CHAPTER 2: RENAL FUNCTION
RENAL BLOOD FLOW Glomerulus consists of a coil of approximately eight capillary
Renal artery supplies blood to the kidney. Blood enters
the capillaries of the nephron through the afferent arteriole. It
then flows through the glomerulus and into the efferent
arteriole. The varying sizes of these arterioles help to create
the hydrostatic pressure differential important for glomerular
filtration and to maintain consistency of glomerular capillary
pressure and renal blood flow within the glomerulus.
Blood from the efferent arteriole enters the peritubular
capillaries and the vasa recta and flows slowly through the
cortex and medulla of the kidney close to the tubules. The
peritubular capillaries surround the proximal and distal
convoluted tubules, providing for the immediate reabsorption of
essential substances from the fluid in the proximal convoluted
tubule and final adjustment of the urinary composition in the
distal convoluted tubule. The vasa recta are located adjacent to
the ascending and descending loop of Henle in juxtamedullary
nephrons. In this area, the major exchanges of water and salts
take place between the blood and the medullary interstitium.
This exchange maintains the osmotic gradient (salt
concentration) in the medulla, which is necessary for renal
concentration.
GLOMERULAR FILTRATION
lobes referred to collectively as the capillary tuft. It is located within
Bowman’s capsule, which forms the beginning of the renal tubule.
Although the glomerulus serves as a nonselective filter of plasma
substances with molecular weights of less than 70,000, several
factors influence the actual filtration process. These include the
cellular structure of the capillary walls and Bowman’s capsule,
hydrostatic and oncotic pressures, and the feedback mechanisms of
the reninangiotensin-aldosterone system.
CELLULAR STRUCTURE OF THE GLOMERULUS
Plasma filtrate must pass through three cellular layers: the
capillary wall membrane, the basement membrane (basal lamina),
and the visceral epithelium of Bowman’s capsule. The endothelial
cells of the capillary wall differ from those in other capillaries by
containing pores and are referred to as fenestrated.
GLOMERULAR PRESSURE
This pressure is necessary to overcome the opposition of
pressures from the fluid within Bowman’s capsule and the oncotic
pressure of unfiltered plasma proteins in the glomerular capillaries.
By increasing or decreasing the size of the afferent arteriole, an
autoregulatory mechanism within the juxtaglomerular apparatus
maintains the glomerular blood pressure at a relatively constant rate
regardless of fluctuations in systemic blood pressure.
1
 TUBULAR REABSORPTION
Body cannot lose 120 mL of water-containing essential
RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM
Renin-angiotensin-aldosterone system (RAAS) controls substances every minute. When plasma ultrafiltrate enters the
the regulation of the flow of blood to and within the glomerulus. proximal convoluted tubule, the nephrons, through cellular transport
The system responds to changes in blood pressure and mechanisms, begin reabsorbing these essential substances and
plasma sodium content that are monitored by the water.
juxtaglomerular apparatus, which consists of the REABSORPTION MECHANISMS
For active transport to occur, the substance to be reabsorbed
juxtaglomerular cells in the afferent arteriole and the macula
must combine with a carrier protein contained in the membranes of
densa of the distal convoluted tubule.
the renal tubular cells. Active transport is responsible for the
Renin, an enzyme produced by the juxtaglomerular cells,
reabsorption of glucose, amino acids, and salts in the proximal
is secreted and reacts with the blood-borne substrate
convoluted tubule, chloride in the ascending loop of Henle, and
angiotensinogen to produce the inert hormone angiotensin I.
sodium in the distal convoluted tubule. Passive transport is the
As angiotensin I passes through the lungs, angiotensin
movement of molecules across a membrane as a result of
converting enzyme (ACE) changes it to the active form
differences in their concentration or electrical potential on opposite
angiotensin II. Angiotensin II corrects renal blood flow in the
sides of the membrane.
following ways: causing vasodilation of the afferent arterioles
TUBULAR CONCENTRATION
and constriction of the efferent arterioles, stimulating
Excessive reabsorption of water as the filtrate passes
reabsorption of sodium in the proximal convoluted tubules, and
through the highly concentrated medulla is prevented by the water-
triggering the release of the sodium-retaining hormone
impermeable walls of the ascending loop. This selective
aldosterone by the adrenal cortex and antidiuretic hormone by
reabsorption process is called the countercurrent mechanism and
the hypothalamus.
serves to maintain the osmotic gradient of the medulla.
Maintenance of this osmotic gradient is essential for the final
concentration of the filtrate when it reaches the collecting duct.

2
COLLECTING DUCT CONCENTRATION
The process is controlled by the presence or absence of
ADH, which renders the walls of the distal convoluted tubule
and collecting duct permeable or impermeable to water. A high
level of ADH increases permeability, resulting in increased
reabsorption of water, and a low-volume concentrated urine.
Likewise, absence of ADH renders the walls impermeable to
water, resulting in a large volume of dilute urine
TUBULAR SECRETION
Tubular secretion involves the passage of substances
from the blood in the peritubular capillaries to the tubular filtrate.
Tubular secretion serves two major functions: elimination of
waste products not filtered by the glomerulus and regulation of
the acid-base balance in the body through the secretion of
hydrogen ions.
ACID-BASE BALANCE
To maintain the normal blood pH of 7.4, the blood must
buffer and eliminate the excess acid formed by dietary intake
and body metabolism. The buffering capacity of the blood
depends on bicarbonate (HCO3 - ) ions, which are readily
filtered by the glomerulus and must be expediently returned to
the blood to maintain the proper pH.
As a result of their small molecular size, hydrogen ions
are readily filtered and reabsorbed. Therefore, the actual
excretion of excess hydrogen ions also depends on tubular
secretion. A disruption in the secretory functions can result in
metabolic acidosis or renal tubular acidosis, the inability to produce
an acid urine.
RENAL FUNCTION TESTS
GLOMERULAR FILTRATION TESTS
Standard test used to measure the filtering capacity of the
glomeruli is the clearance test; a clearance test measures the rate
at which the kidneys are able to remove (to clear) a filterable
substance from the blood.
The earliest glomerular filtration tests measured urea
because of its presence in all urine specimens and the existence of
routinely used methods of chemical analysis. At present, the use of
urea as a test substance for glomerular filtration has been replaced
by the measurement of other substances including creatinine, inulin,
beta2 microglobulin, cystatin C, or radioisotopes.
Inulin Clearance
Inulin, a polymer of fructose, is an extremely stable
substance that is not reabsorbed or secreted by the tubules. A test
that requires an infused substance is termed an exogenous
procedure and is seldom the method of choice if a suitable test
substance is already present in the body (endogenous procedure).
Creatinine Clearance
Creatinine, a waste product of muscle metabolism that is
normally found at a relatively constant level in the blood, provides
the laboratory with an endogenous procedure for evaluating
glomerular function.
3
 When interpreting the results of a creatinine clearance filtered by the glomerulus and reabsorbed and broken down by the
test, the GFR is determined not only by the number of renal tubular cells. Monitoring levels of cystatin C is recommended
functioning nephrons but also by the functional capacity of for pediatric patients, persons with diabetes, the elderly, and
these nephrons. In other words, even though half of the critically ill patients.
available nephrons may be nonfunctional, a change in the GFR TUBULAR REABSORTPION TESTS
The loss of tubular reabsorption capability is often the first
will not occur if the remaining nephrons double their filtering
function affected in renal disease.
capacity.
Osmolarity
Calculated Glomerular Filtration Estimates
Specific gravity depends on the number of particles present
The most frequently used formula was developed by
in a solution and the density of these particles, whereas osmolarity
Cockcroft and Gault. It is also used to document eligibility for
is affected only by the number of particles present. When evaluating
reimbursement by the Medicare End Stage Renal Disease
renal concentration ability, the substances of interest are small
Program and for evaluating patient placement on kidney
molecules, primarily sodium (molecular weight, 23) and chloride
transplant lists.
(molecular weight, 35.5).
l Disease (MDRD) system, utilizes additional variables
An osmole is defined as 1 g molecular weight of a substance
and does not include body weight. The variables include
divided by the number of particles into which it dissociates. A
ethnicity, blood urea nitrogen, and serum albumin.
nonionizing substance such as glucose (molecular weight, 180)
Good correlation between the GFR and plasma levels of
contains 180 g per osmole, whereas sodium chloride (NaCl)
beta2 microglobulin has been demonstrated. Beta2
(molecular weight, 58.5), if completely dissociated, contains 29.25 g
microglobulin (molecular weight 11,800) dissociates from
per osmole.
human leukocyte antigens at a constant rate and is rapidly
The osmolarity of a solution can be determined by measuring
removed from the plasma by glomerular filtration. Sensitive
a property that is mathematically related to the number of particles
methods using enzyme immunoassay are available for the
in the solution (colligative property) and comparing this value with
measurement of beta2 microglobulin.
the value obtained from the pure solvent. Because water is the
Cystatin C is a small protein (molecular weight 13,359)
solvent in both urine and plasma, the number of particles present in
produced at a constant rate by all nucleated cells. It is readily

