Ortho Clinical Diagnostics (OCD), in partnership with the National Association of Chronic Disease Directors (NACDD), published consumer-survey results and a report revealing the state of blood test health literacy in the United States, with strategies to support patient education and empowerment. The survey and report are part of the Know Your Numbers campaign, which was launched in conjunction with National Health Literacy Month last year.
Among the survey’s findings were that nearly 90 percent of people would prefer to discuss blood test results during a doctor visit, yet only about 40 percent have actually discussed results in person. And, although eight in ten people who reported having a recent blood test said they understood the results, roughly half of those people did not know their own cholesterol levels. Nearly two-thirds of them, or 65 percent, did not know their blood-glucose level.
The Know Your Numbers campaign seeks to help patients realize the importance of blood test results in maintaining their health and encourage them to take a more active role in engaging with their healthcare providers to understand those results. Key to achieving the goals of the campaign, says OCD, is ensuring that laboratories can get results directly to patients and their healthcare providers-currently a limiting factor in most states.
Getting Started. Stephanie Fagan, vice president of communications, OCD, explains the impetus for the campaign this way: “When we at OCD stepped back and asked what our role was in improving health literacy, it was really striking because we, along with other manufacturers of blood tests, are the starting point for healthcare decisions for both the physician and the consumer. And it’s the information contained in our tests that is either the starting point for wellness or the starting point for understanding sickness and what to do about it. We asked, ‘How do we make inroads to health literacy with the work that we do in healthcare?’”
Taking Steps Toward Change. The campaign, she says, can so far be viewed as three steps: The first was doing the survey and understanding the results. The second was holding a Fundamental to Wellness summit in the early part of 2011 that brought together many of the stakeholders who “touch the healthcare continuum,” Fagan says. These included industry representatives, physicians, nurse practitioners, and members of organizations that serve minority populations. “We brought together this diverse group of stakeholders,” Fagan says,  “to take a look at those survey results and determine the barriers to health literacy as it relates to people understanding their own blood tests. We compiled the discussion points and guidelines into a summit report.” OCD is making that report available to “a diverse group of healthcare stakeholders to first bring awareness and then hopefully foster understanding,” Fagan says.
The third step was to make some educational materials available to consumers. “We wanted to give consumers a simple call to action,” says Fagan. That call to action is as follows: Make sure you have an annual health exam. As part of that exam, make sure your blood test results are captured, and make sure that there is follow-up with your healthcare provider to have a conversation about those results. “We know from the survey that [consumers’ receiving test results and discussing them] was the missing link,” Fagan says.
Future Plans. Now that the survey results are in and have been examined by the relevant stakeholders, OCD is working on future plans. “We are working to come up with a measurable definition of success” for the campaign, Fagan says. “One option is, now that we’ve held the summit, done market research, talked with various stakeholders, do we go into disadvantaged communities next? Do we pick one or two, for example, in the United States and do some sort of market-research community-based project where we partner with nurses and physicians in a community setting? And take the tools that we’ve developed so far and put them to action?” She asks, “What might we see, for example, in a community clinic if patients get their lab tests done first? Do we see an improvement in outcomes? What do we see if we also have a control arm that doesn’t do that?” OCD is now looking at ways to put the data it has to use to see what kind of differences it can make in healthcare settings when blood tests are “top of mind and become an integral part of the provider-patient conversation,” Fagan says.
“What’s really exciting about this,” she adds, “is taking healthcare information that typically has been relegated to the lab and finding a new voice for it by getting consumers involved.”
 Ultimately this campaign will be a success, Fagan says, if OCD can get consumers to turn their blood-test results into action and make a difference in their own healthcare. “If they know a number and they know enough to take control of a glucose or cholesterol level and avoid a path to chronic disease, that’s the ultimate goal,” she says.

