MODULE 5: CHEMICAL EXAMINATION OF URINE

This module aims to focus on the principle of reagent strip method for the
determination of the chemical characteristics of a urine sample. The principle,
components, indicators, end color results and clinical importance of the different
parameters on the reagent strip will also be discussed.

LEARNING OBJECTIVES:

At the end of the module, the student will be able to:

1. Describe the proper technique for performing reagent strip testing

2. List four causes of premature deterioration of reagent strips, and describe how to
avoid them
3. Identify quality-control procedures routinely performed with reagent strip testing.
4. Discuss the principle of 11 parameters in the reagent strip
5. Discuss the clinical indications of the end colors of each parameters
6. Correlate physical and chemical urinalysis results

Lesson 1: Chemical Examination of Urine

● Reagent strips

Reagent strips currently provide a simple, rapid means for performing medically
significant chemical analysis of urine, including pH, protein, glucose, ketones, blood,
bilirubin, urobilinogen, nitrite, leukocytes, and specific gravity. The two major types of
reagent strips are manufactured under the trade names Multistix (Siemens Healthcare
Diagnostics, Deerfield, IN) and Chemstrip (Roche Diagnostics, Indianapolis, IN). These
products are available with single-or multipletesting areas, and the brand and numbers
of tests used are a matter of laboratory preference.

Reagent strips consist of chemical-impregnated absorbent pads attached to a

plastic strip. A color-producing chemical reaction takes place when the absorbent pad
comes in contact with urine. The reactions are interpreted by comparing the color
 produced on the pad within the required time frame with a chart supplied by the
manufacturer. Several colors or intensities of a color for each substance being tested
appear on the chart. By careful comparison of the colors on the chart and the strip, a
semiquantitative value of trace, 1+, 2+, 3+, or 4+ can be reported. An estimate of the
milligrams per deciliter present is available for appropriate testing areas. Automated
reagent strip readers also provide Système International units.

Reading Time Parameter Principle Positive Color

Glucose Double sequential enzyme Green to brown
30 secs reaction
Bilirubin Diazo reaction Tan or pink to violet
40 secs Ketones Sodium nitroprusside reaction Purple
(Legal’s test)
45 seconds Specific pKa change of a polyelectrolyte Blue (SG 1.000) to Yellow
Gravity (SG 1.030)
Protein Protein (Sorensen’s) error of Blue
indicators
pH Double indicator system Orange (pH 5.0 to Blue (pH
9.0)
Blood Pseudoperoxidase activity of ● Uniform green/Blue
60 seconds hemoglobin (Hemoglobin or
Myoglobin)
● Speckled/Spotted
(Hematuria)

Urobilinogen Erhlich Red

Nitrite Greiss reaction Uniform pink
120 secs Leukocytes Leukocyte esterase Pink
10 secs (C-stix) Ascorbic Molybdenum blue
60 secs (Stix) Acid
 ● pH

Along with the lungs, the kidneys are the major regulators of the acid–base content in
the body. They do this through the secretion of hydrogen in the form of ammonium ions,
hydrogen phosphate, and weak organic acids, and by the reabsorption of bicarbonate
from the filtrate in the convoluted tubules.

Causes of Acid and Alkaline Urine

Acid Urine Alkaline Urine Alkaline Urine
✔ Emphysema ✔ Hyperventilation
✔ Diabetes mellitus ✔ Vomiting Starvation
✔ Starvation ✔ Renal tubular
✔ Dehydration acidosis
✔ Diarrhea ✔ Presence of urease-
✔ Presence of producing bacteria
acid-producing bacteria ✔ Vegetarian diet
(Escherichia coli) ✔ Old specimens
✔ High-protein diet
✔ Cranberry juice
✔ Medications:
- Methenamine
mandelate [Mandelamine]
- Fosfomycin
tromethamine [Monurol])

A healthy individual usually produces a first morning specimen with a slightly acidic pH
of 5.0 to 6.0; a more alkaline pH is found following meals (alkaline tide). The pH of
normal random samples can range from 4.5 to 8.0. Consequently, no normal values are
assigned to urinary pH, and it must be considered in conjunction with other patient
information.

Clinical Significance

1. Primarily as an aid in determining the existence of systemic acid–base

disorders of metabolic or respiratory origin and in the management of urinary
conditions that require the urine to be maintained at a specific pH.
 Examples:
▪ Respiratory or metabolic acidosis (not related to renal function
disorders)
- The urine is acidic
▪ Respiratory or metabolic alkalosis
- The urine is alkaline
2. The precipitation of inorganic chemicals dissolved in the urine forms urinary
crystals and renal calculi. This precipitation depends on urinary pH and can
be controlled by maintaining the urine at a pH that is incompatible with the
precipitation of the particular chemicals causing the calculi formation.
Knowledge of urinary pH is important in the identification of crystals observed
during microscopic examination of the urine sediment.
▪ Calcium oxalate
- A frequent constituent of renal calculi, precipitates primarily in
acidic and not alkaline urine. Therefore, maintaining urine at an
alkaline pH discourages formation of the calculi.
▪ Maintaining an acidic urine can be valuable in treating urinary tract
infections
- It is caused by urea-splitting organisms because they do not
multiply as readily in an acidic medium. These same organisms are
also responsible for the highly alkaline pH found in specimens that
have been allowed to sit unpreserved for extended periods.
3. Identification of freshly voided and accepted urine sample
● The pH of freshly excreted urine does not reach above 8.5 in normal or
abnormal conditions. A pH above 8.5 is associated with an improperly
preserved specimen and indicates that a fresh specimen should be
obtained to ensure the validity of the analysis.
Reagent Strip Reactions

● Reagent strips measure urine pH in 0.5 or 1-unit increments between pH 5 and 9.

● To differentiate pH units throughout this wide range, they use a double-indicator
system of methyl red and bromthymol blue.
● Methyl red produces a color change from red to yellow in the pH range 4 to 6,
and bromthymol blue turns from yellow to blue in the range of 6 to 9.
● Therefore, in the pH range 5 to 9 measured by the reagent strips, one sees
colors progressing from orange at pH 5 through yellow and green to a final deep
blue at pH 9.
Methyl red + H+ →→→→ Bromthymol blue – H+
(Red-orange → Yellow) (Green → Blue)
(4.0→6.0) (6.0→9.0)

● Protein

The most indicative of renal disease is the protein determination. Proteinuria is often
associated with early renal disease, making the urinary protein test an important part of
any physical examination. Normal urine contains very little protein: usually, less than 10
mg/dL or 100 mg per 24 hours is excreted.

