CHEMICAL TESTS

IN ROUTINE
URINALYSIS
 REAGENT STRIPS
Reagent strips provide a rapid, simple means
for performing medically significant chemical
analysis including pH, protein, glucose, ketones,
blood, bilirubin, urobilinogen, nitrite, leukocytes and
specific gravity. These are manufactured under the
tradenames,Multistix and Chemstrip and may
possess single or multiple testing areas.

Reagent strips are consist of chemically

impregnated absorbent pads attached to a plastic
strip. A color is produced after it is dipped into a
urine sample and the result is compared to a
colored chart provided by the manufacturer.
The results may be read visually or in special
instruments that read specific reagent strips.
Some areas are used for screening tests and
others are used to estimate the amount(semi-
quantitative).

Advantages of dry reagent strip tests over

traditional chemical tests:
1. Convenience 5. Disposability
2. Cost effectiveness 6. Smaller sample vol.
3. Stability 7. Space saving
4. Relative ease in learning to use
 CARE OF REAGENT STRIPS:
1.Store w/ dessicant in a tightly closed opaque
container.
2.Store below 30oC; do not freeze.
3. Do not expose to volatile fumes.
4. Do not use past expiration date.
5. Do not use if chemical pads are discolored.
6. Remove strips immediately prior to use.
ERRORS DUE TO IMPROPER TECHNIQUE:
1. Formed elements such as red and white blood cells sink to
the bottom of the specimen and will be undetected in an
unmixed specimen.
2. Allowing the strip to remain in the urine for an extended
period may cause leaching of reagents from the pads.
3. Excess urine remaining on the strip after its removal from
the specimen can produce a run-over between chemicals
on adjacent pads, producing distortion of the colors.
4. The timing for reactions to take place varies between tests
and manufacturers, and ranges from an immediate
reaction for pH to 120 seconds for leukocyte esterase.
5. A good light source is essential for accurate interpretation
of color reactions.
 Reagent strips must be checked with both
Positive and Negative controls a minimum
of once every 24 hours.
 Many laboratories perform this check at
the beginning of each shift.
 CHEMICAL TESTS IN URINE
I. pH
-pH is a unit that describes the acidity or
alkalinity of a solution. Together with the lungs, the
kidneys are the major regulators of the acid-base
content in the body. This is made possible by the
secretion of hydrogen in the form of ammonium ions,
hydrogen phosphate, & weak organic acids and by
the reabsorption of bicarbonate from the filtrate in
the convoluted tubules.
-A healthy individual will usually produce a first
morning urine w/c is slightly acidic(5.0-6.0) and an
alkaline pH following meals(alkaline tide).
pH of random sample = 4.5 to 8.0

CLINICAL SIGNIFICANCE:
1.Respiratory or metabolic acidosis – diabetic acidosis
2.Respiratory or metabolic alkalosis
3. Renal tubular acidosis – inability of the kidney to
produce an acid urine in the presence of metabolic
acidosis.
4. Treatment of urinary tract infection
5. Management of renal calculi formation
6. Identification of urinary crystals
7. Determination of unsatisfactory specimen
 Urinary pH is controlled primarily by
dietary regulation.
 The pH of freshly excreted urine does not
reach 9.0 in normal or abnormal
conditions. A pH of 9.0 is associated with
improperly preserved specimen and
indicates that a fresh specimen should be
obtained to ensure the validity of the
analysis.
CAUSES OF ACID OR ALKALINE URINE:
ACID URINE ALKALINE URINE
Emphysema Hyperventilation
Diabetes mellitus Vomiting
Starvation Renal tubular acidosis
Dehydration Presence of urease-produ
Diarrhea cing bacteria
Presence of acid- Vegetarian diet,milk
producing bacteria(E. coli) Old specimens
High protein diet Medications(e.g.sodium
Cranberry juice bicarbonate)
Medications(e.g.Mandelamine)
DETERMINATION OF URINE pH:
Reagent strip test
-Uses methyl red & bromthymol blue double
indicator system that measures urine pH in a
range from 5 to 9. Methyl red detects pH ranging
4.4 to 6.2 w/ a color change from red to yellow
while bromthymol blue detects pH range from 6.0
to 7.6 w/ a color change from yellow to blue.

