Clinical Laboratory Assistance:
as part of doctor’s routine
Urine analysis
Clinical microscopy - One of the oldest
laboratory Method that is an axillary
branch of laboratory medicine which
deals with the study of physical,
chemical and microscopic study of body
fluids.
HISTORY
• Analyzing urine was actually the
beginning of laboratory medicine.
• References to the study of urine can
be found in the drawings of cavemen
and in Egyptian hieroglyphics, such
as the Edwin Smith Surgical
Papyrus.
• Pictures of early physicians
commonly showed them examining a
bladder-shaped flask of Urine. Often
these physicians never saw the
patient,only the patient’s urine.
INTRODUCTION TO URINE
• Hippocrates- “uroscopy” 5th Century
• 1140AD – color charts was
developed (20 color) where chemical
testing progressed from and the
“TASTE TESTING “ for glucose and
“Pisse Prophets”
• Frederik Dekkers – albuminuria by
boiling 1694
• 17th Century – examination of urine
sediments through the invention of
microscope.
• Thomas Addison – method for
quantitating urinary sediments
Richard Bright – 1827 introduced urinalysis
Two unique characteristics of a urine
specimen account
for this continued popularity:
1. Urine is a readily available and easily
collected specimen
2. Urine contains information, which can be
obtained by inexpensive laboratory tests,
about many of the body’s major metabolic
functions
• Various diseases/ disorder can be
monitored by urinalysis due its
composition such as
• Body metabolism diseases
• Endocrine function
• Renal function
A significant amount of information can be
obtained through the examination of urine.
Careful examination enables the detection of
disease processes intrinsic to the urinary
system, both functional (physiologic) and
structural (anatomic), and sometimes
unsuspected.
Components of Basic (Routine) Urinalysis
The basic (routine) urinalysis consists of
four parts: specimen evaluation,
gross/physical examination, chemical
screening, and sediment /microscopic
examination.
URINE COMPOSITION
§ 95% water and 5% solutes (conc. of
solutes influenced by: dietary
intake, physical activity, body metabolism,
endocrine functions
IMPORTANT NOTE:
§ Primary organic component: Urea
§ Primary inorganic component: Chloride
SPECIMEN COLLECTION to tubes containing preservatives
for microbiology testing and tubes
§ The urine analysis also helps in with conical bottoms for sediment
(1) monitoring wellness analysis or round bottoms for
(2) determination and treatment of urinary automated reagent strip testing.
tract infections
(3) detection and monitoring progress of
treatment in metabolic diseases
(4) determination of the effectiveness of an
administered therapy as well as checking if
the patients exhibited any therapy-related
complications.

§The accuracy of urine analysis results will

largely depend on several factors such as:
1. collection method
2. container use
3. transportation and handling of specimen
4. timeliness of the testing

CONTAINERS
§ Clear, clean, dry, leak-proof
containers: Disposable containers
§ wide mouth to facilitate
collections from female patients
and a wide, flat bottom to prevent
overturning
§ Individually packaged sterile
containers with secure closures
should be used for microbiologic
urine studies
§ bags with adhesive for the
collection of pediatric specimens
IMPORTANT NOTE:
and large containers for 24-hour
specimens
• Urine from infants - Pliable
polyethylene bags with adhesive
• 24-hour urine collection - Large,
The transfer straw has a needle
wide-mouthed plastic containers are
and an evacuated tube holder. used
• Urine for bacterial culture – sterile
Urine can be sterilely transferred plastic containers
 SPECIMEN LABELS

• patient’s name
• identification number
• date and time of collection
• additional information such as the
patient’s age location and the
healthcare provider’s name

• labels must be attached to the

container

• Laboratory Requisition form

• The information on the form must
match the information on the
specimen label.
• § Method of collection or type of
specimen, possible interfering
medications, and the patient’s
clinical information.
• § The time the specimen is received
in the laboratory should be recorded
on the form