Last October, the European Diagnostic Manufacturers Association (EDMA; Brussels) hosted the European IVD Forum 2011. The primary purpose of the forum was to facilitate discussions among high-level stakeholders on the challenges faced by healthcare systems. To this end, the forum was attended by members of the IVD industry, key policymakers from European and national institutions, and representatives from patient and professional healthcare groups.
According to EDMA officials, the key global policy issues discussed at the forum were the following: the convergence of legislation at a global level and the importance of intra-association cooperation in achieving solutions to common global challenges; the importance of health technology assessment; and the crucial role of IVDs in active and healthy aging.
As an offshoot of the forum, representatives of six IVD associations from around the world held a meeting to discuss a number of mutually important global policy issues. Officials from AdvaMedDx (United States), IVD Australia, the Camara Brasileira de Diagnostico Laboratorial (Brazil), EDMA, the Japan Association of Clinical Reagents Industries, and the Japan Analytical Instruments Manufacturers exchanged views on the ongoing policy debates in their respective regions and engaged in forward-looking discussions. The meeting built on the longstanding relationships among the international partners and aimed to form the basis for even greater cooperation in the future in order to find solutions to common global challenges and ensure the place of IVDs at the forefront of twenty-first-century healthcare.
“We have come together to further strengthen the ties between our organizations and to enhance the effectiveness of the sector by contributing to policies that will advance public health through the development and use of innovative diagnostics all around the globe,” said Dr. Jurgen Schulze, EDMA’s president. He further pointed out that “this relationship will take our coordination, collaboration, and alignment of information and policies on mutually important issues to a new level.”
As priority issues, the delegates identified the need to raise awareness of the value of IVD testing, facilitate the timely access of patients to good diagnostic testing, and cooperate with regulators and policymakers on issues such as companion diagnostics, clinical evidence for IVDs, and the convergence of regulatory requirements around the world.
Andy Fish, executive director at AdvaMedDx, attended the meeting and said that a good part of the time the associations spent together was taken up providing briefings on key policy issues, the status of those issues, what was going on with regulators, and how the IVD industry was engaged with regulators and policymakers in each of their respective marketplaces.
“Clearly one of the key takeaways from that discussion is that we are all facing essentially the same issues,” said Fish. “And that is the need for regulatory reform to ensure faster and timely patient access to new tests and technologies as well as the need for reimbursement reform to more fully recognize the full value of diagnostics. Beyond those core policy areas, we also continue to underscore our mutual recognition that it is incumbent on all of us as industry representatives to continue our work around educating policymakers and other stakeholders about the technologies and the value of the technologies that we all employ, and to continue to expand those efforts to support our overall advocacy work.”
Fish also said that AdvaMedDx and its sister IVD associations continue to recognize that not all policymakers are as versed in diagnostics as they should be.
“We all have individually and increasingly collectively taken on this task of educating policymakers about how IVDs contribute to all major healthcare systems, both for individual patients and to healthcare as a whole,” said Fish. “So we recognize that in order to maximize our value as advocates for this industry, we need to make sure that the policymakers we’re talking to are as educated as possible about our products. We’ve all taken that on individually, and increasingly we’re sharing ideas and good practices for doing that.”
In addition, Fish said what all of the IVD associations have identified was that they each have their own individual plans for continued advocacy work, resting in part on a robust public affairs outreach.
“I can speak more specifically to both EDMA and AdvaMedDx having well formed plans for continued engagement with a number of stakeholders as we look ahead to the coming year, which would include both increasing engagement with patient advocacy organizations as well as direct briefings for policymakers,” said Fish. “So we didn’t so much identify additional activities for all of us to engage in as a group of associations but rather identified that we’re all working on the same kinds of activities within our own spheres. What we did agree to was to continue to share the kinds of information that we’re all developing to support our overall public affairs and advocacy work. For example, continued health economics studies that may come out of one region or another, case examples of particular technologies and the impact that they’re having on healthcare, and so forth.”
Information about AdvaMedDx can be accessed at www.advameddx.org. Information about EDMA can be accessed at www.edma-ivd.be.
 -Richard Park

