CE UPDATE—INSTRUMENTATION II

Michael J. Pugia, PhD

Technology Behind
Diagnostic Reagent Strips

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ABSTRACT Diagnostic reagent strips are commonly used
in clinical analysis of urine and blood, in particular for
monitoring glucose concentration. Results are obtained
instrumentally or visually as thresholds and quantitative
outputs. Dry reagents are applied in the construction of
strips in a variety of ways. The mechanism used to make
strips operational and practices used by manufacturers to
establish performance are reviewed, and limitations and
benefits of reagent strips are assessed.
This is the second article in a 4-part continuing education series on instrumentation.
On completion of this article, the reader will be able to identify the types of
technology used in reagent strip products.
From Bayer
Diagnostics, History of Reagent Strips
Elkhart, IN. The diagnostic reagent strip, with 1 or more
Reprint requests to reagent pads adhered to a plastic handle, is one of
Dr Pugia, Bayer the most common testing technologies in routine
Diagnostics, 1884 clinical use. The key reason for its acceptance is
Miles Ave, Elkhart, IN
ease of use. One of the first reagent strips contained
46515-0070.
a reagent pad for glucose (Clinistix; Ames Co,
Elkhart, IN; 1956) that could be dipped into urine,
was allowed to react for a minute, and read.1 This
diagnostic method eliminated the necessity of
preparing liquid reagents and was easier to use
than tablet reagents. During the next 40 years many
products were developed that offered the same ease
of use and simplicity, benefits still valued today.
After the urine glucose strips, a series of reagents
were developed, initially with diabetes mellitus as
the disease focus. As dry reagent pad tests for more
analytes became available, testing panels were
defined to assist diagnosis in the areas of kidney
function, liver function, urinary tract infections,
and carbohydrate metabolism.2
The routine urinalysis panel used today tests for
glucose (1956), protein (1957), ketone (1957), pH
(1959), occult blood (1961), bilirubin (1969), uro-
bilinogen (1969), nitrite (1972), specific gravity
(1981), and WBCs (1984) (Fig 1). Multistix 10 SG
(Bayer Diagnostics, Elkhart, IN; 1984) was an early
multiple panel with 10 reagents included on 1
reagent strip.
Aside from urinalysis, strip technology was also
applied to blood analysis with a series of glucose-
monitoring products, starting with Dextrostix
(Miles Laboratories; now Bayer; 1966) and leading
to the current glucose methods. In addition, strip
technology was used for analysis of other blood
analytes, such as lipids, hemoglobin, alanine
aminotransferase (ALT), aspartate aminotrans-
ferase (AST), bilirubin, and uric acid, and for ther-
apeutic drug monitoring. Instrumentation for
these uses included the Reflotron system
(Boehringer Mannheim, Indianapolis, IN; 1985)
and the Seralyzer system (Bayer; 1981).
Early dipsticks relied solely on colorimetric
reagents, and even today most products are col-
orimetric. The reagents change color, with the
intensity of the color proportional to the concen-
tration of the analyte measured in the clinical
specimen. For visual interpretation, the color of
the reagent is matched against 2 or more blocks
on a carefully developed color chart. Each block
represents an assigned analyte concentration
range. Assignments are based on agreement of
reagent strip results with reference methods. The
reagent is read at a specified time after application
of the specimen.

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Instrumentation
Instrumental reading arose from the need for
accuracy and data documentation. It eliminated
the subjectivity of visual interpretation and Glucose
increased convenience. The earliest instrument
was the Ames Reflectance Meter (ARM; Miles
Bilirubin
Laboratories), introduced in 1966 for measuring
blood glucose concentration. During the next 30
years a series of instruments was developed to read Ketone
dry reagent strips. These instruments translated
the reagent colors into numerical output by mea- Urobilinogen