SPECIMEN REJECTION

1. Specimens in unlabeled containers

2. Nonmatching labels and
requisition forms
3. Specimens contaminated with
feces or toilet paper
4. Containers with contaminated
exteriors
5. Specimens of insufficient quantity
6. Specimens that have been
improperly transported
SPECIMEN HANDLING
• § Specimen Preservation
• § The most routinely used method of
preservation is refrigeration at 2°C to
8°C, which decreases bacterial
growth and metabolism.
• § If the urine is to be cultured, it
should be refrigerated during transit
and kept refrigerated until cultured
up to 24 hours.
• § When a specimen must be
transported over a long distance and
refrigeration is impossible, chemical
preservatives may be added.
• § The ideal preservative should be
bactericidal, inhibit urease, and
preserve formed elements in the
sediment.
TYPES OF SPECIMENS
Random Specimen
• This is the most commonly received
specimen because of its ease of
collection and convenience for the
patient.
• Used for routine screening tests but
may also show erroneous results
resulting from dietary intake or
physical activity just before
collection.
First Morning Specimen
• The ideal screening specimen
• Preventing false-negative pregnancy
tests and for evaluating orthostatic
proteinuria
• A concentrated specimen, thereby
assuring detection of chemicals and
formed elements that may not be
present in a dilute random specimen
Glucose Tolerance Specimens
• GTTs may include fasting, half-
hour, 1-hour, 2-hour, and 3-hour
specimens, and possibly 4-hour, 5-
hour, and 6-hour specimens.
• The urine is tested for glucose and
ketones, and the results are reported
along with the blood test results as
an aid to interpreting the patient’s
ability to metabolize a measured
amount of glucose and are
correlated with the renal threshold
for glucose.
24-Hour (or Timed) Specimen
• Measuring the exact amount of a
urine chemical substance
• Many solutes exhibit diurnal
variations such as catecholamines,
17- hydroxysteroids, and
electrolytes
• Diurnal variations and with daily
activities such as exercise, meals,
and body metabolism, 24-hour
collection is required
• A 24-hour specimen must be
thoroughly mixed and the volume
accurately measured and recorded
• If a specimen is collected in two
containers, the contents of the
containers should be combined and
thoroughly mixed before aliquoting.
• Refrigerated or kept on ice during
the collection period and a chemical
preservative may be required to be
added.
Sample 24-Hour (Timed) Specimen
Collection Procedure
§ Provide the patient with written
instructions, and explain the collection
procedure.
§ Provide the patient with the proper
collection container and preservative.
§ E.g Day 1 at 7 a.m.: patient voids and
discards specimen
§ Collects all urine for the next 24 hours.
Refrigerate the specimen.
§ Day 2 at 7 a.m.: patient voids and adds this
urine to previously collected urine.
§ On arrival at laboratory, the entire 24-hour
specimen is thoroughly mixed, and
the volume is measured and recorded.
Catheterized Specimen
• This specimen is collected under
sterile conditions by passing a
hollow tube (catheter) through the
urethra into the bladder.
• The most commonly requested test
on a catheterized specimen in a
bacterial culture.
Midstream Clean-Catch Specimen
• As an alternative to the catheterized
specimen, the midstream clean-catch
specimen provides a safer, less
traumatic method for obtaining urine
for bacterial culture and routine
urinalysis.
• Strong bacterial agents, such as
hexachlorophene or povidone-
iodine, should not be used as
cleansing agents.
• Mild antiseptic towelettes are
recommended.
\
Suprapubic Aspiration
• Collected by external introduction
of a needle through the abdomen
into the bladder.
• It provides a sample for bacterial
culture that is completely free of
extraneous contamination.
• The specimen can also be used for
cytologic examination.
Prostatitis Specimen
(1) Three-Glass Collection
• Prior to collection the area is
cleansed using the male midstream
clean-catch procedure.
• Then instead of discarding the first
urine passed, it is collected in a
sterile container.
• Next, the midstream portion is
collected in another sterile container.
• The prostate is then massaged so
that prostate fluid will be passed
with the remaining urine into a third
sterile container.
• Quantitative cultures are performed
on all specimens, and the first and
third specimens are examined
microscopically.