Blood is the only fluid that flows throughout the entire human body. Blood analysis can detect a wide range of common physiological problems such as anemia, diabetes, autoimmune deficiencies, infections, and cancers. It can also expose genetic information, viruses, organ deficiencies, etc.
Hematology is the study of blood cells. Hematology analyzer instruments can take the form of a relatively simple handheld unit, a more sophisticated point-of-care diagnostic instrument, or a highly complex clinical laboratory analyzer. While all blood analyzers are designed to be accurate and reliable, the primary factors that set these three types of instrumentation apart from each other are sample throughput rate and the number of blood parameters measured.
The most common blood test is a complete blood count (CBC). This test can help to detect blood diseases and disorders such as anemia, infections, clotting problems, blood cancers, and immune system disorders. The specific blood components counted are the following:

•     Red blood cells carry oxygen from the lungs throughout the human body. Abnormalities in this reading could indicate conditions such as anemia, dehydration, or bleeding.
•     White blood cells are the part of the immune system that fights infection and disease. Abnormal levels could indicate infection, blood cancer, or an immune system disorder.
•     Platelets are blood cell fragments that aid in clotting. Abnormal levels could indicate a bleeding disorder, such as hemophilia, in which blood is slow to clot, or a thrombotic disorder, which exhibits too much clotting.
•     Hemoglobin is an iron-rich protein in the red blood cell that carries oxygen. Abnormal levels could indicate anemia, sickle cell anemia, or other blood disorders.
•     Hematocrit is a measure of how much space red blood cells take up in the blood. A high level might indicate dehydration. A low level might indicate anemia. Abnormal readings may also be a sign of a blood or bone marrow disorder.

Other common blood tests include the following:
•     Blood chemistry tests or basic metabolic panel measure different chemicals in the blood, such as glucose, calcium, and electrolytes.
•     Blood enzyme tests measure enzymes such as troponin and creatine kinase, which can help to identify whether patients have had a heart attack.
•     Blood tests to aid in assessing the risk of heart disease. Such tests measure cholesterol and triglycerides.

Anatomy of a Hematology Instrument
Hematology analyzers count, measure, and characterize blood cells and their components by collecting and calculating results from electrical impedance and/or light-scattering data. Comprised of pumps, motors, syringes, tubing, optics, electronics, and software, the analyzer performs an intricate and delicate task. Multidisciplinary skills in mechanics, chemistry, electronics, software, optics, and fluidics are necessary to engineer and develop such an instrument (see Figure 1).

Figure 1. Comprised of pumps, motors, syringes, tubing, optics, electronics, and software, this hematology analyzer, developed and manufactured by BIT C2 Diagnostics, performs an intricate and delicate task. Multidisciplinary skills in mechanics, chemistry, electronics, software, optics, and fluidics are necessary to engineer and develop such an instrument.

Design and Mechanics. A hematology analyzer contains basic mechanical parts: pumps, syringes, motors, and tubing. These mechanical parts operate in conjunction with reagents, optics, and software to deliver the appropriate test results to clinicians. In order to produce successfully such an instrument, a developer must have an intimate knowledge of the underpinnings of the system, how that system works, and what results it must deliver. Only by having a solid knowledge in place can one develop cost-effective, innovative solutions with maximum optimization for all of the involved technologies. Engineering teams need to work with the latest CAD tools, which enable internet data exchange in many different standards including management of a database of software updates and version upgrades.
Technical specifications of the hematology analyzer are defined jointly between the customer and the developing teams. At the beginning of the project, the teams strive to find the best mechanical design solutions to improve the manufacturing process and reduce future maintenance costs. Models, prototypes, work pieces, assembling process, and tools are designed and digitized using CAD software, and then developed using either quick prototyping or machining techniques. The mounting and assembly of modules or complete instruments is performed in order to check and validate the overall design.
Fluidics. Precise fluidics is crucial to the accurate operation of a hematology analyzer. Blood specimens travel throughout the instrument, being pushed or pulled through tubing by pumps. Specialized techniques must be used to prevent pushing or pulling too hard or too quickly, which could destroy or alter the blood components, thereby causing faulty and inaccurate results.

