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Automated immunoassay systems: a new frontier.

Automated immunoassay systems promise to be one of the most rapidly growing segments of the multibillion dollar clinical laboratory instrument market. If the trend is true, a large volume of proprietary reagents will be sold during the next decade. By some estimates, sales will exceed $300 million within the next few years.

Most manufacturers have introduced or plan to introduce at least one automated immunoassay analyzer in the near future. It seems that just as random-access chemistry analyzers revolutionized laboratory testing in the early 1980s, nonisotopic automated immunoassay systems are destined to change today's lab in radical ways. Workstation consolidation and decreased technical labor may no longer be promises but actually may soon appear. This article will explain why these changes are happening and how laboratorians may benefit from them.

Automating corrects a number of logistical problems involved in using immunoassays, which are valuable adjuncts to the clinical laboratory. Among other functions, immunoassays are commonly used to diagnose serious illnesses, including infectious diseases such as hepatitis and AIDS; to detect infertility problems; and to monitor medications.

Improving on lAs. First, while the potential volume of tests is great, lAs are typically batch oriented and labor intensive, a liability in today's tight-labor marketplace. Second, the tests are frequently done in small batches and are therefore an expensive proposition. The alternative is to send them to a reference facility at potentially much higher cost. Finally, turnaround time is generally far greater than it should be. Diagnosing illness may take long enough to prolong a hospital stay. Automation relieves most of these drawbacks.

Another reason automation will enhance the use of immunoassays is the trend among U.S. hospitals to move into the outpatient environment. In such settings, 24hour TAT is a must. Labs that must refer tests to a competitive facility, thus lengthening TAT, will be at a competitive disadvantage.

Understanding the principles of immunoassay automation will be easier after a quick review of the method itself. This refresher will identify advantages and limitations of the manual and semiautomated systems in widespread use today and of the more fully automated systems of the present and future. * Definition. Immunoassays are typically used to test drugs, hormones polypeptides, tumor markers, or other analytes that are present in quantities too small to be measured by traditional chemical methods. Standard methods tend to be inadequate for such testing because they cannot distinguish the chemical signal from background clutter.

How do immunoassays measure quantities beyond the reach of routine systems? Immunoassay is based on the interaction of an antibody with an antigen. This interaction is extremely specific and often strong. The analyte may be an antigen or an antibody. The reaction is monitored with a tracer-an antigen or antibody that has been tagged with a label, such as an enzyme, a fluorescent material, or a radionuclide. The tracer remains free in solution (free fraction) or binds to the complex or to one of its components (bound fraction). The tracer produces a signal, such as fluorescence. The signal is detected and is eventually converted into a result.

Immunoassay methods are either heterogeneous or homogeneous. In a heterogeneous assay, the bound and free fractions must be separated physically. This is usually done with a solid support, coated with an antigen or antibody, that binds one of the fractions, allowing the other to be washed away. For instance, the solid phase may be a tube, glass bead, or microtiter well. By measuring how much label remains in the bound fraction and comparing it with the values obtained with known standards, one can calculate the concentration of the analyte.

A heterogeneous assay, such as a radioimmunoassay, can be difficult to automate because of the robotics needed to perform the separation and because of the many washing steps. Some of the newer instrument systems simplify the task by using unique reagent systems that are nonisotopic.

What's the benefit of a heterogeneous immunoassay? This extremely sensitive technique is able to detect molecules of high or low molecular weight. Until now, however, automation has been limited by the multiple steps and precise timing required. These conditions differ considerably from one assay to another, even those from the same vendor. The result is increased costs for labor and training.

Homogeneous assays eliminate the need to separate bound from free fractions. The reason is that the free tracer yields a different signal from that of the bound tracer. Since no mechanical separation step is required, the assay can be easily automated on a dedicated instrument or on one that is used for other chemistries. With this technique, specialty immunoassays and routine chemistries can be run on the same instrument.

Homogeneous assays are limited to measuring small molecules, such as those of drugs. The ease of use of homogeneous assays was one reason for the explosive growth of therapeutic drug monitoring in the 1970s and 80s.

