbohydrates, pigments, fatty o Less than 1mL/kg/hr in infants
acids, mucin, enzymes, hormones; may be o Less than 0.5mL/kg/hr in children
present in small amounts depending on diet o Less than 400mL/24hrs adult
and health. <500mL/24hrs (Henry’s)
 Urea – major organic component of urine o Causes: dehydration, vomiting,
(Urganic) diarrhea, perspiration, severe burns
 Chloride – major inorganic component of urine  Polyuria – increased urine output
followed by sodium and potassium o 2.5 to 3 mL/kg/day in children
o ChaNaK – Chloride, Sodium, Potassium o >2L/24hr (Henry’s) > 2.5L/24hrs
– inorganic components (Stras)
 The single most useful substances that o Causes: diabetes mellitus, diabetes
identifies a fluid as a urine is its uniquely insipidus, diuretics, caffeine, alcohol,
high creatinine concentration (approx..50 nervousness
times that of plasma)  Nocturia – increased excretion of urine
Tip: (>500mL) at night.
Should it be necessary to determine whether a o Common among pregnant women, and
particular fluid is urine, the specimen can be tested for its urine has a specific gravity of less than
urea and creatinine content. Because both these 1.018
 Anuria – cessation of urine flow or no urine  The required specimen volume for urine
output. microscopic analysis is 10 to 15mL
o Causes: damage to the kidneys, renal  Containers should stand upright, have an
stones, renal tumors opening of at least 4 to 5 cm, and have the
capacity of 50 to 100 mL
Analysis of Urine in Differentiating between
Diabetes Mellitus and Diabetes Insipidus Labels
 Patient’s name
 Patient identification number
 Date and time of collection
 Additional information such as age, sex, etc.
 Labels must be attached to the body of the
container, not on the lid, and should not
become detached if the container is
refrigerated.

Requisition form
 A requisition form must accompany specimens
delivered to the laboratory.

3Ps (Polyuria, Polydipsia, Polyphagia) - Three

hallmarks of DM and DI (symtptoms)
 Polyuria – increased urine output
 Polydipsia – increased water intake (feeling too much Policy for Handling Mislabeled Specimens
thrist)  Do not assume any information about the
 Polyphagia – increased food intake (hunger) specimen or patient.
DM and DI have the same symptoms but these  Do not relabel an incorrectly labeled specimen.
differ in specific gravity.
 Do not discard the specimen until the
 decreased ADH – Diabetes Insipidus investigation/examination is complete.
 increased specific gravity, decreased  Leave the specimen exactly as you receive it;
function of insulin, increased glucose – put in the refrigerator for preservation until
Diabetes Mellitus errors can be resolved.
 Notify the floor, nursing station, doctor’s office,
urine specific gravity – number of constituents of
etc. of the problem and why it must be
urine (the more na mataas ang specific gravity, mas
corrected for analysis to continue.
mataas ang contents ng urine)
 Identify the problem on specimen requisition
with the date, time and your initials.
Specimen Collection
 Make the person responsible for specimen
 Urine specimen should be delivered to the collection participates in the solution of
laboratory promptly and tested within 2hrs (any problems. Any action take should be
exceeding will be erroneous) documented on the requisition slip.
 Never discard a specimen without checking it  Report all mislabelled specimens to the
with the supervisor appropriate supervisor.
 Characteristics of the urine container
When to reject Specimens?
o clean, dry, leak-proof  Specimens in unlabelled containers
o with screw-top lids – these
 Non-matching labels and requisition forms
are less likely to leak than
 Specimens contaminated with feces or toilet
snap on lids
paper.
o made of sterile material
 Containers with contaminated exteriors
 The recommended container
 Specimens of insufficient quantity
capacity is 50mL
 Specimens that have improperly transported
 Specimens that have not been preserved Specimen Preservation
correctly during a time delay  A specimen that cannot be delivered and
 Specimens for urine culture collected in a non- tested within 2 hours should be refrigerated
sterile container or have an appropriate chemical preservation
 Inappropriate collection for the type of testing added.
needed (for example, midstream clean-catch
specimen for bacterial culture)
Changes in Unpreserved Specimen
↑PBaON-C pH, Bacteria, Odor, Nitrite, Color

if will be used for culture;

specimen should be warm
easiest; most common (not cold anymore) before
processing

preserves protein bacteriostatic at

18g/L of urine

for cellular analysis

prevents glycolysis
 ToPhe - Toluene & Phenol – doesn’t interfere with routine testing

Sodium propionate

o Mix thoroughly upon arrival at the

Types of Urine Specimen laboratory. Accurately measure and
Random Specimen record the total volume.
 Most commonly received specimen o Combine and mix contents if collected in
 Easy to collect and convenient multiple containers before aliquoting.
 For routine screening  Preservation and Storage:
 Can be collected at any time, usually during o Refrigerate or keep on ice during
daytime hours, and without prior patient collection.
preparation o Consider adding a chemical preservative
 Not recommended for culture if needed.

First Morning Specimen or 8-hour Specimen Afternoon urine (2 pm – 4 pm)

 An ideal urine specimen for routine urinalysis  Preferred for urobilinogen measurements
 Most concentrated specimen
12-hr urine specimen
 Ideal for cytology study because the number of
 Ideal for screening microalbuminuria
epithelial cells present can be significant
 For pregnancy testing
Catheterized Specimen
 Orthostatic or postural proteinuria (matagal na
 The specimen is collected under sterile
nakatayo, there’s protein in urine; protein sa ihi
signifies that there’s kidney damage or failure) conditions by passing a hollow catheter
 Glucose monitoring through the urethra into the bladder
 Urine flows from the bladder through the
Second morning/fasting Specimen catheter into a plastic bag
 For glucose or diabetic monitoring and  Specimens can be collected from this urine bag
screening  The test most commonly requested for a
 Refers to second morning voided after rising catheterized specimen is a bacterial culture.
 Fasting specimen (first choice); second choice for
fasting specimen is the first morning Midstream Clean-Catch Specimen
 Safer and less traumatic alternative to
2-hr Postprandial Specimen catheterized specimens for bacterial culture
 Collect urine after 2 hrs of meal  Less contaminated by epithelial cells and
 For glucose or diabetic monitoring bacteria, providing a more representative
sample of the actual urine
Timed Specimen/24-hr Specimen  Avoid using strong bacterial agents like
 Accurate measurement of urine chemicals hexachlorophene or povidone-iodine
requires precise timing  Mild antiseptic towelettes are recommended.
 Diurnal variations (e.g., catecholamines,, 17-
hydroxysteroids, electrolytes) necessitate Suprapubic Aspiration
careful timing  Urine may be collected by introducing a needle
 Collection instructions: through abdomen into the bladder.
o Begin and end with an empty bladder.  Suprapubic aspiration provides a specimen
Calculate substance concentration based that is completely free of extraneous
on the urine volume produced during the contamination.
collection period.  This method is particularly useful for infants or
 24-Hour specimen handling: children.
 The specimen can be used for bacterial
culture and cytological
examination

Pediatric Specimen
 Wee bag
 Soft clear plastic bags with
hypoallergenic skin adhesive to
attach to the genital area of both
boys and girls.

