Originally Published IVD Technology October 2004
Assay system components
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| Polysciences Inc. (Warrington, PA) supplies a wide range of plain, dyed, fluorescent, magnetic, functionalized, and custom synthesized microparticles. |
The IVD field is dynamic, continuously evolving to meet the ever-increasing
demands on clinical and laboratory diagnostic testing. Increasingly common are
panels of tests that are available to the end-user for a more accurate and timely
diagnosis of specific disease processes. Indeed, two of the most important characteristics
of diagnostic platforms are the availability of multiple tests on a single platform
and the extremely high level of sensitivity and specificity with regard to the
analyte in question.
This approach is clinically relevant particularly to pathological disorders
with a high incidence of morbidity and mortality, such as cancer and cardiovascular
diseases. Diagnostic tests for these—and other diseases—are targeted
to markers of risk prior to the clinical onset of the disease, during prognostic
evaluation of the disease process, and for assessing the patient’s current
stage of the disease. With these results in hand, clinicians can intervene appropriately.
For example, in the pathological continuum of acute coronary syndrome (ACS)
it is important to triage the patient to the individual stage of the disease
process: predisposition, thrombosis (blood clotting), ischemia (the result of
reduced blood flow), necrosis (cell death), or fibrinolysis (clot breakdown).
Multiple markers thus have been evaluated for their diagnostic sensitivity and
specificity. They include homocysteine, C-reactive protein, beta natriuretic
peptide (BNP), prothrombin F1.2, soluble fibrin, myoglobin, creatine kinase–MB,
troponin, D-dimer, and others. Logically, the platform that offers the greatest
number of these tests that have demonstrated high diagnostic accuracy would
be the one that provides the most information to the clinician and thereby optimizes
therapeutic intervention.
Unfortunately, such multitest systems have been available only in central-laboratory
settings until recently. They do not lend themselves well to acute-care facilities
such as emergency rooms, ambulances, and trauma centers. However, original and
innovative testing technologies that incorporate multiple tests with high diagnostic
precision on single platforms suitable for near-patient or point-of-care (POC)
testing are now changing the approach to diagnosis. BioSite Inc. (San
Diego), Spectral Diagnostics Inc. (Toronto), i-STAT Corp. (East
Windsor, NJ), and other companies are all developing systems intended to give
the clinician the option to perform several diagnostic tests conveniently at
the POC.
What is additionally interesting to the IVD manufacturer is the fact that these
tests may be developed using traditional assay system components in combination
with emerging technologies, particularly improvements in instrumentation. The
IVD manufacturing community should also recognize the clinical relevance of
an aging population that is being administered cocktail pharmaceutical regimens.
The need to monitor these patients is always growing; therefore, the instrumentation
and tests offered to the market must advance to meet new requirements.
Immunoassay Advances
Antibodies remain the mainstay of IVD test systems, though immunoassays have
evolved considerably since their introduction in the late 1970s. Most immunoassays
consist of three essential components: a solid phase, or surface, where the
reaction occurs; antibodies to bind the target analyte; and some type of signal
that allows quantitation of the concentration of analyte in the sample. By today’s
standards, early immunoassays—although much superior to other diagnostic
tests of the time—were crude preparations with low sensitivity and specificity.
The primitiveness of their capabilities was primarily due to the isolation procedures
used to identify the capturing antibody in the initial stages of technology
development.
Later technology brought about improvements. Now, antibodies can be isolated
rapidly with extremely high specificities. This translates into more-accurate
diagnosis, because these antibodies are able to identify extremely low concentrations
of the target analyte—in the picogram range and better. In concert with
novel detection systems that have replaced traditional colorimetric signals,
including fluorescent and chemiluminescent tags, better antibody performance
has made the newer immunoassay systems highly sensitive.
The use of fluorescent tags in immunoassays has changed with the technology’s
development. Fluorescence is the resulting emission of light at one wavelength
from a chemical that initially absorbed light of another wavelength. Early fluorescent
signals were triggered by ultraviolet wavelengths. These, although welcomed
by the diagnostics community, were not significantly better than colorimetric
signals. Since then, however, fluorescent markers that require lower activation
energies have been developed, resulting in a significant increase in sensitivity.
Panels have been introduced that incorporate multiple fluorescent tags for the
detection of numerous analytes in a single reaction by means of time-gated light
emission. These are the multitest systems defining the future of the IVD industry
as noted above. To best support clinical practice and improve patient triage
and outcome, they should be designed for near-patient testing as well as use
in centralized settings.
The best tagging method is chemiluminescence, whose extremely high sensitivity
makes it commonly exploited as a mode of signal generation. Chemiluminescence
involves the measurement of light from a chemical reaction. It enables the concentration
of an analyte in a sample to be determined by inferring the rate at which light
is emitted. This process is used in more than 50% of laboratory-based analyzers
today, primarily because of its greater sensitivity and its ability to detect
smaller concentrations of target analyte more quickly than other technologies
can. As mentioned, sensitivity is extremely important because it relates directly
to whether a disease state can be detected, how early it can be detected, and
how accurately it may be diagnosed. Studies indicate that existing chemiluminescence
techniques are 100,000 times more sensitive than spectroscopy and 1000 times
more sensitive than fluorescence, two of the more common diagnostic techniques.