Figure 2. Electronic measurement techniques are used to classify cells. Light-scattering events are translated into electrical pulses by opto-electrical devices, and thousands of measurements are performed each second.

Electronics. Different measurement techniques are used to classify cells. Light-scattering events are translated into electrical pulses by opto-electrical devices, and thousands of measurements are performed each second (see Figure 2). Specific capabilities needed to design, prototype, and manufacture embedded system products include the following: programmable logic design, expertise in VHDL (FGPA, CPLD), 8 to 32 bits embedded processor boards for RTOS, analog and digital board design, low noise data acquisition and signal conditioning, low EMI design, motion and positioning systems, and PCB layout.
Reagents. Reagents are solutions used to either dilute the blood that is being tested or differentiate or mark various cell components in the blood sample, which enables the further electrical and optical analyses. Other reagents are used to clean the device between sample analyses to prevent cross-contamination of samples. Reagents are consumables that differ from IVD manufacturer to IVD manufacturer. Each manufacturer uses or designs its instrument to work with different reagents, and these reagents are often integrated into an instrument’s unique design.
Optics. Precision optics are an integral part of a hematology analyzer. The blood cells’ characterization relies on optical systems that measure scattering properties of individual cells. Sophisticated software enables the counting and identifying of various types of blood cells. In practice, a dilute suspension of cells passes through tubing past a laser beam. Light scatter from each cell is analyzed by the software, and the resulting numerical representation is interpreted by clinicians. In this manner, cell populations are described, quantified, and classified. Fluorescent techniques may also be used to improve cell identification (see Figure 3).

Figure 3. A blood cell's characterization relies on optical systems that measure scattering properties of individual cells. The y-axis represents axis loss light (ALL), and the x-axis represents forward side scatter (FSC).

Software. The software in a hematology instrument performs multiple functions. It interprets and carries out the orders of the instrument operator. It monitors and drives the operation of the instrument’s working components. It collects and analyzes the resulting data. Essentially, the software pilots everything. In addition, the software works in conjunction with the graphical user interface (GUI), which is different for every customer and depends on the measurement techniques and parameters measured by the device. Menus with selections are used by the administrator of the instrument to control the underlying processes. The graphical menus serve to enrich the user experience and help to reduce operator error.
Basic functions of a common clinical analyzer might include pipetting (i.e., moving fluid from one place to another), mixing and adding reagents, and incubating the final mixture for a determined amount of time. The software coding for such basic functions controls the speed of the motor, the operation of the pumps, and the direction and precise movement of the loaders and stepper motors in order to minimize jostling of the fluids. In addition, software controls the pipetting function, higher level incubation, and the action of picking up a sample and dropping it off somewhere else inside the instrument for more processing.
For an instrument developer, the handling competencies required for full IVD automation management include the following: design of embedded systems with real-time requirements; motor driving (continuous or step-by-step), inputs, outputs, and system control; data acquisition and signal processing; ergonomic and international GUI (languages, alphabets, unities); data transmission; modules and release downloading; software for verification and validation; and production and control tools.
From a programming perspective, IVD instruments are very complex. The more tasks an instrument performs, the more software code is required. And the more code there is, the greater the chance for software bugs to emerge. Programmers

Figure 4. The graphical user interface (GUI) provides menus with selections used by the administrator of the instrument to control the underlying processes. The graphical menus serve to enrich the user experience and help to reduce operator error.

often think they can write pages of code in a day. But after debugging and making that code functional, the amount of code written is reduced to only a few lines per day. Given the time and expense needed to identify the bugs and eradicate them, it is critical to reuse clean code that has already been validated and certified. This is possible through Software Platform Technology, an innovative approach to IVD instrumentation development in which developers can utilize a layered system: building advanced features on top of a solid, tested foundation of codes dictating fundamental processes. Organizing a library of proven code sequences for reuse can effectively expedite a bug-free design of an IVD instrument (see Figure 4).