Specimen for Prostatitis

Three-glass collection
 First Specimen:
o Discard the first urine passed; it is
collected in a sterile container.
 Midstream Specimen:
o Collect the midstream portion in
another sterile container.
 Prostate Fluid Specimen:
o Massage the prostate to pass prostate
fluid with the remaining urine into a
third sterile container.
 Perform quantitative cultures on all specimens.
 Examine the first and third specimens
microscopically.
 Prostatic Infection Indicators:
o The third specimen will show:
 White blood cell / high-power
field count
 Bacterial count 10 times higher
than that of the first specimen.
 Possible presence of
macrophages containing lipids.
 o Second specimen will be used as a MODULE 3
control for bladder and kidney PHYSICAL EXAMINATION OF URINE
infection.
COLOR
o If the second specimen is positive,
 The normal color of urine includes:
the results from the third specimen
o Pale Yellow: Normal; indicates good
are invalid due to contamination by
hydration.
infected urine.
o Yellow: Normal; typical for well-
o If all three specimens are positive in
hydrated individuals.
WBC and bacteria, the patient is
o Dark Yellow: Concentrated; may
confirmed to have UTI.
indicate dehydration.
Pre and Post Massage Test  The yellow color of urine is caused by the
presence of pigment- Urochrome – the most
 Pre-Massage Specimen: Collect a clean-catch
concentrated pigment.
midstream urine sample.
 The actual amount of urochrome produced in
 Post-Massage Specimen: Collect a second
the body is dependent on the body’s metabolic
urine sample after massaging the prostate.
state.
 A positive result is indicated by significant
 Increased urochrome production:
bacteriuria in the post-massage specimen
o Thyroid conditions
which is greater than 10 times the count in
o Fasting
the pre-massage specimen.
o Urine stands at room temperature
● 2 additional pigments present in urine in much
Stamey-Mears (4 glass)
smaller quantities:
 The prostatic secretions are cultured and
o Uroerythrin – pink pigment, most
examined for WBC. More than 10-20 WBC per
evident in specimens that have been
hpf is considered abnormal.
refrigerated, resulting in the
precipitation of amorphous urates.
o Urobilin – orange-brown color, an
oxidation product of the normal urinary
constituent.
How to check for urine color
VB – Voided bladder ● Care should be taken to examine the specimen
under a good light source, looking down through
Drug Testing Specimen Collection the container against a white background.
 Chain of evidence or chain of custody –
Dark Yellow/Amber/Orange Urine
process that provides documentation of proper
Possible Causes
sample identification from the time of collection
o Bilirubin: Liver dysfunction or hemolysis.
to the receipt of laboratory.
o Detection through chemical tests.
o donor/client – drug testing patients
 Yellow Foam:
 The COC is a standardized form that must Indicates bilirubin
document and accompany every step of drug presence.
testing, from collector to courier to laboratory o Urobilin: Increased
to medical review officer. concentration or photo-
 For urine specimens to withstand legal oxidation.
scrutiny, it is necessary to prove that no o Phenazopyridine: Medication
tampering of the specimen occurred, such as causing bright orange urine.
substitution, adulteration or dilution, the
collector adds bluing agent (dye) to the toilet Red/Pink/Brown Urine
water reservoir to prevent an adulterated
specimen. Possible Causes
 Most common adulterant is water. o Blood (Hematuria): Causes
 Container capacity: 60mL red or pink color; may be
 Urine volume collected: 30 to 45mL accompanied by turbidity,
 Urine temperature: read within 4 minutes, presence of RBCs in the
range of 32.5 to 37.5°C urine.
 The urine color is also inspected to determine o Hemoglobinuria: Clear red
any signs of contamination urine; plasma appears red.
o Hemoglobinuria resulting from the in vivo
breakdown of RBCs is accompanied by red
 plasma. Brown/Black Urine
o Myoglobinuria: Reddish-brown urine; plasma Possible Causes
remains normal. o Melanin: Malignant melanoma.
o Breakdown of skeletal muscle produces o Homogentisic Acid: Alkaptonuria (genetic
myoglobin. metabolic disorder).
o Food/Medications: Beets, blackberries, o Medications/Foods: Certain drugs or dietary
rifampin. factors.
o Distinguishing between hemoglobinuria and
myoglobinuria may be possible by examining Blue/Green Urine
the patient’s plasma. Possible Causes
Identifying the difference between Hemoglobinuria o Bacterial Infections: Pseudomonas infections.
and Myoglobinuria: o Inherited Disorders: Familial benign
Hemoglobinuria – red pasma hypercalcemia.
Myogobinuria – normal colored plasma o Foods/Medications: Clorets, dyes, B vitamins.
URINE CLARITY
 Clarity is the general term that refers to the
transparency or turbidity of a urine specimen
 The specimen should be in a clear container
 The clarity of the specimen certainly provides a
key to the microscopic examination results,
because the amount of the turbidity should
correspond with the amount of material
observed under the microscope.
 Clear urine is not always normal
 Nubecula- faint cloud in the urine after
standing due WBCs, epithelial cells and mucus

Definition and Importance (manner of reporting

clarity)
 Clear: Indicates normal urine without particles.
 Hazy: Slightly cloudy; may be normal or due to
mild contaminants.
 Cloudy/Turbid: Significant presence of
particles; possible infection or other conditions.
 Milky: Presence of lipids, bacteria, or high
levels of mucus.

How to Check for Urine Clarity

 Usually examine the mixed specimen while
holding it in front of a light source. View
through a newspaper print.

Urine must be mixed because unmixed urine

samples can result to prevent false negative blood in
urine strip.