● Albumin
● Due to its low molecular weight, albumin is the major serum protein found
in normal urine.
● Even though it is present in high concentrations in the plasma, the normal
urinary albumin content is low because the majority of albumin presented
to the glomerulus is not filtered, and much of the filtered albumin is
reabsorbed by the tubules.
● Tamm-Horsfall protein (uromodulin)
● It is produced by the renal tubular epithelial cells and proteins from
prostatic, seminal, and vaginal secretions.
● Uromodulin is routinely produced in the distal convoluted tubule. It forms
the matrix of casts formed in the distal convoluted tubule.

Clinical Significance

Demonstration of proteinuria in a routine analysis does not always signify renal disease;
however, its presence does require additional testing to determine whether the protein
represents a normal or a pathologic condition. Clinical proteinuria is indicated at 30
mg/dL or greater (300 mg/L). The causes of proteinuria are varied and can be grouped
into three major categories based on the origin of the protein:

a. Pre-renal Proteinuria
b. Renal Proteinuria
c. Post-renal Proteinuria

Pre-renal Proteinuria

● Caused by conditions affecting the plasma prior reaching the kidney and,
therefore, is not indicative of actual renal disease.
● This condition is frequently transient, caused by increased levels of
low-molecular-weight plasma proteins such as hemoglobin, myoglobin, and the
acute phase reactants associated with infection and inflammation.
● The increased filtration of these proteins exceeds the normal reabsorptive
capacity of the renal tubules, resulting in an overflow of the proteins into the
urine.
● Because reagent strips detect primarily albumin, pre-renal proteinuria is usually
not discovered in a routine urinalysis.

I. Bence Jones Protein

- A primary example of proteinuria due to increased serum protein levels is
the excretion of Bence Jones protein by persons with multiple myeloma.
▪ Multiple myeloma, a proliferative disorder of the
immunoglobulin-producing plasma cells, the serum contains markedly
elevated levels of monoclonal immunoglobulin light chains (Bence
Jones protein). This low molecular-weight protein is filtered in
quantities exceeding the tubular reabsorption capacity and is excreted
in the urine.
▪ Suspected cases of multiple myeloma must be diagnosed by
performing serum electrophoresis and immunoelectrophoresis.
▪ The screening test for Bence Jones protein is not routinely performed,
as cases of multiple myeloma are easily detected by chemical
methods.
⮚ Screening Test for Bence Jones Protein
- Unlike other proteins, which coagulate and remain
coagulated when exposed to heat, Bence Jones protein
coagulates at temperatures between 40°C and 60°C and
dissolves when the temperature reaches 100°C.
 - Therefore, a specimen that appears turbid between 40°C
and 60°C and clear at 100°C can be suspected of containing
Bence Jones protein.
- Interference due to other precipitated proteins can be
removed by filtering the specimen at 100°C and observing
the specimen for turbidity as it cools to between 40°C and
60°C.

Renal Proteinuria

● Proteinuria associated with true renal disease may be the result of either
glomerular or tubular damage.

I. Glomerular Proteinuria
- When the glomerular membrane is damaged, selective filtration is
impaired, and increased amounts of serum protein and eventually red and
white blood cells pass through the membrane and are excreted in the
urine.
- major causes of proteinuria due to glomerular damage:
✔ Amyloid material
✔ Toxic substances
✔ Lupus erythematosus
✔ Streptococcal glomerulonephritis
- Benign proteinuria:
✔ The discovery of protein transiently in random samples
✔ Shown in the following conditions:
o Strenuous exercise
o High fever
o Dehydration
o Exposure to cold
II. Microalbuminuria
- The development of diabetic nephropathy leading to reduced glomerular
filtration and eventual renal failure is a common occurrence in persons
with both type 1 and type 2 diabetes mellitus.
- Onset of renal complications associated with Diabetes Mellitus can first be
predicted by detection of microalbuminuria.
- The presence of microalbuminuria is also associated with an increased
risk of cardiovascular disease.
- The progression of renal disease can be prevented through better
stabilization of blood glucose levels and control of hypertension.
- Microalbuminuria Testing Procedure:
✔ Required collection of a 24-hour urine specimen. Specimens were
tested using quantitative procedures for albumin.
✔ Results were reported in mg of albumin/24 hours or as the albumin
excretion (AER) in µg/min.
✔ With these methods, microalbumin was considered significant when
30 to 300 mg of albumin is excreted in 24 hours or the AER is 20 to
200 µg/min.
III. Orthostatic (Postural) Proteinuria
- aka: Postural Proteinuria
- It is a persistent benign proteinuria occurs frequently in young adults.
- It occurs following periods spent in a vertical posture and disappears when
a horizontal position is assumed. Increased pressure on the renal vein
when in the vertical position is believed to account for this condition.
- Procedure: Patients suspected of orthostatic proteinuria are requested to
empty the bladder before going to bed, collect a specimen immediately
upon arising in the morning, and collect a second specimen after
remaining in a vertical position for several hours.
 - Results: Both specimens are tested for protein, and if orthostatic
proteinuria is present, a negative reading will be seen on the first morning
specimen, and a positive result will be found on the second specimen.
IV. Tubular Proteinuria
- Increased albumin is also present in disorders affecting tubular
reabsorption because the normally filtered albumin can no longer be
reabsorbed.
- Other low-molecular-weight proteins that are usually reabsorbed are also
present.
- Causes of tubular dysfunction include:
✔ exposure to toxic substances and heavy metals
✔ severe viral infections
✔ Fanconi syndrome.
- The amount of protein that appears in the urine following glomerular
damage ranges from slightly above normal to 4 g/day, whereas markedly
elevated protein levels are seldom seen in tubular disorders.

