Business & Marketing

Knowledge is power, they say. So here's a bundle of power in the form of key marketing information for IVD manufacturers.

Each month, the editors of IVD Technology receive dozens of phone calls from people inquiring about the status of the market for particular types of in vitro diagnostics. To help readers keep up with the growth of technologies and market opportunities in the industry, IVDT has compiled a list of current market reports that analyze the industry and its future. A brief summary of each report's contents, gathered from the publisher's catalogs, brochures, or the report's table of contents, is provided, as well as length, price, and date of publication. In addition, a directory of the companies publishing these reports is provided. Contact the companies directly concerning purchase of reports or for additional information contained in the reports or the company's other research.

Changes in Diagnostic Practice
Financial Times
156 pp, $488, May 1996

The clinical diagnostics industry comprises the in vitro and electromedical diagnostic markets. These two markets are converging as automation and close-to-patient testing increase. According to this report, no more than 20 companies will soon account for 90% of sales in the world's IVD and electromedical markets. This report projects market potential by product type to the year 2000 and analyzes the future of the industry, its changing structure and market drivers, and successful and failed technologies. It includes profiles of 26 leading diagnostics companies and discusses the health-care, medical device, and IVD regulatory systems of the key global markets.

The Market for Rapid In Vitro Diagnostic Tests
FIND/SVP
300+ pp, $2950, February 1997

Worldwide, the market for rapid IVD tests grew 9.6% in 1996 to $623 million. Sales in the United States reached $259 million, primed by point-of-care testing and shaped by lab-licensing regulatory changes, managed-care cost-cutting measures, and drug legislation. Rapid-IVD-test marketers face competition from automated labs and managed care; the future of the rapid-test market lies in acceptance at the level of hospitals and independent laboratories.

Medical Testing Chemicals
The Freedonia Group, Inc.
204 pp, $3200, February 1996

Demand for medical testing chemicals will expand more than 8% per year (or 3.7% when adjusted for inflation) to $5.1 billion in the year 2000. The expanding need for pretreatment diagnostics will keep the demand for medical chemical testing concentrated in routine clinical chemistry and x-ray substances. The fastest gains will occur among immunology and hematology chemicals, based on advances in monoclonal antibodies that will enhance the reliability of AIDS testing and lead to new tests for previously hard-to-detect cancers, allergies, and autoimmune disorders. The report includes discussions of the national and international markets and the industry structure.

European Diagnostic Products: Competitive Benchmarking
Frost & Sullivan
489 pp, $1950, May 1997

This study examines 50 companies involved in the development, production, and marketing of diagnostic products and services in Europe, and forecasts trends to 2003. Market sectors analyzed include IVDs, electromedical diagnostic equipment, patient monitoring, and diagnostic enhancement reagents.

European In Vitro Cancer Diagnostic Markets
Frost & Sullivan
231 pp, $3900, August 1996

In vitro cancer diagnostic markets in Europe are highly specialized and extremely competitive. Profitability is declining because of increased competition. This trend is expected to continue, because testing sites across Europe have consolidated to create large laboratories that reduce expenses through economies of scale. As a result, manufacturers must supply automated machinery that can process broad testing parameters with minimum expenditure. This report examines major market and technology trends, revenues and growth rates, and market leaders' strategies for success.

European Non-Isotopic Immunoassay System Markets
Frost & Sullivan
271 pp, $3900, March 1996

This report analyzes the European non-isotopic immunoassay system market in terms of revenues, pricing, and industry trends from 1992 to 2002. Four major system product markets are addressed: manual nonisotopic immunoassay systems, semiautomated systems, automated batch systems, and automated random-access systems, which offer increased accuracy, high-volume throughput, and significantly reduced operator involvement.

European Rapid Microbiology Product Markets
Frost & Sullivan
353 pp, $3950, November 1996

The many segments of the rapid microbiology market do not combine to form a single, coherent industry, and the suppliers are diverse. Competitors bring different backgrounds to the market and range from large, well-rounded multinationals to small companies specializing in one area. This report examines the industry, provides forecasts for the market to 2002, provides revenue, trend, and competitive information for each market segment, and describes current and emerging strategies.

European Research Biochemical Markets
Frost & Sullivan
355 pp, $3950, June 1997

Recent advances in biotechnology have caused the use of biochemical research reagents to spread to laboratories involved in disciplines as disparate as forensic science and archaeology. Biochemical research reagent companies will be serving rapidly growing markets, and the release of new products will be necessary if they are to remain competitive and profitable. This report is divided into product categories examining the molecular biology, cell biology, immunology, protein chemistry, and analytical reagents markets in Europe. Key participants' current marketing, sales, and development strategies are examined, and industry leaders are profiled.

European Spectrometer and Spectrophotometer Markets
Frost & Sullivan
396 pp, $3950, September 1996

The spectrometers and spectrophotometers market has experienced widespread merger and acquisition activity and continued consolidation. The market is expected to follow a price-led recovery from the recession through sales of high-end and application-specific instruments and analyzers. Innovative uses of Fourier transform analyzers are currently of great interest to manufacturers. This study provides revenue, trend, and competitive information for each European market segment and describes the current and emerging strategies of market leaders.

U.S. Biotechnology and Pharmaceutical Instrumentation Markets
Frost & Sullivan
471 pp, $2495, March 1996

Demand for biotechnology and pharmaceutical instrumentation has grown significantly in the past 15 years. In 1995, manufacturer revenues swelled to $560 million in the United States. By 2001, biotechnology and pharmaceutical instrumentation revenues are projected to reach $1 billion. This report analyzes market trends, particularly toward the miniaturization of instrumentation technology, and examines market leaders' strategies for success.

U.S. DNA Probe Markets
Frost & Sullivan
313 pp, $3295, December 1996

DNA probes have historically been used in research to locate and identify particular genes of interest. Major technological advances in recent years have launched this research tool into the clinical laboratory. Using small amounts of DNA, probe technology holds promise as a testing method for more than 4000 genetic diseases. For the next five years, however, infectious disease diagnostics are projected to be the most active market segment. This report analyzes market and technology trends and examines current and emerging competitive strategies.

U.S. Markets for In Vitro Sensor Technologies, 1995­2000
Medical Data International, Inc.
178 pp, $2850, August 1996

Driven by a continued emphasis on point-of-care testing, the market for in vitro sensor-based clinical diagnostic products is expected to experience strong growth, reaching $1.2 billion in sales by 2000--an average annual increase of 20.1%. Sensors are expected to be important in every major segment of the clinical diagnostics market, including clinical chemistry, immunodiagnostics, cellular analysis, and microbiology. The report covers electrochemical sensors, optical sensors, noninvasive optical spectroscopy, electrooptical sensors, mass sensors and microanalytical technology, and the role of sensor technology in clinical testing. It also includes sections on new trends and emerging technologies, market analyses and sales forecasts, and profiles of 23 companies.

U.S. Rapid Microbiology Test Markets
Frost & Sullivan
460 pp, $2995, May 1997

Point-of-care testing allows earlier, accurate diagnosis and treatment, eliminating costly delays. Manufacturers are just beginning to tap into this promising area for clinical microbial test development. This study presents analyses of and forecasts for the market to 2003 and describes current marketing, sales, and development strategies of key market participants and industry leaders.

U.S. Reference Laboratory Testing Markets
Frost & Sullivan
368 pp, $2995, January 1997

The reference laboratory testing industry is under considerable cost pressures. Current trends make it difficult for laboratories to earn profits. Most labs are gambling that they can sacrifice profits for market share or target only profitable niches. This report provides an overview of the clinical chemistry, hematology, immunoassay, and chromatography testing markets. It then examines the current and emerging strategies of the market leaders.

World Biosensor Markets
Frost & Sullivan
250 pp, $2450, March 1997

The world biosensors market is on the verge of significant expansion. This report provides analysis of the biosensor industry and explores key competitive issues, including research and development into new technologies, expansion into new markets, and increasingly stringent international laws. Analysis and forecasts are provided to 2003 for medical, environmental, industrial, and military biosensor market segments.

World Clinical Laboratory Analytical Instrument Markets
Frost & Sullivan
517 pp, $2995, January 1996

For clinical laboratories, increased automation and instrument consolidation eliminates instrument and labor costs by reducing the number of instruments used and operators required. This report provides an overview of the world market and provides complete revenue, trend, and competitive information for the chemistry analyzer, hematology analyzer, immunoassay analyzer, electrolyte analyzer, blood gas analyzer, bilirubinometer, and osmometer markets.