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suring reflectance. The first urine strip instrument
was the Clinilab (Bayer), introduced in 1972. Protein
Today analyzers range from high throughput, fully
automated systems (Clinitek Atlas; Bayer; 1994) to pH
single urine strip analyzers for use in the physi-
cian’s office (Clinitek 50, Bayer, 1996; Urilux S,
Occult blood
Boehringer Mannheim, 1998). Microscopic ana-
lyzers have also made use of reagent strips (Yellow
Iris, Iris, Chatsworth, CA). Specific gravity
Glucose meters were the first instruments with
low enough cost and ease of operation for con- Nitrite
sumer use. With the advance of low-cost digital
processing, in 1981 Bayer introduced its Glucome- WBCs
ter, and today a number of companies offer glu-
cose meters to meet a variety of individual needs Negative Positive
(eg, One Touch II, Lifescan, Milpitas, CA; Com-
panion II, Medisense, Cambridge, England;

Scientific Communications
Reflolux, Boehringer Mannheim, Mannheim,
Germany).
Dry reagent strip technology involves the con- Fig 1. Multiple panels
trolled application of reagents in the manufacture with 4 reagent areas (Fig 2). The first area at the tip of dry reagents are
of the reagent pads. These reagents contain indica- of the strip is for sample application and overlaps used for urinalysis.
tor dyes, metals, enzymes, polymers, antibodies, the next reagent area, providing for transfer of the Each reagent detects
and various other chemicals dried onto carriers.3 patient sample (urine) to the third reagent area. a specific analyte
through the use of
Carriers commonly used are papers, membranes, The treated sample then migrates across a third colorimetric
or polymers with various sample uptake and layer, where reactants are immobilized for color reactions.
transporting properties. development. This migration is driven by a fourth

Reagent Strip Design
In the 1980s, reagent strip design began to include
multiple reagent layers to measure 1 analyte. This
allowed chemical reagent systems to be placed in
distinct reagent layers and provided for reaction
separation steps such as chromatography and fil-
tration. Immunochromatography strips are con-
structed so that chemical reactions occur in
distinct areas of reagents.
The Clinitek hCG strip test (Bayer) for human
chorionic gonadotropin is a dry reagent strip test
4
area that takes up excess specimen.
Section
The chromatography reaction takes place in the
third area, called the test or capture zone, typically
a nitrocellulose membrane. In the first and second
areas, an analyte-specific antibody reacts with the
analyte in the specimen and is chromatographi-
cally transferred to the nitrocellulose membrane.
The antibody is bound to colored latex particles as
a label.4 If the sample contains the analyte, it reacts
with the labeled antibody. In the capture zone a
second antibody is immobilized in a band and