Business of Hematology Instrumentation Development
Automated blood analyzers, point-of-care instrumentation, and patient-operated handheld units have become high-tech and complex in operation yet simple to use. Research advancements have led to professional demands for more capabilities in the testing units. The business of developing and manufacturing such precise, flexible technologies quickly and within budget has become a specialized discipline. Hematology instrumentation developers must be equipped to meet the challenges of high technology coupled with customers’ demands for speed-to-market and cost efficiency. Research advancements continually drive IVD manufacturers to enhance designs, adding new features and capabilities while making the units easier to use and service.
Since speed-to-market and competitive cost structures are primary drivers when it comes to outsourcing the development of diagnostic instruments, more IVD manufacturers are considering platform technology. BIT Companies has found that by exploiting the fact that many instruments share the basic building blocks with regard to both hardware and software, a manufacturer can repurpose a foundation that is proven and validated, and spend development dollars on the proprietary technologies and features that make the instrument unique. With the availability of a virtual library of basic certified components that are scalable and updateable, platform technology has changed the way many IVD manufacturers do business.
With the development process time measured in years, and the average cost in the millions of dollars, IVD manufacturers are eager to fast-forward their projects by repurposing fundamental, proven technology in the shape of existing, validated platform modules. Platform modules are previously designed, developed components that have been tested, proven, certified, and used in other IVD systems over time. They are composed of selections from a developer’s portfolio of existing hardware, electronics, and software systems that can be scaled up or down to suit the purpose. All of the components comprising the platform module are specifically designed for operation within a complex medical diagnostic instrument, and all systems are developed on the lowest level possible to allow for the highest vertical and horizontal integration.

High-End Hematology Analyzer Market
In the high-end hematology analyzer market, there are five to six major corporate players. A high-end blood analyzer can analyze more than one hundred samples per hour and measure a multitude of parameters. Such automated instruments are very complex and usually take more than seven years to develop, validate, and release to market. IVD manufacturers nearly

Figure 5. Low-range segment models of blood analyzers use technology similar to the high-end models' but offer lower throughput rates and fewer parameters measured.

always choose to develop these instruments internally within their companies rather than outsource the development to an outside IVD instrumentation development firm.
However, these same IVD manufacturers usually offer both low- and mid-range models of blood analyzers which, although using similar technologies as the high-end models, offer lower throughput rates and fewer parameters measured (see Figure 5). Mid-range models generally provide a sample throughput rate of 60-80 samples per hour and are equipped with or without an autosampler. Low-range models provide a sample throughput rate of fewer than 50-60 samples per hour.
Due to the lower throughput rates and parameter offerings (and therefore lower price and profit margin), IVD manufacturers find it more efficient and more cost-effective to outsource the low- and mid-range hematology instruments to companies that specialize in IVD instrumentation development and manufacturing. The technologies used in the low- and mid-range models offer the same reliability as the high-end models and are often based on the same underlying technologies (i.e. the same measurement techniques and reagents). However, as opposed to the high-end model’s seven-year development period, the slower, simpler models can be developed and released to market in about two years.
Due to the competitive nature among the major corporate players in the high-end hematology instrument segment, it is rare for a contract manufacturer to develop a line of instrumentation for more than one of the large IVD manufacturers.