AIDS Diagnostics and Therapeutics Markets
Theta Reports
100+ pp, $995, June 1997

The home market for AIDS testing expanded in mid-1996, and the clinical reference laboratory testing segment is expected to increase significantly. The automation of test processes, incorporation of gp120 testing capabilities, and development of less-expensive DNA probe testing procedures to determine viral load will affect the market for AIDS diagnostics. The report analyzes clinical, competitive, demographic, and technological trends affecting the current and future expansion of the AIDS diagnostics and therapeutics markets. Revenues and growth rates are included for the market segments, with projections to 2002.

Biotechnology Laboratory Products Market
Theta Reports
100+ pp, $1295, June 1997

The market for laboratory products currently exceeds $2 billion. Bioseparations, cell handling, cell culturing, sequencing/synthesizing, and life-science consumables are assessed. The report includes discussion of liquid chromatography, electrophoresis, tissue culture incubators, fermentors, capillary electrophoresis, biosensors, disposable glassware, restriction enzymes, PCR, separation resins, columns, detectors, and nucleic acid and amino acid synthesizers and sequencers.

Blood Gas and Electrolyte Instruments Markets
Theta Reports
116 pp, $995, January 1997

The U.S. market for blood gas and electrolyte analyzers is projected to grow from $314 million in 1995 to $343 million in 1998. By 1998, worldwide sales will exceed $1 billion. The report examines market trends and new developments in dedicated electrolyte analyzers and blood gas instruments marketed to hospital labs and to clinics and doctors' offices performing medium- to high-volume automated clinical chemistry testing. Sales volume and market size are projected by segment to the year 2000.

Cancer Diagnostics and Therapeutic Markets
Theta Reports
161 pp, $995, August 1996

Competition is increasing in the cancer diagnostics and therapeutic products market. Among diagnostics, tumor markers are expected to grow 10% annually, led by growth in the sales of PSA tests. Sales by genetic testing services are expected to reach $1 billion by the next decade. The report provides an overview of the market, offers insight into new cancer drugs and diagnostic techniques and systems, and provides market forecasts and company profiles.

Cytologic Diagnostics Markets
Theta Reports
100+ pp, $995, March 1997

The cytometry systems market totals more than $500 million worldwide and is expected to reach $685 million by the year 2000. The market is expected to grow 5 to 8% annually, with the market for reagents growing faster than that for instrumentation. The report examines the cytometry systems market, including research and clinical segments, through 2000.

Diagnostic Market and Technology Trends--Worldwide
Theta Reports
100+ pp, $1295, April 1997

By the year 2000, hospital consolidations and the related emphasis on preventive medicine, testing guidelines, and optimized treatment scheduling will create an increasing need for more timely delivery of diagnostic test information. The result will be greater use of point-of-care testing and an increase in the number of sophisticated diagnostic tests allowing for customized patient therapeutics. The report examines the evolution of IVD technologies in major world markets through 2000. Key categories include immunoassays, general chemistries, hematology/coagulation, microbiology, DNA probe assays, and cytology.

DNA Diagnostics and Gene Therapy Markets
Theta Reports
120+ pp, $995, March 1997

This report examines the maturing diagnostic market for nonamplified assays for pathogenic bacteria, where combination tests for chlamydia and gonorrhea dominate. Technologies based on biochips and oligonucleotide arrays have received financial support from venture capitalists and government funding agencies. PCR and other target amplification technologies applied to high-sensitivity assays for bacteria and viruses are entering the market, as is genetic screening. The report tracks the progress, prospects, and players in this growing market.

Laboratory Disposables Markets
Theta Reports
100+ pp, $995, September 1997

The report forecasts total and market segment revenues through 2002 for disposable products that are used for automated and nonstandard testing in general laboratory, hematology, immunology, microbiology, pathology, and toxicology procedures. The report also provides a series of company profiles that describe the top competitors in the lab disposables market, and a competitive analysis evaluates consolidation and restructuring strategies.

Point-of-Care Testing in Hospital Markets
Theta Reports
100+ pp, $995, March 1997

This report considers the advantages and disadvantages of point-of-care testing in hospital settings. It discusses the internal and external influences, facilitators, and impediments to hospital adoption of POC testing methods. Market size, shares, and growth are provided for glucose monitoring, critical care, coagulation, cardiac risk, and other product areas, including urinalysis and drug-of-abuse testing. Company information is provided for market leaders and niche companies, and a directory of companies active in the market is included.

Point-of-Care Testing: Physician's/Home
Theta Reports
145 pp, $995, December 1996

This report analyzes consumer self-testing and physician-office markets for point-of-care testing. The leading products covered are blood glucose test strips, pregnancy and ovulation tests, and blood pressure monitors. Fecal occult blood testing, cholesterol tests, and HIV tests are also covered. The home-test market is projected to grow 14% per year over the next five years. By 1999, several new test categories will be available in the home segment, with noninvasive glucose monitors expected to be the largest.

Separations: Chromatography and Electrophoresis
Theta Reports
90 pp, $995, July 1996

The report examines the size and growth of the $1.3-billion separations market in biotechnology, biopharmaceutical, biomedical, research, and industrial applications. HPLC systems, columns, and detectors; low-pressure LC, chromatography supplies and reagents; electrophoresis; and star capillary electrophoresis are covered. The report projects growth rates to 2001, gives current market shares of major competitors, and profiles 10 companies. Among other results, the report concludes that computerization and software will be key to market success, and that HPLC will offer the largest market segment through 2001.

U.S. Biotechnology Research Reagent Market
Theta Reports
97 pp, $995, October 1996

The biotechnology research reagents and kits market is divided into three segments: molecular biology, immunochemistry, and peptides. The report projects sales by user, application, and manufacturer for each of these segments through 2001. Only the laboratory research market is covered. The report predicts continued double-digit market growth through 2001.

Market-Study Publishers

Financial Times
14 East 60th St., Ste. 1206, Penthouse
New York, NY 10022
212/888-3469
 

FIND/SVP
625 Avenue of the Americas, Dept. FNF
New York, NY 10011
800/346-3787
 

The Freedonia Group, Inc.
3570 Warrensville Center Rd., Ste. 201
Cleveland, OH 44122-5226
216/921-6800
 

Frost & Sullivan
2525 Charleston Rd.
Mountain View, CA 94043
415/961-9000
 

Medical Data International, Inc.
2 Park Plaza, Ste. 1200
Irvine, CA 92614
800/826-5759
 

Theta Reports
1775 Broadway, Ste. 511
New York, NY 10019
212/262-8230
 

Note: this is the second part of a two-part article. Part 1 is also available for on-line reading.

In recent years, glucose monitors using biosensor technologies have enjoyed tremendous commercial success. Despite their seeming potential for use in other areas, however, biosensors for other analytes have so far met with limited endorsement. Advancing the use of this emerging technology will require manufacturers to have an understanding of both the opportunities and limitations it presents.

The first installment of this article provided a brief introduction to biosensors, with emphasis on amperometric enzyme electrodes (IVDT, July/August 1997, pp 39­45). Amperometric biosensors combine the selectivity of an enzyme reaction with the sensitivity of amperometric detection. In operation, these biosensors use an enzyme to convert an analyte into an electroactive product, which is then transduced into a quantifiable amperometric response by an electrode.

The level of sophistication associated with such biosensors can be defined by the manner in which the enzyme reaction is transduced to the amperometric response. The latest generation of biosensors is characterized by "wired" enzymes, in which the enzymatic reaction is directly transduced to the amperometric response by means of a molecular wire that connects the enzyme to the electrode.

Below, the second installment of this article describes the use of wired-enzyme technology with peroxidase enzymes to detect H^₂O^₂. Although H^₂O^₂ is rarely an analyte of primary interest, diagnostic assays often require H^₂O^₂ detection. Equally important, the response characteristics of H^₂O^₂ biosensors can facilitate several unique applications, including the adaptation of wired-enzyme sensors to an electrochemical affinity assay.

Wiring of Peroxidase

Peroxidases (POD) include a broad group of enzymes able to catalyze the following reactions:

The first reaction is highly selective for peroxides, primarily H^₂O^₂ and a few small organic peroxides. The second and third reactions are much less selective for electron donors (HA).

Amperometric peroxidase-based H^₂O^₂ sensors have been made by using fast reversible redox couples. In these, the reducing member of the redox couple (essentially species HA in the reactions above) donates electrons to H^₂O^₂ and is oxidized:

The oxidized redox couple is then cathodically reduced at the electrode surface:

The most commonly used enzyme in these biosensors is horseradish peroxidase (HRP), a small (44-kD) heme peroxidase, but others have been used as well. Detection schemes vary in their method of enzyme immobilization, mediator, and type of enzyme. At one extreme are systems based on the direct transfer of electrons from the electrode surface through surface-bound mediators to HRP redox centers contacting the surface. At the other extreme are systems with freely diffusing mediators and enzyme.