4
a sample can be determined by comparing a colligative
property value of the sample with that of pure water.
Freezing Point Osmometers
These osmometers determine the freezing point of a
solution by supercooling a measured amount of sample to
approximately 27C. The supercooled sample is vibrated to
produce crystallization of water in the solution. The heat of
fusion produced by the crystallizing water temporarily raises
the temperature of the solution to its freezing point. A
temperature-sensitive probe measures this temperature
increase, which corresponds to the freezing point of the
solution, and the information is converted into milliosmoles.
Clinical osmometers use solutions of known NaCl
concentration as their reference standards because a solution
of partially ionized substances is more representative of urine
and plasma composition.
Vapor Pressure Osmometers
Samples are absorbed into small filter paper disks that
are placed in a sealed chamber containing a temperature-
sensitive thermocoupler. The sample evaporates in the
chamber, forming a vapor. When the temperature in the
chamber is lowered, water condenses in the chamber and on
the thermocoupler. The heat of condensation produced raises
the temperature of the thermocoupler to the dew point
temperature. This dew point temperature is proportional to the
vapor pressure from the evaporating sample. Temperatures are
compared with those of the NaCl standards and converted into
milliosmoles. The vapor pressure osmometer uses microsamples of
less than 0.01 mL; therefore, care must be taken to prevent any
evaporation of the sample prior to testing.
Technical Factors
Falsely elevated values owing to the formation of lactic acid
also occur with both methods if serum samples are not separated or
refrigerated within 20 minutes. Vapor pressure osmometers do not
detect the presence of volatile substances, such as alcohol, as they
become part of the solvent phase; however, measurements
performed on similar specimens using freezing point osmometers
will be elevated.
Free Water Clearance
The free water clearance is determined by first calculating
the osmolar clearance using the standard clearance formula and
then subtracting the osmolar clearance value from the urine volume
in mL/min.
5
TUBULAR SECRETION AND RENAL BLOOD FLOW TESTS
The test most commonly associated with tubular
secretion and renal blood flow is the p-aminohippuric acid
(PAH) test. Historically, excretion of the dye
phenolsulfonphthalein (PSP) was used to evaluate these
functions.
PAH Test
The principle is the same as in the clearance test for
glomerular filtration. However, to ensure measurement of the
blood flow through the entire nephron, the substance must be
removed from the blood primarily in the peritubular capillaries
rather than being removed when the blood reaches the
glomerulus.
Titratable Acidity and Urinary Ammonia
Measurement of urine pH, titratable acidity, and urinary
ammonia can be used to determine the defective function. The
tests can be run simultaneously on either fresh or toluene-
preserved urine specimens collected at 2-hour intervals from
patients who have been primed with an acid load consisting of
oral ammonium chloride. By titrating the amount of free H
(titratable acidity) and then the total acidity of the specimen, the
ammonium concentration can be calculated as the difference
between the titratable acidity and the total acidity

6
 INTRODUCTION TO URINALYSIS
HISTORY AND IMPORTANCE development by Thomas Addis of methods for quantitating the
 Analyzing urine was actually the beginning of laboratory microscopic sediment.
medicine.  Richard Bright introduced the concept of urinalysis as part of a
 Pictures of early physicians commonly showed them doctor’s routine patient examination in 1827.
examining a bladder-shaped flask of urine. They were able  Two unique characteristics of a urine specimen account for this
to obtain diagnostic information from such basic continued popularity:
observations as color, turbidity, odor, volume, viscosity, and 1. Urine is a readily available and easily collected
even sweetness (by noting that certain specimens attracted specimen.
ants). 2. Urine contains information, which can be obtained by
 Many well-known names in the history of medicine are inexpensive laboratory tests, about many of the body’s
associated with the study of urine, including Hippocrates, major metabolic functions.
who in the 5th century BC wrote a book on “uroscopy.”  The Clinical and Laboratory Standards Institute (CLSI) (formerly
 By 1140 AD, color charts had been developed that NCCLS) defines urinalysis as “the testing of urine with
described the significance of 20 different colors procedures commonly performed in an expeditious, reliable,
 Chemical testing progressed from “ant testing” and “taste accurate, safe, and cost-effective manner.”
testing” for glucose to Frederik Dekkers’ discovery in 1694  Reasons for performing urinalysis identified by CLSI include
of albuminuria by boiling urine. aiding in the diagnosis of disease, screening asymptomatic
 The credibility of urinalysis became compromised when populations for undetected disorders, and monitoring the
charlatans without medical credentials began offering their progress of disease and the effectiveness of therapy
predictions to the public for a healthy fee. These charlatans, URINE FORMATION
called “pisse prophets”  The kidneys continuously form urine as an ultrafiltrate of plasma.
 The invention of the microscope in the 17th century led to Reabsorption of water and filtered substances essential to body
the examination of urinary sediment and to the function converts approximately 170,000 mL of filtered plasma to
the average daily urine output of 1200 mL.

7

URINE COMPOSITION
 In general, urine consists of urea and other organic and
inorganic chemicals dissolved in water.
 Urine is normally 95% water and 5% solutes, although
considerable variations in the concentrations of these
solutes can occur owing to the influence of factors such as
dietary intake, physical activity, body metabolism, endocrine
functions, and even body position.
 Urea, a metabolic waste product produced in the liver from
the breakdown of protein and amino acids, accounts for
nearly half of the total dissolved solids in urine.
 Other organic substances include primarily creatinine and
uric acid.
 The major inorganic solid dissolved in urine is chloride,
followed by sodium and potassium.
 Dietary intake greatly influences the concentrations of these
inorganic compounds, making it difficult to establish normal
levels.
 Other substances found in urine include hormones,
vitamins, and medications.
 Although not a part of the original plasma filtrate, the urine
also may contain formed elements, such as cells, casts,
crystals, mucus, and bacteria.
 Increased amounts of these formed elements are often
indicative of disease.
ANGELA TANTEO
 A high urea and creatinine content can identify a fluid as urine.
URINE VOLUME
 Urine volume depends on the amount of water that the kidneys
excrete.
 Water is a major body constituent; therefore, the amount
excreted is usually determined by the body’s state of hydration.
 Factors that influence urine volume include:
1. Fluid intake,
2. Fluid loss from nonrenal sources,
3. Variations in the secretion of antidiuretic hormone, and
4. Need to excrete increased amounts of dissolved solids,
such as glucose or salts.
 The normal daily urine output is usually 1200 to 1500 mL, a
range of 600 to 2000 mL is considered normal.
 Oliguria, a decrease in urine output, which is less than 1
mL/kg/hr in infants, less than 0.5 mL/kg/hr in children, and less
than 400 mL/day in adults, is commonly seen when the body
enters a state of dehydration as a result of excessive water loss
from vomiting, diarrhea, perspiration, or severe burns.
 Oliguria leading to anuria, cessation of urine flow, may result
from any serious damage to the kidneys or from a decrease in
the flow of blood to the kidneys.
 The kidneys excrete two to three times more urine during the
day than during the night.
 An increase in the nocturnal excretion of urine is termed
nocturia.
8
 Polyuria, an increase in daily urine volume (greater than
2.5 L/day in adults and 2.5–3 mL/kg/day in children), is
often associated with diabetes mellitus and diabetes
insipidus; however, it may be artificially induced by diuretics,
caffeine, or alcohol, all of which suppress the secretion of
antidiuretic hormone.
 Diabetes mellitus and diabetes insipidus produce polyuria
for different reasons, and analysis of the urine is an
important step in the differential diagnosis
 Diabetes mellitus is caused by a defect either in the
pancreatic production of insulin or in the function of insulin,
which results in an increased body glucose concentration.
 The kidneys do not reabsorb excess glucose, necessitating
excretion of increased amounts of water to remove the
dissolved glucose from the body. Although appearing to be
dilute, a urine specimen from a patient with diabetes
mellitus has a high specific gravity because of the
increased glucose content.
 Diabetes insipidus results from a decrease in the
production or function of antidiuretic hormone; thus, the
water necessary for adequate body hydration is not
reabsorbed from the plasma filtrate. In this condition, the
urine is truly dilute and has a low specific gravity. Fluid loss
in both diseases is compensated by increased ingestion of
water (polydipsia), producing an even greater urine volume.
ANGELA TANTEO
Polyuria accompanied by increased fluid intake is often the first
symptom of either disease
SPECIMEN COLLECTION
 Urine is a biohazardous substance that requires the observance
of Standard Precautions.
 Gloves should be worn at all times when in contact with the
specimen.
 Specimens must be collected in clean, dry, leak-proof containers.
 Disposable containers are recommended because they
eliminate the chance of contamination due to improper washing.
 Properly applied screw-top lids are less likely to leak than snap-
on lids.
 Containers for routine urinalysis should have a wide mouth and
flat bottom to prevent overturning.
 They should be made of a clear material to allow for
determination of color and clarity.
 The recommended capacity of the container is 50 mL, which
allows 12 mL of specimen needed for microscopic analysis,
additional specimen for repeat analysis, and enough room for
the specimen to be mixed by swirling the container.
 All specimens must be labeled properly with the patient’s name
and identification number, the date and time of collection, and
additional information such as the patient’s age and location and
the physician’s name, as required by institutional protocol.
9
 Labels must be attached to the container, not to the lid, and  Additional information on the form can include method of
should not become detached if the container is refrigerated collection or type of specimen, possible interfering medications,
or frozen. and the patient’s clinical information.
 A requisition form (manual or computerized) must  The time the specimen is received in the laboratory should be
accompany specimens delivered to the laboratory. recorded on the form.