Post-renal Proteinuria

● Protein can be added to a urine specimen as it passes through the structures of

the lower urinary tract (ureters, bladder, urethra, prostate, and vagina).
● Bacterial and fungal infections and inflammations produce exudates containing
protein from the interstitial fluid.
● The presence of blood as the result of injury or menstrual contamination
contributes protein, as does the presence of prostatic fluid and large amounts of
spermatozoa.

Reagent Strip Reactions

- Principle: protein error of indicators

- Contrary to the general belief that indicators produce specific colors in
response to particular pH levels, certain indicators change color in the
 presence of protein even though the pH of the medium remains constant.
This is because protein (primarily albumin) accepts hydrogen ions from
the indicator.
- The test is more sensitive to albumin because albumin contains more
amino groups to accept the hydrogen ions than other proteins.
- Depending on the manufacturer, the protein area of the strip contains
either tetrabromophenol blue (Multistix) or 3',3",5',5"-tetrachlorophenol,
3,4,5,6-tetrabromosulfonphthalein (Chemstrip), and an acid buffer to
maintain the pH at a constant level.
- At a pH level of 3, both indicators appear yellow in the absence of protein;
however, as the protein concentration increases, the color progresses
through various shades of green and finally to blue.
- Readings are reported in terms of negative, trace, 1+, 2+, 3+, and 4+; or
the semiquantitative values of 30, 100, 300, or 2000 mg/dL corresponding
to each color change. Trace values are considered to be less than 30
mg/dL.

Indicator + protein →→→→→→→→→→→ (+) Blue Green

(Yellow) (-) Yellow

Reaction Interference

- The major source of error with reagent strips occurs with highly buffered
alkaline urine that overrides the acid buffer system, producing a rise in pH
and a color change unrelated to protein concentration.
- A technical error of allowing the reagent pad to remain in contact with the
urine for a prolonged period may remove the buffer. False-positive
readings are obtained when the reaction does not take place under acidic
conditions.
 - Highly pigmented urine and contamination of the container with quaternary
ammonium compounds, detergents, and antiseptics also cause
false-positive readings.
- A false-positive trace reading may occur in specimens with a high specific
gravity

Tests for determination of Proteinuria

I. Sulfosalicylic Acid Precipitation Test

- The sulfosalicylic acid (SSA) test is a cold precipitation test that reacts
equally with all forms of protein.
- Various concentrations and amounts of SSA can be used to precipitate
protein, and methods vary greatly among laboratories.
- All precipitation tests must be performed on centrifuged specimens to
remove any extraneous contamination.
- Procedure:
1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine.
2. Mix by inversion and observe for cloudiness.
3. Grade the degree of turbidity.
- Note: Since reagent strip is sensitive to Albumin, if the reagent strip is
negative for protein but positive in SSA, it indicates that proteins other
than Albumin are present.

Reporting SSA Turbidity

Grade Turbidity Protein Range (mg/dL)

Negative No increase in turbidity Less than 6

Trace Noticeable turbidity 6-30

1+ Distinct turbidity, no granulation 30-100

 2+ Turbidity, granulation, no flocculation 100-200

3+ Turbidity, granulation, flocculation 200-400

4+ Clumps of protein Greater than 400

II. Testing for Microalbuminuria

- Several semiquantitative reagent strip methods have been developed so
that patients at risk for renal disease can be monitored using random or
first morning (recommended) specimens. These methods are based on
immunochemical assays for albumin or albumin-specific reagent strips
that also measure creatinine to produce an albumin:creatinine ratio.
a. Micral-Test (Roche Diagnostics, Indianapolis, IN)
- Contain a gold-labeled antihuman albumin antibody-enzyme
conjugate.
- Strips are dipped into the urine up to a level marked on the
strip and held for 5 seconds.
- Albumin in the urine binds to the antibody. The bound and
unbound conjugates move up the strip by wicking action.
Unbound conjugates are removed in a captive zone by
combining with albumin embedded in the strip. The urine
albumin–bound conjugates continue up the strip and reach
an area containing enzyme substrate. The conjugated
enzyme reacts with the substrate, producing colors ranging
from white to red.
- The amount of color produced represents the amount of
albumin present in the urine.
- The color is compared with a chart on the reagent strip bottle
after 1 minute. (+) Red; (-) White
 - Results range from 0 to 10 mg/dL.

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▪ The Immunological test principle with monoclonal antibodies is highly specific for human
albumin
▪ The urine sample is absorbed by the test strip and transferred through the following two
zones before reaching the detection pad:
Zone 1 – Conjugate Fleece contains free gold-labelled antibodies
Zone 2 – Capture Matrix Fleece with fixed human serum albumin (HSA)

b. ImmunoDip reagent strip

- Uses an immunochromographic technique.
- Strips are individually packaged in specially designed
containers.
- The container is placed in the urine specimen for 3 minutes.
A controlled amount of urine enters the container through a
vent hole.
- The urine encounters blue latex particles coated with
antihuman albumin antibody. Albumin in the urine binds with
 the coated particles. The bound and unbound particles
continue to migrate up the strip.
- The migration is controlled by the size of the particles;
unbound particles do not migrate as far as the bound
particles.
- First a blue band is formed by the unbound particles. The
bound particles continue to migrate and form a second blue
band further up the strip.
- The top band therefore represents the bound particles (urine
albumin) and the bottom band represents unbound particles.
- The color intensity of the bands is compared against the
manufacturer’s color chart.
- Interference: Diluted urine (False negative)
- Interpretation of Results:

Result Band Albumin level (mg/dL)

Negative darker bottom band less than 1.2 mg/dL

Borderline equal band colors 1.2 to 1.8 mg/dL

Positive darker top band 2.0 to 8.0 mg/dL

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c. Albumin:Creatinine Ratio (Clinitek Microalbumin and the

Multistix Pro reagent strips)
- Simultaneous measurement of albumin/protein and
creatinine that permits an estimation of the 24-hour
microalbumin excretion.
- Principle: Sensitive albumin tests related to creatinine
concentration to correct the patient’s hydration
 Albumin Creatinine
Reagents diiodo-dihydroxydinitrophenyl - Copper sulfate
tetrabromosulphonphthalein - tetramethylbenzidine (TMB)
(DIDNTB) - diisopropyl benzene
dihydroperoxide (DBDH)

Sensitivity 80-150mg/L 10-300 mg/dL (0.9-26.5 mmol/L)

Interferences - highly buffered alkaline - visibly bloody urine and
urine abnormally colored urines
- polymethyl vinyl ether - presence of the gastric
- visibly bloody urine and acid–reducing medication
abnormally colored cimetidine (Tagamet)
urines
 ● Glucose

The glucose test is the most frequently performed chemical analysis on urine because
of its value in the detection and monitoring of diabetes mellitus. Early diagnosis of
diabetes mellitus through blood and urine glucose tests provides a greatly improved
prognosis.