The redox polymers designed and used for oxidase wiring are able to transfer redox equivalents in the reverse direction from electrode to POD active sites, making wired-enzyme H^₂O^₂ biosensors (Figure 1).^1,2 When the electrode is poised at 0.0 mV versus SCE, the H^₂O^₂ flux is measured as a cathodic current. The sensitivity is 1 A cm^­2 M^­1 over the linear range extending from 0.1 to 100 µM. The limit of detection for a steady-state measurement is 10 nM. Using a flow system, injections of less than 100 pM have been detected.³

Figure 1. The redox cycles occurring at a three-dimensional redox epoxy wired enzyme electrode. The wired enzyme is a heme-containing peroxidase (POD).

 

Amperometric biosensors have used platinum electrodes for H^₂O^₂ detection since they were first developed. The platinum electrodes continue to be used because of their excellent performance. Although feasible, POD-based amperometric H^₂O^₂ detection is not commercially used except in a flow-injection analysis detector sold by Bioanalytical Systems (West Lafayette, IN).

With the development of the wired POD biosensor for H^₂O^₂ detection, the supremacy of H^₂O^₂ detection on platinum was challenged. The H^₂O^₂ biosensor also facilitated several applications achievable only due to the enhanced performance or unique characteristics of enzyme wiring. These include sensitive flow-system detectors,^3,4 selective-scanning electrochemical probe tips,^5,6 enhanced sensitivity for oxidase sensors,⁷ amperometric NADH detection,¹ and use in affinity sensors.^8,9

Electrochemical Affinity Biosensors

Affinity sensors detect the coupling reaction between the selective binding unit (SBU) (e.g., avidin, antibody, single-stranded DNA, lectin, and host artificial molecular recognition species) and its complementary component (e.g., biotin, antigen, complementary single-stranded DNA, sugar sequence, and guest target compound). Affinity sensors' design ensures that binding of SBU and complement takes place on the transducer surface. The sensors are thus implicitly heterogeneous. The transducer converts the binding event into a measurable response.

The sensors can be divided into two categories: nonlabeled and labeled. Nonlabeled affinity sensors directly detect the affinity complex by measuring physical changes at the transducer induced by complex formation. In contrast, labeled affinity sensors incorporate a sensitively detectable label, and the presence of the affinity complex is then determined through measurement of the label.

Typically, detection of the binding event is not a direct measurement. Labeling of either SBU or complement aids in signaling the binding event. Enzyme labels are particularly useful for providing signal amplification. Their incorporation yields higher sensitivity. Since enzyme electrodes effectively coupled redox enzymes with amperometric detection, there was a natural progression to coupling enzyme-labeled affinity reactions and amperometric detection.

Heineman pioneered the use of alkaline phosphatase­antibody conjugates to perform sandwich immunoassays.¹⁰ In these assays, aminophenyl phosphate is used as a substrate (in place of nitrophenyl phosphate), and the aminophenol product is detected anodically with a flow-injection analysis system.

Aizawa devised a host of sensors with a classic Clark-type O^₂ electrode as the base sensor and catalase as the enzyme label.¹¹ Catalase is used to decompose H^₂O^₂ to O^₂ and H^₂O. When the enzyme label is immobilized at the sensor surface by an affinity reaction, an increase in O^₂ signal is observed in an H^₂O^₂ solution.

Rishpon, Bourdillon, and others have developed electrode-based affinity sensors incorporating enzyme labels and the immobilization of an affinity component at the electrode surface.^12­15 Although excellent sensitivities were obtained, these assays were hampered by the need to wash the working electrode and change the incubation and test solutions. The chief problem was the difficulty in distinguishing enzyme-catalyzed reactions in bulk solution from surface-associated reactions.

A goal of affinity sensor engineers has been the development of nonseparation methods where wash steps (a source of irreproducibility) are not necessary. In a recent article, Duan and Meyerhoff proposed a scheme where the substrate, which is converted into electroactive product, is brought into the cell from behind the electrode.¹⁶ This approach allowed for measurement of the binding reaction without the usual washing steps. However, a specially designed cell and electrodes were necessary.

Wired-Enzyme Affinity Biosensors

Previously, sensitive H^₂O^₂ electrodes built by covalently immobilizing HRP in a redox hydrogel were described.^1,2 The redox hydrogel was formed of HRP and water-soluble poly(vinylpyridine) that was quaternized partly with 2-bromoethylamine and partly with osmium bipyridine redox centers (PVP-NH^₂-Os), and cross-linked with poly(ethylene glycol diglycidyl ether) on vitreous carbon.

The sensitivity of these electrodes, on which H^₂O^₂ was electrocatalytically reduced by the sequence shown in Figure 1, was remarkably high: 1 A cm^­2 M^­1. Catalytic electroreduction of H^₂O^₂ was observed with as little as 1 µg/cm² HRP incorporated in the hydrogel. Modification of the catalytic behavior of the hydrogel by addition of minute amounts of HRP led to the hypothesis that the specific binding of HRP-labeled affinity reagents to an electrode could be selectively detected and that the resulting amperometric affinity sensors would not require washings or separation of reagents.

Based on this hypothesis, fast, compact, inexpensive, and separation-free amperometric affinity sensors for biotin and avidin were developed.⁸ The sensor was constructed by immobilizing an SBU (avidin) into a three-dimensional electron-conducting redox hydrogel (enzyme wiring) on a 3-mm vitreous carbon electrode. The SBU provided the electrode affinity for the SBU's complementary component (biotin). Incubation of the affinity sensor with its complementary component led to selective uptake of the complement from the solution. If the complement was first labeled with a redox enzyme, incubation led to binding of that enzyme to the wiring gel on the electrode. The principles of detection in such a sensor for the avidin-biotin system are presented schematically in Figure 2.

Figure 2. Direct transduction of biotinylated-HRP (B-HRP), avidin (X), and biotin (*) concentrations to currents in a PVI-Os "wire" and avidin-modified electrode. When B-HRP binds with avidin in the HRP-wiring hydrogel (A), a current flows. The current is inhibited if (B) the B-HRP binds to dissolved rather than surface avidin or (C) the binding sites of avidin in the wiring hydrogel are occupied by free biotin.

 

The biotin/avidin affinity electrode was used to directly detect redox enzyme­labeled complement in a test sample (pictorially described in Figure 2, path A). Here the avidin is immobilized at the electrode in the hydrogel, and the conjugate biotin is labeled with a POD redox enzyme. In this method, the affinity sensor is incubated in a solution containing redox-labeled complement (B-HRP). Binding of the complement selectively immobilizes redox enzyme in the hydrogel. Addition of redox enzyme substrate generates an electrical signal detectable at the electrode.

In the case of POD labels, electrons generated at the electrode are relayed to the POD enzyme through the hydrogel network to which the POD is selectively bound by avidin. In the presence of the enzyme's substrate H^₂O^₂, the electrons are then transferred from the reduced POD to hydrogen peroxide, generating the flow of an electrical current. This current is a function of the concentration of biotinylated peroxidase immobilized at the electrode by the SBU. As shown in Figure 3, electrons are relayed from the electrode through the wire and the POD enzyme to H^₂O^₂, which is electroreduced to water. Measurement is generally at +100 mV (Ag/AgCl). PVI-Os is a polymer with a polyvinylimidazole backbone and osmium bipyridine redox sites coordinated to 20% of the imidazole groups. PVI-Os serves the same redox wiring function as PVP-Os-NH^₂.

Figure 3. Time dependence of the current of the polyvinylimidazole with coordinated osmium bipyridine (PVI-Os) avidin-modified electrode (2 µg avidin, 3.3 µg PVI-Os, and 0.83 µg polyethylene glycol diglycidyl ether [PEGDGE]) after injecting H^₂O^₂ to 100 µM and injecting B-HRP to 1 µg/ml concentration. Conditions: 5 ml PBS; 1000 rpm; +0.1 V Ag/AgCl.

 

The affinity electrode can also detect SBU by a competitive process. An unknown concentration of avidin, free in the solution, is allowed to compete with electrode-immobilized avidin for a limited number of enzyme-labeled complement molecules. This process is pictorially represented in Figure 2, path B. The free avidin effectively prevents the complement from complexing with the avidin in the wiring hydrogel. The current resulting from the electrocatalytic reduction of H^₂O^₂ is higher when fewer complement SBUs are present in the solution. The limit of detection is below 5 µg/ml.

In a similar assay, biotin was detected by allowing a fixed number of labeled complement molecules to compete with an unknown concentration of biotin (not redox enzyme­labeled) for the limited number of SBUs immobilized at the electrode. The process is pictorially presented in Figure 2, path C. The current generated from reduction of enzyme substrate is inversely related to the amount of unlabeled complement. The limit of detection is below 10 nM. All of these assays were accomplished without washing of the electrodes.