ANGELA TANTEO 10
 Improperly labeled and collected specimens should be
rejected by the laboratory, and appropriate personnel
should be notified to collect a new specimen.
 Unacceptable situations include specimens in unlabeled
containers, nonmatching labels and requisition forms,
specimens contaminated with feces or toilet paper,
containers with contaminated exteriors, specimens of
insufficient quantity, and specimens that have been
improperly transported.
ANGELA TANTEO
SPECIMEN HANDLING
SPECIMEN INTEGRITY
 Following collection, specimens should be delivered to the
laboratory promptly and tested within 2 hours.
 A specimen that cannot be delivered and tested within 2 hours
should be refrigerated or have an appropriate chemical
preservative added.
SPECIMEN PRESERVATION
 The most routinely used method of preservation is refrigeration
at 2 degrees C to 8 degrees C, which decreases bacterial
growth and metabolism.
 If the urine is to be cultured, it should be refrigerated during
transit and held refrigerated until cultured up to 24 hours.2
11
 Refrigeration can increase the specific gravity, when  At the same time, the preservative should not interfere with

measured by urinometer, and the precipitation of chemical tests.

amorphous phosphates and urates, which may obscure the
microscopic sediment analysis.
 The specimen must return to room temperature before
chemical testing by reagent strips. This will correct the
specific gravity and may dissolve some of the amorphous
urates.
 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
 To obtain a specimen that is representative of a patient’s
metabolic state, regulation of certain aspects of specimen
collection is often necessary.
 These special conditions may include time, length, and method
of collection and the patient’s dietary and medicinal intake.
RANDOM SPECIMEN
 The random specimen may be collected at any time, but the
actual time of voiding should be recorded on the container.
 The random specimen is useful for routine screening tests to
detect obvious abnormalities. However, it may also show

ANGELA TANTEO 12
 erroneous results resulting from dietary intake or physical
activity just before collection.
FIRST MORNING SPECIMEN
 This is the ideal screening specimen.
 It is also essential for preventing false-negative pregnancy
tests and for evaluating orthostatic proteinuria.
 The first morning specimen, or 8-hour specimen, is a
concentrated specimen, thereby assuring detection of
chemicals and formed elements that may not be present in
a dilute random specimen.
 The patient should be instructed to collect the specimen
immediately on arising and to deliver it to the laboratory
within 2 hours.
FASTING SPECIMEN (SECOND MORNING)
 A fasting specimen differs from a first morning specimen by
being the second voided specimen after a period of fasting.
 This specimen will not contain any metabolites from food
ingested before the beginning of the fasting period.
 It is recommended for glucose monitoring.
2-HOUR POSTPRANDIAL SPECIMEN
 The patient is instructed to void shortly before consuming a
routine meal and to collect a specimen 2 hours after eating.
 The specimen is tested for glucose, and the results are
used primarily for monitoring insulin therapy in persons with
diabetes mellitus.
ANGELA TANTEO
 A more comprehensive evaluation of the patient’s status can be
obtained if the results of the 2-hour postprandial specimen are
compared with those of a fasting specimen and corresponding
blood glucose tests.
GLUCOSE TOLERANCE SPECIMENS
 Glucose tolerance specimens are sometimes collected to
correspond with the blood samples drawn during a glucose
tolerance test (GTT).
 The number of specimens varies with the length of the test.
GTTs may include fasting, halfhour, 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.
 Collection of these specimens is an institutional option.
24- HOUR OR TIMED SPECIMEN
 A carefully timed specimen must be used to produce accurate
quantitative results.
 Many solutes exhibit diurnal variations such as catecholamines,
17-hydroxysteroids, and electrolytes in which the lowest
concentration is in the early morning and the highest
concentration occurs in the afternoon.
 When the concentration of the substance to be measured
changes with diurnal variations and with daily activities such as
13
 exercise, meals, and body metabolism, 24-hour collection is
required. If the concentration of a particular substance
remains constant, the specimen may be collected over a
shorter period. Care must be taken, however, to keep the
patient adequately hydrated during short collection periods.
 To obtain an accurate timed specimen, the patient must
begin and end the collection period with an empty bladder.
 The concentration of a substance in a particular period
must be calculated from the urine volume produced during
that time.
 Addition of urine formed before the start of the collection
period or failure to include urine produced at the end of the
collection period will produce inaccurate results.
 On its arrival in the laboratory, a 24-hour specimen must be
thoroughly mixed and the volume accurately measured and
recorded.
 If only an aliquot is needed for testing, the amount saved
must be adequate to permit repeat or additional testing.
 If a specimen is collected in two containers, the contents of
the containers should be combined and thoroughly mixed
before aliquoting.
 All specimens should be refrigerated or kept on ice during
the collection period and may also require addition of a
chemical preservative.
 The preservative chosen must be nontoxic to the patient
and should not interfere with the tests to be performed.
ANGELA TANTEO
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
is a bacterial culture.
 If a routine urinalysis is also requested, the culture should be
performed first to prevent contamination of the specimen.
 A less frequently encountered type of catheterized specimen
measures functions in the individual kidneys. Specimens from
the right and left kidneys are collected separately by passing
catheters through the ureters of the respective kidneys.
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.
 It provides a specimen that is less contaminated by epithelial
cells and bacteria and, therefore, is more representative of the
actual urine than the routinely voided specimen.
 Patients must be provided with appropriate cleansing materials,
a sterile container, and instructions for cleansing and voiding.
 Strong bacterial agents, such as hexachlorophene or povidone-
iodine, should not be used as cleansing agents.
 Patients are instructed to wash their hands before beginning the
collection.
14
 Male patients should clean the glans, which begins at the
urethra, and withdraw the foreskin, if necessary.
 Female patients should separate the labia and clean the
urinary meatus and surrounding area.
 When cleansing is complete, patients are to void first into
the toilet, then collect an adequate amount of urine in the
sterile container, and finish voiding into the toilet.
 As with a catheterized specimen, if a routine urinalysis is
also requested, the culture should be performed first to
prevent contamination of the specimen.
SUPRAPUBIC ASPIRATION
 Occasionally urine may be collected by external
introduction of a needle through the abdomen into the
bladder.
 Because the bladder is sterile under normal conditions,
suprapubic aspiration provides a sample for bacterial
culture that is completely free of extraneous contamination.
 The specimen can also be used for cytologic examination.
PROSTATITIS SPECIMEN
 Similar to the midstream clean-catch collection, the
threeglass collection procedure is used to determine
prostatic infection.
 Instead of discarding the first urine passed, it is collected in
a sterile container. Next, the midstream portion is collected
in another sterile container.
ANGELA TANTEO
 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.
 The second specimen is used as a control for bladder and
kidney infection.
 If it is positive, the results from the third specimen are invalid
because infected urine has contaminated the specimen.
 Variations of the procedure include the Stamey-Mears four-glass
localization method and the preand postmassage test (PPMT).
 The four-glass method consists of bacterial cultures of the initial
voided urine (VB1), midstream urine (VB2), expressed prostatic
secretions (EPS), and a postprostatic massage urine specimen
(VB3).
 Urethral infection or inflammation is tested for by the VB1, and
the VB2 is tested for urinary bladder infection.
 The prostatic secretions are cultured and examined for white
bood cells. More than 10 to 20 white blood cells per high-power
field is considered abnormal.
 In the PPMT test, a clean-catch midstream urine specimen is
collected. A second urine sample is collected after the prostate
is massaged.
15
 A positive result is significant bacteriuria in the  The specimen must be handled securely, with a guarantee that
postmassage specimen of greater than 10 times the no unauthorized access to the specimen was possible.
premassage count.  Proper identification of the individual whose information is
PEDIATRIC SPECIMEN indicated on the label is required.
 Collection of pediatric specimens can present a challenge.  The decision to obtain a witnessed collection is indicated when it
Soft, clear plastic bags with hypoallergenic skin adhesive to is suspected that the donor may alter or substitute the specimen
attach to the genital area of both boys and girls are or it is the policy of the client ordering the test.
available for collecting routine specimens.  If a witnessed specimen collection is ordered, a same-gender
 Sterile specimens may be obtained by catheterization or by collector will observe the collection of 30 to 45 mL of urine.
suprapubic aspiration.  Witnessed and unwitnessed collections should be immediately
 Specimens for culture also may be obtained using a clean- handed to the collector.
catch cleansing procedure and a sterile collection bag. Care  The urine temperature must be taken within 4 minutes from the
must be taken not to touch the inside of the bag when time of collection to confirm the specimen has not been
applying it. adulterated. The temperature should read within the range of
 For quantitative testing, bags are available that allow a tube 32.5C to 37.7C.
to be attached and excess urine transferred to a larger
container.
DRUG SPECIMEN COLLECTION
 The chain of custody (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.
 For urine specimens to withstand legal scrutiny, it is
necessary to prove that no tampering of the specimen
occurred, such as substitution, adulteration, or dilution.
 All personnel handling the specimen must be noted.

ANGELA TANTEO 16
 If the specimen temperature is not within range, the  The urine color is inspected to identify any signs of contaminants.
temperature should be recorded and the supervisor or  The specimen is labeled, packaged, and transported following
employer contacted immediately. laboratoryspecific instructions.