In prostatic infection, the third specimen will
have a white blood cell/ high- power field
count and a bacterial count 10 times that of
the first specimen. Macrophages containing
lipids may also be present.
(2) Pre- and Post-Massage Test
• In the pre- and post-massage test
(PPMT), a clean-catch midstream
urine specimen is collected.
• A second urine sample is collected
after the prostate is massaged.
• A positive result is significant
bacteruria in the post-massage
specimen of greater than 10 times
the pre-massage count.
Stamey-Mears Test for Prostatitis
• The four-glass method consists of
bacterial cultures of the initial
voided urine (VB1), midstream
urine (VB2), expressed prostatic
secretions (EPS), and a post-
prostatic massage urine specimen
(VB3).
• Urethral infection or inflammation is
tested for by the VB1, and the VB2
tests for urinary bladder infection.
• The prostatic secretions are cultured
and examined for white blood cells.
Pediatric Specimens
• Use of soft, clear plastic bags with
hypoallergenic skin adhesive.
• Sterile specimens may be obtained
by catheterization or by suprapubic
aspiration.
• For routine specimen analysis
ensure the area is free of
contamination. Attach the bag firmly
over the genital area avoiding the
anus.
• For microbiology specimens clean
the area with soap and water and
sterilely dry the area, removing any
residual soap residue. Firmly apply
a sterile bag.
Drug Specimen Collection
• The chain of custody (COC) is the
process that provides this
documentation of proper sample
identification from the time of
collection to the receipt of
laboratory results.
• The COC is a standardized form that
must document and accompany
every step of drug testing, from
collector to courier to laboratory to
medical review officer to employer.
• Proper identification of the
individual whose information is
indicated on the label is required.
• Urine specimen collections may be
“witnessed” or “unwitnessed.”
• If a witnessed specimen collection is
ordered, a same-gender collector
will observe the collection of 30 to
45 mL of urine.
• The urine temperature must be taken
within 4 minutes from the time of
collection to confirm the specimen
has not been adulterated.
• The temperature should read within
the range of 32.5°C to 37.7°C.
• Urine temperatures outside of the
recommended range may indicate
specimen contamination.
• The urine color is also inspected to
identify any signs of contaminants.
COMMON URINE TESTS
Routine urinalysis (UA)
• This frequently ordered urine test
screens the patient for any urinary or
systematic disorders.
• This can be ordered during
hospitalization and also form part of
the physical examination procedure.
• The analysis is not limited to
physical analysis (color, clarity and
odor) but also covers the chemical
(ph, specific gravity, detection of
bacteria, etc.) and microscopic
analysis (urine components such as
cells, crystals and microorganisms)
of the specimen.
Culture & sensitivity (C&S)
• The Culture & sensitivity (C&S)
urine test is requested if the patient
has symptoms of a urinary tract
infection (UTI).
• The urine specimen used should be
midstream clean-catch collection
which is placed in a sterile container.
• A measured portion of urine is
cultured on a special nutrient
medium for 18 to 24 hours.
• If a microorganism is present and
identified, a sensitivity or antibiotic
susceptibility test is performed to
determine the most effective
antibiotic that should be prescribed
for treatment.
Urine drug testing
Drug screening is performed using urine
since many drugs are detected in the urine
but not in the blood.
This test is performed to detect:
(1) illicit use of recreational drugs
(2) use of anabolic steroids to enhance
performance in sports
(3) unwarranted use of prescription drugs. It
also monitors therapeutic drug use to
minimize the symptoms associated with the
withdrawal and also confirm drug overdose.
Urine glucose and ketone testing
• The urine glucose and ketone testing
is performed to screen diabetes and
determine the glucose level for
patients who are already diabetics.
• The urine ketone level test is used to
determine if the patient is suffering
from diabetic ketoacidosis.
Urine pregnancy testing
• The urine pregnancy test is used to
confirm pregnancy which can be
detected 8 to 10 days from
conception.
• The first morning urine specimen is
preferred because it has the highest
concentration of human
gonadotropin (HCG).