If both color and clarity ang iche-check, use a white

background with a good light source.
Color only – white background
Clarity only – good lighting
 URINE SPECIFIC GRAVITY
 Specific gravity is defined as the density of a
solution compared with the density of a similar
volume of distilled water (SG 1.00) at a similar
temperature.
 Specific gravity is influenced by the number of
particles present, and the size of the particles.
 The evaluation of the urine concentration is
included in the routine urinalysis by measuring
the specific gravity.

 The specific gravity of the plasma filtrate

entering the glomerulus is 1.010
o Isosthenuric- term to describe urine
with SG 1.010
Amorphous urate precipitate o Hyposthenuric/ diluted urine term to
describe urine with SG below 1.010
URINE ODOR o Hypersthenuric / concentrated urine-
 Normal Odor term to describe urine with SG above
o Fresh urine: Slightly aromatic 1.010
o After standing: Stronger ammonia odor due  SG of normal random urine: 1.002 > 1.035
to urea breakdown  Most of the random specimen falls between
 Abnormal Odors 1.015-1.030
o Bacterial Infections: Strong, unpleasant  Abnormally high SG results- >1.040 – are
smell seen in patients who have recently undergone
o Diabetic Ketones: Sweet or fruity odor an intravenous pyelogram (ex. Radiographic
o Maple Syrup Urine Disease: Sweet, maple contrast dye)
syrup-like odor
o Foods: Asparagus, garlic, onions
 Genetic Factors
o Variations in odor perception

Methods for Detection of Urine S.G.

 Direct methods – hydrometer, harmonic
oscillation, densitometer, falling drop method
 Indirect methods – refractometer, reagent strip
  Calibrating fluid
o Potassium sulphate = SG should be
read at 1.015
o Water = SG should be read at 1.00

Reagent strip
 The reagent strip reaction is based on the
change in the pKa (dissociation constant) of a
polyelectrolyte in an alkaline medium
 S.G. reading is not affected by radiographic
dye, protein and glucose.

Hydrometer (urinometer)
The urinometer consists of a weighted float attach
to a scale that has been calibrated in terms of REFRACTOMETER (TS meter/total solid meter)
urine gravity
 When using urinometer, an
adequate amount of urine is
poured into proper-sized
container and the urinometer
is added with a spinning
motion. The scale reading then
taken at the bottom of the
Urine meniscus.
 A major disadvantage of using
a urinometer to measure
specific gravity is that it
requires a 10-15 ml of
specimen.
 It determines the concentration of dissolved
 It needs to be corrected for temperature,
particles in a specimen. It does this by
glucose and protein,
measuring refractive index.
 Less accurate than other methods and not o Refractive index is a comparison of the
recommended by CLSI. velocity of light in air with the velocity of
 The calibrated temperature on the instrument light in a solution (urine)
is usually about 20C o It needs only small volume of specimen
 To correct for the S.G.: (memorize) (one or 2 drops)
o Add 0.001 for every 3degC above the o Temp correction is not necessary
calibration temp. calculated
o Subtract 0.001 for every 3degC below o Temp is compensated bet 15degC and
the calibration temp. 38degC
o Subtract 0.004 for every 1 gram of
glucose (4G)
o Subtract 0.003 for every 1 gram of
protein (Pro3n)
 Correction for glucose and protein
o glucose= subtract 0.004/gram
o protein= subtract 0.003/gram
 Calibrating fluid
o Water = SG should be 1.000
o 3% NaCl = 1.015 +/- 0.001
o 5% NaCl = 1.022 +/- 0.001
o 9% sucrose = 1.034 +/- 0.001
o Triple distilled water = 1.000 (board exam
must know)
 Calibration of the refractometer is performed
using a calibration screw.
 MODULE 4  To prevent chemical run-over between adjacent
CHEMICAL EXAMINATION pads and distortion of colors, blot the edge of
REAGENT STRIPS the strip with absorbent paper and hold the
 Reagent strips offer a simple and rapid strip horizaontally, facing downward, when
method for performing clinically significant comparing it to the color chart.
urine analyses.  Hold the strip close to the color chart without
 These strips feature chemically placing it directly on the chart.
impregnated absorbent pads attached to a  Allow refrigerated specimens to return to room
plastic strip. When the pad comes into temperature before testing, as enzymatic
contact with urine, a color-producing reactions on the strips are temperature-
chemical reaction occurs. Testing is dependent.
conducted using a fresh, well-mixed,  Ensure that reactions are allowed to proceed
uncentrifuged urine sample. The strips test for the proper amount of time for accurate
for 10 parameters: pH, protein, glucose, results.
ketones, blood, bilirubin, urobilinogen,
nitrite, leukocytes, and specific gravity Handling and Storing Reagent Strips
(S.G.).  Reagent strips are packaged in opaque, tightly
 An additional parameter, not included in sealed containers with a desiccant to protect
the list is ascorbic acid/Vitamin C. them from light and moisture.
 Store the strips at temperatures below 30°C
Technique/Procedure (room temperature) and do not freeze.
1. Briefly dip the reagent strip (for no more  Remove strips just before use and immediately
than 1 second) into a well-mixed, reseal the bottle tightly.
uncentrifuged urine sample at room  Avoid exposing the strips to volatile fumes.
temperature (RT).  Do not use strips past their expiration date.
2. Remove excess urine by touching the edge  Do not use strips if the chemical pads appear
of the strip to the side of the container as discolored.
you withdraw it.  Discard any strips that show signs of
3. Gently blot the edge of the strip on a deterioration, contamination, or improper
disposable absorbent pad. storage.
4. Allow the strip to sit for the specified  Ensure refrigerated specimens return to room
reaction time. temperature before testing, as enzymatic
5. Compare the color reaction on the strip to reactions on the strips are temperature-
the manufacturer’s color chart under good dependent.
lighting conditions.
Quality Control of Reagent Strips
 Reagent strips should be tested with both
positive and negative controls at least once
every 24 hours. Many laboratories perform this
quality check at the start of each shift.
 Distilled water is not suitable as a negative
control because reagent strip reactions are
optimized for ionic concentrations similar to
those in urine.
 All negative control readings must show
negative results.
 Positive control readings should match the
published values for the control.