 Urine temperatures outside of the recommended range may

indicate specimen contamination.
 Recollection of a second specimen as soon as possible will
be necessary.

ANGELA TANTEO 17
ANGELA TANTEO 18
 CHAPTER 4. PHYSICAL EXAMINATION OF URINE
COLOR
➢ Normal Urine Color
-Urochrome is a product of endogenous metabolism, and
under normal conditions the body produces it at a constant
rate.
-Urochrome also increases in urine that stands at room
temperature
-The presence of uroerythrin, a pink pigment, is most evident
in specimens that have been refrigerated, resulting in the
precipitation of amorphous urates.
-Urobilin, an oxidation product of the normal urinary
constituent urobilinogen, imparts an orange-brown color to
urine that is not fresh
➢ Abnormal Urine Color
➔ Dark Yellow/Amber/Orange
- Normal urine produces only a small amount of rapidly
disappearing foam when shaken, and a large amount
of white foam indicates an increased concentration of
protein.
➔ Red/Pink/Brown
- A fresh brown urine containing blood may also indicate
glomerular bleeding resulting from the conversion of
hemoglobin to methemoglobin

ANGELA TANTEO 19
 - When RBCs are present, the urine is red and ➔ Blue/Green
cloudy; however, if hemoglobin or myoglobin is - Pathogenic causes of blue/green urine color are limited
present, the specimen is red and clear. to bacterial infections, including urinary tract infection
- Urine specimens containing porphyrins also may ● Clarity
appear red resulting from the oxidation of - Clarity is a general term that refers to the
porphobilinogen to porphyrins. They are often transparency/turbidity of a urine specimen.
referred to as having the color of port wine.
- Fresh urine containing myoglobin frequently
exhibits a more reddish-brown color than
hemoglobin.

➢ Normal Clarity
➔ Brown/Black
-Freshly voided normal urine is usually clear, particularly if it is
- Melanin is an oxidation product of the colorless
a midstream clean-catch specimen.
pigment, melanogen, produced in excess when a
-Precipitation of amorphous phosphates and carbonates may
malignant melanoma is present.
cause a white cloudiness.
- Homogentisic acid, a metabolite of phenylalanine,
➢ Nonpathologic Turbidity
imparts a black color to alkaline urine from
-Refrigerated specimens frequently develop a thick turbidity
persons with the inborn-error of metabolism,
caused by the precipitation of amorphous phosphates,
called alkaptonuria.
carbonates, and urates.

ANGELA TANTEO 20
-Amorphous phosphates and carbonates produce a
white precipitate in urine with an alkaline pH, whereas
amorphous urates produce a precipitate in acidic
urine that resembles pink brick dust due to the
presence of uroerythyrin.
ANGELA TANTEO
➢ Pathologic Turbidity
● Specific Gravity
- Specific gravity is defined as the density of a solution
compared with the density of a similar volume of distilled
water at a similar temperature.
➢ Urinometer
-The weighted float displaces a volume of liquid equal to its
weight and has been designed to sink to a level of 1.000 in
distilled water.
-The additional mass provided by the dissolved substances in
urine causes the float to displace a volume of urine smaller
than that of distilled water.
➢ Refractometer
-Refractometry, like urinometry, determines the concentration
of dissolved particles in a specimen. It does this by
measuring refractive index.
-Refractive index is a comparison of the velocity of light in air
with the velocity of light in a solution.
-The concentration of dissolved particles present in the
solution determines the velocity and angle at which light
passes through a solution.
The concentration of the specimen determines the angle at which
the light beam enters the prism.
21
ANGELA TANTEO 22
➢ Harmonic Oscillation Densitometry -Normal random specimens may range from 1.003 to 1.035,
-Harmonic oscillation densitometry is based on the depending on the patient’s amount of hydration.
principle that the frequency of a sound wave entering Specimens measuring lower than 1.003 probably are not
a solution changes in proportion to the density of the urine.
solution.
-Shifts in harmonic oscillation are measured, and relative
density is calculated.
-A portion of the urine sample enters a U-shaped glass
tube with an electromagnetic coil at one end and a
motion detector at the other end.
-An electric current is applied to the coil, which causes
the sound wave to pass (oscillate) through the urine
sample. ● Odor
-Its frequency is altered by the density of the specimen.
-A microprocessor at the other end of the tube measures
the change in sound wave frequency, compensates
for temperature variations, and converts the reading
to specific gravity that closely correlates with
gravimetric measurement
➢ Clinical Correlations
-The specific gravity of the plasma filtrate entering the
glomerulus is 1.010. The term isosthenuric is used to
describe urine with a specific gravity of 1.010.
Specimens below 1.010 are hyposthenuric, and those
above 1.010 are hypersthenuric.

ANGELA TANTEO 23
 CHAPTER 5: CHEMICAL ANALYSIS OF URINE 1. Store reagent strips in their bottle with lid closed at room
Introduction to reagent strip testing
temperature.
 A reagent strip, also called a dipstick, is a narrow strip of
2. Do not expose strips to:
plastic with small pads attached to it.
 Sunlight
 Each pad contains reagents for a different reaction, thus
 Heat
allowing for the simultaneous determination of several
 Cold
tests. Routine urinalysis includes chemical testing for pH,
 Volatile substances
protein, glucose, ketones, occult blood, bilirubin,
 Moisture (each bottle contains a desiccant)
urobilinogen, nitrite, leukocyte esterase, and strip test
3. Only remove the number of strips you need for immediate
method for specific gravity, which is covered in another
testing.
lecture.
4. Do not use strips that have discolored pads.
 The colors generated on each reagent pad vary
5. Do not use expired reagent strips.
according to the concentration of the analyte present.
6. Test urine which is brought to room temperature.
Dipping method
 The manual method for using a reagent strip to test
 Reagent strips that have been exposed to air, moisture, or
urine calls for dipping the entire strip into the specimen
are expired may show discoloration of some of the reagent
and withdrawing it in one continuous motion while
pads.
removing excess urine by dragging across the edge of
 Using discolored reagent strips makes interpretation of
the specimen container.
reactions impossible and can lead to false results.
Reading method
Urine pH Kidneys regulate acid-base balance
 Colors generated by each pad are visually compared
 pH changes demonstrate and assess this function
against a range of colors on brand-specific color charts.
 Must be evaluated in clinical context
 A critical requirement is that the reactions be read at the
 Kidneys help to regulate acid-base balance by excreting
prescribed time after dipping and then compared closely
acids and alkalis.
with the color chart provided by the manufacturer.
For accurate results:

ANGELA TANTEO 24
  This function is reflected in the pH of urine, the urine’s
hydrogen ion concentration
 Urine pH normally fluctuates between acidic and
alkaline and does not remain constant for a long period
of time.
 There is no abnormal range as such so, it is important
for the physician to correlate the urine pH with other
information to determine whether there is a problem.
Urine pH pH color chart Acid indicator – methyl red
 Alkaline indicator – bromthymol blue
 Read 60 seconds after dipping pH ranges from 5.0 to
8.5 in half units Indicators, methyl red and bromthymol Urine pH: Significance
blue, and measure a range of pH from 5.0 to 8.5. The To know if there is:
results may be reported in whole units or interpolated to
half units. If a more precise reading is needed,
measurement may be made using a pH meter with a
glass electrode.
 Some laboratoriesreport the reaction as “acid,” “neutral,”
or “alkaline,” instead of giving numerical values.
Urine pH
 pH must be read immediately as this will prevent
misreading due to thephenomenon of “run-over” effect.
This term is used to describe what happens when
excess urine is left on the stick after dipping, and so the
acid buffer from reagent in the protein pad runs onto the
pH area. On the brand of dipstick illustrated here the
reagent adjacent the pH pad is specific gravity and is causing
run-over effect.
 Contamination from the protein pad can cause a false
lowering of the pH reading, especially in the case of alkaline
or neutral urine. Specific gravity reagent run-over may falsely
raise the pH reading as seen here.
 Run-over can sometimes be recognized by the technologist,
because the edge nearest the interfering pad will usually
change first. However, if the strip is not observed constantly
after dipping, this occurrence can be overlooked as the entire
pH pad will eventually be affected.
 Persistent Acidity
 Acidifying drugs
 Dehydration
 Diabetes
 Diarrhea
 Fever
 Gout
 High protein diets
 Pulmonary emphysema
 There is no abnormal range as such, since the urine can
normally vary from acid to alkaline. For this reason, it is