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Fig 2. The immuno-
chromatography strip
has 4 distinct areas:
sample application
area; reagent area;
capture zone; and
absorbent area.
Test Your
Knowledge!
Look for the CE
Update exam on
Instrumentation (002)
in the April issue of
Laboratory Medicine.
Participants will earn
4 CMLE credit hours.
94 L A B O R ATO RY M E D I C I N E
Blood
separation
area
captures particles when analyte is present. A col-
ored test line is formed (Fig 3). A second band of
reagent is also immobilized in the capture zone to
allow a control line to react with particles, forming
color. Color at the control line is always formed
when the test system is working properly, even in
the absence of hCG in the patient sample (Fig 3).
Whole blood glucose strips often use multiple
reagents to trap intact RBCs that interfere with the
color generation area. For example, the Encore
glucometer (Bayer) uses a trapping area placed
directly over the color-generating area. The color
is read from the bottom of the strip through a
transparent window. Other designs allow the sam-
ple to migrate to a color-generating area aside
from the trapping area, and color is read from the
top of the strip. Whole blood test strips often
make use of plastic cassettes to hold the reaction
areas in place.
Multiple layers of reagent have also been
applied to film slides, such as the reagent system
used with the Ektachem analyzer (Vitros) devel-
oped by Eastman Kodak in 1980. Slides enabled
multiple separating, spreading, and color-forming
areas to enhance colors.
Typical chemical reactions occurring in dry
reagent strips can be grouped as dye binding,
enzymatic, immunologic, and redox catalysis.3
Dye binding to analytes such as albumin leads to
color changes at micromolar levels. Indicator
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Antibody-latex
conjugate
Analyte
Test band
Control band
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Absorbent/ Nitrocellulose Absorbent Polystyrene
conjugate area membrane area backing
dyes can be covalently bound to the analyte (dia-
zonium compounds binding bilirubin) or tightly
associated with the analyte (sodium-sensing
indicators). Enzymatic reactions can be used to
detect enzymes at micromolar levels through
reactions with color-forming substrates. Enzy-
matic reactions can also be used to detect mole-
cules, such as glucose, through reactions with
enzymes to yield colored end products. Particle-
labeled antibodies are the primary reagents that
provide for the detectable reaction of immuno-
logic strips based on chromatography. Redox
catalysis uses metal chelates to oxidize or reduce
indicators in the presence of specific analytes
such as hemoglobin, and can detect molecules
down to the nanomolar level.
Reagent Strip Performance
Reagent strip technology has been used to provide
either threshold or quantitative results for many
analytes. Both applications are well described for
many clinical uses. Threshold tests are applied to
analytes with well-established clinical decision
limits, based on large outcomes studies (eg, albu-
minuria, hCG), and have well-defined differences
between positive and negative test results. Quanti-
tative tests are more often applied to analytes that
require measuring of the specific levels present
over a continuous range of values (eg, glucose or
therapeutic drug monitoring) or do not have
well-established clinical outcomes or have smaller
differences between decision levels (eg, cholesterol
risk assessment).
Reagent strips that provide threshold results
are usually calibrated by the manufacturer, based
on agreements with quantitative reference meth-
ods and accounting for as many sources of errors
as possible. The dynamic range of the reagent sys-
tem is centered on the clinical decision threshold
to allow optimal clinical decision making. There-
after, the instrument automatically self-adjusts
during first-time power-up and during each assay.
In the event the system is unable to make appro-
priate internal adjustments, an error message is
displayed.
Before the manufacturer releases reagent strips,
each lot of reagent paper and finished reagent
strip package undergoes thorough analysis and
characterization. Reagents are released with fac-
tory calibration after meeting requirements that
account for as many sources of variation as possi-
ble. When reagents are stored and used properly,
acceptable performance is ensured until the expi-
ration date.
Major manufacturers ensure performance with
tight process controls specific for their analyzers.
However, the operating instructions on dipping
and placing the strip on the analyzer must be fol-
lowed. Read times are typically set for conve-
nience. Reading at incorrect times can cause errors
in some systems, as not all tests are read
time–independent. Samples are usually applied by
dipping the strip into the specimen with immedi-
ate removal or by placing a drop of sample on the
dry reagent pad. Prolonged exposure to the sam-
ple can leach chemical from the reagent pad and
alter results. Although most potential interfer-
ences are known and mentioned in the labeling,
contamination of specimens with any additive
may affect results and should be avoided.
Quantitative results with reagent strips have
required more carefully controlled calibration
processes, either through use of calibration solu-
tions or a lot-specific calibration number or cod-
ing. Calibration numbers are factors used in the
standard curve to increase accuracy, and are
derived from characterization of manufacturing
Control band
Test band
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Negative Low Medium High
positive positive positive
Fig 3. The
lots. Calibration solutions reduce ease of use and
progression of
provide additional sources of analytical error (the results for an
process of doing calibration), but provide greater immuno-
accuracy potential. Lot-specific calibration cannot chromatography
offer as much user control of accuracy and strip occurs through
Scientific Communications
changes in color
requires greater analysis, characterization, and
intensity at the test
process control by the manufacturer. band.
The challenge of providing quantitative results
with reagent strip technology has prompted sev-
eral manufacturers to introduce liquid systems for
quantitative applications. This is particularly true
with analytes that require measurement over a
wide continuous range or without large degrees of
difference between clinical results (positive and
negative). In many cases, point-of-care blood
4
chemistry analyzers also require CLIA [Clinical
Section
Laboratory Improvement Amendments of 1988]
waived status (highest degree of accuracy with
essentially error-free use). Liquid reagent systems
can be factory calibrated yet automatically treat
specimens with the diluents needed to reduce
matrix affects and improve accuracy. One point-
of-care system is the hemoglobin A1c assay system
on the DCA 2000+ Analyzer (Bayer).
The use of reagent strips for threshold results
has required products to be more specific to dis-
ease conditions. Manufacturers have addressed
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COMING UP IN MARCH
Feature
interference through development of new chem-
istry tests. Greater sensitivity is also being
addressed with tests that detect new analytes that
increase earlier in disease progression. For example,
Market Your Laboratory for urinary protein assessment, the move is away
Experienced laboratory leaders cite customer service, monitoring from using the presence of Ä300 mg/L protein as a
costs, and keeping abreast of volume demands as key marketing decision limit and toward using trace levels of albu-
musts for a successful outreach program. Review how marketing min (eg, Ä30 mg/L, or microalbuminuria) for
impacts outreach business in the next feature article to appear in detection of nephropathy risk. The Clinitek
Laboratory Medicine. Microalbumin Reagent Strip (Bayer) is an example
of a more sensitive and specific technology that