As yet, no wash solution has been found that effectively separates biotin from avidin without destroying the ability of avidin to bind biotin or changing the redox characteristics of the PVI-Os films. Such a solution is bound to be elusive, considering that the couple does not separate even at extremes in pH.

The lack of reversibility makes it necessary to use multiple electrodes when establishing calibration curves. Preliminary work with an antibody to biotin incorporated in PVI-Os gels on electrodes has shown that, like the PVI-Os-avidin films, the binding of B-HRP can be tracked by the increase in H^₂O^₂ reduction current. However, unlike the PVI-Os-avidin films, where binding is practically irreversible, the B-HRP binds reversibly to the antibiotin-containing film. In three cycles of binding and separation, the current increased and decreased reproducibly, showing that the film did not degrade upon brief cycling (Figure 4). With any multiple-use affinity biosensor, a washing sequence is required, at least for the separation and removal of the initially bound complement.

Figure 4. Three biotin-labeled horseradish peroxidase binding (B-HRP) cycles (A, B, and C) are shown for an immunosensor made with a 1-µl loading of solution containing 2.5 mg/ml PEGDGE, 1 mg/ml goat antibiotin, and 10 mg/ml PVI-Os mixed in a 1:5:1 ratio. The B-HRP binding event was carried out in 5 mL pH 7.4 PBS. The H^₂O^₂ concentration was 0.1 mM and the B-HRP concentration was 1 µg/ml. The electrode was rotated at 1000 rpm and poised at 100 mV versus Ag/AgCl. The binding was reversed by washing the electrode in pH 2 PBS for 2 hours.

 

Conclusion

This previous work described a generic approach for direct electrical detection of the occurrence of an affinity reaction. The sensitivity and detection limits were adequate for some widely performed assays. The microampere currents measured were a thousandfold higher than those routinely measured with simple and inexpensive ($50) potentiostats. They were a millionfold higher than currents measured in Faraday cages with state-of-the-art low-noise current amplifiers and potentiostats.

While sensitivity in a competitive assay is typically based on the shape of the displacement curve, the electrochemical assays were actually limited by the electrodes' size and binding capacity. Considering that all the affinity reagent was stripped from a large (5-ml) volume, no obstacle can be seen to detecting thousandfold and even millionfold smaller amounts of affinity reagents, simply by using smaller electrodes. For example, by using standard 10-µm-diameter ultramicroelectrodes, the sensitivity could be increased by a factor of 10⁵.

Over the past decade, biosensors have been touted as the future of chemical sensors. Academic and basic research efforts have flourished. At a recent symposium on biosensing and biosensors sponsored by the American Chemical Society, 250 papers were presented. However, with the exception of blood glucose monitoring, the gap between R&D and development of actual commercial products has rarely been bridged.

The nature of the biosensor limits the opportunities for commercial success. Affinity biosensors will have a difficult time competing with techniques such as standard enzyme-linked immunosorbent assays, which can be fully automated and operated in multiplexed batches of 96 and even 384 samples. Biosensors seem best suited for limited-use and point-of-care applications.

The personal blood glucose­monitoring business is the prime example of a market requiring immediate on-site analysis without requiring high throughputs. To successfully commercialize affinity biosensors, a similar niche market will have to be identified. Likely targets include infectious disease detection, military applications for immediate detection of hazardous chemicals/microbes, food safety monitoring for bacteria, and possibly genome testing.

One hurdle to tackling the limited markets is the difficulty in recovering product development costs. The blood glucose market is several billion dollars strong and can sustain major R&D efforts. The market for an affinity biosensor is only a fraction of this market. A biosensor strategy that is adaptable to multiple analytes will have the distinct advantage of spreading development costs over several products.

Glossary

amperometry: Measurement of the current resulting from a redox reaction.

anode: Electrode at which oxidation occurs.

avidin: A glycoprotein having four subunits. Each subunit has one binding site for biotin.

biotin: A 244-molecular-weight vitamin found in tissue and blood. It binds with a high affinity to avidin.

cathode: Electrode at which reduction occurs.

mediator: Any chemical species able to transfer electrons between an enzyme's active site and an electrode.

oxidation: A redox reaction involving the loss of electrons.

oxidoreductase: An enzyme that catalyzes an electron transfer reaction.

potential (electrochemical potential): The tendency of a species to give off (oxidize) or take up (reduce) electrons. The value is always relative to another reaction.

reduction: A redox reaction involving the addition of electrons.

selective binding unit (SBU): The biological recognition element in the affinity biosensor. It may be an antibody, antigen, DNA sequence, lectin, avidin, or biotin.

References

1. Vreeke MS, Maidan R, and Heller A, "Hydrogen Peroxide and Beta-Nicotinamide Adenine Dinucleotide Sensing Amperometric Electrodes Based on Electrical Connection of Horseradish Peroxidase Redox Centers to Electrodes through a Three-Dimensional Electron Relaying Polymer Network," Anal Chem, 64:3084­3090, 1992.

2. Vreeke MS, Yong KT, and Heller A, "A Thermostable Biosensor of Hydrogen Peroxide," Anal Chem, 67:4247­4249, 1995.

3. Yang L, Janle E, Huang T, et al., "Application of 'Wired' Peroxidase Electrodes for Peroxide Determination in Liquid Chromatography Coupled to Oxidase Immobilized Enzyme Reactors," Anal Chem, 67:1326­1330, 1995.

4. Huang T, Yang L, Gitzen J, et al., "Detection of Basal Acetylcholine in Rat Brain Microdialysate," Chromatog B, 670:323­327, 1996.

5. Horrocks BR, Schmidtke D, Heller A, et al., "Scanning Electrochemical Ultramicroelectrodes for the Measurement of Hydrogen Peroxide at Surfaces," Anal Chem, 65:3605­3614, 1993.

6. Sakai H, Baba R, Hashimoto K, et al., "Local Detection of Photoelectrochemically Produced H^₂O^₂ with a 'Wired' Horseradish Peroxidase Microsensor," J Phys Chem, 99:11896­11900, 1995.

7. Ohara TJ, Vreeke MS, Battaglini F, et al., "Bioenzyme Sensors Based on Electrically Wired Peroxidase," Electroanal, 5:825­831, 1993.

8. Vreeke MS, Rocca P, and Heller A, "Direct Electrical Detection of Dissolved Biotinylated Horseradish Peroxidase, Biotin, and Avidin," Anal Chem, 67:303­306, 1995.

9. Vreeke MS, and Rocca P, "Biosensors Based on Cross-Linking of Biotinylated Glucose Oxidase by Avidin," Electroanal, 8:55­60, 1996.

10. Xu Y, Halsall HB, and Heineman WR, "Heterogeneous Enzyme Immunoassay of Alpha-fetoprotein in Maternal Serum by Flow Injection Amperometric Detection of 4-aminophenol," Clin Chem, 36:1941­1944, 1990.

11. Aizawa M, "Enzyme-Linked Immunosorbent Assays Using Oxygen-Sensing Electrode," in Electrochem Sensors Immunolog Anal, Ngo TT (ed), New York, Plenum, pp 269­278, 1987.

12. Hadas E, Soussan L, Margalit IR, et al., "A Rapid Sensitive Heterogeneous Immunoelectrochemical Assay Using Disposable Electrode," J Immunoassay, 13:231­252, 1992.

13. Bourdillon C, Demaille C, Gueris J, et al., "A Fully Active Monolayer Enzyme Electrode Derivatized by Antibody Attachment," J Am Chem Soc, 115:12264­12269, 1993.

14. Gleria KD, Hill HAO, McNeil CJ, et al., "Homogeneous Ferrocene-Mediated Amperometric Immunoassay," Anal Chem, 58:1203­1205, 1986.

15. Willner I, Blonder R, and Dagan A, "Application of Photoisomerizable Antigenic Monolayer Electrodes as Reversible Amperometric Immunosensors," J Am Chem Soc, 116:9365­9366, 1994.

16. Duan C, and Meyerhoff ME, "Separation-Free Sandwich Enzyme Immunoassay Using Microporous Gold Electrodes and Self-Assembled Monolayer/Immobilized Capture Antibodies," Anal Chem, 66:1369­1377, 1994.

Mark S. Vreeke, PhD, is a product development scientist at TheraSense, Inc. (Alameda, CA). This work was completed at the Department of Chemical Engineering and Materials Science and Engineering Center of the University of Texas at Austin. Support was provided by an H. H. Dow Memorial Award, a Welch Fellowship, NSF, NIH, and the Department of Defense.

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In contrast to some researchers who are able to take a more leisurely approach to their studies, the industrial scientist is under constant pressure to produce products that meet exacting specifications with the fewest resources. To adhere to rigorous product requirements, the industrial statistician must employ those design and analytical tools that allow the most information to be squeezed from the least effort. This need applies particularly to development of technical products such as medical diagnostics, where reagent lots must be constantly available and produced to rigid specifications.