Urinary pH 60 seconds
Importance:
Errors from Improper Technique
 Identification: Helps in identifying urinary
 Formed elements like white blood cells (WBC)
crystals and determining whether specimens
and red blood cells (RBC) can settle at the
are satisfactory
bottom of the specimen and may not be
 Systemic Acid-Base Balance: Assists in
detected if the urine is not well mixed.
assessing systemic acid-base balance
 Avoid leaving the strip in the urine for too
disorders. Urinary pH is primarily regulated by
long, as this may cause the reagents to
diet but can also be influenced by medications.
leach out from the pads.
 Clinical Significance Source of Error/Interference:
o Aidds in the treatment of urinary tract  No known interfering substances
infections  Run-over from adjacent pads
o Assists in managing and preventing  Old specimens
renal calculi (kidney stones)
 Normal urine pH: 4.5 – 8.0 Specific Gravity 45 seconds
 First morning urine pH: 5.0 – 6.0  Definition: Specific gravity measures the
 Abnormal pH values: density of a urine sample compared to the
o improperly preserved specimen: pH>9 density of an equal volume of distilled water at
or >8.5 the same temperature. It reflects the
o Note: presence of detergent in the urine concentration of solutes in the urine.
container can lead to alkalization of the  Influencing Factors: The specific gravity is
urine. affected by the number and size of particles in
the solution.
CAUSES OF ACIDIC URINE  Measurement: The reagent strip specific
 Emphysema gravity test does not measure the total solute
 Diabetes mellitus content but only those solutes that are ionic.
 Starvation  Normal Random Urine SG: Typically ranges
 Dehydration from 1.002 to 1.035
 Cranberry juice
 Special Cases:
 High protein diet o Radiographic contrast dye: SP can
 Foods rich in fats/lipids exceed 1.040
 Presence of acid-producing bacteria (e.g., E. o Diluted urine: SP is less than 1.002
coli)
 Additional Notes:
 Medications such as Mandelamine and
o A double indicator system is used
Fosfomycin
for accurate measurement in both
 Renal tubular acidosis
acidic and alkaline urine.
o Consistency: Urine is generally
CAUSES OF ALKALINE URINE
acidic, with pH levels falling in the
 Hyperventilation
acidic range (pH 5-8).
 Vomiting
 Vegetarian diet
Reagent Strip Reaction (45 seconds)
 Old specimens
 Presence of urease-producing bacteria (e.g.,
Principle: The test measures changes in the pKa
Proteus spp. and Pseudomonas spp.)
(dissociation constant) of a polyelectrolyte in
 Alkaline tide (following meals)
response to urine's ionic strength. The color
 Citrus foods such as pomelo, lime, orange,
change from blue to green to yellow reflects
lemons, and grapefruits
increasing urine density and specific gravity:
 Blue: Low SP (dilute urine)
Trivia:
Cranberry juice is known to produce acidic urine  Green: Intermediate SP
and has traditionally been used as a home remedy  Yellow: High SP (concentrated urine)
for minor bladder infections. It helps inhibit the
colonization of certain urinary pathogens. Reagents:
Individuals who frequently experience urinary tract  Multistix: Poly (methyl vinyl ether/maleic
infections are often advised to drink cranberry juice anhydride) with bromthymol blue
or take over-the-counter cranberry supplements to  Chemstrip: Ethylene glycol diamineoethyl
help manage and prevent these infections. ether tetra-acetic acid with bromthymol blue

Reagent Strip Reaction (60 seconds) Sensitivity:

 Ranges from 1.000 (Blue) to 1.030 (Yellow)
Principle: Double Indicator System
The reagent strip uses two indicators, Methyl Red Interference:
and Bromthymol Blue, to measure pH levels.  False positive: High protein concentration
Methyl Red changes color in acidic conditions (pH (100-500mg/dL) or ketoacidosis
4.0-6.0), shifting from red to yellow. Bromthymol  False negative: Highly alkaline urines (pH
Blue changes color in alkaline conditions (pH 6.0- greater than 6.5) may result in an artificially
9.0), shifting from green to blue. low

Reagents: Methyl Red and Bromthymol Blue

 Protein 60 seconds Protein Levels: The amount of protein in the
Significance: urine due to glomerular damage can
 Protein in urine is a key indicator of renal range from slightly above normal to as
disease. It is often used to assess kidney much as 4 g/day
function and detect potential issues.
A. Diabetic Nephropathy (Kimmelstiel-
Normal Levels: Wilson’s Disease)
 Urine typically contains very little protein: less  Characteristics:
than 10 mg/dL or 100 mg per 24 hours o Decreased Glomerular Filtration
(Strasinger). Rate (GFR).
 According to Henry’s, normal urine contains o Often associated with renal
less than 150 mg of protein per 24 hours failure in individuals with Type I
and II diabetes mellitus.
Major Protein: Albumin is the primary protein
found in normal urine due to its low molecular  Indicator:
weight. o Microalbuminuria: Presence of
albumin in the urine that is
Other Proteins typically found: above normal levels but below
 Serum and tubular microglobulins the detectable range of
 Tamm-Horsfall protein (Uromodulin) conventional urine dipstick
 Proteins from prostatic and vaginal methods.
o Microalbuminuria is also linked
Appearance: Proteinuria can produce a white with an increased risk of
foam in the urine. cardiovascular disease
Clinical Proteinuria: Defined as >30 mg/dL or B. Amyloidosis
>300 mg/L  A condition where abnormal protein
deposits accumulate in the kidneys,
impairing their function.
Pre-Renal or Overflow Proteinuria
This type of proteinuria results from conditions C. Immune Complexes:
affecting plasma before it reaches the kidneys,
 Systemic Lupus Erythematosus (SLE):
leading to an overflow of proteins into the urine.
Autoimmune disease where immune
complexes deposit in the kidneys.
Causes:
 Streptococcal Glomerulonephritis: A
 Hemoglobin: Resulting from intravascular
kidney condition following streptococcal
hemolysis.
infections, where immune complexes affect
 Myoglobin: Due to muscle injury. glomerular function.
 Acute Phase Reactants: Associated with
inflammation and infections. D. Toxic Substances
 Bence Jones Protein: Found in multiple  Certain toxins can damage the glomeruli
myeloma and cause proteinuria

E. Pre-eclampsia and Eclampsia

 Pregnancy-related conditions that can lead
to significant proteinuria and renal
impairment.