ANGELA TANTEO 25
 important for the physician to correlatethe urine pH with
other information to determine whether there is a
problem.
 Persistent alkalinity
 Alkaline drugs
 Acute and chronic renal failure
 Diuretics
 Renal tubular acidosis
 Urinary tract infections
Urine protein
 Small amounts of low–molecular weight protein are
filtered at the glomerulus
 Most of this protein is reabsorbed in the tubules
 Less than 150 mg/24 h (or 20mg/dL) is excreted
 Mucoprotein Tamm–Horsfall is secreted by the renal
tubules is also excreted
 In the normal kidney, only a small amount of low–
molecular weight protein is filtered at the glomerulus.
The structure of the glomerular membrane prevents the
passage of high–molecular weight proteins including
albumin (mol wt 69,000). After filtration, most of the
protein is reabsorbed in the tubules with less than 150
mg/24 h (or 20 mg/dL) being excreted. In a child, the
normal excretion is less than 100 mg/m2/24 h.
ANGELA TANTEO
 The protein that is normally excreted includes a mucoprotein
called Tamm–Horsfall protein, which is not contained in the
plasma but is secreted by the renal tubules. This protein
forms the matrix of most urinary casts.
Protein color chart
 Principle: “protein error of indicators”
 Tetrabromphenol blue most common indicator
 Color ranges from yellow to blue
 Detects primarily albumin
 Negative does not rule out other significant proteins
 This colorimetric method used in dipsticks is based on the
concept known as the “protein error of indicators,” a
phenomenon which means that the point of color change of
some pH indicators is different in the presence of protein
from that observed in the absence of protein, because
proteins act as hydrogen ion acceptors at a constant pH.
 Usually, the indicator changes from yellow to blue (or green)
between pH 3 and pH 4, but in the presence of protein, this
color change will occur between pH 2 and pH 3. Therefore, in
the presence of protein an “error” occurs in the behavior of
the indicator.
 Most brands of reagent stips use Tetrabromphenol blue. Be
aware that reagent strips detect primarily albumin and are
less sensitive to globulins.
 Protein that is primarily excreted as the result of glomerular
damage or disease. Other urine proteins such as gamma
26
 globulin, glycoprotein, ribonuclease, lysozyme, some dipsticks during treatment with phenazopyridine and
hemoglobin, Tamm–Horsfall mucoprotein, and Bence- after the infusion of polyvinylpyrrolidon as a plasma expander.
Jones protein are much less readily detected than  Chlorhexidine gluconate, found in skin cleansers, may
albumin. produce false-positive results. In addition, specimens
 Therefore, a negative urinary dipstick result does not containing blood may cause a false-positive protein reaction.
necessarily rule out the presence of these proteins. Urine protein: false negative
Urine protein: false positive  Dilute urines
 Highly buffered alkaline urine (medications or old urine)  Elevated amounts of proteins other than albumin
 Prolonged exposure to the sample  False-negative protein results can occur in dilute urines and
 Container cleaning compounds (quaternary ammonia) when proteins other than albumin are present in slightly
 Phenazopyridine elevated concentrations.
 Plasma expander polyvinylpyrrolidon  The various acid precipitation tests that also screen for
 Chlorhexidine gluconate (skin cleansers) urinary proteins are not routinely performed in most clinical
Blood laboratories.
 False-positive protein results may occur in a highly Urine protein: significance
buffered alkaline urine, which may result from alkaline Physiology causes:
medication or stale urine. The alkaline pH can overcome  Exercise
the acid buffer in the reagent and the area may change  Emotional stress
color in the absence of protein.  Exposure to heat or cold
 If the dipstick is left in the urine for too long, the buffer  Fever
will be washed out of the reagent, the pH will increase,  Pregnancy
and the strip will turn blue or green even if proteinis not  Physiologic conditions such as exercise, emotional stress,
present. exposure to heat and cold, fever, and pregnancy that can
 Quaternary ammonium compounds that may be used to lead to increased protein excretion in the urine in the
clean the urine containers will alter the pH and result in absence of renal disease.
a false-positive reaction. False positives may occur on
ANGELA TANTEO 27
Pathologic causes:
 Glomerular nephritis
 Pyelonephritis
 Malignant hypertension
 The presence of increased amounts of protein in the
urine can be an important indicator of renal disease. It
may be the first sign of a serious problem and may
appear long before other clinical symptoms. Pathologic
causes include glomerular nephritis, pyelonephritis and
malignant hypertension.
 There are also some renal disorders in which proteinuria
are absent.
Urine glucose Glucose color chart
Reaction A: glucose oxidase
Glucose + O gluconic acid + H2O2 Urine glucose: false negative
Reaction B: peroxidase Decreased sensitivity in:
H2O2 + chromogen oxidized chromogen
 Reagent strips that are impregnated with the enzyme
glucose oxidase detect only glucose. These strips use a
double sequential enzyme reaction as seen here.
 The chromogen that is used varies among the different
reagent strips; the most common being potassium
iodide.
 Glucose results are read at 30 or 60 seconds,
depending on the manufacturer.
 The results are reported as negative to 4 (negative to 2000
mg/dL).
Urine glucose: false positive
 Oxidizing cleaning agents
 Peroxide
 Hypochorite
 Elevated urobiliongen with some automated methods
 If the urine specimen is contaminated with strong oxidizing
cleaning agents peroxide or hypochlorite, a false-positive
reaction may occur.
 In urines positive for glucose, a falsely elevated glucose may
result in the presence of elevated urobilinogen when using
automated methods for some brands of reagent
 strips.
 Cool urine
 Urine with high specific gravity
 Alkaline urine
 High ketone levels
 Ascorbic acid (Vitamin C) high doses
 Sensitivity for glucose may be affected by temperature,
specific gravity, and pH. Reactivity for glucose can vary with
temperature because of the effect temperature can have on
enzymatic reactions.

ANGELA TANTEO 28
 An elevated specific gravity may decrease the sensitivity
of glucose oxidase.
 Alkaline pH may decrease sensitivity to glucose.
 The combination of high specific gravity and alkaline pH
may result in false negatives at low concentrations of
glucose.
 Moderately high ketone levels (40 mg/dL) may reduce
the sensitivity and may cause false negatives with
glucose levels of 100 mg/dL.
 High urinary concentrations of ascorbate (ascorbic acid
or vitamin C) can inhibit the enzymatic reaction which
will result in a reduced or false-negative reading.
 The ascorbic acid will be oxidized by the hydrogen
peroxide in the second part of the enzyme reaction, and
will, therefore, compete with the oxidation of the
chromogen, resulting in the inhibition of the color
formation.
 The ingestion of a normal amount of vitamin C usually
presents no problem, but the recent interest in the self-
prescription of large doses of vitamin C (2–15 g/day) to
prevent or cure the common cold has created a potential
problem. Large concentrations of urinary ascorbic acid
can also occur with the parenteral administration of
vitamin C or antibiotics that contain ascorbic acid as a
stabilizing agent (e.g., tetracycline). If vitamin C
interference is suspected, a repeat test should be
ANGELA TANTEO
performed at least 24 hours after the last intake of ascorbic
acid.
Urine with reducing substances
 Other sugars
 Fructose
 Galactose
 Lactose
 Maltose
 Dextrins
 Homogentisic acid
 Glucuronates
 In addition to glucose, other sugars that may be found in
urine, such as fructose, galactose, lactose, and maltose, are
reducing substances. Procedures, which are based on the
ability of glucose to reduce copper, will also detect
thesesugars if they are present. Any other reducing
substances which can occasionally be found in the urine
such as dextrins, homogentisic acid, and glucuronates will
also give positive reduction tests.
Clinitest color chart
 Clinitest (Benedict’s Test), a copper reduction test, can be
used to test for glucose but is usually used to screen for
other reducing substance which may be present. This test is
based on the fact that in strongly alkaline solutions and in the
presence of heat, reducing sugars will reduce cupric ions to
cuprous oxide. The reaction produces a color change of blue
29
 through green to orange depending upon the amount of closely. If measurement beyond 2% is medically desirable,
reducing substances present in the urine. an alternate two-drop method is available.
 Clinitest is a self-heating method for the  This method involves adding only 2 drops of urine to 10
semiquantitative determination of reducing substances drops of water, but a special color chart must be used.
in the urine. The tablet contains the following reagents:  The two-drop method will allow for quantitation up to 5% but
copper sulfate, citric acid, sodium hydroxide, and the “pass-through” phenomenon may still occur.
sodium carbonate. When placed in a mixture of water Clinitest: false positive
and urine, the tablet is rapidly dissolved by the action of  Ascorbic acid
sodium carbonate and citric acid which act as an  Nalidixic acid
effervescent. The sodium hydroxide provides the  Cephalosporins
alkaline medium necessary for the reaction, and the  Probenecid
heat required is provided by the reaction of sodium Urinary preservatives:
hydroxide with water and citric acid. The reducing  formalin
substances in the urine then react with the copper  formaldehyde
sulfate to reduce the cupric ions to cuprous oxide.
 High concentrations of ascorbic acid have been considered
 The test is reported as negative, 1/4 % (or trace), 1/2% to give false-positive results, but recent studies question
(1), 3/4% (2), 1% (3), or 2% (4). whether this is really a problem.
 During the reaction, if the color should rapidly  Other substances cause a false positive Clinitest include
“passthrough” bright orange to a dark brown or Nalidixic acid, cephalosporins, probenecid, and the urinary
greenish-brown, report the result as being greater than preservatives such as formalin and formaldehyde if present
2%. Clinitest is a very accurate procedure if the in large quantities.
manufacturer’s directions are carefully followed. Failure Clinitest: false negative
to observe the reaction as it takes place will result in a Technique errors
falsely low reading. The “pass-through” phenomenon
can occur so rapidly that it can be missed ifnot observed

ANGELA TANTEO 30
  If all directions for the procedure are followed closely, no Urine reducing sugars: significance
false-negative results will occur. Clinitest is an accurate  Routinely performed on urine from children 2 years old and
and reliable test for reducing substances. younger
Urine glucose: significance  Early detection of galactosemia
 Normal renal threshold – mg/dl  A test for reducing substances should be included in the
 Glycosuria dependent on: routine urinalysis of all pediatric patients. This will provide for
 Blood glucose levels the early detection of those metabolic defects which are
 Glomerular filtration rate characterized by the excretion of reducing sugars such as
 Tubular reabsorption galactose, which is present in the urine in patients with
 Seen in diabetes and congenital forms of glucosuria galactosemia.