Recent past. Traditionally, most immunoassays have been performed with radioisotopes, which provide highly sensitive assays for measuring molecules of any size. Radioimmunoassay (RIA), however, has substantial limitations. Since radioactivity decays, for example, radionuclides have a relatively short shelf life, often no longer than a month or two-as contrasted with the more typical six- to 12-month shelf life of other chemistry reagents. Second, RIA poses safety concerns that require specially designed areas and monitoring of staff. Also needed are special waste disposal procedures, which are not easy to provide in every location. Finally, RIA is a labor-intensive batch technique that is difficult to automate. It is therefore unsuited to small laboratories in which the volume of any given test is low. For many facilities that have large test volumes, such as commercial labs, the technique remains efficient. . Immediate past. In the late 1970s, enzyme-linked immunosorbent assay (ELISA) techniques were introduced as a substitute for RIA tests. ELISA tests had longer shelf lives and were not radioactive, a major consideration as environmental and safety concerns grew. Without certain devices, which make them relatively easy to perform, they were hard to automate.

In any case, ELISA tests remain batch procedures. The tests are performed on a schedule that meets the lab's need to process a large number of specimens efficiently rather than the physician's and patient's need for a rapid result.

Homogeneous EIA tests were the one bright spot in view. Dedicated instruments performed these assays rapidly and in very small batches. In fact, individual assays could be run with little penalty. Certain tests could be put on random -access analyzers and run in this manner. Although the concept was attractive, the low volume of some tests often made it difficult to implement. Homogeneous assays worked well only for smaller molecules. The development of homogeneous immunoassay instruments created a demand for automation of other procedures. Laboratories were quickly alerted to the benefits of automation and wanted to change the way they performed all immunoassays. In response, manufacturers have been working to make the technological improvements that had to precede such a transition. * Breakthroughs. Advances over the last decade have contributed to the new wave of diagnostic immunoassay analyzers. These changes have made it possible for assays to progress from the research type to the clinical laboratory.

Monoclonal antibodies. Developed for diagnostic assays in the early 1980s, monoclonal antibodies permitted the creation of nonisotopic assays that were almost as sensitive and specific as radioimmunoassay. Some of the first assays, such as (Beta)-hCG and (Beta)-strep, provided rapid test results in an easy-to-read single-test icon. Some of these tests have proved so popular that they have replaced more automated but batch-oriented systems. New detection modes. A generation of nonisotopic labels was developed that form the basis of the detection system for most automated immunoassay instruments.

1. Enzymes. The most commonly used label, enzymes catalyze reactions that produce a color change read by a photometer. This progression avoids the problems involved with radioactivity.

2. Luminescence. Chemiluminescence is observed when light is emitted from a chemical reaction. If the reaction occurs in a living system or is derived from one, the process is called bioluminescence. A general characteristic of luminescence is that light intensity increases rapidly at first and then trails off quickly. The key to the amount of light emitted is the number of photons (light packets) produced by the chemical reaction.

Classic luminescence consists of a bright, short endurance light, much like an electronic flash but of far less intensity. One of the best emitters is the compound luminol and its derivatives. The reaction typically occurs when luminol is mixed with an alkaline solution of hydrogen peroxide and a catalyst (often Mn + + complexed with horseradish peroxidase).

More than 15 years ago, investigators observed that the light emitted was of strong intensity relative to the amount of compound present and proposed luminescent assays as nonisotopic replacements for traditional RIA. The typical gamma counter also serves as a light detector. The difference is that the light is produced by a gamma ray striking a special sodium iodide crystal rather than emerging from a chemical process.

3. Fluorescence. In a typical assay, the fluorescent compound is tagged to the antigen or antibody or the compound is produced by an enzyme-catalyzed reaction. A variation on the theme is time-resolved fluorescence, in which the emitted light appears after a measurable time delay. This delay helps to eliminate background problems.

Instrumentation. The final major technological changes that led to immunoassay automation involved aspects of instrumentation. Sophisticated computer systems enhanced the capability of instruments to read bar codes and to do calibrations and store the resulting data. Advances in computer software permitted the development of precisely controlled robotic movements. With these new devices, each step of a complex heterogeneous assay could be emulated mechanically-and this could be done correctly every time.

Combining robotics with the new reagent technologies enabled manufacturers to create test packs that could accommodate the entire test procedure. Often these packs contain not only reagents but also magnetic particles for separation and specialized miniature columns for separating bound from free fractions.

