Reagent Strip

Reagent Strip
• Multistix (Siemens Healthcare Diagnostics, Deerfield,
IN)
• Chemstrip (Roche Diagnostics, Indianapolis IN)
• 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
• A semiquantitative value of trace. 1+ 2+ 3+ 4+ can be
reported.
 Errors Caused by 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. To ensure against run-over, blotting the edge of
the strip on absorbent paper and holding the strip
horizontally while comparing it with the color chart is
recommended.
 Errors Caused by Improper Technique
4. The timing for reactions to take place varies
between tests and manufacturers, and rangers
from an immediate reaction for pH to 120
seconds for leukocyte esterase. Manufacturers
recommended that reactions be read between
60 and 120 seconds, with the leukocyte esterase
reaction read at 120 seconds.

5. A good light source is essential for accurate

interpretation of color reactions.
 Errors Caused by Improper Technique
6. The strip must be held close to the color chart
without actually being placed on the chart.
Automated reagent strip instrument
standardize the color interpretation and
timing of the reaction and are not subject to
room lighting deficiencies or inconsistency
among laboratory personnel.
7. Reagent strip and color charts from different
manufacturers are not interchangeable
8. Specimen that have been refrigerated must
be allowed to return to room temperature
prior to reagent strip testing, as the enzymatic
reactions on the strips are temperature
dependent.
 Handling and Storing Reagent Strips
• Reagent strip are packaged in opaque containers
with a desiccant to protect them from light and
moisture
• Strips are removed just prior to testing, and the
bottle is tightly resealed immediately.
• Bottles should not be opened in the presence of
volatile fumes.
• Manufacturers recommended that reagent strips
be stored at room temperature below 30˚C (but
never refrigerated)
 Handling and Storing Reagent Strips
• All bottles are stamped with an expiration
date that represents the functional life
expectancy of the chemical pads.
• Reagent strips must not be used past the
expiration date.
• Care must be taken not to touch the chemical
pads when removing the strips.
 Quality Control of Reagent Strips
• Reagent strips must be checked with both
positive and negative controls a minimum of
once every 24 hours.
• Distilled water is not recommended as a
negative control because reagent strip
chemical reactions are designed to perform at
ionic concentrations.
Confirmatory Testing
• Non reagent strip testing procedures using
tablets and liquid chemicals may be available
when questionable results are obtained or
highly pigmented specimens are encountered.
pH
• A healthy individual usually produces a first
morning specimen with a slightly acidic -> pH of
5.0 – 6.0
• More alkaline pH is found following meals
(alkaline tide)
• The pH of normal random samples can range
from -> 4.5 – 8.0
• Collecting specimens in containers other than the
single-use laboratory-supplied containers can
produce a pH above 8.5 if alkaline detergent
remains in the container
Reagent Strip Reactions
• The Multistix and Chemstrip brands of reagent strips
measure urine pH in 0.5 or 1-unit increments
between pH 5 and 9
• Double-indicator system of methyl red and
bromthymol blue
• Methyl red produces a color change from red to
yellow in the pH range 4-6
• Bromthymol blue turns from yellow to blue in the
range of 6-9

Methyl red + H + -> bromthymol blue – H+

(Red-orange -> yellow) (green -> blue)
Protein
• Normal urine contains very little protein: usually,
less than 10mg/dL or 100mg per 24 hours is
excreted.
• Albumin is the major serum protein found in
normal urine.
• Other proteins include small amounts of serum
and tubular microglobulins; Tamm-Horsefall
protein (uromodulin) produced by the renal
tubular epithelial cells; and proteins from
prostatic, seminal and vaginal secretions.
Reagent Strip Reactions
• Traditional reagent strip testing for protein uses
the principle of the Protein error of indicators to
produce a visible colorimetric reaction.
• 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)