Low- and Mid-Range Hematology Analyzer Market
The IVD manufacturers specializing in marketing low- and mid-range hematology analyzers include 10-15 major players. Some of these companies employ their own design teams to work in conjunction with contract manufacturers to produce the instruments, while others outsource all design, development, and manufacturing and simply market the instrument.
Since all blood analyzer instruments currently operate on the same basic principles developed more than fifty years ago, the IVD manufacturers compete on the basis of unique measurement techniques, reagents used, and the number of parameters offered for the best price, style, and service. While the low-range instruments are easier and quicker to market, there are lower barriers to entry, and therefore there are more competitors. There is always pressure on the developer to reduce the price of development and manufacturing, and speed up the launch of the instrument to market.
Contract manufacturers must move quickly, be innovative, and have a fountain of knowledge, experience, and specialized skills. Prevalidated, proven components and software, which are modified to adapt to the needs of a specific system, can save the developer valuable time. As the impetus for hematology instruments to be smaller continues to grow, a specialty in injected plastic parts helps to reduce costs because smaller size translates directly into manufacturing cost savings. For example, ten years ago, an optical bench measured up to 50-60 centimeters long and 10 centimeters in diameter and was very expensive. Today, due to the creation of specialized injected plastic parts resulting from the drive toward miniaturization, that same optical bench measures 10-15 centimeters long and 3 centimeters in diameter.
Reagents also play a key role in hematology instrument development. Driven by economy of scale and a desire to recoup high-end segment instrument investment costs, IVD manufacturers often require that the same reagents used in their high-end analyzers also be used in the low- and mid-range systems. This requirement can present a challenge to developers as they simultaneously strive to respond to demands by implementing new techniques to increase reliability and reduce price. In such cases, the secret to success may lie in the process of reagent development and integration (i.e., the method by which a compound mixture is blended, the timing, the temperature, etc.). Such valuable know-how is fiercely protected. However, due to the revealing nature of the patent process, developers do not often apply for patents on this technology.

Future Demands and Advancements
The customers’ unrelenting demands to reduce the size, cost, and development time of hematology instruments while increasing value through reliability, flexibility, ease of use, and the number of parameters tested requires developers to be innovative and highly specialized in multiple areas with systems and processes that are streamlined and organized for efficiency.
There has recently been a movement in the IVD industry to re-invent the technology for analyzing blood samples. If successful, this significant undertaking will revolutionize the industry by increasing the ability to measure more parameters in blood with greater precision and reliability at a lower cost. With the last great technology leap occurring more than fifty years ago, such an advancement would present great changes and new opportunities in the hematology analyzer business.
 

Henri Champseix is Chief Technology Officer and Director of Research and Development at BIT C2 Diagnostics, a BIT Company located in Montpellier, France. He can be reached at h.champseix@bit-c2d.com.

Eric Jolain is Chief Operating Officer of BIT C2 Diagnostics, a BIT Company, located in Montpellier, France. He can be reached at e.jolain@bit-c2d.com.