Clinical Significance

● Normal circumstances
- Almost all the glucose filtered by the glomerulus is actively reabsorbed in
the proximal convoluted tubule; therefore, urine contains only minute
amounts of glucose. Tubular reabsorption of glucose is by active transport
in response to the body’s need to maintain an adequate concentration of
glucose.

● Hyperglycemia (as occurs in diabetes mellitus)

- The tubular transport of glucose has reached its renal threshold, and
glucose appears in the urine. The blood level at which tubular
reabsorption stops (renal threshold) for glucose is approximately 160 to
180 mg/dL.
- Blood glucose levels fluctuate and a non-fasting normal person may have
glycosuria (presence of sugar in urine) following a meal containing high
glucose content.
- The most informative glucose results are obtained from specimens
collected under controlled conditions. Fasting prior to the collection of
samples for screening tests is recommended.
- For purposes of diabetes monitoring, specimens are usually tested 2
hours after meals. A first morning specimen does not always represent a
fasting specimen because glucose from an evening meal may remain in
 the bladder overnight, and patients should be advised to empty the
bladder and collect the second specimen.

● Gestational diabetes
- Hyperglycemia that occurs during pregnancy and disappears after
delivery.
- The onset of the hyperglycemia and glycosuria is normally around the
sixth month of pregnancy, although glycosuria may occur sooner.
- Hormones secreted by the placenta block the action of insulin, resulting in
insulin resistance and hyperglycemia.
- Detection of gestational diabetes is important to the welfare of the baby,
because glucose crosses the placenta whereas insulin does not. The baby
develops high glucose levels, causing the baby’s pancreas to produce
more insulin. The excess glucose presented to the baby is stored as fat,
resulting in a large baby (macrosomia) at risk for obesity and later type 2
diabetes. Women who have gestational diabetes also are prone to
developing type 2 diabetes mellitus in later years.

● Hyperglycemia (Non-diabetic origin)

- Some disorders that also produces glycosuria:
✔ Pancreatitis
✔ Acromegaly
✔ Cushing syndrome
✔ Hyperthyroidism
✔ Pheochromocytoma
✔ Thyrotoxicosis
- The hormones glucagon, epinephrine, cortisol, thyroxine, and growth
hormone, which are increased in these disorders, work in opposition to
insulin, thereby producing hyperglycemia and glucosuria.
 ▪ Primary function of insulin:
✔ to convert glucose to glycogen for storage (glycogenesis)
✔ these opposing hormones cause the breakdown of glycogen
to glucose (glycogenolysis)
✔ resulting in increased levels of circulating glucose
- Epinephrine is also a strong inhibitor of insulin secretion and is increased
when the body is subjected to severe stress, which accounts for the
glucosuria seen in conjunction with cerebrovascular trauma and
myocardial infarction

● Glycosuria (occurs in the absence of hyperglycemia)

- It occurs when the reabsorption of glucose by the renal tubules is
compromised.
- This is frequently referred to as “renal glycosuria” and is seen in:
✔ End-stage renal disease
✔ Cystinosis
✔ Fanconi syndrome.
- Glycosuria not associated with gestational diabetes is occasionally seen
as a result of a temporary lowering of the renal threshold for glucose
during pregnancy.

Summary of Clinical Significance of Urine Glucose

Hyperglycemia-Associated Renal-Associated

Diabetes mellitus Fanconi syndrome

Pancreatitis Advanced renal disease
Pancreatic cancer Osteomalacia
Acromegaly Pregnancy
Cushing syndrome
Hyperthyroidism
 Pheochromocytoma
Central nervous system
damage
Stress Gestational diabetes

Reagent Strip (Glucose Oxidase) Reaction

The glucose oxidase procedure provides a specific test for glucose. Reagent strips
employ the glucose oxidase testing method by impregnating the testing area with a
mixture of glucose oxidase, peroxidase, chromogen, and buffer to produce a double
sequential enzyme reaction. In the first step, glucose oxidase catalyzes a reaction
between glucose and room air (oxygen) to produce gluconic acid and peroxide. In the
second step, peroxidase catalyzes the reaction between peroxide and chromogen to
form an oxidized colored compound that is produced in direct proportion to the
concentration of glucose.

1. Glucose + O2 (air) →→→→→→→→Gluconic Acid + H 2O2

2. H2O2 + Chromogen →→→→→→→→ Oxidized Colored Chromogen + H2O2

Reagent strip manufacturers use several different chromogens, including potassium

iodide (green to brown) (Multistix) and tetramethylbenzidine (yellow to green)
(Chemstrip). Urine glucose may be reported in terms of negative, trace, 1+, 2+, 3+, and
4+; however, the color charts also provide quantitative measurements ranging from 100
mg/dL to 2 g/dL, or 0.1% to 2%.
 Glucose Reaction Interference
False Positive False Negative
✔ If containers become ✔ Substances that interfere with
contaminated with peroxide or the enzymatic reaction or
strong oxidizing detergents. strong reducing agents, such
✔ High specific gravity and low as ascorbic acid, that prevent
temperature may decrease the oxidation of the chromogen
sensitivity of the test. may produce false-negative
results.
▪ Incorporating additional
chemicals into the test
pads. An example is iodate
that oxidizes ascorbic acid
so that it cannot interfere
with the oxidation of the
chromogen
✔ Allowing specimens to remain
unpreserved at room
temperature for extended
periods, subjecting the
glucose to bacterial
degradation.
Copper Reduction Test (Clinitest)

● Measurement of glucose by the copper reduction method was one of the

earliest chemical tests performed on urine.
● The test relies on the ability of glucose and other substances to reduce
copper sulfate to cuprous oxide in the presence of alkali and heat. A color change
progressing from a negative blue (CuSO4) through green, yellow, and orange/red
(Cu2O) occurs when the reaction takes place.