Surfaces and System Components
Traditionally, many IVD systems employed microtiter plates of different consistency
as the solid phase for reagent immobilization, whereas others used specialty
reaction chambers. Developments since then have been considerable. One such
advance was the introduction of spherical microparticles as the solid phase.
These include polymer, latex, paramagnetic, and polystyrene beads that vary
in diameter and density with the test for which they are used. Advantageous
features of microparticles are that their surface chemistry can be specially
modified to create linkers for antibody or antigen binding and that they can
carry labels and dyes for detection purposes.
The use of paramagnetic particles has been ever increasing and will continue
to do so. However, advancing the traditional chemistries used in IVD applications
involving these innovative materials is a great and important challenge. One
group has succeeded by combining immunoassay development with the employment
of modified polystyrene microparticles and fluorescent tagging to make possible
the detection of more than 100 analytes in a single specimen. Others have attached
antibodies of extremely high sensitivity and specificity to paramagnetic particles.
This innovation has led to accelerated binding between the analyte and the antibody
with electromagnet-driven mixing; excellent active-phase separation by electromagnetic
means; and adaptability of the technique to all biological fluids, specifically,
whole blood, plasma, serum, urine, and cerebrospinal fluid.
While the demand is growing for several analytes, such as multiple cardiac markers,
to be evaluated in one sample, it has been recognized that very low sample volumes
for each test—in the microliter range—are a necessity. This has resulted
in the development of specialized and miniaturized sample-processing systems,
including automated sample-handling equipment. Such systems not only increase
the number of tests that can be performed from a single patient sample, but
they also significantly improve the accuracy and precision of the tests. These
benefits, in addition to reducing turnaround times, should enhance diagnostic
accuracy and enable the period from diagnosis to treatment to be shortened.
Lab-on-a-Chip Systems
Although POC test platforms have found a niche owing to clinicians’ needs
for more-sensitive and more-rapid assays to optimize patient triage, a huge
research and development investment has been made in clinical diagnostic systems
incorporating innovative new lab-on-a-chip technologies. These technologies,
more formally referred to as micro–total analysis systems (µTAS),
have enormous potential for clinical diagnosis. Exhibiting the features clinical-care
professionals are requiring of POC platforms, many of these µTAS systems
incorporate full analytical functionality. They are the site of sampling and
sample pretreatment, necessary dilution and/or sample separation (e.g., whole
blood to plasma), mixing and incubation, chemical reaction, and signal detection.
Moreover, these novel µTAS systems are readily adaptable to immunodiagnostics.
Sample volumes had previously been reduced to low levels. Now, lab-on-a-chip
systems are capable of testing microvolume samples. Additionally, these systems
provide for the evaluation of multiple analytes in parallel. Chips with advanced
microtechnologies are small, relatively inexpensive to manufacture—albeit
in bulk quantities only—and disposable. However, although µTAS systems
generally are becoming more common, issues have arisen in certain cases when
they were challenged to perform multiple analyses on complex clinical samples
such as nonanticoagulated whole blood and urine.
Regardless of their current limitations, what is very impressive about these
systems is the rapidity of the immunoassay. Turnaround times of less than 30
seconds make them ideal for acute clinical settings where speedy diagnosis can
significantly reduce morbidity and mortality, for example, in cases of acute
myocardial infarction and stroke. The ultimate degree of acceptance of µTAS
systems in clinical environments will be determined by economic considerations
of cost versus benefits. System developers cannot ignore such an important point.
Optimizing Assay Systems
As noted earlier, IVD tests are being optimized by combining emerging technologies
with traditional assay system components. Some companies have indeed successfully
produced the ideal IVD system: they have taken monoclonal antibodies with extremely
high sensitivity and specificity, incorporated the optimal tag for the assay,
and developed a system suitable for near-patient, or POC, acute clinical environments.
A sector that has learned from advances in glucose testing is the at-home clinical
diagnostic market. Glucometers for self-management of diabetic conditions have
evolved to the point where systems based on reverse iontophoresis facilitate
noninvasive glucose monitoring—no fingerstick—that purportedly provides
a more complete picture of the patient's glucose levels than before. These systems
allow the patient’s blood glucose levels to be determined every 10 minutes.
Another novel glucose monitor entered the clinical arena that can take up to
288 glucose measurements over a 24-hour period. Compared with four-times-a-day
fingersticks, it provides potentially 72 times more information to healthcare
providers. Home-use cholesterol, pregnancy, and HIV tests that follow the glucose
model currently are available. It may be that, in response to the recognized
U.S. epidemic of congestive heart failure affecting some 5 million people, BNP
tests of this type will be developed for home or outpatient testing.
In summary, key features of the best current IVD assay systems include the utilization
of state-of-the-art technologies to optimize the diagnostic test; the option
to perform multiple analytical tests on a single platform through use of a variety
of cartridges; selection of a method of signal detection that provides for high
sensitivity and specificity; and, significantly, a high degree of user-friendliness.
Rapid acceptance in the clinical arena, of course, is a must for competitive
success. Other important upcoming assay system components are powerful software
programs to allow system troubleshooting; onboard calibration and quality control;
and data handling, processing, and storage.
Diagnostic assays with all these characteristics—and that are able to provide
rapid, accurate results at the point of care—will, when combined with future
improvements in the management of pathological disease, make a considerable
contribution to the general advance of human healthcare.
Kirk Guyer,
Cascade Technologies (South Bend, IN)
Copyright ©2004 IVD Technology