This brief introduction to the design of experiments (DOE) focuses on design tools that minimize resource requirements and optimize product specifications. Part 2, which will appear in a subsequent issue, presents an immunodiagnostic application of these tools as well as a brief analysis to illustrate the methodology.

DOE and Design Types

Formal experimental design has several advantages over the classic, one-step-at-a-time approach that has been favored by academe. These advantages include improved performance characteristics, reduced costs, and shortened development and testing times.

The formal experimental design examines changes in output variables for a process, given any combination of input variables. In the most basic immunochemical designed experiment, various concentrations of biochemical reactants (for example, an antigen and an antibody) are mixed and the resulting reaction rate or concentration of product is measured. The researcher can then ensure a desired rate or concentration to within acceptable specifications by setting the antigen and antibody concentrations to those values derived by the analysis.

By isolating and better understanding those factors that most affect the outcomes, the researcher can devote fewer resources to investigating the less-important factors. In testing design strategies or troubleshooting established ongoing processes, the experimental design process may be used to gain additional knowledge about the relationships among input and output variables. Formal design also allows the researcher to mathematically model the process and optimize the response.

The goal is to fill a design space with the least number of points (data collection units), yet still fully characterize the system. Researchers accomplish this goal by employing a number of well-defined design types that are standard in the literature. For more adventurous experiments or for modeling a space sufficiently different from the norm, special models are available that are easily implemented in several commercial software programs.

In all of these experiments, the strategy is essentially the same. The first experiment is a screening run. This run isolates those input factors that are most important to the outputs. Tools available for this experiment include statistical output from the software and the researchers' own knowledge of the system, including the cost factors.

The second step is exclusion of those factors deemed to be of minor significance to the desired output, based on the results of the screening experiment. With the remaining factors, researchers can use a response surface design to determine the changes in outputs for any combination of changes in inputs. Software analysis allows the response to be targeted to a given value within the limits of experimental error. The outputs may also be simultaneously maximized or minimized. For instance, cost may be set as a factor to be minimized. In addition, many software packages allow visualization of this three-dimensional response surface, rotation of it, and examination of specific points.

The last experiment is a verification run, whereby the outputs are checked against the predictions of the response surface design. Once the suitable input settings have been verified, the process may run at these settings until any inputs are changed.

To visualize the conversion of measurable, real-world inputs and outputs to a response surface, consider the following example. Typical immunochemical input factors include antigens, antibodies, and fluorescently labeled probes. In Figure 1, a simple relationship between the antigen and antibody concentrations (the inputs) and the reaction rate being measured (the output) is illustrated. Since we can envision only three dimensions, the concentration of the probe molecule has been held constant. Readily measured concentrations of the inputs are plotted on the x- and z-axes, while the resultant reaction rate is given on the y-axis.

Figure 1. A response surface. Axis labels represent arbitrary units.

 

The response surface consists of the three-dimensional collection of all experimental points measured and plotted (as well as the areas defined by these points) and is bounded by the limits of the actual experiment. These bounds are called the design space.

Screening Designs

The screening run may be designed using one of the following models:

Linear. A linear screening design consists of a line connecting end points that represent only those values near the highest and lowest settings of each input. The limitation of this design lies in what may happen in the center of the design space, perhaps a bend or curvature in the area where no data have been taken. This design is illustrated in Figure 2, in which the reaction rate is plotted against the change in antigen concentration while the antibody concentration is held constant. In statistical terms, the antibody is held at a single level.

Figure 2. Linear screening design.

 

Linear with Center Point. Although the linear-with-center-point design is linear, it does require data collection at the center (see Figure 3). This requirement eliminates the drawback of the strictly linear design. These are among the most popular and widely used screening designs.

Figure 3. Linear-with-center-point screening design.

 

Plackett-Burman. Two-level screening strategies, Plackett-Burman designs are mathematically derived in multiples of four trials (that is, 8, 12, 16, 20 trials and up). The term level here may be thought of as the different concentrations of the reagents used. Therefore, researchers may enter the concentrations within a software package as either high or low, or use the actual numerical values (for instance, 10 µM/dl and 20 µM/dl). If one trial is allowed for a constant, the remaining number of trials is 7, 11, 15, 19, or more. To use a design for variables not occurring in this sequence, the researchers may choose a larger design and ignore the unused variable points in the design space.

These designs have two main drawbacks. Lack of fit is difficult to assess, and first-order effects may be confounded with interaction effects (e.g., it may be impossible to separate an antigen effect from an antigen-antibody interaction effect).

Response Surface Designs

A variety of response surface designs are available for visualization of the effects of changes in inputs.

Factorial Designs. These occur as two types, full and fractional. Full factorial designs are used to estimate all possible effects, including interactions, of the input variables. The number of runs for a design that uses k variables and n levels is nk. This equation means that the number of runs increases dramatically as the numbers of levels and factors increase. The advantages of the full factorial design include orthogonality (ability to exclude confounding, among other properties), lack of aliasing (identical columns in the design), and evaluation of all main effects and interactions. The disadvantages include time, cost, and resource commitment.

Fractional factorial designs retain orthogonality while requiring fewer runs. However, doing fewer runs means acquiring less information. To ensure that subsequent runs capture the most important information, experimenters usually eliminate runs for interactions that are deemed insignificant.

Quadratic Designs. These are based on a standard quadratic model:

They therefore contain linear, interactive, and quadratic terms. These designs are easily implemented with software, and the mathematical results are most readily associated with physical events or response surface curvature.

Partial Cubic Designs. The partial cubic model includes all the terms in a quadratic model but also includes terms having cubic interactions:

It does not contain pure cubic terms. Since cubic interactions occur only rarely in chemical products development work, this exclusion is usually justified.

Box-Behnken Designs. An efficient three-level design that uses embedded 2k designs, the Box-Behnken design holds certain factors at their center points. Tables that specifiy the number of runs to be used are published and readily available. The model drops all corner points in the design space. This elimination of data may be of concern to those who need measurements at the extremities. These designs are close to orthogonal and may be used to estimate main and quadratic effects as well as all linear two-way interactions. Least-squares regression compensates for the nonorthogonality.

Central Composite Designs. These symmetrical, space-filling designs are flexible and efficient. Also known as Box-Wilson designs, these multidimensional cubes are face-centered and may be easily rotated by choice of appropriate factors. This easy rotation means that the predicted response may be estimated with equal variance regardless of the direction from the center of the design space. The only drawback in these designs is that the number of runs required for the larger designs rapidly becomes very large.

Designing an Experiment

The following brief outline of steps illustrate the design and interpretation of a response surface experiment.

Choose the Design Type. In immunology it is usually important to fully understand the contributions of each reagent to the final reaction. In such a case, the researcher would select a full factorial model in order to include each input variable. A fractional factorial design might be chosen if only a subset of the main variables were considered important.

A quadratic design is usually chosen for the initial response surface experiment, since by mathematical design and historical use they have been found the most effective for immunologic experiments. Such a design considers not only the main effects (in this case the individual reagents) but also the interactions (for example, does antigen x react in any way with antibody y) and the quadratic terms. These last are the squared terms and describe the curves or bends in the response surface that help fit the calculated surface to the data. No physical meaning is generally ascribed to the quadratic terms (what does "antigen squared" mean in the real world?).

The cubic, Box-Behnken, and central composite designs are somewhat specialized and are included here for completeness. Rarely are cubic and higher-order designs needed. By experience, quadratic designs have been demonstrated to accurately describe physical data sets to within generally accepted ranges. Furthermore, the mathematics are greatly simplified by restricting the model to first- and second-order terms, and the interpretation of individual factors and interactions is more meaningful.

Implement the Design. In the second step, the laboratory data are collected and observed for possible exclusion of outliers. Portions of the experiment may be repeated. The data are then analyzed in the design software.

Interpret the Results. A typical outcome would be as follows: At x concentration of antigen and y concentration of antibody, the largest reaction rate is observed. If the degree of certainty surrounding this rate is acceptable, the laboratory's standard operating procedures will be written to specify mixing these levels of antigen and antibody to ensure the stated reaction rate plus or minus system error.

Conclusion

This discussion by no means exhausts the types of designs available, both for screening and response surfaces. It is, however, a representative sample of the more commonly used design types. The example provided above demonstrates the relationship between the mathematical abstractions and the actual process being measured and manipulated.

Part 2 of this article (which will be posted here at the end of October, 1997) will further demonstrate the practical application of the principles outlined here. The screening and response surface designs for master lot testing for a serum protein will be explored in detail, and commercially available software packages will be described.

Bibliography

Atkinson AC, and Donev AN, Optimum Experimental Designs, Oxford, Clarendon Press, 1992.