F. Orthostatic / Cadet / Postural Proteinuria

 Definition: Proteinuria that occurs when
Renal Proteinuria / True Renal Disease standing and resolves when lying down.
I. Glomerular Proteinuria  Urine Specimen
Definition: It occurs when the glomerular o Orthostatic Proteinuria:
membrane is damaged, impairing Protein levels are normal in the
selective filtration. This damage allows first morning urine and elevated
increased amounts of serum proteins, 2 hours after standing
and potentially red and white blood
cells, to pass through the membrane II. Glomerular Proteinuria
and appear in the urine. Definition: : Tubular proteinuria occurs when
normally filtered proteins, such as
 albumin, cannot be reabsorbed by the
renal tubules and thus appear in the Tests for Microalbuminuria
urine. 1. Albumin Normal: 0 – 20ug/min
Examples: Excretion Microalbuminuria: 20-200
 Fanconi’s Syndrome: A disorder affecting Rate (AER) ug/min or 30-300mg/24hrs
the renal tubules' ability to reabsorb 2. Immunologic Principle: Enzyme immunoassay
nutrients and proteins. Micral-Test
Sensitivity: 0 to 10mg/dL
 Toxic Agents/Heavy Metals: Exposure to Reagents: Gold-labeled antibody,
substances like cadmium can damage the B-galactosidase, Chlorophenyl red
renal tubules. galactoside
 Severe Viral Infections: Certain viral Procedure:
infections can impair tubular function.  Dip the strip into the urine up
to the marked level and hold
Post – Renal Proteinuria for 5 seconds.
Post-renal proteinuria arises from conditions  Reading time is 1 minute.
affecting the urinary tract after the urine has left Results:
the kidneys. This type of proteinuria results from  Negative: White color
contamination or issues in the lower urinary tract.  Positive: Red color
Interferences:
Causes:  False-positive: Strong
 Lower Urinary Tract Infections (UTI): oxidizing agents (e.g., soap).
Infections in the bladder or urethra can lead to  False-negative: Dilute urine.
the presence of proteins in the urine. 3. ImmunoDi Principle: Immunochromatographic
 Inflammations: Inflammatory conditions in the p assay
lower urinary tract can cause protein leakage. Sensitivity: 1.2 to 8.0 mg/dL.
 Injury/Trauma: Physical injury or trauma to Reagents: Antibody-coated blue
the lower urinary tract can result in the latex particles.
presence of proteins in the urine. Procedure:
 Menstrual Contamination: Blood from  Strips are individually
menstruation can contaminate the urine packaged and placed in the
sample, leading to proteinuria. urine specimen for 3 minutes.
 Prostatic Fluid/Spermatozoa: In males, Appearance and Interpretation:
proteinuria can occur due to the presence of  Darker bottom band: <1.2
prostatic fluid or spermatozoa in the urine. mg/dL (Negative).
 Vaginal Secretions: In females, proteinuria  Equal band colors: 1.2 to 1.8
may be due to vaginal secretions contaminating mg/dL (Borderline).
the urine sample.  Darker top band: 2 to 8 mg/dL
(Positive).
Benign Proteinuria Interference:
The presence of protein in the urine is not always  False-negative: Dilute urine
indicative of a pathological condition. Benign
proteinuria can occur due to various non-disease-
related factors. Albumin: Creatinine Ratio
The Clinitek Microalbumin reagent strips and the
Characteristics: Multistix Pro reagent strips provide simultaneous
 Transience: Benign proteinuria is often measurement of albumin and creatinine. This
temporary and resolves when the underlying allows for the estimation of 24-hour microalbumin
cause is addressed. excretion.
Procedure
Common Causes:  Strips can be read manually or on automated
 Strenuous Exercise: Intense physical activity Clinitek instruments, which automatically
can lead to temporary proteinuria. calculate results.
 High Fever: Elevated body temperature can  Reporting
cause a transient increase in urinary protein. o Abnormal Albumin
 Dehydration: Reduced fluid intake or loss can o Ratio: 30 to 300 mg/g or 3.4 to
concentrate urine and increase protein levels. 33.9 mg/mmol
 Exposure to Cold: Cold temperatures can
cause temporary proteinuria due to
physiological stress on the body.
 Albumin Strip  Sensitivity: The test is more sensitive to
Principle: Uses the dye bis (3',3"-diiodo-4',4"- albumin due to its higher content of amino
dihydroxy-5',5"-dinitrophenyl)-3,4,5,6- groups, which readily accept hydrogen ions.
tetrabromosulfonephthalein (DIDNTB),  pH Stability: The test maintains a constant pH
which provides high sensitivity and (pH 3, buffered with citrate) to ensure accurate
specificity for albumin. results
Measurement Range: 8 to 15 mg/dL (80 to 150  Specificity: The reagent strip is sensitive
mg/L) of albumin, minimizing interference specifically to albumin
from other proteins.
Color Range: From pale green (low albumin) to Reagents
aqua blue (high albumin).  Multistix: Tetrabromphenol blue
Interferences:  Chemstrip:
 Highly Buffered Alkaline Urine: Controlled by Tetrachlorophenoltetrabromosulfonapthalein
using paper treated with bis-(heptapropylene
glycol) carbonate. Grading and Quantity
 Falsely Elevated Results: Can be caused by  Trace: <30 mg/dL
visibly bloody urine or abnormally colored  1+: 30 mg/dL
urine.  2+: 100 mg/dL
 Polymethyl Vinyl Ether: Reduces nonspecific  3+: 300 mg/dL
binding of polyamino acids to the albumin pad.  4+: 2000 mg/dL

Creatinine Strip Interferences:

Principle: Measures pseudoperoxidase activity of False Positive:
copper-creatinine complexes.  Highly buffered alkaline urine.
Reagents: Copper sulfate (CuSO4), 3,3',5,5'-  Pigmented specimens (e.g., phenazopyridine).
tetramethylbenzidine (TMB), and  Quaternary ammonium compounds
diisopropyl benzene dihydroperoxide (detergents).
(DBDH).
Color Range: From orange (negative) to green and False Negative:
blue (positive).  Loss of buffer due to prolonged exposure of the
Measurement Range: Reported as 10, 50, 100, strip to the specimen.
200, 300 mg/dL, or 0.9, 4.4, 8.8, 17.7, 26.5  High specific gravity.
mmol/L.  Proteins other than albumin.
Purpose: The creatinine measurement is used to  Microalbuminuria, which may not be detected
normalize the albumin concentration by this test due to its lower sensitivity for small
according to urine concentration, allowing amounts of albumin.
calculation of a semi-quantitative albumin
(A:C) ratio. Note:
Interferences:  The specific gravity of the urine specimen
 False Increased: Visibly bloody urine, presence should be considered when evaluating protein
of the gastric acid-reducing medication levels. A trace amount of protein in a dilute
cimetidine (Tagamet), and abnormally colored sample is more clinically significant than the
urines same amount in a concentrated sample.