II. PROTEIN
- This is the most significant test in routine
urinalysis that indicate a renal disease. Normal
urine contains very little protein usually less than
10 mg/dL or 100 mg/24 hours.
-this is consist of primarily of low molecular weight
serum proteins that have been filtered by the
glomerulus(e.g. albumin) and those produced in the
genitourinary tract.(e.g. Tamm-Horsfall
protein/Uromodulin, prostatic and vaginal secretions)
Proteinuria – occurrence of protein in the urine.
Albumin – major serum protein found in normal urine
due to its low molecular weight, however not all are
filtered out, majority are reabsorbed by the tubules.

Three categories of proteinuria:

A. Prerenal proteinuria
- caused by conditions affecting the plasma
prior to its reaching the kidney.
 -not indicative of actual renal disease.
- frequently transient caused by increased
level of hemoglobin, myoglobin and acute phase
reactants associated w/ infection & inflammation.
-since reagent strip tests for protein detect
only albumin, these can not be discovered in routine
urinalysis.
Bence Jones Protein
- a monoclonal immunoglobulin light chain
that is produced in increased amounts in Multiple
myeloma(a proliferative disorder of the
immunoglobulin-producing plasma cells). Its
elevated level in the plasma exceeds the tubular
reabsorption capacity & is excreted in urine.
 -Unlike other proteins that coagulate when heated,
Bence Jones coagulate at temperatures between 40oC
and 60oC and dissolves when the temperature reaches
100oC.
**Not all persons w/ Multiple myeloma excrete
detectable levels of Bence Jones protein, they must be
diagnosed by serum electrophoresis.

B. Renal proteinuria
- associated with true renal disease and is a result of
either glomerular or tubular damage. Selective
filtration is impaired due to damage in the glomerular
membrane leading to excretion of increased albumin
& blood cells in urine.
Glomerular disorders include:
a.Immune complex disorders f. Strenuous exercise
b.Amyloidosis g.Hypertension
c.Toxic agents h. Pre-eclampsia
d.Diabetic nephropathy i. Orthostatic or pos-
e.Dehydration tural proteinuria

Tubular disorders include:

a. Fanconi’s syndrome
b. Severe viral infections
c. Toxic agents/heavy metals
Microalbuminuria
-proteinuria that can not be detected by
routinely used reagent strips.
- useful in detecting early development of
renal complications in patients w/ diabetes mellitus.
- can be detected by Micral test(a reagent
strip test employing antibody-enzyme conjugate to
bind human albumin).

C. Postrenal proteinuria
- protein is usually derived from the lower
urinary tract such as the ureter, bladder, urethra,
prostate & vagina.
This is usually associated with the following
conditions:
a. Lower urinary tract infection
b. Injury or trauma
c. Menstrual contamination/Vaginal secretions
d. Prostatic fluid/spermatozoa

DETERMINATION OF URINE PROTEIN:

1.Reagent strip test
- uses the principle of the “protein error of
indicators”. This test is sensitive to albumin w/c
is believed to accept ions from the indicator. The
indicator on the strip may either be tetrabrom-
phenol blue or tetrachlorophenol or tetrabromosulfon-
Phthalein and an acid buffer to maintain the pH. At
pH 3, the indicator will appear yellow in the absence
of protein and change to various shades of green to
blue in the presence of protein.
Sources of error in strip test:
(False positive)
1.Highly buffered alkaline urine
2.Allowing the reagent pad to remain in contact w/
urine for a prolonged period-remove the buffer
3. Highly pigmented urine and contamination w/
ammonium compounds, detergent & antiseptic
4. High specific gravity urine
(False Negative)
1.Proteins other than albumin

2.Microalbuminuria

2. Sulfosalicylic acid precipitation test

- a cold precipitation semi-
quantitative test that reacts equally with all
forms of protein. This must be performed
on centrifuged specimen to avoid any
extraneous contamination.
III. GLUCOSE
- is the most commonly found sugar in urine. This is
the most frequently performed chemical analysis on
urine. Glucose in urine is used to diagnose and
monitor a metabolic condition rather than renal or
urinary tract condition. Normally, almost all glucose
filtered by the glomerulus are reabsorbed in the
proximal convoluted tubule, therefore urine contains
only minute amounts of glucose or none at all.
- However, if blood glucose level is elevated and if it
exceeds the renal threshold for glucose, glucose will
start to appear in urine.
 Glycosuria(glucosuria) – presence of detectable
amounts of glucose in urine. The chief cause is
diabetes mellitus. This is sometimes observed in
some pregnant women due to placental hormones
that block insulin action.