● Care must be taken to observe the reaction closely as it is taking place, because
at high glucose levels, a phenomenon known as “pass through” may occur.
✔ When this happens, the color produced passes through the orange/red
stage and returns to a green-brown color, and if not observed, a high
glucose level may be reported as negative.
✔ An alternate method using two drops instead of five drops of urine can
minimize the occurrence of “pass through.” A separate color chart must be
used to interpret the reaction. This chart provides values up to 5 g/dL,
whereas the five-drop method is limited to 2 g/dL.
Clinical Significance

● In addition to glucose, commonly found reducing sugars include galactose,

fructose, pentose, and lactose, of which galactose is the most clinically
significant.
✔ Galactose in the urine of a newborn represents an “inborn error of
metabolism” in which lack of the enzyme galactose-1-phosphate uridyl
transferase prevents breakdown of ingested galactose
✔ Results in failure to thrive and other complications, including death.
✔ All states have incorporated screening for galactosemia into their required
newborn screening programs because early detection followed by dietary
restriction can control the condition.
 ● Ketones
● “Ketones” represents three intermediate products of fat metabolism, namely:
✔ Acetone (2%)
✔ Acetoacetic acid (20%)
✔ β -hydroxybutyrate (78%)
● Normally, measurable amounts of ketones do not appear in the urine, because all
the metabolized fat is completely broken down into carbon dioxide and water.
● However, when the use of available carbohydrate as the major source of energy
becomes compromised, body stores of fat must be metabolized to supply energy.
Ketones are then detected in urine.

Clinical Significance

● Ketonuria
▪ Testing for urinary ketones is most valuable in the management and
monitoring of insulin-dependent (type 1) diabetes mellitus.
▪ Ketonuria shows a deficiency in insulin, indicating the need to regulate
dosage.
▪ It is often an early indicator of insufficient insulin dosage in type 1 diabetes
and in patients with diabetes who experience medical problems in addition to
diabetes.
▪ Increased accumulation of ketones in the blood leads to electrolyte
imbalance, dehydration, and, if not corrected, acidosis and eventual diabetic
coma.
● Clinical reasons for increased fat metabolism includes:
▪ The inability to metabolize carbohydrate, as occurs in diabetes mellitus
▪ Increased loss of carbohydrate from vomiting
▪ Inadequate intake of carbohydrate associated with starvation and
malabsorption.
Reagent Strip Reactions

● The three ketone compounds are not present in equal amounts in urine. Both
acetone and β -hydroxybutyric acid are produced from acetoacetic acid. The
proportions of 78% β -hydroxybutyric acid, 20% acetoacetic acid, and 2%
acetone are relatively constant in all specimens.
● Reagent strip tests use the sodium nitroprusside (nitroferricyanide) reaction to
measure ketones.
● In this reaction, acetoacetic acid in an alkaline medium reacts with sodium
nitroprusside to produce a purple color.
● The test does not measure β -hydroxybutyrate and is only slightly sensitive to
acetone when glycine is also present; however, in as much as these compounds
are derived from acetoacetic acid, their presence can be assumed, and it is not
necessary to perform individual tests.
● Results are reported qualitatively as negative, trace, small (1+), moderate (2+),
or large (3+), or semiquantitatively as negative, trace (5 mg/dL), small (15
mg/dL), moderate (40 mg/dL), or large (80 to 160 mg/dL).

→→→→→ purple color

Reaction Interference

Atypical color reactions False positive False decrease

✔ Large amounts of levodopa ✔ improperly ✔ Volatilization of
and medications containing timed acetone and the
sulfhydryl groups, including readings breakdown of
mercaptoethane sulfonate acetoacetic acid by
sodium (MESNA) and bacteria are seen in
captopril improperly
✔ Reactions with interfering preserved specimens
substances frequently fade on
standing, whereas color
development from
acetoacetic acid increases,

Acetest Tablets

● Acetest Tablets Acetest (Siemens Healthcare Diagnostics Inc., Deerfield, IL)

provides sodium nitroprusside, glycine, disodium phosphate, and lactose in tablet
form.
● The addition of lactose gives better color differentiation.
● Acetest tablets are hygroscopic; if the specimen is not completely absorbed
within 30 seconds, a new tablet should be used.
● It has been used as a confirmatory test for questionable reagent strip results;
however, it was primarily used for testing serum and other bodily fluids and
dilutions of these fluids for severe ketosis.
http://downloads.lww.com/wolterskluwer_vitalstream_com/samplecontent/978158

2558752_Mundt/samples/Chapter_04.pdf

● Blood
● Blood may be present in the urine either in the form of intact red blood cells
(hematuria) or as the product of red blood cell destruction, hemoglobin
(hemoglobinuria). Blood present in large quantities can be detected visually;
hematuria produces a cloudy red urine, and hemoglobinuria appears as a clear
red specimen.
 ● Because any amount of blood greater than five cells per microliter of urine is
considered clinically significant, visual examination cannot be relied upon to
detect the presence of blood.
● Microscopic examination of the urinary sediment shows intact red blood cells, but
free hemoglobin produced either by hemolytic disorders or lysis of red blood cells
is not detected. Therefore, chemical tests for hemoglobin provide the most
accurate means for determining the presence of blood. Once blood has been
detected, the microscopic examination can be used to differentiate between
hematuria and hemoglobinuria.

Clinical Significance

The finding of a positive reagent strip test result for blood indicates the presence of red
blood cells, hemoglobin, or myoglobin.