Indicator + protein -> protein + H+

(Yellow) Indicator – H+
(blue – green)
Glucose
Reagent Strip(Glucose Oxidation) Reaction
• Mixture of glucose oxidase, peroxidase,
chromogen and buffer to produce a double
sequential enzyme reaction.
• Glucose oxidase catalyzes a reaction between
glucose and room air (oxygen) to produce
gluconic acid and peroxide
• 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.
• Reagent strip manufacturers use several
different chromogens, including potassium
iodide (green to brown) (Multistix) and
tetramethylbenzidine (yellow to green)
(Chemstrip) Glucose oxidase
» 1. Glucose + O2 (air) gluconic acid +H2O2
Peroxidase
» 2. H2O2 + chromogen oxidized colored
chromogen + H2O
Copper Reduction Test (Clinitest)
• 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
 Heat
CuSO₄ (cupric sulfide) + reducing substance
Alkali

Cu₂O (cuprous oxide) + oxidized substance color

(blue/green orange/red)
Ketones
• The term “ketones” represents three
intermediate products of fat metabolism,
namely:
Acetone (2%)
Acetoacetic acid (20%)
b-hydroxybutyrate (78%). However, when the use
of available carbohydrate as the
 Ketones
• Normally, measurable amounts of ketones do
not appear in the urine, because of all the
metabolized fat is completely broken down
into carbon dioxide and water.

• Major source of energy becomes

compromised, body stores of fat must be
metabolized to supply energy; Ketones are
then detected in urine
Reagent Strip Reactions
• The three ketones compounds are not present in
equal amounts in urine.
• Both acetone and b-hydroxybutyric acid are
produced from acetoacetic acid
• The proportions of 78% b-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
• Acetoacetic acid in an alkaline medium reacts
with sodium nitroprusside to produce a purple
color

Acetoacetate (acid acetone) + sodium nitroprusside

alkaline
+ (glycine) purple color
Acetest Tablets
• Acetest (Siemens Healthcare Diagnostic 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.
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).

• Hematuria produces a cloudy red urine, and

hemoglobinuria appears as a clear red
specimen
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
Hemoglobin
H₂O₂ + chromogen oxidized chromogen + H₂O
Peroxidase
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
Hepatitis – Other liver disorders
Cirrhosis – Biliary obstruction (gallstones,
carcinoma)
Reagent Strip (Diazo) Reactions
• Routine testing for urinary bilirubin by reagent
strip uses the diazo reaction.
• Bilirubin combines with 2,4-dichloroaniline
diazonium salt or 2,6-dichlorobenzene-
diazonium-tetrafluoroborate in an acid
medium to produces an azodye, with colors
ranging from increasing degrees of tan or pink
to violet Acid
Bilirubin glucoronide + diazonium salt azodye
Ictotest Tablets
• A confirmatory test for bilirubin is the Ictotest
(Siemens Healthcare Diagnostics Inc.,
Deerfield, IL).
• Ictotest kits consist of testing mats and tablets
containing p-nitrobenzenediazonium-p-
toluenesulfonate, SSA, sodium carbonate, and
boric acid.
Ictotest Tablets
• A blue-to-purple color appears on the mat
when bilirubin is present
• Color 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.
Urobilinogen
• When conjugated bilirubin is excreted
through the bile duct into the intestine, the
intestinal bacteria convert the bilirubin to a
combination of urobilinogen and
stercobilinogen.
• 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.
• A small amount of urobilinogen – less than
1mg/dl or Ehrlich unit – is normally found in
the urine.
Reagent Strip Reactions and Interference

• Differ between Multistix and Chemstrip

Multistix uses Ehrlich’s aldehyde reaction
In which urobilinogen reacts with p-
dimethylaminobenzaldehyde (Ehrlich reagent) to
produce colos ranging from light to dark pink.
Chemstrip
An azo-coupling (diazo) reaction using 4-
methoxybenzenediazonium-tetrafluoroborate to react
with urobilinogen, producing colors ranging from white
to pink.
This reaction is more specific for urobilinogen than the
Ehrlich reaction.