Reagent Strip Reaction for PROTEIN (60 Sulfosalicylic Acid (SSA) Precipitation Test
seconds) Principle:
 A cold precipitation test that reacts with all
Principle: The protein test uses the Sörensen’s forms of protein equally, providing an overall
error of indicator principle. The indicator measure of proteinuria.
reacts with proteins, primarily albumin, Procedure:
resulting in a color change. 1. Mix 3 mL of 3% SSA (Exton’s reagent) or 7%
Reaction SSA with 3 mL of centrifuged urine.
 Indicator + Protein → Protein + Hydrogen 2. Observe for cloudiness, which indicates the
 Color Change: From yellow (negative) to blue- presence of protein.
green (positive)

Details
 Proteins: Mainly albumin accepts hydrogen
ions from the indicator.
  Normal blood glucose, Increased Urine glucose
 Causes:
o Fanconi’s syndrome, Advanced renal
disease, Osteomalacia, Pregnancy,
ESRD (End stage renal disease),
Cystinosis

Reagent Strip Reaction for Glucose (30 seconds)

Interferences:
Principle: Double sequential enzyme reaction
False Increase/Positive:
 Radiographic contrast dye/x-ray film. Glucose oxidase
Glucose + O2 Gluconic acid + H2O2
 Drugs such as Tolbutamide, Penicillin,
Sulfonamide, Cephalosporin. H2O2 + Chromgen Oxidized chromgen + H2O
 Para-amino-salicylic acid/Salicylates.
False Decrease/Negative: STEP: In the first step, glucose oxidase catalyzes a
 Highly alkaline urine. reaction between glucose and room air (oxygen) to
 Quaternary ammonium compounds (e.g., produce gluconic acid and peroxide. In the second
detergents, soap). step, peroxidase catalyzes the reaction between
peroxide and chromogen to form an oxidized
Microscopic Patterns: colored compound that is directly proportional to
 If Proteins Cause a Positive Reaction: The SSA the concentration of glucose.
pattern will show amorphousprecipitation.
 If Drugs and Radiographic Contrast Dye Cause Reagents
a Positive Reaction: The SSA pattern will show  Multistix = glucose oxidase, peroxidase,
crystalline precipitation Potassium iodide (blue to green to brown)
 Chemstrip = glucose oxidase, peroxidase,
Glucose tetramethylbenzidine (yellow to green)
 Most frequently performed chemical analysis  Other chromogens: Aminopropylcarbazole
on urine (due to monitoring of DM). (yellow to orange brown) and O-toluidine (pink
 Renal threshold- plasma concentration of a to purple)
substance at which tubular reabsorption stops
 Renal threshold for glucose = 160-180 mg/dl Grading
 Specimens used:
o Fasting or second morning /8-Hours
urine sample or First morning urine /
2hr post prandial
 A first morning specimen does not always
represent a fasting specimen because glucose Interferences
from an evening meal may remain in the False positive:
bladder overnight, and patients should be  Contamination of oxidizing agents and
advised to empty the bladder and collect the detergents
second specimen False negative:
 High levels of ascorbic acid
Clinical Significance of Urine Glucose  High levels of ketones
Hyperglycemia – Associated  High SG
 Increased blood glucose, increased urine  Low temp
glucose normal blood glucose, increased urine  Improperly preserved specimens
glucose
 Causes: Iodate- It is added by the manufacturers in the
o Diabetes Mellitus and Gestational glucose reagent strip to minimize interference by
Diabetes Mellitus, Pancreatitis and ascorbic acid. This chemical oxidizes ascorbic acid
Pancreatic Cancer, Pheochromocytoma, so that it cannot interfere with the oxidation of the
Acromegaly, Cushing syndrome, chromogen
Hyperthyroidism, Liver disease,
Cerebrovascular accident / stroke Copper Reduction Test
(Clinitest/Benedict’s Test)
Renal – Associated  Non-specific test for reducing sugars such as
 Glucose, galactose, fructose, maltose, lactose, cannot be used as a confirmatory test for
pentoses glucose
 Sucrose – a non-reducing sugar and cannot be
detected by this test Interferences
False Positive:
Clinical significance:  Reducing agents such as vitamin C, formalin,
For the detection of inborn error of metabolism uric acid, and some drugs
especially galactosemia in newborns in which there (cephalosporin)
is a lack of enzyme galactose -1- phosphate False Negative:
uridyltransferase  Oxidizing agents such as detergent

Component of the Tablet Grading

 Copper sulfate- main reacting agent
 Sodium carbonate – eliminates interfering O2
(room air)
 Sodium citrate /citric acid – for heat
production
 Sodium hydroxide – for heat production

The tablet, when placed in a mixture of water and

urine, the tablet is rapidly dissolved by the action
of sodium carbonate and citric acid which act as
an effervescent. The sodium hydroxide provides the
alkaline medium necessary for the reaction, and
the heat required is provided by the reaction of
sodium hydroxide with water and citric acid

Procedure:
5 gtts urine + 10gtts distilled H2O + Clinitest tablet
---→ observe the reaction Ketones
Note: Wait 15 seconds after boiling (there is  Result from increased fat metabolism. They
effervescent formation) has stopped and gently are formed from beta oxidation of fats.
shake the contents of the tube  Inability to metabolize or utilize available
carbohydrate –DM type1
 Upon addition of the tablet to water and urine,  Increased loss of carbohydrates – Vomiting
heat is produced by the hydrolysis of sodium  Inadequate intake of carbohydrate – Starvation
hydroxide and its reaction with sodium citrate, and malabsorption/pancreatic disorder
and carbon dioxide is released from the sodium  Overuse of available carbohydrates – Frequent
carbonate to prevent room air from interfering strenuous exercise
with the reduction reaction.  Ketone Bodies:
 Clinitest tablets are very hygroscopic and o 78% Beta Hydroxybutyric acid –
should be stored in their tightly closed major ketone but not detected in
packages. A strong blue color in the unused reagent strip
tablets suggests deterioration due to moisture o 20% Acetoacetic acid (AAA) /
accumulation, as does vigorous tablet fizzing. Diacetic acid – parent ketone (primarily
detected)
Pass through phenomenon: o 2 % Acetone – detected only when
 Occurs when greater than 2 g/dl sugar is glycine is present
present When the blood ketone concentration exceeds 70
 From blue > green > yellow > orange/brick red mg/dL (the renal threshold level), ketones are
> green brown excreted in the urine
 To prevent pass through, use 2 gtts urine
Reagent Strip Reaction for Ketones (40
Principle: Copper Reduction seconds)
CuSO4(cupric sulfide) + reducing substance Principle: sodium nitroprusside reaction (legal’s
Cu2O (cuprous oxide) + oxidized substance test)
color Acetoacetate and acetone + sodium nitroprussidee
 The sensitivity of Clinitest to glucose is reduced + glycine (+) Purple
to a minimum of 200 mg/dL so the Clinitest
Reagents: Blood
Sodium nitroprusside (nitroferricyanide), glycine  The finding of a positive reagent strip test
(Chemstrip) result for blood indicates the presence of red
blood cells, hemoglobin, or myoglobin.
Reporting / Grading  Any amount of blood greater than five cells
per microliter of urine is considered clinically
significant