● Hematuria
- Most closely related to disorders of renal or genitourinary origin in which
bleeding is the result of trauma or damage to the organs of these systems.
- Major causes of hematuria include renal calculi, glomerular diseases,
tumors, trauma, pyelonephritis, exposure to toxic chemicals, and
anticoagulant therapy.
- The laboratory is frequently requested to perform a urinalysis when
patients presenting with severe back and abdominal pain are suspected of
having renal calculi.
- In such cases, hematuria is usually of a small to moderate degree, but its
presence can be essential to the diagnosis.
- Hematuria of non-pathologic significance is observed following strenuous
exercise and during menstruation.
● Hemoglobinuria
- Hemoglobinuria may result from the lysis of red blood cells produced in
the urinary tract, particularly in dilute, alkaline urine. It also may result from
 intravascular hemolysis and the subsequent filtering of hemoglobin
through the glomerulus.
- Lysis of red blood cells in the urine usually shows a mixture of
hemoglobinuria and hematuria, whereas no red blood cells are seen in
cases of intravascular hemolysis.
- Under normal conditions, the formation of large hemoglobin-haptoglobin
complexes in the circulation prevents the glomerular filtration of
hemoglobin. When the amount of free hemoglobin present exceeds the
haptoglobin content—as occurs in hemolytic anemias, transfusion
reactions, severe burns, brown recluse spider bites, infections, and
strenuous exercise— hemoglobin is available for glomerular filtration.
- Reabsorption of filtered hemoglobin also results in the appearance of
large yellow-brown granules of denatured ferritin called hemosiderin in the
renal tubular epithelial cells and in the urine sediment.
● Myoglobinuria
- Myoglobin, a heme-containing protein found in muscle tissue, not only
reacts positively with the reagent strip test for blood but also produces a
clear red-brown urine.
- In myoglobinuria, the presence of myoglobin rather than hemoglobin
should be suspected in patients with conditions associated with muscle
destruction (rhabdomyolysis).
- Examples of these conditions include trauma, crush syndromes,
prolonged coma, convulsions, muscle-wasting diseases, alcoholism,
heroin abuse, and extensive exertion.
- The development of rhabdomyolysis has been found to be a side effect in
certain patients taking the cholesterol-lowering statin medications.
- The heme portion of myoglobin is toxic to the renal tubules, and high
concentrations can cause acute renal failure.
 - The massive hemoglobinuria seen in hemolytic transfusion reactions also
is associated with acute renal failure.

Hemoglobin vs. Myoglobin

Test Hemoglobin Myoglobin
Plasma examination Red/Pink Plasma Pale Yellow Plasma
(↓ Haptoglobin) (↑ CK, Aldolase)
Blondheim’s test
(Ammonium sulfate)

Procedure:
Urine + 2.8g NH4Sulfate
(80% saturated)
↓
(Filter/Centrifuge)
↓
Test supernatant for blood
with reagent strip
The principle of this screening test is based on the fact that the larger hemoglobin
molecules are precipitated by the ammonium sulfate, and myoglobin remains in the
supernatant.
Therefore, when myoglobin is present, the supernatant retains the red color and gives a
positive reagent strip test for blood. Conversely, hemoglobin produces a red precipitate
and a supernatant that tests negative for blood.
Reagent Strip Reactions

Chemical tests for blood use the pseudoperoxidase activity of hemoglobin to catalyze a
reaction between the heme component of both hemoglobin and myoglobin and the
chromogen tetramethylbenzidine to produce an oxidized chromogen, which has a
green-blue color.

H2O2 + Chromogen →→→→→→→→ Oxidized Chromogen + H2O

● Reagent strip manufacturers incorporate peroxide, and tetramethylbenzidine, into

the blood testing area. Two color charts are provided that correspond to the
reactions that occur with hemoglobinuria, myoglobinuria, and hematuria (RBCs).
▪ In the presence of free hemoglobin/myoglobin, uniform color ranging from a
negative yellow through green to a strongly positive green-blue appears on
the pad.
▪ In contrast, intact red blood cells are lysed when they come in contact with
the pad, and the liberated hemoglobin produces an isolated reaction that
results in a speckled pattern on the pad.
● Reagent strip tests can detect concentrations as low as five red blood cells per
microliter.
● The terms trace, small, moderate, and large or trace, 1+, 2+, and 3+ are used for
reporting.

Reaction Interference

False-positive False-negative
✔ Menstrual contamination ✔ Ascorbic acid (vitamin C)
✔ Strong oxidizing detergents ✔ Urine with a high specific gravity
are present in the specimen ▪ Contains crenated red blood
container cells that do not lyse when they
✔ Vegetable peroxidase and come in contact with the reagent
bacterial enzymes, including pad.
an Escherichia coli ✔ When formalin is used as a
peroxidase, may also cause preservative
false-positive reactions ✔ When the hypertension medication
▪ Therefore, sediments captopril or high concentrations of nitrite
containing bacteria (greater than 10 mg/dL) are present.
should be checked ✔ Failure to mix the specimen prior to
closely for the presence testing
of red blood cells. ▪ Red blood cells settle to the
bottom of the specimen
container
 ● Bilirubin

The appearance of bilirubin in the urine can provide an early indication of liver disease.
It is often detected long before the patient exhibits jaundice. Bilirubin, a highly
pigmented yellow compound, is a degradation product of hemoglobin.

Bilirubin Production
Clinical Significance

● Conjugated bilirubin can appear in the urine when bile duct obstruction
(post-hepatic jaundice e.g., gallstones or cancer) occurs
● It can also appear when the integrity of the liver is damaged (hepatic jaundice),
allowing leakage of conjugated bilirubin into the circulation.
● Hepatitis and cirrhosis are common examples of conditions that produce liver
damage, resulting in bilirubinuria.
● Detection of urinary bilirubin provides an early indication of liver disease and also
its presence or absence can be used in determining the cause of clinical
jaundice.
 Urine Bilirubin and Urobilinogen in Jaundice
Urine Bilirubin Urine Urobilinogen
Bile duct obstruction +++ Normal

Liver damage + or – ++

Hemolytic disease Negative +++

Reagent Strip

● Principle: Diazo reaction.

● Bilirubin combines with 2,4-dichloroaniline diazonium salt or
2,6-dichlorobenzene-diazonium-tetrafluoroborate in an acid medium to produce
an azodye, with colors ranging from increasing degrees of tan or pink to violet,
respectively.
● Qualitative results are reported as negative, small, moderate, or large, or as
negative, 1+, 2+, or 3+.