CLINICAL SIGNIFICANCE:
Hyperglycemia associated:
*Diabetes mellitus *Pancreatitis
*Acromegaly *Cushing’s syndrome
*Hyperthyroidism *Pheochomocytoma
*Gestational diabetes *CNS damage
*Pancreatic Ca *Stress
Renal associated
*Fanconi’s syndrome *Osteomalacia
*Advanced renal disease *Pregnancy

DETERMINATION OF URINE GLUCOSE:

1. Reagent strip test
- a specific test for glucose; uses glucose
oxidase.
- the test area is impregnated w/ a mixture of
glucose oxidase, peroxidase, chromogen and
buffer to produce a double sequential enzyme
reaction.
 Step by step reaction:
1. Glucose + O2(air)glucose oxidase Gluconic acid + H2O2

2. H2O2 + chromogen peroxidase oxidized colored + H2O

chromogen
** Chromogen used may either be potassium
iodide(green to brown) or tetramethylbenzidine
(yellow to green). Colored chart provide
quantitative measurements ranging from 100mg/dL
to 2 g/dL or 0.1% to 2%.

Sources of error:
1.Container contaminated w/ peroxide or strong
oxidizing detergents.(false positive)
2.Ascorbic acid- prevents oxidation of chromogen.
2. Copper reduction test (Benedict’s test)
- relies on the ability of glucose and other
substances to reduce copper sulfate to cuprous
oxide in the presence of an alkali and heat. A color
change is observed progressing from blue(negative)
to green, yellow, and finally to orange/red(Cu2O).
CuSO4(Cu+3) + glucose heat Cu2O(Cu+2)
alkali

Cu2O + oxidized substance color

(blue/green-orange/red)

- nonspecific test for glucose, since any

reducing sugar will also yield a (+) result.
Clinitest tablet
- a more convenient method w/c uses the
same principle as Benedict’s.
- the tablet contains copper sulfate, sodium
carbonate, sodium citrate & sodium hydroxide.
- Upon addition of the tablet to water & urine,
heat is produced by the hydrolysis of sodium
hydroxide & its reaction w/ sodium citrate and CO2
is released from sodium carbonate to prevent room
air from interfering w/ the reduction reaction. The
color result is then compared w/ a colored chart.
- the sensitivity of the test is 200mg/dL
glucose
 Comparison of Clinistix and Clinitest
1. Clinitest is not specific while Clinistix is specific.
2. Clinistix is more sensitive than Clinitest.
3. A negative Clinistix with a positive Clinitest is the
most significant discrepancy. This may be due to
interfering substances or a metabolic disorder.

IV. KETONES
- intermediate products of fat metabolism w/c
include acetone, acetoacetic acid(diacetic acid) &
beta-hydroxybutyric acid. Normally, these do not
appear in urine since all metabolized fat are
broken down into carbon dioxide & water.
- Ketone bodies will only appear in urine when fat
instead of carbohydrate is utilized as the source of
energy. Increased fat metabolism will lead to
increased ketone in the blood(Ketonemia) and in
urine(Ketonuria).
-Acetone & beta-hydroxybutyric acid are produced
from acetoacetic acid and their proportion in urine
is as follows:
78% beta-hydroxybutyric acid
20% acetoacetic acid
2% acetone.
CLINICAL SIGNIFICANCE:
1. Diabetic acidosis
2. Insulin dosage monitoring
3. Starvation
4. Malabsorption/pancreatic disorders
5. Strenuous exercise
6. Vomiting
7. Inborn errors of amino acid metabolism

DETERMINATION OF URINARY KETONES:

1. Reagent strip test
- uses the sodium nitroprusside reaction.
 - Acetoacetic acid in an alkaline medium will react w/
sodium nitroprusside to produce a purple color. This
test does not measure beta-hydroxybutyric acid
and is only slightly sensitive to acetone.
2. Acetest
- a tablet test for acetone and can also be used for
serum and other body fluid.
Interferences:
False Positive:
Phthalein dyes
Highly pigmented red urine
Levodopa
Medications containing free sulfhydryl groups
False Negative:
Improperly preserved specimen

V. BLOOD
- together with protein and
microscopic examination of the sediment, test
for blood in urine is an indicator of the state of
the kidney & urinary tract.
- Blood may appear as intact red blood
cells or as free hemoglobin.
Hematuria – presence of intact red blood cells;
produces a cloudy red urine.
Hemoglobinuria – presence of free hemoglobin in
urine; produces clear red specimen.