 Usually, glucose will not be present in the urine until the Urine ketones: normal formation

blood level exceeds 160–180 mg/dL, which is the  Ketones result from the breakdown of fat.
normal renal threshold for glucose.  One of the intermediate products of fatty acid
 The presence of significant amounts of glucose in the  Breakdown is acetyl CoA.
urine is called glycosuria (or glucosuria). The quantity of  Acetyl CoA enters the citric acid cycle (Krebs cycle) in the
glucose that appears in the urine is dependent upon the body if fat and carbohydrate degradation are appropriately
blood glucose level, the rate of glomerular filtration, and balanced.
the degree of tubular reabsorption.  The first step in the Krebs cycle is the reaction of acetyl CoA
 When the blood glucose exceeds the renal threshold, with oxaloacetate to yield citrate.
the tubules cannot reabsorb all of the filtered glucose, Urine ketones: excessive formation
and so glycosuria occurs. Normally, this level is not  Oxaloacetate will be used to form glucose when
exceeded even after the ingestion of a large quantity of carbohydrates are absent or improperly used.
carbohydrate. A small amount of glucose may be  No oxaloacetate available for condensation with acetyl CoA.
present in the normal urine, but the fasting level in an  CoA cannot enter the Krebs cycle and is diverted to the
adult is only about 2–20 mg of glucose per 100 mL of formation of ketone bodies.
urine.
ANGELA TANTEO 31
  The ketone bodies are acetoacetic acid (diacetic acid),
B-hydroxybutyric acid, and acetone. Acetoacetic acid is
the first ketone that is formed from acetyl CoA, and the
other ketones are formed from acetoacetic acid as
shown in hereaction.
 B-Hydroxybutyric acid is formed by reversible reduction,
and acetone is formed by a slow spontaneous
decarboxylation. Acetoacetic acid and B-hydroxybutyric
acid are normal fuels of respiration and are important
sources of energy.
Urine ketone: false positive or atypical color
 Highly pigmented urines
 Combination of high specific gravity and a low pH
 Levodopa metabolites
 Sulfhydryl groups
 Phenylketones
 Phthalein compounds
 Positive and questionable results may be confirmed with
a tablet test.
 False-positive results may occur when the urine
specimen is highly pigmented or when it contains large
amounts of levodopa metabolites.
 Some specimens that have both a high specific gravity
and a low pH may give false-positive reactions.
Compounds that contain sulfhydryl groups may cause a
ANGELA TANTEO
false-positive or atypical color reactions. Phenylketones may
cause a red–orange coloration. Phthalein compounds used
in liver and kidney function tests produce a reddish coloration
due to the alkalinity of the test zone. These colors, however,
are easily distinguishable from the colors obtained with
ketone bodies.
Urine ketone: false negative
 Controls solutions that use acetone.
 Because of the specificity of Multistix and DiaScreen for
diacetic acid, these brands of dipstick will not give a positive
ketone result with controls that contain acetone.
Urine ketones confirmatory test
 The Acetest tablet contains sodium nitroprusside, glycine, a
strong alkaline buffer (disodium phosphate), and lactose.
Acetest can be used to test urine, serum, plasma, or whole
blood.
 Diacetic acid and acetone react with sodium nitroprusside
and glycine in an alkaline medium to form a purple color. The
lactose in the tablet helps enhance the color.
 Acetest is about 10 times more sensitive to diacetic acid than
to acetone. However, Acetest will not react with B-
hydroxybutyric acid.
 Results are reported as “small, moderate, or large.” For urine,
the small color block corresponds to approximately 5–10
mg/dL of diacetic acid, the moderate block is 30–40 mg/dL,
and the large block is about 80–100 mg/dL.
32
Acetest color chart  Strenuous exercise
 Urine ketone Ketone blood level are normally very low  Vomiting
(2-4 mg/dl).  The heart muscle and the renal cortex prefer to use
 20% acetoacetic acid acetoacetate instead of glucose. But glucose is the major
 2% acetone fuel of the brain in well-nourished individuals, even though
 78% B-hydroxybutyric acid. them brain can adapt to utilize acetoacetate in the absence
 Acetone is lost into the air at room temperature of glucose. The odor of acetone may be detected in the
 Normally small amounts of ketones are present in the breath of an individual who has a high level of ketones in the
blood, 2–4 mg/dL.23 The relative proportion of each is blood because acetone is eliminated via the lungs.
approximately 20% acetoacetic acid, 2% acetone, and  Carbohydrate utilization problems can occur in other
78% B-hydroxybutyric acid. conditions besides diabetes.
 There may, however, be considerable proportional Urine blood/hemoglobin
variation among individuals.  Hematuria
 Acetone is lost into the air if a sample is left standing at  Glomerulus
room temperature. Therefore, urines should be tested  Renal tubules
immediately or refrigerated in a closed container until  Ureters
testing.  Bladder
Urine ketone: significance  Hemoglobinuria
 Diabetes mellitus  Glomerular filtrate
 Diarrhea  Myoglobinuria
 Exposure to cold  The presence of blood in the urine can occur as a result of
 Fasting damage to the upper or lower urinary tracts.
 Fever  The presence of intact RBCs is termed hematuria. However,
 Insufficient carbohydrate intake RBC may not visiable in urinary sediment of very dilute
 Malnutrition urines or urines with very acid or alkaline pH.

ANGELA TANTEO 33
  The hemoglogin in RBCs causes a positive reagent strip
reaction.
 Free hemoglobin will also yield a positive result. Free
hemoglobin from intervascular hemolysis enters the
urine as a filtrate of plasma.
 Another substance yielding a positive blood reaction is
myoglobin, which enters the urine as a filtrate of plasma
after an occurrence of muscle damage.
Urine blood/hemoglobin
Blood color chart
 The dipstick procedure is based on the peroxidaselike
activity of hemoglobin and myoglobin which catalyzes
the oxidation of a chromogen by an organic peroxide as
in the reaction.
 Most dipsticks are capable of detecting
erythrocytes as well as free hemoglobin and myoglobin.
 Intact RBCs in the urine will hemolyze on the test pad.
The freed hemoglobin will react with the reagent and will
result in green spots on a yellow or orange background.
Thus, the presence of intact red cells will give a spotted
green reaction,whereas free hemoglobin and myoglobin
will give a uniform green or green to dark blue color.
 Blood is usually read at 60 seconds, and the color
change is from orange to green to dark blue. There are
two separate color scales for erythrocytes
hemoglobin. Intact RBCs may display a speckle-pattern
reaction in the absence of free hemoglobin. The results are
reported as trace or moderate numbers of intact RBCs or
trace through 3 (large) amount of hemoglobin.
Urine blood/hemoglobin: false positive
 Oxidizing contaminants
 Hypochlorites
 Bacterial peroxidases
 Menstrual blood
 Povidoneiodine (Betadine)
 Most dipsticks will give false-positive results in the presence
of certain oxidizing contaminants such as hypochlorites
which may be used to clean urine-collection containers.
 When the urine is contaminated with a high bacterial content,
a false-positive reaction may occur because of bacterial
intact peroxidases.
 False positives will result if the urine is contaminated with
menstrual blood.
 False-positive reactions may occur if the urine or test strip is
contaminated with povidoneiodine (Betadine)
Urine blood/hemoglobin: false negative
 Technique errors inadequate mixing
 Formalin preservative
 High concentrations of ascorbic acid (Vitamin C)
 Captoprin (Capoten)
and  High specific gravity
ANGELA TANTEO 34
  Nitrites
 Proteins
 The test is slightly more sensitive to free hemoglobin
and myoglobin than to intact RBCs. If the urine sample
is not mixed well before testing, a false-negative result
can occur because the red cells tend to settle in the
bottom of the container.
 Some dipsticks give lower or false-negative readings in
the presence of high levels of ascorbic acid. If
necessary, the test should be repeated at least 24 hours
after the last dose of vitamin C.
 Captopril (Capoten) may reduce the reagent pad’s
sensitivity.
 Sensitivity is less in urines with high specific gravity,
nitrites, or protein.
 In addition, specimens preserved using formalin will
yield a false-negative result.
Urine blood/hemoglobin: significance
 Hematuria
 Hemoglobinuria
 Myoglobinuria
 The urine is normally free of all of these substances;
therefore, a positive test for occult blood should be
followed by determination of the exact cause and origin
of this abnormal finding. A correlation must also be
ANGELA TANTEO
made with the microscopic examination, and this may be
done by asking the following questions:
 Are there red cells present?
 Does the number of red cells agree with the intensity
of the chemical test?
 Are there red cell casts or hemoglobin casts?
 Are there empty red cell membranes (ghost cells)?
 Are there numerous squamous epithelial cells present
(possible menstrual contamination)?
 It should be noted that hematuria,hemoglobinuria, and
myoglobinuria can occur either individually or together.
Urine blood/hemoglobin: hematuria
 Intact RBCs
 If lysed in urine, “ghosts” may be present in sediment
 Urine may appear normal with small amounts
 Urine is red with greater amounts of blood
 Hematuria is the presence of blood or intact RBCs in the
urine. A urine that is highly alkaline or has a very low specific
gravity (1.007) can cause the red cells to lyse, thus releasing
their hemoglobin into the urine. The presence of this type of
hemoglobin is still considered to be hematuria as far as the
origin is concerned, but it is very difficult to distinguish from
true hemoglobinuria. When lysing occurs, the microscopic
examination may show the empty red cell membranes which
are often referred to as “ghost” cells.
35
  In microhematuria there is such a small amount of blood  Electric shock
in the urine that the color of the specimen is unaffected  Lysis only in urine does not carry the same significance
and the hematuria can only be detected chemically or  Hemoglobinuria is the presence of free hemoglobin in the
microscopically. On the other hand, gross hematuria urine as a result of intravascular hemolysis.
alters the color of the urine and is easily visible  The hemolysis that occurs in the urine while in the urinary
macroscopically. tract or after voiding because of a low specific gravity or
Urine blood/hemoglobin: hematuria highly alkaline pH may be considered to be hemoglobinuria,
Can indicate renal diseases: but it does not bear the same significance as true
 Glomerular hemoglobinuria.
 Tubular  Hemoglobinuria without hematuria occurs as a result of
 Interstitial hemoglobinemia and, therefore, it has primarily nothing to do
 Vascular with the kidneys even though it may secondarily result in
Also present in patients with: kidney damage.
 Lithiasis (kidney stones) Urine blood/hemoglobin: myoglobinuria
 Urinary tract infections  Damage to cardiac or skeletal muscle
 Urinary tract tumors  Crush injuries
 Electric shock
 Myocardial infarct
Urine blood/hemoglobin: hemoglobinuria  Myoglobin is the heme protein of striated muscle. It serves as
 Free hemoglobin a reserve supply of oxygen and also facilitates the movement
 Intervascular hemolysis of oxygen within muscle. Injury to cardiac or skeletal muscle
 Incompatible blood transfusions results in the release of myoglobin into the circulation.
 Hemolytic anemia  Even just subtle injury to the muscle cells can bring about the
 Burns release of myoglobin.
 Cold auto-immune anemia