Bilirubin glucuronide + diazonium salt →→→→→→→→ Azodye

Reaction Interference

False-positive False-negative
✔ Primarily due to urine pigments ✔ Testing of specimens that are not
▪ Particular concern are the fresh
yellow orange urines from ✔ Bilirubin is an unstable compound
persons taking that is rapidly photo-oxidized to
phenazopyridine biliverdin when exposed to light
compounds, because the ▪ Biliverdin does not react with
thick pigment produced may diazo tests
be mistaken for bilirubin on ✔ High concentrations of ascorbic
initial examination acid (greater than 25 mg/dL) and
 ✔ Presence of indican and nitrite may lower the sensitivity of
metabolites of the medication the test
Lodine ▪ Because they combine with
the diazonium salt and
prevent its reaction with
bilirubin.

Ictotest Table (Siemens Healthcare Diagnostics Inc., Deerfield, IL)

● A confirmatory test for bilirubin.

● Ictotest kits consist of testing mats and tablets containing
p-nitrobenzenediazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric
acid.
● Ten drops of urine are added to the mat, which has special properties that cause
bilirubin to remain on the surface as the urine is absorbed.
● Following the chemical reaction, a blue-to-purple color appears on the mat when
bilirubin is present. Colors other than blue or purple appearing on the mat are
considered to be a negative result.
● If interference in the Ictotest is suspected, it can usually be removed by adding
water directly to the mat after the urine has been added. Interfering substances
are washed into the mat, and only bilirubin remains on the surface.
 ● Urobilinogen

Urobilinogen appears in the urine because, as it circulates in the blood back to the liver,
it passes through the kidney and is filtered by the glomerulus. Therefore, a small
amount of urobilinogen—less than 1 mg/dL or Ehrlich unit—is normally found in the
urine.

Clinical Significance

● Increased urine urobilinogen (greater than 1 mg/dL) is seen in liver disease and
hemolytic disorders. Measurement of urine urobilinogen can be valuable in the
detection of early liver disease.
▪ However, studies have shown that when urobilinogen tests are routinely
performed, 1% of the non-hospitalized population and 9% of a hospitalized
population exhibit elevated results due to constipation.
● Impairment of liver function decreases the ability of the liver to process the
urobilinogen recirculated from the intestine. The excess urobilinogen remaining in
the blood is filtered by the kidneys and appears in the urine.
● The clinical jaundice associated with hemolytic disorders results from the
increased amount of circulating unconjugated bilirubin. This unconjugated
bilirubin is presented to the liver for conjugation, resulting in a markedly
increased amount of conjugated bilirubin entering the intestines. As a result,
 increased urobilinogen is produced, and increased amounts of urobilinogen are
reabsorbed into the blood and circulated through the kidneys where filtration
takes place. In addition, the overworked liver does not process the reabsorbed
urobilinogen as efficiently, and additional urobilinogen is presented for urinary
excretion.
● Although it cannot be determined by reagent strip, the absence of urobilinogen in
the urine and feces is also diagnostically significant and represents an
obstruction of the bile duct that prevents the normal passage of bilirubin into the
intestine.
● The production of pale stools as the result of the lack of urobilin.

Different tests for Urobilinogen

1. Watson-Schwartz Test
- Differentiate urobilinogen (UBG), porphobilinogen (PBG), and other
Erlich-reactive compounds (ERC)
- Uses extraction with organic solvents: Chloroform and Butanol
2. Hoesch Test (Inverse Erlich reaction)
- Rapid screening test for Porphobilinogen (equal or greater than 2mg/dL)
- Procedure:

Red Color

Reagent Strip Reactions

Multistix Chemstrip
Reaction Ehrlich’s aldehyde reaction azo-coupling
principle (diazo) reaction
 Reagent p-dimethylaminobenzaldehy 4-methoxybenzenediazoniu
de (Ehrlich reagent) m-tetrafluoroborate
End color Light to dark pink White to pink
Reporting Ehrlich units (EU), mg/dL
which are equal to mg/dL
Specificity Less specific More speicific

Multistix Reagent Strip Principle:

Urobilinogen + p-dimethylaminobenzaldehyde →→→→→→→→ Red color

Chemstrip:

Urobilinogen + diazonium salt →→→→→→→→ Red azodye

Reagent strip Interference

False-positive False-negative
✔ Ehrlich-reactive compounds ✔ Improperly preserved
▪ Porphobilinogen ✔ Allowing urobilinogen to be
▪ Indican photo-oxidized to urobilin.
▪ p-aminosalicylic acid ✔ High concentrations of nitrite
▪ Sulfonamides interfere with the azo-coupling
▪ Methyldopa reaction on Chemstrip.
▪ Procaine ✔ When formalin is used as a
▪ chlorpromazine compounds preservative.
✔ Sensitive in high temp.
 ▪ Testing should be performed
at room temperature

● Nitrite
● It provides a rapid screening test for the presence of urinary tract infection (UTI).
The test is designed to detect cases in which the need for a culture may not be
apparent but it is not intended to replace the urine culture as the primary test for
diagnosing and monitoring bacterial infection.
● The nitrite test also can be used to evaluate the success of antibiotic therapy and
to periodically screen persons with recurrent infections, patients with diabetes,
and pregnant

Clinical Significance:

● Detection of Urinary Tract Infections (UTI)

 - Believed to start in the bladder as a result of external contamination and, if
untreated, progress upward through the ureters to the tubules, renal
pelvis, and kidneys.
● Detection of Cystitis
- Initial bladder infection
● Detection of Pyelonephritis
- An inflammatory process of the kidney and adjacent renal pelvis
- A frequent complication of untreated cystitis and can lead to renal tissue
damage, impairment of renal function, hypertension, and even septicemia.