Causes of Hematuria:
1. Renal calculi 6. Toxic chemicals
2.Glomerulonephritis 7. Anticoagulants
3. Pyelonephritis 8. Strenuous exercise
4. Tumors
5. Trauma
Causes of Hemoglobinuria:
1.Transfusion reaction 4. Infections/malaria
2. Hemolytic anemia 5. Strenuous exercise
3. Severe burns

Myoglobinuria
- presence of myoglobin(heme-containing
protein found in muscle tissue). This reacts w/
reagent strip test for blood and produces a clear
red-brown urine.
Causes of Myoglobinuria:
1. Muscular trauma/crush syndrome
2. Prolonged coma
3. Convulsions
4. Muscle wasting disease
5. Alcoholism/overdose
6. Drug abuse
7. Extensive exertion
8. Cholesterol-lowering statin medications

Identification of hemoglobinuria from myoglobinuria:

Hemoglobinuria Myoglobinuria
Normal CK & LDH CK & LDH
Plasma-normal appearance Plasma- red color
Hemoglobin can be pre- Myoglobin cannot be
cipitated by ammonium by ammonium sulfate
sulfate
DETERMINATION OF BLOOD IN URINE:
1. Reagent strip test
- utilizes the pseudoperoxidase activity of
hemoglobin to catalyze a reaction between hydrogen
peroxide and the chromogen tetramethylbenzidine to
produce an oxidized chromogen, w/c has a blue green
color.
H2O2 + chromogen hemoglobin oxidized chromogen +H2O
peroxidase
***In hemoglobinuria/myoglobinuria color range is
from yellow(negative) through green to a strongly po-
sitive green-blue.
***In hematuria, a speckled pattern appears on the
pad. Sensitivity is 5RBCs/uL of urine.
Sources of error:
False positive reactions are due to:
*Menstrual contamination *Vegetable peroxidase
*Strong oxidizing detergents *Bacterial enzymes

False negative reactions are due to:

*High specific gravity urine containing crenated rbcs
*Formalin is used as preservative
*Hypertensive drug such as captopril
*High nitrite conc.(>10 mg/dL)
*Ascorbic acid(>25mg/dL)
*Unmixed specimen
VI. BILIRUBIN
- a highly pigmented yellow compound w/c is
a degradation product of hemoglobin.
- not a normal urine constituent.
Hemoglobin degradation
RBC(120 days) Biliverdin

Hemoglobin Unconjugated bilirubin

Plasma albumin
Globin Heme Liver(conjugation)

Amino Iron Protoporphyrin Conjugated bilirubin

Acid
Pool BM Tissues Biliverdin Bile duct
 Bile duct

Intestines
Intestinal bacteria
Urobilinogen
1/2 1/2
Feces Reabsorbed & recirculated through liver

Fecal Small amount escapes liver & goes to kidney

Urobilinogen

Urine urobilinogen
CLINICAL SIGNIFICANCE:
1. Obstruction of the bile duct(e.g. gallstones or
cancer)
2. Liver disease – Hepatitis & cirrhosis
3. Determines the cause of clinical jaundice

Urine Bilirubin Urine

urobilinogen
Bile duct obstruc- +++ Normal
tion
Liver damage + or - ++
Hemolytic disease Negative +++
DETERMINATION OF URINE BILIRUBIN:
1. Reagent strip test
- uses the diazo reaction.
- Bilirubin combines w/ 2,4 dichloroaniline
diazonium salt or 2,6 dichlorobenzene diazonium
tetrafluoroborate in an acid medium to produce an
azodye with colors ranging from tan or pink to violet.

2. Ictotest
- higher sensitivity as compared to strip test.
- purple color on the mat is a positive result.
- used as confirmatory test.
Sources of error:
False positive: Urine pigments such as indican &
those caused by some drugs
False negative: Specimen is not fresh, high
concentration of ascorbic acid & nitrite

VII. UROBILINOGEN
- by-products of rbc degradation; formed from
bilirubin by bacterial action in the intestine &
excreted in the feces as stercobilin.
- Normally, a small amount of urobilinogen is present
in urine; less than 1 mg/dL or 1 Ehrlich unit.
CLINICAL SIGNIFICANCE:
1. Increased in liver disease and hemolytic disorders
2. Absent in urine & feces in complete obstruction of
the bile duct.