ANGELA TANTEO 36
  Myoglobin has a molecular weight of approximately  Unconjugated bilirubin is insoluble in water and cannot be
17,000 and so it is easily filtered through the glomerulus filtered through the glomerulus.
and excreted in the urine.  Conjugated with glucuronic acid in the liver to form bilirubin
 Because myoglobin is cleared so rapidly from the diglucuronide.
circulation, the plasma is left uncolored even though the  Conjugated bilirubin (direct) is water soluble and is excreted
urine may be red to brown to black, depending on the by the liver through the bile duct and into the duodenum.
degree of myoglobinuria.  Bilirubin is formed from the breakdown of hemoglobin in the
Urine blood/hemoglobin vs myoglobin reticuloendothelial system. It is then bound to albumin and
Both will produce positive reagent strip blood results: transported through the blood to the liver. This free or
Marcoscopic differentiation unconjugated bilirubin is insoluble in water and cannot be
 red plasma plus red urine equals filtered through the glomerulus. In the liver, bilirubin is
hemoglobin removed by the parenchymal cells and is conjugated with
 clear plasma plus red urine equals glucuronic acid to form bilirubin diglucuronide.
myoglobin  This conjugated bilirubin, which is also called direct bilirubin,
Chemical differentiation is water soluble and is excreted by the liver through the bile
 Ammonium sulfate test duct and into the duodenum.
 Hemoglobinuria and myoglobinuria can be
rapidly differentiated by the following Urine bilirubin
screening criteria red plasma plus red  small amounts of conjugated bilirubin regurgitate back from
urine equals hemoglobin; clear plasma the bile duct and into the blood system.
plus red urine equals myoglobin.  filtered through the glomerulus and excreted in the urine
 Another screening procedure is the whenever the plasma level is increased.
ammonium sulfate test.  Normally, no detectable amounts are present in urine
Urine bilirubin formation  Normally, very small amounts of conjugated bilirubin
 Formed by hemoglobin degradation bound to albumin regurgitate back from the bile duct and into the blood system.
and transported through the blood to the liver.

ANGELA TANTEO 37
  Therefore, very small amounts of conjugated bilirubin
can be found in the plasma, but not in concentrations
higher than 0.2–0.4 mg/dL.
 Because conjugated bilirubin is not bound to protein, it is
easily filtered through the glomerulus and excreted in
the urine whenever the plasma level is increased.
Normally, no detectable amount of bilirubin (sometimes
referred to as “bile”) can be found in the urine.
Urine bilirubin Bilirubin color chart acid
 Most dipsticks are based on the coupling reaction of a
diazonium salt with bilirubin in an acid medium as show
by this reaction:that is used and the color that develops.
 Bilirubin results are read from 30 to 60 seconds,
depending on the manufacturer and display a range of
colors from buff through various shades of tan or
tannish-purple.
Urine bilirubin: false positive
Technique errors
Reading after the prescribed time atypical color
reactions produced by:
 Indican
 Metabolites of etodolac (Lodine)
 Chlorpromazine (Thorazine)
 Metabolites of phenazopyridine
 Confirm results with Ictotest
ANGELA TANTEO
 If the bilirubin pad is observed after the prescribed
amount of time, it may develop other colors that may
interfere with the reading of bilirubin reactions. Several
compounds may produce atypical color reactions on
the bilirubin pad. Indican and metabolites of etodolac
(Lodine) can produce an interfering color reaction.
Patients receiving large doses of chlorpromazine
(Thorazine) may have false-positive results.
 Metabolites of drugs such as phenazopyridine give a
red color at an acid pH and cause misinterpretation of
results that could lead to false-positive reports.
 The Ictotest should be used to confirm bilirubin results
on urines that generate a positive or atypical color
reaction.
Urine bilirubin: false negative
 Large amounts of ascorbic acid decrease the sensitivity
 High levels of nitrite
 Exposure to light and room temperature
 Bilrubin oxidizes to biliverdin
 Large amounts of ascorbic acid decrease the sensitivity of
this test.
 Repeating the test at least 10 hours after the last dose of
vitamin C will produce more accurate results.
 Elevated levels of nitrite will lower the bilirubin result.
38
  A false negative result will be obtained if the bilirubin
has been oxidized to biliverdin, as occurs when
specimens are exposedto room temperature and light.
Urine bilirubin confirmatory test
 Ictotest is a tablet test that is based on the same diazo
reaction as the dipsticks. However, Ictotest is much
more sensitive than the dipsticks, being able to detect
as little as 0.05 mg/dL. Because of this sensitivity,
Ictotest is the recommended procedure when a test for
just bilirubin is ordered. It also serves as a good
confirmatory test for a positive dipstick.
 The tablet contains 2,6-dichlorobenzene-
diazoniuimtetrafluoroborate, sulfosalicylic acid,
sodium bicarbonate. The mats that are used in the
procedure are made of an asbestos–cellulose mixture.
When the urine is placed on the mat, the absorbent
qualities of the mat cause the bilirubin to remain on the
outer surface. The sulfosalicylic acid provides the acid
environment for the reaction. It also acts with the sodium
bicarbonate to provide an effervescence which helps
partially dissolve the tablet. The diazonium salt then
couples with the bilirubin on the mat, giving a blue or
purple reaction product.
 Ictotest color reactions.
Urine bilirubin: significance
Normally, no bilirubin is present in urine
ANGELA TANTEO
Present in urine:
 when bile flow to colon is obstructed
 liver damage
 Hepatitis
 cholestasis
 Normally, very small amounts of conjugated bilirubin
regurgitate back from the bile duct and into the blood
system. Therefore, very small amounts of conjugated
bilirubin can be found in the plasma, but not in
concentrations higher than 0.2–0.4 mg/dL. However, no
detectable amount of bilirubin (sometimes referred to as
“bile”) can be found in the urine.
and  Because conjugated bilirubin is not bound to protein, it is
easily filtered through the glomerulus and excreted in the
urine whenever the plasma level is increased such as in
biliary obstruction, liver damage, hepatitis and cholestasis.
Urine urobilinogen formation
 Formed from bilirubin in the intestines
 Urobilinogen (colorless) oxidizes to urobilin (brown)
 Most lost in the feces
 About 10–15% reabsorbed into the bloodstream, returns to
the liver, and reexcreted into the intestines
 A small amount of this urobilinogen is excreted by the
kidneys into the urine
 Normal level: 1–4 mg/24 h or less than 1.0 Ehrlich unit
39
 In the intestines, bacterial enzymes convert bilirubin,  p-Dimethylaminobenzaldehyde + urobiligen = azo dye
through a group of intermediate compounds, to several Urine urobilinogen: false positive
related compounds which are collectively referred to as  p-aminosalicylic acid
urobilinogen. Most of the urobilinogen (a colorless  sulfonamides,
pigment) and its oxidized variant, urobilin (a brown  p-aminobenzoic
pigment), are lost in the feces. About 10–15% of the  Phenazopyridine
urobilinogen is reabsorbed into the bloodstream, returns  Prophobilionogen
to the liver, and is reexcreted into the intestines. A small  May use Watson-Shwartz test to differentiate
amount of this urobilinogen is also excreted by the  Several interfering substances may react with the
kidneys into the urine, with a normal level of about 1–4 urobilinogen test pad to produce atypical colors. These
mg/24 h or less than 1.0 Ehrlich unit/2 h. interfering substances include p-aminosalicylic acid,
sulfonamides, and p-aminobenzoic acid.
 Urine urobilinogen Urobilinogen color chart  Reagent strips using p-dimethylaminobenzaldehyde may
 Screening tests for urobilinogen are based on the react with prophobilionogen, although this is not a reliable
Ehrlich Aldehyde Reaction method for detecting porphobilinogen. Urine from patients
 This is a simple color development reaction in which receiving phenazopyridine may show a false-positive
aldehyde or diazonium compounds react with reaction.
urobilinogen to produce a pink to red color in an acid Urine urobilinogen: false decrease
environment.  A true absence of urobilinogen is not detectable.
 Urobilinogen results are read at 30 or 60 seconds,  Broadspectrum antibiotics
depending on the manufacturer and display a range of  Nitrite
colors in the pink spectrum from light to dark. Most
 Formalin
brands of dipsticks show two blocks on the color chart
 Improper storage of specimen
for normal levels of urobilinogen of 0.1 and 1 mg/dL.
 oxidation of urobilinogen to urobilin
The other color blocks range from 2 to 8 or 12,
depending onthe manufacturer.