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CE Updates indicates clinically significant analyte levels through
Instrumentation—Hematology Analyzers new chemistry tests.5
Offer a User-Friendly New Technology
This third article in a 4-part continuing education series on Conclusion
instrumentation incorporates the principles, applications, and Diagnostic reagent strips have been used for more
user-friendliness of hematology analyzers. The once labor-intensive than 40 years, and they continue to serve clini-
manual procedure for blood-cell analysis can now be performed cians because of their ease of use and conve-
with highly automated instruments capable of measuring many nience. Manufacturers have continued to apply
disease-associated parameters, while incorporating nontraditional and improve the technology to adapt to market
flow cytometry, robotics, and expert system technologies. needs. Because of ease of use and acceptable reli-
ability, reagent strip technology remains the pre-
Waste—Medical Waste Management: ferred choice for many applications, particularly
Where Does the Garbage Go? for threshold tests (eg, urinalysis) and tests per-
The modern landfill is designed to control emissions and minimize formed in the physician’s office (eg, hCG,
adverse effects on the environment. This first article in a 3-part microalbuminuria, drugs of abuse, group A Strep-
continuing education series on current waste management tococcus, Helicobacter pylori, Chlamydia, and C-
practices addresses how careful management of medical waste reactive protein).l
minimizes the spread of infection and what happens to medical
waste once it is placed in a municipal solid waste landfill. References
1. Free A, Free H. Urinalysis in Clinical Laboratory Practice.
Boca Raton, FL: CRC Press; 1975:217-224.
Special Report 2. Henry JB, ed. Clinical Diagnosis and Management by Labo-
Medical Technology and Clinical Laboratory Science Faculty Survey ratory Methods, 19th ed. Philadelphia, PA: Saunders;
A questionnaire regarding salary compensation for those teaching 1996:164;411-456.
3. Burtis CA, Ashwood ER. Reference information for the
in medical technology (MT) and clinical laboratory science (CLS) clinical laboratory. In Tietz NW, ed. Tietz’ Textbook of Clinical
was sent to program directors at 126 university-based MT and CLS Chemistry, 3rd ed. Philadelphia, PA: Saunders; 1999:483-484,
programs to determine if there have been changes since previous 798-801, 1218, 1799-1839.
4. Bangs LB. New developments in particle-based immunoas-
surveys. Find out the results compiled by academic rank according says: introduction. Pure Appl Chem. 1996;68:1873-1879.
to geographic location and type of program in the March issue of 5. Pugia MJ, Lott JA, Luke KE, et al. Comparison of instru-
Laboratory Medicine. ment-read reagent strips for albumin and creatinine in urine
with visual results and quantitative methods. J Clin Lab Anal.
1998;12:280-284.
Science
Central Laboratory Analysis
of Intact PTH Using Intraoperative Samples
A more convenient and less expensive alternative to point-of-care
methods has been developed to test chemiluminescent assays of
plasma intact parathyroid hormone (PTH). Evaluated by collecting
venous blood samples in EDTA tubes, transporting them to the
laboratory by pneumatic tube or courier, and processing and
assaying them as stat tests, the central laboratory method achieved
results within a time interval equivalent to previous point-of-care
tests. In addition, the new procedure did so at a fraction of the cost
of bedside procedures and proved diagnostically valuable.l

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