Different packs, distinguished by the reagents they contained, could therefore be processed by the same analyzer. Assay protocols were designed to minimize differences between test requirements, when possible.

Thanks to the technological advances of the last few years, various new immunoassay analyzers have been introduced. Some can run homogeneous as well as heterogeneous assays, while others run only the latter. Photometric, fluorogenic, and luminescent assays are available from several vendors. Some of the analyzers are no larger than standard benchtop analyzers. Others are small freestanding units designed to hold large volumes of supplies. Advantages. How can these new instruments benefit the laboratory? Advantages, summarized in Figure 1, are many.

Consolidation. Different test types can be performed at a single workstation. Institutions that perform immunoassays in different locations can consolidate their efforts. For instance, a single workstation could produce the B 12 normally done in hematology, the hCG in routine chemistry, and the FSH and LH in endocrinology. As the new analyzers do the work of several lab sections, they save space and technologist labor.

Larger menu. Test menu expansion is one of the first goals of the leading vendors. Eventually, laboratories will be able to run certain high-volume therapeutic drugs, hormones, B 12 and folate, tumor markers, and serologies on the same analyzer. Depending on instrument design, it may be possible to run more than one of these tests at the same time.

Ease of use. The instruments use simplified nonisotopic reagent systems. Heterogeneous assays, for example, are often run with single-test size packs. Each pack may be multiwelled and thus able to hold all reagents needed for a given test on one specimen, including the cuvette.

To separate the bound tracers from the free ones, the instrument may wash the cuvette or use a unique design to trap free tracer in an area where it cannot be detected. Because the pack is bar coded, the instrument can determine which test it is running and calculate results accordingly. Even large modified batch systems use easy-to-load reagent containers.

Ease of training. Each system runs the different tests on its menu in a similar way. It is therefore easy to train technologists. Long reagent shelf life and stable calibration will permit labs of any size to perform assays once reserved for larger hospitals and reference facilities.

High volume. Unlike RIA, which is strictly a batch testing system and can be laborious for running a small number of specimens, these instruments will process tests in a random-access or fully automated rapid-batch method. With high-volume random access instruments, the operator selects tests from an on-board menu of liquid reagents. The instrument robotizes the steps necessary for performing the assay.

Lower volume random-access analyzers use the unitized test cartridges described above. The operator loads only the tests desired. Although batch systems run only one test at a time, they provide results rapidly. Changing reagents is simple.

Disadvantages. No system is perfect. One problem with automated immunoassay systems is that most of them lock the user into purchasing reagents from a single vendor-not a good position for competitive pricing. Second, the laboratory may have to settle for good methods for all tests and forgo a few superior assays that are not available on the instrument acquired. Any test not automated by the chosen instrument will have to be performed with a manual method or referred to a commercial facility. Therefore, the system selected will largely determine the ability to expand the lab's menu. Figure I reviews the few disadvantages of automated immunoassay systems. 0 Saving time and money. A system is on the market to meet the needs of just about any laboratory, regardless of the volume of tests performed. Special benefits will accrue to the small lab that currently has insufficient test volume to justify performing many such tests in-house. The ability to perform tests in-house that were previously sent to a reference laboratory helps reduce send-out bills, no small consideration in our increasingly cost-conscious health care environment. The cost advantage can be gained without adding staff or space or reorganizing the laboratory structure.

Since automated immunoassay instruments do not require extensive calibration with each group of specimens, they can be run throughout the day. Test results can be reported more quickly than with other instrumentation-often on the same shift.

The instrumentation provides the opportunity to distribute work more evenly as well. A lab manager may decide, for example, that running most immunoassays during the less busy evening or night shifts is more efficient than doing them during the day. Implementing this preference is no problem, since the analyzers are so easy to use and training staff takes little time.

Immunoassay automation is a trend to watch for in the decade ahead. The laboratory needs these systems; manufacturers have the necessary technology. In the end, a single instrument may serve as the immunoassay analyzer for the vast majority of testing in each institution or hospital system. n
COPYRIGHT 1990 Nelson Publishing
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Author:De Cresce, Robert P.; Lifshitz, Mark S.
Publication:Medical Laboratory Observer
Date:Jul 1, 1990
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