Myers RH, and Montgomery DC, Response Surface Methodology: Process and Product Optimization Using Designed Experiments, New York, John Wiley, 1995.

Schmidt SR, and Launsby RG, Understanding Industrial Designed Experiments, 3rd ed, Colorado Springs, CO, Air Academy Press, 1992.

Wheeler B, ECHIP Reference Manual, Hockessin, DE, ECHIP, 1993.

John A. Wass is a mathematical analyst in the scientific support group at Abbott Laboratories (Abbott Park, IL).

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In a recent interview, Arun Gandhi, grandson of the archetypal pacifist and founder of modern India, Mahatma Gandhi, identified "science without humanity" as one of the eight basic causes of violence. Unfortunately, in the minds of technically trained personnel, the connections among scientific research, clinical practice, and patients are usually distant and diffuse.

This fact is frequently reflected in the professional training available to industry personnel. Out of the 138 sessions at June's BIO '97 International Biotechnology Meeting and Exposition, for instance, just one directly addressed the social and ethical impacts of biotechnology development; five others related to the topic only peripherally. To secure ethical practice in the in vitro diagnostics industry--and thereby the opening and development of new IVD markets and continued public support for genetic research--it is crucial that all professionals along the pipeline, from the laboratory to the marketing department, be sensitive to the human impact of discoveries and technological advancements in this field.

It is by no means inevitable that professional training should perpetuate a separation between science and humanity. An example to the contrary is provided by two concurrent conferences that I recently attended at the University of Malta (Msida, Malta). At the first conference--the Sixth International Conference on Thalassaemia and the Haemoglobinopathies--speakers concentrated primarily on the clinical and scientific aspects of thalassemia research, particularly on advances in genetics and prenatal diagnostics. Meanwhile, the second conference--the Eighth Annual Thalassaemia Parent and Thalassaemics International Conference--offered a forum for patients, their families, and their caregiving support groups. Throughout these concurrent meetings, the diverse audience of researchers, clinicians, and those afflicted with or affected by this serious inherited disorder wandered the same hallways, shared coffee breaks, and occasionally attended joint sessions.

Thalassemia is characterized by the reduced ability of the blood to transport oxygen; the condition results from variant hemoglobin subunits that do not assemble properly. Treatment of the disorder involves blood transfusions every 2­6 weeks and iron chelation therapy with desferrioxamine. When they have access to the current medical practices in economically developed societies, patients can expect normal growth, development, and lifespan. In fact, several speakers at the patient and parent conference claimed that, when proper medical care is available, thalassemics can experience a higher quality of life, self-esteem, and emotional well-being than otherwise healthy individuals, perhaps owing to the family and social support systems developed to care for those afflicted with the disorder.

For more than a decade, clinics throughout the Mediterranean region, Asia, Western Europe, and the United States have offered carrier screening, prenatal diagnosis, and, more recently, preimplantation diagnosis of in vitro fertilized embryos to detect thalassemic mutations in hemoglobin genes. Because advances in prenatal diagnostic technology have preceded advances in treatments for this disorder (true of most genetic tests), in some countries the information derived from such tests has been used to justify the use of abortion to prevent the birth of unhealthy babies. In Sardinia, for example, births of thalassemic infants dropped from 120 in 1975 to 0 in 1995 because of the availability of and strong public advocacy for prenatal testing and pregnancy termination.

But countries that choose this course of action face a delicate ethical dilemma. To prevent the suffering of an affected individual and family, they must be prepared to recommend the abortion of a fetus that would perhaps lead a happier-than-normal life if born. At the patient and parent conference, several thalassemia patients from the Mediterranean region openly stated that they were delighted with their lives. Nevertheless, because it is commonly the governments of Mediterranean nations that must bear the burden of the expensive medical treatment required for thalassemics, and because most of the societies involved are not wealthy, it is unlikely that their health authorities would willingly allow thalassemics to be born. In cases such as this, genetic testing not only provides health information, but also introduces economic considerations that can be used to reduce health-care costs by limiting the distribution of care.

Throughout the world, the development of molecular diagnostics is happening at a frenetic pace, usually in advance of effective treatments. Under these circumstances, the moral question is whether the development and marketing of genetic tests will improve patients' health and satisfy their right to personal information, or segregate patients into market niches, apportioning scarce health-care resources on the basis of DNA sequences.

In the United States, the potentially abusive impact of genetic test data is very much on the minds of public policy makers. Signed into law in August 1996, the Health Care Portability and Accountability Act contains some modest restrictions on the use of genetic test data by health insurers. The Equal Employment Opportunity Commission has issued guidelines prohibiting employment discrimination on the basis of genetic tests. President Clinton voiced support for genetic privacy--the restriction of genetic test results to patients and their physicians unless authorized in writing by the individual--in his commencement address at Morgan State University in May. About a dozen bills having to do with the privacy of genetic test results are pending before Congress, including Senate bills called the Genetic Fairness Act (Feinstein, D­CA), the Genetic Privacy and Nondiscrimination Act (Hatfield, R­OR), and the Genetics Confidentiality and Nondiscrimination Act (Domenici, R­NM). These bills have in common a strong inclination to protect individuals against unauthorized access to their genetic test results or the use of such results to discriminate in the areas of insurance, employment, or education.

Generally speaking, however, the formation of policies to regulate the availability of genetic test results and safeguard the public interest has proven much more difficult than the technical design of genetic tests themselves. Since 1989, scientists funded by the human genome project have successfully sequenced the entire genomes of several model viruses and microorganisms, and uncovered several thousand human genes. Meanwhile, the program's Working Group on Ethical, Legal, and Social Implications of Human Genome Research--a subcommittee of the National Institutes of Health­Department of Energy Task Force on Genetic Testing--has managed to generate only a single document, a set of policy recommendations released earlier this year.

Public policy will be strongly influenced by the recommendations of this prestigious working group, a collection of scientists, ethicists, sociologists, and medical and legal professionals who unanimously support the right of patients to control access to their own genetic test information. Perhaps most significant is the task force's recommendation to establish a new government agency, the Genetics Advisory Committee, that would report to the secretary of Health and Human Services and have authority to oversee genetic testing and regulate the availability of test data. Within this agency, one subcommittee would advise FDA on ensuring the validity and utility of new genetic tests and another would advise the Clinical Laboratory Improvement Advisory Committee (CLIAC) on ensuring the quality of laboratories performing and interpreting the tests.

This last point is critical for the future of genetic IVDs. A study recently published in the New England Journal of Medicine showed that approximately one-third of genetic test results were misinterpreted by doctors or genetic counsellors.¹ There is a tremendous need for education and training of health-care providers before genetic tests become widespread and, until that time, genetic testing should be closely regulated. Although I agree with those who support the public's right to obtain medical information, the average patient will not understand the implications of genetic test results. The establishment of professional education, training programs, and regulatory safeguards is necessary to ensure that the public receives genetic test information that is both correct and well understood.

The experience of Myriad Genetics, Inc. (Salt Lake City), in marketing BRCA analysis kits for breast cancer illustrates that there are clear economic incentives for mindfulness of public sentiment in sensitive areas like genetic testing. Although the product may perform well technically, investors' concern about the public understanding and acceptance of the product, as well as the extremely murky regulatory climate surrounding genetic testing, probably caused the withdrawal of Myriad's $43-million stock offering last November.²

There are similarly strong and clear economic incentives for IVD manufacturers to support federal legislation and regulations, since the existance of such legislation and regulations reassures the public of the safety and utility of these products. But IVD makers should get involved now while laws and policies are in formulation. A good starting point for manufacturers of IVDs that perform genetic testing is to obtain a copy of the recommendations of the Task Force on Genetic Testing.³

The ability to determine DNA sequences from the moment of conception has given medical professionals the tools to define "perfection" in human offspring. The public generally does not understand genetics and is largely afraid, with good reason, of the eugenic implications of the widespread availability of genetic test results. It is in the best interests of IVD manufacturers, and of their customers, to practice science with humanity.

References

1. Giardello FM, "The Use and Interpretation of Commercial APC Gene Testing for Familial Adenomatous Polyposis," N Engl J Med, 336(12): 823­827, 1997.

2. Marshall E, "Gene Tests Get Tested," Sci, 275:782, 1997.

3. "Proposed Recommendations of the Task Force on Genetic Testing," Federal Register, 62 FR: 4539­4547.