DETERMINATION OF URINE UROBILINOGEN

1. Reagent strip test
- uses Ehrlich’s aldehyde reaction wherein
urobilinogen reacts w/ p-diethylaminobenzaldehyde
(Ehrlich’s reagent) to produce colors ranging from
light to dark pink.
Interferences:
False (+):
Highly pigmented urine Indican
Porphobilinogen Sulfonamides
Mehthyldopa Procaine
False (-):
Old specimen
Preservation in formalin

2. Ehrlich’s tube test

- a cherry red color is produced in the
presence of urobilinogen. Other Ehrlich reactive
compounds will also yield a positive result.
Porphobilinogen
- normal, colorless precursor of porphyrins and
related to urobilinogen.
- eliminated from the body in urine & feces as
coproporphyrin I and small amount of
coproporphyrin III.
- can also be detected by Ehrlich’s aldehyde
reaction.

Watson-Schwartz test
- a classic test for differentiating urobilinogen &
porphobilinogen.
Chloroform extraction Butanol extraction
Urobilinogen Urobilinogen & other
Ehrlich reactive
compds.

U B U B U B

C U C U
C U

Urobilinogen Porphobilinogen Ehrlich reactive

Hoesch test
- a rapid screening or monitoring test for urinary
porphobilinogen.
- a red color is a positive result.

VIII. NITRITE
- the test for nitrite is a rapid method of
detecting urinary tract infection.
- most useful when combined with leukocyte
esterase test.
-nitrate is a normal constituent of urine w/c is
converted by certain bacteria to nitrite(not normally
found in urine).
Gram negative bacteria(Enterobacteriaceae) – can
reduce nitrate to nitrite.
Gram positive bacteria(Enterococci) & yeasts – cannot
reduce nitrate to nitrite.

CLINICAL SIGNIFICANCE:
1. To detect initial bladder infection(cystitis)
2. Pyelonephritis – inflammatory process of the
kidney & adjacent renal pelvis.
3. Evaluation of antibiotic therapy
4. Monitor patients who are at high risk for UTI
5. Screening of urine culture specimen
DETERMINATION OF NITRITE
Reagent strip test
- involves the Greiss reaction in w/c
nitrite
at an acidic pH reacts with an aromatic amine
(para-arsanilic acid or sulfanilamide) to form a
diazonium compound that reacts w/ tetrahydro-
benzoquinolin compound to produce a pink
colored azo dye.
- sensitivity is 100,000 organisms/mL.

Bacteriuria – presence of significant number of

bacteria in urine(100,000 or more/mL)
Sources of error:
False Positive: Sample is not fresh;highly
pigmented urine
False Negative:
1. If the bacteria present do not have reductase
enzymes.
2. Sample is randomly collected.
3. Lack of dietary nitrate.
4. Large number of bacteria present – nitrite is further
reduced to nitrogen.
5. Presence of antibiotics, high conc.of ascorbic acid,
and high specific gravity urine.
IX. LEUKOCYTE ESTERASE
- this test detects esterase released from the
primary granules of granulocytic leukocytes as well
as monocytes. Esterases are also present in
Trichomonas & histiocytes.
- this can also detect leukocytes that have
been lysed particularly in dilute alkaline urine and
would not appear in the microscopic examination.

CLINICAL SIGNIFICANCE:
1.Bacterial and non bacterial UTI
2.Inflammation of the urinary tract(e.g. interstitial
nephritis)
3. Screening of urine culture specimen
DETERMINATION FOR LEUKOCYTE ESTERASE
Reagent strip test
- utilizes the action of leukocyte esterase in
catalyzing the hydrolysis of an acid ester imbedded
on the reagent pad to produce an aromatic
compound and acid. The aromatic compound then
combines with a diazonium salt to produce a purple
azo dye.
Sources of error:
False Positive: *Strong oxidizing agents
*Highly pigmented urine
*Nitrofurantoin
*Formalin
False Negative:
*High concentration of protein, glucose, ascorbic
acid and oxalic acid
*Crenation of leukocytes
*Antibiotics such as Gentamicin, Cephalexin,
Cephalothin & Tetracycline
*High specific gravity