HEMATURIA
 Cloudy red urine
 Presence of an intact RBC
 Produces a speckled/spotted pattern on
reagent pad
 Seen in cases of:
o Glomerulonephritis
o Renal calculi
Interference o Pyelonephritis
False positive: o Tumors
 Phthalein dyes o Trauma
 Highly pigmented red urine o Anticoagulants
 Levodopa o Strenuous exercise
 Medications containing free sulfhydryl groups o Hypertension
including mercaptoethane sulfonate sodium o Cystitis
(MESNA) and captopril o Exposure to toxic chemical
False negative:
 Improperly preserved specimens HEMOGLOBINURIA
 Clear red urine
ACETEST TABLET  Uniform green / blue color in reagent strip pad
 Composition:  Hemoglobinuria may result from the lysis of red
o Sodium nitroprusside blood cells produced in the urinary tract,
o Disodium phosphate particularly in dilute, alkaline urine
o Lactose –gives better color  Seen in cases of:
differentiation o Transfusion reactions
 The Acetest tablet test has been used as a o Hemolytic anemias - intravascular
confirmatory test for questionable reagent hemolysis
strip results; however, it was primarily used o Severe burns
for testing serum and other bodily fluids and o Infections: malaria, syphilis,
dilutions of these fluids for severe ketosis mycoplasma, and C.perfringens
 Read for 30 seconds o Strenuous exercise
 Report as negative, small (5 to10mg/dl), o Brown recluse spider bites
moderate (30 to 40mg/dl), or large (80 to Note
100mg/dl). Hemoglobin must be present in the urine in an
 Acetest tablets are hygroscopic; if the amount exceeding 10 mg/dL before it is detected
specimen is not completely absorbed within 30 by routine protein reagent strip test
seconds, a new tablet should be used.
 Acetest can be used to test urine, serum, MYOGLOBINURIA
plasma, or whole blood  Clear red urine
 It is 10 times more sensitive to diacetic acid  Heme portion of the myoglobin is toxic to the
than to acetone renal tubules
 Uniform green / blue color in reagent strip pad
ACETEST Procedure:  Seen in cases of:
1. Remove the Acetest tablet from the bottle and o Rhabdomyolysis- muscle disease
place on a clean, dry piece of white paper. o Prolonged coma
2. Place 1 drop of urine on top of the tablet. o Convulsions
3. Wait 30 seconds. o Extensive exertion
4. Compare the tablet color with the o Muscle wasting diseases
manufacturer-supplied color chart. o Cholesterol- lowering statin medications
5. Report as negative, small, moderate or large. o Muscle ischemia: carbon monoxide
poisoning
 o Muscle infection(myositis) False positive:
o Trauma  Strong oxidizing agents b.
o Crush syndrome  Vegetable and Bacterial peroxidases (e.g.,
o ALCOHOLISM Escherichia coli)
o Heroin abuse  Menstrual contamination

The heme portion of myoglobin is toxic to the False negative:

renal tubules, and high concentrations can cause  High SG / Crenated cells
acute renal failure

 Formalin
Reagent Strip Reaction for Blood (60 seconds)  Captopril
 Ascorbic acid (>25mg/dl)
Principle: Pseudoperoxidase activity of  Unmixed specimen / failure to mix the
Hemoglobin specimen prior to testing
Hemoglobin
 High concentration of nitrite (>10mg/dl)
H2O2 + chromogen oxidized chromogen +
H2O Pseudoperoxidase Bilirubin
 The appearance of bilirubin in the urine can
(-) Yellow; (+) Green to Blue
provide an early indication of liver disease.
Note:
 It is associated with:
 Reagent strip tests can detect concentrations
o Hepatic jaundice = Hepatitis and
as low as five red blood cells per microliter.
Cirrhosis
 Through pseudoperoxidase activity of the heme
o Post hepatic jaundice = Biliary
moiety, peroxide is reduced and the chromogen
obstruction (gallstones, carcinoma)
becomes oxidized, producing a color change on
 Only the B2 or conjugated bilirubin is water
the reaction pad from yellow to green
soluble thus can be seen in urine and can be
detected.
Reagents
Multistix  It produces an amber urine with yellow foam
 Conjugated bilirubin is normally excreted in
 diisopropylbenzenedehydroperoxidetetramet
the bile into the duodenum, and normal adult
hylbenzidine
Chemstrip urine contains only 0.02 mg of bilirubin per
deciliter. This small amount is not detected by
 dimethyldihydroperoxyhexanetetramethylbe
the usual testing methods.
nzidine
 Excretion of bilirubin is enhanced by alkalosis
Interferences:
Bilirubin
 highly pigmented yellow compound
 a degradation product of hemoglobin
 Normal conditions: Life span of RBC is 120
days, at which time they are destroyed in the
spleen and liver by the phagocytic cells of the
reticuloendothelial system
 Liberated hemoglobin -> broken down into its
component parts:
o iron
o protein Reagent Strip Reaction for Bilirubin (30
o protoporphyrin seconds)
Principle: Diazo reaction
 Body reuses iron and protein while
reticuloendothelial system convert
protoporphyrin to bilirubin
 Bilirubin is then released into the circulation,
where it binds with albumin & is transported Reagents
to the liver Multistix
 At this point, the kidneys cannot excrete the  2,4-dichloroaniline diazonium salt
circulating bilirubin because not only is it Chemstrip
bound to albumin, but it is also water insoluble  2,6-dichlorobenze diazonium tetrafluoroborate
(unconjugated bilirubin)
Interference
False positive:
 Highly pigmented urines such as
phenazopyridine
 Indican
 Metabolites of Lodine Reagent Strip Reaction for Urobilinogen (60
False negative: seconds)
 Specimen exposure to light Principle: Ehrlich’s reaction
 Ascorbic acid Multistix: Uses Ehrlich reagent
 High concentration of nitrite
Urobilinogen + p-dimethylaminobenzaldehyde ->
Reagent strip color reactions for bilirubin are more red color
difficult to interpret than other reagent strip
reactions and are easily influenced by other Chemstrip: Uses 4-methyloxybenzene-diazonium-
pigments present in the urine tetrafluoroborate (more specific than ehrlich’s rxn)