For further information

Biotechnology Industry Organization (BIO)
1625 K St. N.W., Ste. 1100
Washington, DC 20006
Phone: 202/857-0244
Web site: http://www.bio.org/bio/bioinfo.html
 

National Human Genome Research Institute
National Institutes of Health
Bldg. 31, Rm. 4B09
9000 Rockville Pike
Bethesda, MD 20892
Phone: 301/496-0844
Web site: http://www.nhgri.nih.gov
 

National Center for Genome Research
1800 Old Pecos Trail
Santa Fe, NM 87505
Phone: 505/982-7840
Web site: http://www.ncgr.org
 

National Human Genome Research Institute
National Institutes of Health
Bldg. 31, Rm. 4B09
9000 Rockville Pike
Bethesda, MD 20892
Phone: 301/496-0844
Web site: http://www.nhgri.nih.gov

David F. Betsch, PhD, is an assistant professor of science and technology at Bryant College (Smithfield, RI), and a member of the IVD Technology editorial advisory board. He is also president of Biotechnology Training Programs, Inc. (Providence, RI).

NCCLS has recently issued two new guidelines for manufacturers of alpha-fetoprotein assays and coagulation factor assays, and for clinical laboratory personnel who perform the tests.

Determination of Factor Coagulant Activities; Approved Guideline (NCCLS document H48-A) provides guidance on the performance, quality control, and reporting of one-stage coagulation factor assays based on APTT and PT coagulation tests. The guideline recommends techniques to minimize the effects of sources of error and improve inter- and intralaboratory precision.

Assessing the Quality of Systems for Alpha-Fetoprotein (AFP) Assays Used in Prenatal Screening and Diagnosis of Open Neural Tube Defects; Approved Guideline (NCCLS document I/LA17-A) presents preanalytical and analytical considerations necessary to ensure the reliability of AFP testing during the second trimester of pregnancy.

To order copies of the guidelines, contact NCCLS at 610/688-0100.

When physicians need to order diagnostics for detecting tuberculosis, direct amplification tests still take a back seat to acid-fast bacillus stains. What will it take to put these rapid diagnostics in the driver's seat?

Proof of cost-effectiveness, if the presentations at a recent TB workshop held in Los Angeles are any indication. The workshop was sponsored by the Los Angeles County chapter of the American Thoracic Society.

"Los Angeles has the most up-to-date tuberculosis laboratory in the state, if not in the country," said Sydney Harvey, PhD, director of the county's public health laboratories. But in her presentation, she observed that the county's labs are using virtually all the latest technologies for confirming TB, from liquid chromatography to fluorescent staining--but not amplification.

The reasons for the county's decision have more to do with the current cost of the tests than with a lack of need for them.

For TB screening in schools and physician offices, the time-honored PPD remains the gold standard. But the consensus among health-care professionals is that for many populations this method isn't fast enough. At the Los Angeles County jail, for example, the first step in triage for jail-house bookings is that old standby, the chest x-ray, reported Paul Davidson, MD, director of tuberculosis control for the Los Angeles County Department of Health Services. Chest x-rays have the advantage that they can give a positive result in the time it takes to develop film.

But there are problems with that method, too, asserted Antonino Catanzaro, MD, professor of medicine at the University of California, San Diego, School of Medicine. "The chest x-ray is an excellent tool for identifying patients with pulmonary disease," he notes. "However, whether that disease is due to TB and whether or not the TB is active are very important questions that x-rays do not address." By contrast, direct amplification tests have shown a specificity of 100% and sensitivity of 95 and 96% in studies conducted by FDA (Am J Respir Crit Care Med, 155:18104­18118, 1997).

Catanzaro concluded that tests that rapidly identify RNA and DNA panels--such as those produced by Gen-Probe (San Diego) and Roche Diagnostic Systems (Branchburg, NJ)--constitute a significant improvement.

Harvey didn't disagree. But in a study of more than 1400 samples using these nucleic acid­based tests, county investigators found that the cost of using them was prohibitive.

"At the time of the study, the cost of each PCR test was $20--just for the reagent, not the labor involved," she said. Putting these rapid tests into the county's TB screening protocol would mean "major, major cost increases." The county's conclusion: they won't be put into the protocol "until someone decides to give us a whole lot more money."

In July, 1997, FDA's Center for Devices and Radiological Health (CDRH) issued two public health advisories relating to the use of diagnostics for toxoplasmosis and Lyme disease. Both advisories resulted from recommendations of the device center's microbiology devices advisory panel.

Noting that FDA has "recently become aware of inaccurate test results from clinical reference laboratories," the toxoplasmosis advisory instructs physicians that they "should not use the result from any one Toxoplasma IgM commercial test kit as the sole determinant of recent Toxoplasma infection when screening a pregnant patient." An algorithm is provided for the interpretation of test results. The agency is now working with test-kit manufacturers to provide appropriate labeling information.

The Lyme disease advisory observes that "the results of assays for detecting antibody to Borrelia burgdorferi (anti-Bb) "may be easily misinterpreted." It advises physicians to base their diagnoses on the patient's history, physical findings, and laboratory data other than anti-Bb results. A two-step algorithm is provided for interpretation of test results.

The full text of both advisories is available on the device center's Web site, http://www.fda.gov/cdrh.

A looming battle over patent rights has manufacturers of immunochromatographic strip tests worried. Since at least 1992, the diagnostic and medical products maker Becton Dickinson and Co. (Franklin Lakes, NJ) has sued or sent warning letters to more than a dozen test makers, accusing them of infringing its patents. In June, BD's position was bolstered when Quidel Corp. (San Diego) agreed to pay it $1.9 million a year in licensing and royalties to settle an infringement claim against its strep infection test.

Meanwhile, the Anglo-Dutch firm of Unilever (London and Rotterdam) has also jumped into the patent fray. That firm has held European immunochromatography patents for more than a decade, including recent ones relating to the successful Clear Blue One Step pregnancy test, released through subsidiary Unipath. In April, Unilever received a new U.S. patent, and has since then quietly begun contacting test makers about possible infringement.

At stake is a growing, $600-million worldwide industry, with broad applications in agriculture and environmental assessment, drug abuse testing, and pregnancy, fertility, and infectious disease detection. More than 100 firms are involved in supplying test media and injection devices as well as in test manufacture.

The tests, also known as lateral-flow tests, offer the beauty of simplicity. All that's necessary is to apply a sample of blood, urine, or saliva to a small handheld device and wait to eyeball results, typically a color change on the test strip.

Test makers are worried about Unilever's patent, which experts say is soundly reasoned and well written. But many are resisting Becton Dickinson's claims. Some contend that the BD patents are weak, while others insist they aren't broadly applicable. Some experts say the BD and Unilever patents overlap one another and up to a dozen earlier patents, and are therefore vulnerable to court challenge.

Even Quidel questioned BD's patent rights. The settlement between the two companies was a purely financial decision, says Steven C. Burke, Quidel's chief financial officer. "We don't believe we infringed their patent. This was an economic settlement. We were looking at $2 million a year in legal fees."

Quidel has other business relationships with BD, and the company's pact does not necessarily set a precedent. But in a closely watched patent infringement case in North Carolina, Carter-Wallace, Inc. (Cranbury, NJ), also settled with BD. Although its financial terms remain confidential, the agreement included a stipulation by Carter-Wallace that the BD patent was valid and enforceable. Many in the industry have found the settlement puzzling, since Carter-Wallace is reported to be paying Unilever a licensing fee to distribute its products in the United States.

Citing "ongoing litigation in this area," Carter-Wallace chief counsel Stephen Lang declined to discuss the settlement. Unilever did not respond to repeated requests for information.

BD's chief patent counsel, Richard Rodrick, says his company believes that "the majority" of immunochromatographic strip tests on the market are covered by either or both of its two patents. The latest, known as the Rosenstein patent, was issued in January and will remain in force for 161/2 years, Rodrick says. The company's 1987 Campbell patent has seven to eight years to go, he adds.

"We feel very strongly that both our patents are not only presumed valid, but would withstand any legal challenge," says Rodrick. "The widespread licensing by many parties is itself an acknowledgment of the strength of the patents."

As long as BD and Unilever stick together, smaller manufacturers may be forced to capitulate. The courts presume that U.S. patents are valid. Any challenge must be sustained by clear and convincing evidence, a high legal standard. And few companies have the resources to fight billion-dollar companies like BD and Unilever.

Says Quidel's Burke: "There's a high degree of uncertainty in putting a highly technical issue before a jury. And these lawsuits can take two, maybe three years to fight. If they had won, we could have been forced to cease selling. Or they could have demanded very onerous terms."

Consolidation in the IVD industry has reached a fever pitch. Before year's end, two megamergers will have taken place--Roche Holding, Ltd., with Boehringer Mannheim GmbH, and Dade International with the Behring Diagnostics business unit of Hoechst Behring. A third merger significant for its impact on the IVD industry is also in the works, between multinational giant Becton Dickinson and relatively small PharMingen.

The mergers appear to be driven by the growing cost-consciousness of the marketplace. For the parties involved, each of the deals promises greater cost efficiencies, increased market share, and complementary technologies. For other companies, the desire to obtain the same advantages could accelerate an already booming interest in consolidation. "The super-big companies are getting so big that to compete effectively against one another they have to buy up all the technology they possibly can," says Thomas Tsakeris, president of Devices and Diagnostics Consulting Group (Rockville, MD).