ANGELA TANTEO 40
  A true absence of urobilinogen is not detectable.  The nitrite test is a rapid, indirect method for the early
Several substances may decrease the color reaction of detection of significant and asymptomatic bacteriuria.
this test. Patients receiving broadspectrum antibiotics  Nitrates, which are usually present in normal diets, are water
and other substances which will alter the normal soluble and present in urine. During a urinary tract infection
bacterial flora in the intestines will excrete little or no of nitrate-reducing bacteria, nitrates are converted to nitrite.
urobilinogen in their urine because urobilinogen Common organisms that can cause urinary tract infections,
cannotbe formed in the intestines. such as Escherichia coli, Enterobacter, Citrobacter,
 Urines containing nitrites or those preserved with Klebsiella, and Proteus species, produce enzymes that
formalin may produce false-negative results. False reduce urinary nitrate to nitrite.
negatives may also occur in improperly stored samples  For this to occur, the urine must have incubated in the
allowing the oxidation of urobilinogen to urobilin. bladder for a minimum of 4 hours. Hence, the first morning
Urine urobilinogen: significance urine is the specimen of choice.
 Normally present in low amounts Urine nitrite Nitrite color chart
 Peak levels between 2-4 pm Reaction A:
Elevated in:  Nitrite + p-arsanilic acid diazonium compound
 Liver disease Reaction B:
 Intestinal obstruction  3-Hydroxyl-1,2,3,4 tetrahydrobenz-(h)-quinoline +diazonium
 Hemolytic anemia compound = pink color
 Hemolysis
Urine nitrite formation  Reagent strips for the detection of nitrite in the urine
 Nitrates normally present commonly use p-arsanilic acid and a quinoline compound.
 Nitrates converted to nitrite by bacteria  Nitrite reacts with p-arsanilic acid to form a diazonium
 Reaction takes up to 4 hours to complete compound. This compound then couples with the quinoline

 UTIs of only non-nitrate reducers will be negative compound to produce a pink color as in the reaction.
 Nitrite results are read at 30 or 60 seconds, depending on
the manufacturer. Any degree of uniform pink color should be

ANGELA TANTEO 41
 interpreted as a positive nitrite test suggesting the
presence of 105 or more organisms per milliliter. The
color development is not proportional to the number of
bacteria present. Pink spots or pink edges should not be
considered a positive result. If the uniform pink color is
very light, it may best be seen by placing the strip
against a white background. The test is reported as
positive or negative.
Urine nitrite: false positive
 Specimen left at room temperature causing bacteria to
multiple.
Phenazopyridine
 The urine should be tested shortly after being voided,
because if the urine is allowed to stand at room
temperature for several hours, organisms may grow in
the specimen and generate nitrite.
 Results may be misinterpreted as positive in urines that
appear red or contain phenazopyridine and other
substances that turn red in acid.
Urine nitrite: false negative
 Specimen containing non-nitrate reducing pathogens
 Insufficient time in the bladder
 Low or no nitrate diet
 Nitrite was to nitrogen
 Elevated urobilinogen
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Ascorbic Acid
The sensitivity of the test is reduced in urine with a high specific
gravity or elevated level of ascorbic acid. A negative test should
never be interpreted as indicating theabsence of bacterial infection.
There are several reasons forthis:
1. There may be pathogens present in the urine that do not form
nitrite.
2. The urine may not have remained in the bladder long enough for
the nitrate to be converted to nitrite.
3. There are cases in which the urine does not contain any nitrate,
so bacteria may be present but the dipstick will be negative.
4. Under certain circumstances, the bacterial enzymes may have
reduced nitrate to nitrite and then converted nitrite to nitrogen,
which will give a negativenitrite result.
 False-negative nitrite determinations or negative
interferences can be the result of abnormally high levels of
urobilinogen, the presence of ascorbic acid levels as low as5
mg/dL, or acidic urine (pH is 6.0 or less).
 The nitrite test is not meant to take the place of other routine
bacteriology studies such as cultures and smears. The
dipstick procedure is just used as a screening test which is
capable of detecting bacteriuria even when not clinically
suspected. If there are clinical symptoms, then regular
bacteriology tests should be performed, even if the nitrite test
is negative.
42
Urine nitrite: significance
 Screening method only
 Not to replace microbiology procedures
 If there are clinical symptoms, then regular bacteriology
tests should be performed, even if the nitrite test is
negative.
 The nitrite test is not meant to take the place of other
routine bacteriology studies such as cultures and
smears. The dipstick procedure is just used as a
screening test which is capable of detecting bacteriuria
even when not clinically suspected. If there are clinical
symptoms, then regular bacteriology tests should be
performed, even if the nitrite test is negative.
Urine leukocyte esterase
 Leukocyte esterase is present only in neutrophils.
 Few neutrophils can be seen in normal urine.
 Increased numbers of neutrophils usually indicate the
presence of a urinary tract infection.
 Screening for UTI also includes pH, protein, and nitrite.
 Mix specimen well and test at room temperature.
 White blood cells can be present in any body fluid
depending on a cause for their presence. The most
common white blood cell seen in a urine sample is the
neutrophil, which is normally present in low numbers.
Increased numbers of neutrophils usually indicate the
ANGELA TANTEO
presence of a urinary tract infection; and their presence is
indicated by a positive leukocyte esterase test.
 Screening for urinary tract infections also includes evaluation
of pH, protein, and nitrite.
 Most accurate results are obtained on fresh, uncentrifuged,
well-mixed specimens at room temperature.
Urine leukocyte esterase
Leuk. Esterase color chart
Reaction A:
 Indoxyl granulocytic indoxyl or carbonic acid ester orpyrole
esterase pryole
Reaction B:
 Indoxylor diazonium salt = purple pyrole
 Neutrophils contain enzymes known as esterases. These
esterases can be detected by reagent strips that contain an
appropriate substrate such as indoxylcarbonic acid ester and
is based on the reaction shown. The reagents used for this
reaction vary by manufacturer.
 Leukocyte esterase results are read at 2 minutes. A positive
reaction produces a lavender to purple color with a reporting
range of values from trace to large. Values reflecting cell
numbers from negative to 500 may be reported.
 These results may not correlate with the numbers of
neutrophils seen during microscopic examination.
43
Urine leukocyte esterase: false positive  Cephalexin
 Strong oxidizing agents  Cephalothin
 Contamination by vaginal discharge  Gentamicin
 Formalin used as preservative  Tetracycline
 Drugs containing imipenem, meropenem, or clavulanic  False-negative results may occur with high specific gravity
acid and in urines containing glucose and protein. Significantly
 Nitrofurantoin contributes a color to urine that may high levels of protein or glucose can contribute to increased
cause misinterpretation specific gravity. In such an environment, white blood cells will

 Strong oxidizing agents cause a false-positive leukocyte crenate and be unable to release esterase.

esterase result. This occurs when strong detergents  Various drugs and chemicals interfere with this test. Check
used to clean the collection container remain present. the packaging insert of the reagent strip manufacturer for

 False-positive results may also be obtained on females specifics concerning interfering substances. Some drugs and

due to contamination of the urine with vaginal discharge. chemicals that may cause false-negative results include

 Some preservatives such as formalin will cause a false- ascorbic acid, oxalic acid, cephalexin, cephalothin,

positive result. gentamicin, and tetracycline.

 False-positive results may be caused by drugs that Additional urine chemistry parameters

contain imipenem, meropenem, and clavulanic acid.  Calcium

 Nitrofurantoin contributes a color to urine that may  Creatinine

cause misinterpretation of this test.  Microalbumin

Urine leukocyte esterase: false negative  Ascorbic acid

 High specific gravity and in urines containing glucose  Some brands of reagent strips are offering additional test
and protein. parameters including calcium, creatinine, and microalbumin.

 WBCs creanate and cannot release esterase  Excess ascorbic acid can interfere with the chemical
 Ascorbic acid reactions for bilirubin, blood, and glucose and may result in
 Oxalic acid false low or negative results in these parameters.

ANGELA TANTEO 44