Reagent Strip Reactions

● The chemical basis of the nitrite test is the ability of certain bacteria to reduce
nitrate, a normal constituent of urine, to nitrite, which does not normally appear in
the urine.
● Nitrite is detected by the Greiss reaction, in which nitrite at an acidic pH reacts
with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a diazonium
compound that then reacts with tetrahydrobenzoquinolin compounds to produce
a pink-colored azodye.
● To prevent false-positive reactions in externally contaminated specimens, the
sensitivity of the test is standardized to correspond with a quantitative bacterial
culture criterion of 100,000 organisms per milliliter.
● Although different shades of pink may be produced, the test does not measure
the degree of bacteriuria, and any shade of pink is considered to represent a
clinically significant amount of bacteria.
● Results are reported only as negative or positive.
Reaction Interference

The reliability of the nitrite test with negative results in the presence of even vaguely
suspicious clinical symptoms should always be repeated or followed by a urine culture.

1. Bacteria that lack the enzyme reductase do not possess the ability to reduce
nitrate to nitrite. Reductase is found in the gram-negative bacteria
(Enterobacteriaceae) that most frequently cause UTIs. Non–nitrate-reducing
grampositive bacteria and yeasts, however, cause a significant number of
infections, and the nitrite test does not detect the presence of these organisms.
2. Bacteria capable of reducing nitrate must remain in contact with the urinary
nitrate long enough to produce nitrite. Therefore, nitrite tests should be
performed on first morning specimens or specimens collected after urine has
remained in the bladder for at least 4 hours. The correlation between positive
cultures and positive nitrite test results is significantly lower when testing is
performed on random samples.
3. The reliability of the test depends on the presence of adequate amounts of nitrate
in the urine. This is seldom a problem in patients on a normal diet that contains
green vegetables; however, because diet usually is not controlled prior to testing,
the possibility of a false-negative result owing to lack of dietary nitrate does exist.
4. Further reduction of nitrite to nitrogen may occur when large numbers of bacteria
are present, and this causes a false-negative reaction.
5. Other causes of false-negative results include inhibition of bacterial metabolism
by the presence of antibiotics, large quantities of ascorbic acid interfering with the
diazo reaction, and decreased sensitivity in specimens with a high specific
gravity. Large amounts of ascorbic acid compete with nitrite to combine with the
diazonium salt, therefore preventing a true nitrite measurement.
 ● Leukocyte Esterase

Detection of increased urinary leukocytes required microscopic examination of the urine

sediment. The test is not designed to measure the concentration of leukocytes, and the
manufacturers recommend that quantitation be done by microscopic examination. An
additional advantage to the chemical LE test is that it detects the presence of
leukocytes that have been lysed, particularly in dilute alkaline urine, and would not
appear in the microscopic examination. Screening urine specimens using the LE and
nitrite chemical reactions to determine the necessity of performing urine cultures can be
a cost-effective measure. The LE test contributes significantly more to the reliability of
this practice than does the nitrite test.

Clinical Significance

● Normal values for leukocytes are based on the microscopic sediment

examination and vary from 0 to 2 to 0 to 5 per highpower field.
● Women tend to have higher numbers than men as a result of vaginal
contamination.
● Increased urinary leukocytes are indicators of UTI.
● The LE test detects the presence of esterase in the granulocytic white blood cells
(neutrophils, eosinophils, and basophils) and monocytes, but not lymphocytes.
▪ Neutrophils are the leukocytes (WBCs) most frequently associated with
bacterial infections.
▪ Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain
esterases.
▪ A positive LE test result is most frequently accompanied by the presence
of bacteria, which may or may not produce a positive nitrite reaction.
▪ Esterases also are present in Trichomonas and histiocytes.
▪ Infections caused by Trichomonas, Chlamydia, yeast, and inflammation of
renal tissues (i.e., interstitial nephritis) produce leukocyturia without
bacteriuria.

Reagent Strip Reaction

The reagent strip reaction uses the action of LE to catalyze the hydrolysis of an acid
ester embedded on the reagent pad to produce an aromatic compound and acid. The
aromatic compound then combines with a diazonium salt present on the pad to produce
a purple azodye.
 →→→→ indoxyl + acid indoxyl + diazonium salt→→→ purple azodye

The LE reaction requires the longest time of all the reagent strip reactions (2 minutes).
Reactions are reported as trace, small, moderate, and large or trace, 1+, 2+, and 3+.
Trace readings may not be significant and should be repeated on a fresh specimen.

Reaction Interference

False-positive False-negative
✔ Presence of strong oxidizing ✔ High concentrations of:
agents or formalin in the ▪ protein (greater than 500
collection container causes mg/dL)
false-positive reactions. ▪ glucose (greater than 3
g/dL)
▪ oxalic acid
▪ ascorbic acid
 ● Specific Gravity

Clinical Significance

✔ Monitoring patient hydration and dehydration

✔ Loss of renal tubular concentrating ability
✔ Diabetes insipidus
✔ Determination of unsatisfactory specimens due to low concentration

Reagent Strip Reaction

● The reagent strip reaction is based on the change in pKa (dissociation constant)
of a polyelectrolyte in an alkaline medium.
● The polyelectrolyte ionizes, releasing hydrogen ions in proportion to the number
of ions in the solution. The higher the concentration of urine, the more hydrogen
ions are released, thereby lowering the pH.
● Incorporation of the indicator bromthymol blue on the reagent pad measures the
change in pH. As the specific gravity increases, the indicator changes from blue
(1.000 [alkaline]), through shades of green, to yellow 1.030 [acid]).

Reaction Interference

The reagent strip specific gravity measures only ionic solutes, thereby eliminating
the interference by the large organic molecules, such as urea and glucose, and by
radiographic contrast media and plasma expanders that are included in physical
measurements of specific gravity. Elevated concentrations of protein slightly
increase the readings as a result of protein anions. Specimens with a pH of 6.5 or
higher have decreased readings caused by interference with the bromthymol blue
indicator (the blue-green readings associated with an alkaline pH correspond to a
low specific gravity reading).
 ● Ascorbic Acid
● According to the 21st Edition of Henry, ascorbic acid is considered as the 11th
parameter of reagent strip.
● Presence of Ascorbic Acid causes false negative reactions on blood, bilirubin,
leukocytes, nitrite and glucose.
● The reading time of this parameter depends on the manufacturer of the reagent
strip.
▪ C-stix: 10 seconds
▪ Stix: 60 seconds

Ascorbic Acid + Phosphomolybdate →→→→→→→→ (+) Molybdenum blue