Acquisition of technology was a major reason behind Becton Dickinson's purchase of PharMingen (San Diego) for an undisclosed amount. PharMingen's 260 employees have crafted about 2500 biotechnologies ranging from antibodies to cell-cycle proteins. In an open letter to customers of Becton Dickinson Immunocytometry Systems (BDIS; San Jose), BDIS president Deborah Neff emphasized the natural synergy between the people, products, technologies, and services of the two firms. "By integrating our collective research capabilities, we strengthen our ability to rapidly commercialize and provide the broadest range of innovative products," Neff wrote. "This allows us to accelerate the transfer of technologies for clinical assay development."

Technology was also a major part of Roche's strategy in 1991, when the Swiss company paid $300 million to buy the polymerase chain reaction (PCR) gene amplification technology from Cetus Corp. (Emeryville, CA), prior to its current bid for Boehringer Mannheim. Since then, Roche has invested about $1 billion more in PCR development. As a result, it brings to the table its significant investment in PCR technology and test kits; Boehringer Mannheim brings its clinical laboratory equipment and IVD tests. Together Roche Holding, Ltd. (Basel, Switzerland), and Boehringer Mannheim (Mannheim, Germany) will be the largest diagnostics company in the world, eclipsing even Abbott Laboratories in annual revenues with more than $3 billion from diagnostics sales. To make the deal work, Roche is spending an estimated $11 billion to buy the Bermuda-based holding company of Corange, Ltd., which owns Boehringer Mannheim, as well as 84.2% of the stock in DuPuy, Inc. (Warsaw, IN), a manufacturer of orthopedic equipment.

But if diagnostic companies' future success depends upon the depth and breadth of their product offerings and distribution systems, the global diagnostics industry could be in for some dramatic changes in the next few years. According to some analysts, the result will be an industry very different from today's, one that will be dominated by a half dozen or so large, multinational companies.

"Not every company that goes into a period of consolidation and restructuring comes out again," says Scott Garrett, chairman and CEO of Dade International. "And those that emerge are very different from what they were before. They become consolidated organizations with radically redefined approaches toward competing and toward serving customers, as well as toward managing operations and controlling costs."

Dade Behring, the combination of Dade International (Deerfield, IL) and Behring Diagnostics (Frankfurt, Germany), will be very different. For starters, the new company, whose merger should be completed by early fall, will be king of the clinical laboratories industry, sporting annual revenues in excess of $1.5 billion. The new company will combine Dade's clinical chemistry, microbiology, and hemostasis products with Behring's drug monitoring and testing products as well as its plasma-protein testing, infectious disease, and coagulation technologies. "Dade Behring will have unexcelled breadth in the products and services we offer to laboratories," Garrett says.

Synergies may also be achieved by merging two globally complementary organizations. Dade sales are now about 70% in the United States and 30% in the rest of the world. If the proposed merger with Behring Diagnostics is finalized, the combined company will make half its sales in the United States and the other half spread around the world.

The merger of Roche and Boehringer Mannheim, which will not be final until at least this fall, will strengthen Roche in Italy, Spain, France and the UK, as well as in smaller European and Latin American markets. "The main reason for the merger was to gain market share in the diagnostics division," says Peter Wullschleger, a spokesperson for F. Hoffmann­LaRoche, Ltd.

Achieving these competitive strengths are essential for survival, according to Dade International's Garrett. He believes there are only two choices for companies--at least those that serve clinical laboratories. "Either you find a way to take part in the consolidation or you get left behind and forgotten, particularly by customers," he says.

The prospect of a future in which survival depends on consolidation sends a shiver down the collective spine of an industry whose long-term future depends on continued innovation. Dave Casal was a research scientist at Hybritech when it was bought by Eli Lilly for more than $300 million 12 years ago. Lilly sold Hybritech to Beckman Instruments in 1996 for about $10 million. "A lot of pharmaceutical companies have bought diagnostic concerns thinking they complement the pharmaceutical technology, only to find they're completely different," says Casal, who is now an officer at IVD maker Metra Biosystems (Mountain View, CA). "Diagnostic concerns do not necessarily respond well to the same structures and motivations as pharmaceutical concerns."

There is, in fact, a very strong presence of pharmaceuticals in the current consolidations. About 65% of the annual revenues racked up by Roche Holding comes from pharmaceuticals. Boehringer Mannheim is also heavily involved in this sector, garnering about 40% of its revenues from drug sales and the other 60% from diagnostics.

Both major companies, however, already have strong diagnostics businesses. When device businesses are present as well, mergers can be in the best interests of all parties, large and small. PharMingen, for example, is expected to integrate its biotechnologies with advanced instrumentation held by BDIS. In return, it can expect to gain extended access to overseas markets through Becton Dickinson's distribution channels.

Casal, now Metra Biosystems' senior director for clinical, regulatory, and quality affairs, believes that such synergies result from a greater sophistication on the part of large companies. They are already familiar with the character of diagnostics companies, he says, often through collaborative research agreements. For example, Metra Biosystems, which has about a half dozen IVD technologies for assessing bone turnover, is collaborating with several large pharmaceutical and diagnostics companies. "Large companies use firms like Metra as incubators for research, letting us either thrive or fall on our merits," Casal says. "If the bone marker field were to take off, one of those companies might be interested in buying us because we have done all the groundwork and they can then develop the market."

However, there remains the threat that unbridled consolidation will depopulate the corporate landscape of innovators. Not to worry, says consultant Tsakeris. Acquisitions are part of a natural cycle--like evaporation and rainfall. Large companies may absorb small innovators, but inevitably those same companies spin off the next generation of entrepreneurs. "At least half of our small clients were started by individuals who came from other major companies with the objective of getting big enough to get gobbled up," says Tsakeris, whose consulting firm serves mostly small start-up companies.

If the now-pending megamergers go through as expected, there could be plenty of opportunity for new small businesses. Roche Boehringer Mannheim Diagnostics will have 13,500 employees. The combined Dade Behring will have 8700--5500 from Dade and another 3200 from Behring.

But the kind of world in which disenfranchised entrepreneurs will try to build their companies may differ from the one in which earlier entrepreneurs prospered. David Carville has been knocking on the doors of big diagnostics companies, hoping to raise interest in his start-up IVD firm Causeway Scientific (Mishawaka, IN). "It's difficult to convince larger entities that you have something that may be a resource for them," Carville says. "Their idea is that if you want to help them, you should join them. But don't try to go in as a third-party independent--at least not until you have something solid to put on the table."

Diagnostics manufacturers are accustomed to the notion that it is expensive to bring cutting-edge technologies to market. There are substantial costs involved in creating and testing new products and in meeting essential regulatory requirements. But for IVD firms doing business in Canada­the world's fourth-largest market for diagnostics­it's about to become a whole lot more expensive.

That was the message delivered in Toronto last month by Steve Sanders, vice president for quality assurance and regulatory affairs at bioMérieux Vitek (Hazelwood, MO), at the annual meeting of the Clinical Laboratory Management Association. The new expenses come as part of a cost-recovery scheme in Canada's newly proposed system for regulating medical devices and IVDs, scheduled to become effective on February 1, 1998. The preliminary schedule of fees, delivered to manufacturers in July, calls for annual establishment registration fees as well as annual fees to renew the registration of each medical product on the Canadian market. For a company of moderate size with even a few products, Sanders estimated, the total cost of complying with the new system could be as much as $50,000 per year.

"If I were a Canadian citizen, I'd be very upset about this proposal," Sanders said. "Manufacturers will almost certainly attempt to recover their increased costs from the Canadian national health service, Health Canada, which means that the costs will simply be passed from one branch of the government to another."

Sanders predicted that the effects of the new scheme will include increased time to market, greater regulatory affairs costs, higher breakeven points for new products, and squeezed profits. He also suggested that many manufacturers will be reviewing their current product lines to determine whether their Canadian business can remain profitable.

Industry associations, including Medical Devices Canada and the Association of Medical Diagnostics Manufacturers (AMDM), have submitted comments on the proposed regulatory system. But overall, industry reaction to the proposed fee-recovery scheme has been modest. There is some chance that the scheme will be modified before its scheduled final publication in February 1998, but without concerted industry effort to remove the proposed fees, it seems likely they will remain.

I'm looking forward to Sanders' update on the Canadian proposal at the AMDM annual meeting, which will also feature more than a half-dozen speakers from FDA's Center for Devices and Radiological Health. The meeting will be held in Rockville, MD, on October 20­21. Manufacturers that export to Canada should make a point of attending this meeting and becoming informed on the changing regulatory system. For further information, contact AMDM at 202/637-6837.