Urine cytology studies

• The urine cytology study on urine is
requested by the physician to detect
cancer, cytomegalovirus, & other
viral and inflammatory diseases in
the urinary system.
• A fresh clean-catch specimen is
required.
• A smear from the cells of the lining
of the urinary tract is stained using
the Papanicolaou (PAP) method and
examined under the microscope for
abnormal cells.
• ODOR
• The collected specimen should be
examined immediately. – Not evaluated, but may provide clue to
constituents
– Associations
▪ Fruity
▪ Ammonia
 ▪ Mousy
▪ Maple syrup
• CLARITY
– Normal urine: clear
– Any of the urinary sediments (cells, casts,
crystals or bacteria) may make urine cloudy
• COLOR
– Normal urine: pale yellow (straw) to
yellow color
– Urochrome
• Includes determination of: urine color,
clarity, specific gravity (odor NOT part of
routine PE)
• IMPORTANCE:
– Provides preliminary information
concerning disorders (glomerular bleeding,
liver disease, inborn error of metabolism,
UTI)
– Aids in the evaluation of renal tubular
function
– Confirm or explain clinical findings in
chemical and microscopic analysis
URINE COLOR
• Common descriptions: pale yellow,
yellow, dark yellow
• Variation of urine color may be due to:
– Normal metabolic functions
– Physical activities
– Dietary intake
– Pathologic disorders
• Urochrome:
– Responsible for the normal color of urine
– Named by Thudichum in 1864
– Product of endogenous metabolism and is
dependent in body̕s metabolic state
– urochrome is dependent on the body’s
metabolic state, with increased
amounts produced in thyroid conditions and
fasting
– increases in urine that stands at room
temperature (color of urine becomes darker)
• Other pigments that contribute to urine
color:
– Uroerythrin – pink pigment, indicator that
the specimen is refrigerated (binds
to amorphous urates leading to its
precipitation)
– Urobilin – oxidation product of
urobilinogen, Imparts Orange brown color
of urine that is not fresh
ABNORMAL URINE COLOR
• Dark yellow –concentrated specimen
• Amber – dehydration from fever and burns
• Yellow orange
– Bilirubin
▪ Yellow foam appears when
the specimen is shaken
– Urobilinogen
▪ Due to photo-oxidation of
urobilinogen to urobilin
– Phenazopyridine (Pyridium)
▪ Drug use for UTI
▪ Thick orange pigment that
interferes in chemical tests
that are based in color
reactions
• Yellow green
– Due to photo-oxidation of bilirubin to
biliverdin
• Brown/Black
– RBC remaining in an acidic urine produce
a brown color due to oxidation of
hemoglobin to methemoglobin.
– Glomerular bleeding
– Melanin - oxidation product of the
colorless pigment melanogen, produced in
excess when a malignant melanoma is
present
– Homogentisic Acid- metabolite of
phenylalanine, black color in alkaline urine
from person with IEM called alkaptonuria
• Blue
– Medications like methocarbamol (Ex.
Robaxin-muscle relaxant), methylene blue,
and amitriptyline (Ex. Elavil-antidepressant)