ICOTEST (Tablet) for BILIRUBIN Urobilinogen + diazonium salt -> red azodye
 A confirmatory test for bilirubin is the Ictotest (commonly used nowadays)
 Ictotest is much more sensitive than the
dipsticks, being able to detect as little as 0.05 Note:
mg/dL  Ehrlich-reactive compounds:
porphobilinogen, indican, p-aminosalicylic
Components: acid, sulfonamides, methyldopa, procaine,
 p-nitrobenzene-diazonium p- chlorpromazine --- also gives positive
toluenesulfonate reaction for Ehrlich’s reaction
 SSA
Interference
 Sodium carbonate
False positive:
 Boric acid
 Other Ehrlich’s compound
Positive reaction (+): Blue to purple color  Highly pigmented urine
False negative:
 Old specimens
 Preservation in formalin
 Improperly preserved, allowing urobilinogen
to be photo-oxidized to urobilin.
 High concentration of nitrite

WATSON-SCHWARTZ TEST
 Used to differentiate urobilinogen,
porphobilinogen, and other Ehrlich reactive
compounds
 Uses extraction with organic solvents
chloroform and Butanol

Urobilinogen
 A bile pigment that results from hemoglobin
degradation
 Conjugated bilirubin is reduced by intestinal
bacteria into urobilinogen
 A small amount of urobilinogen – less than
1mg/dl or Ehrlich unit – is normally found in
the urine.
 Clinical significance: urine urobilinogen greater
than 1 mg/dl is seen in; Pre hepatic jaundice
such as hemolytic disorders and hepatic
jaundice such ash liver disease

Constipation can RAISE Urobilinogen level

1% of the non-hospitalized population and 9% of a
hospitalized population exhibit elevated results.
This is frequently caused byconstipation HOESCH TEST (Inverse Ehrlich Reaction)
  A Rapid screening test for porphobilinogen solution. The test detects
(>2mg/dl) approximately 2 mg/dL of
 Procedure: porphobilinogen
o 2 gtts urine + 2mL Hoesch reagent o Urobilinogen is inhibited by the
(Ehrlich’s reagent dissolved in 6M or highly acidic pH
6N HCL)- →(+) Red on top of o High concentrations of methyldopa
solution and indican, and highly pigmented
 Interpretation and comments: urines, may produce false positive
o When the tube is shaken, the red results.
color is seen throughout the

Note:
Nitrite  Positive result should uniform/Homogenous
Principle: Greiss reaction pink
 Pink spots/edge is considered as NEGATIVE
 Results are reported only as negative or
positive.
Note:  Positive nitrite corresponds to 100,000
Nitrite at an acidic pH reacts with an aromatic organisms/ml (1x10^5 CFU/ml)
amine (para-arsanilic acid or sulfanilamide) to form o It is for gram negative bacteria/bacilli
a diazonium compound that then reacts with which are mostly nitrite positive
tetrahydrobenzoquinolin compounds to produce a o Enterobacteriaceae/coliform gives
pink-colored azodye nitrite positive result

Reagents Leukocytes
Multistix  Significance:
 p-arsanilic acid, tehtrahydrobenzoquinolin- o UTI/inflammation
3-ol o Screening of urine culture specimen
Chemstrip o Bacterial and non-bacterial infection
 sulfanilamide,  It detects the presence of leukocyte that have
hydroxytetrahydrobenzoquinoline been lysed, particularly in dilute alkaline urine
 It offers a more standardized means for
Interference detection of leukocytes
False positive:  The test is not designed to measure the
 Improperly preserved specimens concentration of leukocytes, and it is
 Highly pigmented urine recommended that quantitation should be done
False Negative by microscopic examination.
 Non reductase containing bacteria  LE test detects esterase found in:
 Insufficient contact time between bacteria o Neutrophil
and urinary nitrate o Basophil
 Large quantities of bacteria converting o Eosinophil
nitrite to nitrogen o Monocytes
 Presence of antibiotics o Trichomonas
 Ascorbic acid o Chlamydia
 High specific gravity o Yeast
o Histiocytes
 NEGATIVE FOR LYMPHOCYTES Sensitivity
o Screening urine specimens using LE  Multistix = 5 to 15 WBC/hpf
test should be correlated with nitrite  Chemstrip = 10 to 25 WBC/hpf
chemical reactions
o Lymphocytes, erythrocytes, bacteria, Interference
and renal tissue cells do not contain False positive:
esterases  Strong oxidizing agents
o Infections involving trichomonads,  Formalin
mycoses (e.g., yeast), chlamydia,  Highly pigmented urine, nitrofurantoin, beets,
mycoplasmas, viruses, or tuberculosis phenazopyridine
cause leukocyturia or pyuria without  False-positive results for leukocyte esterase are
bacteriuria most often obtained on urine specimens
contaminated with vaginal secretions
Reagent Strip Reaction for Leukocytes (120 False negative:
seconds)  High concentration of protein (Greater than
500 mg/dl), high glucose (≥3g/dl), oxalic
Principle: Leukocyte Esterase acid(in acidified urine that has 4.4pH or below)
Indoxylcarbonic acid ester---------→indoxyl + acid and ascorbic acid
indoxyl + Diazonium salt -→ (+) Purple azodye  Antibiotics such as gentamicin, cephalosporins,
tetracyclines,
The reagent strip reaction uses the action of LE to
 Inaccurate timing
catalyze the hydrolysis of an acid ester
embedded on the reagent pad to produce an
Ascorbic Acid
aromatic compound and acid. The aromatic
compound then combines with a diazonium salt  It is the 11th parameter
present on the pad to produce a purple azodye.  Causes False Negative result to BBLNG (Blood,
Bilirubin, Leukocyte, Nitrite, Glucose)
Reagent  Causes False Positive result to Clinitest
Multistix  Ascorbic acid level that causes a negative
 Diazonium salt, derivatized pyrrole amino reaction to Bilirubin and Nitrite - ≥25mg/dl
acid ester  Ascorbic acid level that causes a negative
Chemstrip reaction to glucose- ≥50mg/d
 Diazonium salt, Indoxyl Carbonic acid ester