• Green
– Clorets, phenol derivatives
– found in IV medications
– Pseudomonas species
• Purple
– Indicanuria, Bacterial infection (may occur
in catheter bags and is caused by
– indican in the urine or a bacterial infection,
frequently caused by Klebsiella or
Providencia species
• Red
– RBC -Cloudy urine, positive in chemical
test for blood, rbc, observed microscopically
– Hemoglobin - clear urine w positive chem
test, intravascular hemolysis
– myoglobin - clear urine w/ positive
chemical test, muscle damage
– menstrual contamination, rifampin,
phenolphthalein, phenindione,
phenothiazines
– beets – red in alkaline urine
– blackberries – red in acidic urine
URINE CLARITY
• is the general term that refers to
transparency/turbidity of a urine specimen
• freshly voided urine is normally CLEAR
particularly if it is midstream clean-catch
specimen
• precipitation of amorphous phosphates and
carbonates may cause a white cloudiness
• IMPORTANT NOTE:
– Clear urine is not always normal.
However, with the increased sensitivity of
the routine chemical tests, most
abnormalities in clear urine will be detected
prior to the microscopic analysis.
• Common terminology used:
– Clear: no visible particulates, transparent
– Hazy: few particulates, print easily seen
– Cloudy: many particulates, print blurred
– Turbid: print cannot be seen through urine
– Milky: may precipitate or be clotted
URINE SPECIFIC GRAVITY
• is defined as the density of a solution
compared with the density of a similar
volume of distilled water (SG 1.000) at a
similar temperature
• Used to evaluate urine concentration – Calibrators:
(ability of the kidney to concentrate the
▪ Distilled water – SG is 1.000
glomerular filtrate by selectively
▪ 5% NaCl – SG is 1.022 +/- 0.001
reabsorbing essential chemicals and water
▪ 9% Sucrose – 1.034 +/- 0.001
• Normal specific gravity of random
– Advantages:
specimens: 1.003 to 1.035 (most between
1.015-1.030) ▪ Requires small amount of sample
▪ No temperature correction
• Terms used:
– Isosthenuric: 1.010 (SG of plasma filtrate
entering the glomerulus)
– Hyposthenuric: <1.010
– Hyperstenuric: >1.010
– IMPORTANT NOTE:
▪ If SG is below 1.003: not a urine
specimen
▪ If SG is above 1.035: seen in IV
pyelogram (radiographic contrast
media) and dextran
▪
INSTRUMENTS USED TO MEASURE • CORRECTION FOR URINOMETER
AND REFRACTOMETER
URINE SPECIFIC GRAVITY:
– Correction for both urinometer and
A. Urinometer (Hydrometer) refractometer when large amount of protein
– Consist of a weighted float that displaces a or

volume of liquid equal to its weight glucose is present in the urine

– PARTS: Float, weight, stem ▪ For every 1 g/dL of protein: subtract

– Requires 10-15 mL of urine
– DISADVANTAGES:
▪ Less accurate than other available
methods (NOT CLSI
recommended)
▪ Large volume needed
▪ Temperature correction needed
▪ Calibration temperature: 200C
▪ Cold temperature – 0.001 is
subtracted for every 3 degrees
▪ Above calibration temp – 0.001 is
added for every 3 degrees
B. Refractometer
– Refractometry: determines the
concentration of dissolved particles in a
specimen by measuring refractive index
– Parts: Daylight plate, screw calibration,
rubber grip, eyepiece
0.003
▪ For every 1 g/dL of glucose:
subtract 0.004
EXAMPLE:
v Specimen containing 1 g/dL protein and 1
g/dL glucose has a specific gravity of
1.030
v1.030 – (0.003) protein = 1.027 – (0.004)
glucose = 1.023
C. Harmonic Oscillation Densitometry
(HOD)
– It is based on the principle that the
frequency of sound wave entering a solution
changes in proportion to the density of the
solution
D. Reagent strip specific gravity
– reagent strip reaction is based on the
change in pKa (dissociation constant) of a
polyelectrolyte in an alkaline medium
– Indicator: Bromthymol blue (As the
specific gravity increases, the indicator
changes from blue (1.000 [alkaline]),
through shades of green, to yellow (1.030
[acid])
– Readings can be made at 0.005 intervals
with comparison with a color chart
• Errors caused by improper technique:
– Unmixed specimens – cells will
shrink and will be undetected
– Allowing the strip to remain in the
urine for an extended period may cause
leaching of reagents from the pads
– 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 the
colors

URINE ODOR
• it is seldom of clinical significance and is
not a part of the routine urinalysis
• Normal odor: aromatic odor (breakdown of
urea to ammonia is responsible for the
characteristic odor)

CHEMICAL EXAMINATION OF URINE

REAGENT STRIPS
• provide a simple, rapid means for
performing medically significant chemical
analysis of urine (pH, protein, glucose,
ketones, blood, bilirubin, urobilinogen,
nitrite, leukocytes esterase, and specific
gravity)
• Reagent strips consist of chemical-
impregnated absorbent pads attached to a
plastic strip
• Commercially available reagent strips:
– Siemens Medical Solutions
Diagnostics – multistix
– Roche Diagnostics – chemstrip
MICROSCOPIC EXAMINATION OF
URINE