In September of last year, Sony Corp. acquired venture corporation Micronics Inc. (Redmond, WA) through its wholly owned subsidiary, Sony Corp. of America. Sony bought Micronics to boost its own R&D in the area of point-of-care diagnostic equipment and to accelerate commercialization of these products.
“We don’t have a specific business plan to announce at this time,” says Mack Araki, public affairs, Sony Corp. of America. “Our hope is that the acquisition of Micronics will accelerate commercialization of our point-of-care devices.”
Sony, well known worldwide for everything from consumer electronics to hospital equipment, has so far dabbled in IVD research only. It has not commercialized any IVD technology. “Our goal is to commercialize point-of-care diagnostic devices based on both Micronics’ and Sony’s technologies,” says Araki. “Micronics has globally recognized microfluidics technology and has an impressive patent portfolio,” he adds. “The company has also accumulated a wealth of expertise through its comprehensive product development for third-party clients.”
Araki says that Micronics was the first company to obtain FDA clearance for a microfluidics device for rapid blood typing, the ABORhCard. It is a closed-system, sample-to-result test that provides both ABO and Rh blood type from a fingerstick blood sample. Once the blood sample is applied, the test is performed in less than one minute. That device was cleared in April 2010.
Another product Micronics is focused on, which may be of high interest to Sony, is its PanNAT molecular diagnostic platform and the related assays. The PanNAT instrument is a battery- or main-powered device capable of processing discrete cartridges, each designed to perform a single or multiplexed nucleic acid amplification assay.  Each assay is fully integrated into the disposable cartridge and includes all necessary reagents. A small volume of biological sample is required, and there is no sample prep, according to a product description on Micronics’ Web site. The system provides a sample-on/result-off answer in 30 minutes and is configured for use in decentralized environments. PanNAT assays currently in development include malaria and Shiga-toxin-producing E. coli.
Micronics also holds two patents for a rapid thermocycling methodology for molecular diagnostics. The second patent is called “System and method for heating, cooling and heat cycling on a microfluidic device” and has broad utility across the life sciences sector, with particular application in point-of-care molecular diagnostics, the company says.
Further Extending Its Life Sciences Reach. In a move that appears to support Araki’s contention that Sony is serious about working to extend its reach into diagnostics, on December 5, 2011, the company announced that Sony DADC Biosciences was collaborating with Maven biotechnologies on the development and manufacture of smart consumables for Maven’s LFIRE detection platform. LFIRE, which stands for “label-free internal reflection ellipsometry,” is a real-time imaging technology for performing quantitative, label-free, cell-based assays and multiplexed biomarker detection. In 2010, the National Institute of Health awarded Maven a nearly $2-million grant to commercialize the technology. In a press release on this particular endeavor, Ali Tinalzli, PhD, director of business development & sales North America for Sony DADC said that this collaboration “provides a perfect example” of how Sony DADC can translate its experience in micro-structured engineering, gained through its “pioneering optical disc work,” to the needs of the life sciences industry. “We are particularly pleased,” he said, “to work with Maven at such an early stage, which ensures solutions that will be scalable and traceable, to smooth the way to future regulatory approvals in the clinical space.”
First TVs, Then Medical Equipment, Now Molecular Diagnostics? In a press release about the Micronics acquisition, Keiji Kimura, EVP and executive officer, said, “For some time, Sony has applied its consumer electronics technology to contribute to research and development in the medical and healthcare fields. We believe that the combination of Micronics’ development capabilities in the medical diagnosis domain and our consumer electronics and IT technologies, such as in optical discs, will enable us to offer innovative solutions that are responsive to the rapidly escalating needs of point-of-care diagnostics worldwide.”
Araki points out that Sony has quite a presence in the medical-electronics market already, with products such as printers, recorders, OLED displays, and imagers. “For example,” he says, “our image sensors have been widely used for consumer and professional cameras, and our OLED display was first introduced as a television for general consumer use.”

The primary election issue for IVD companies is the evolution of the clearance and approval system. My belief is that the rules and regulations are fine, and OIVD management needs to make improvements to clarify, standardize, and speed up the clearance and approval processes. Compliance issues need to be dealt with outside of the clearance/approval system, and the IVD industry needs to lobby on that. However, since legislators like to legislate, there is a threat of new laws that will require changes at FDA. In fact, there are already a series of bills that have been introduced by House Republicans. What the IVD industry needs to decide is whether to evaluate, support (or not), and comment on such draft bills, or assume that they are just campaign material.
—Glen P. Freiberg, president, RCQ Consulting (San Diego)

There are many important regulatory issues facing the IVD industry, such as regulatory consistency, predictability, ensuring that the level of regulation is congruent with the level of risk, and promoting innovation. These areas will have an important effect on the delivery of healthcare, costs, and the ability to stimulate new jobs in corporations and academic research. Unfortunately, it appears that no candidate is prepared to talk about these issues at all, let alone discuss them in a meaningful way.—Jeffrey N. Gibbs, JD, director, Hyman, Phelps & McNamara (Washington, DC)

OIVD specifically, and FDA in general, appear currently to be struggling between supporting the development of novel technologies, like companion diagnostics and personalized treatments, and protecting the public from untried and unproven technologies, like direct-to-consumer genetic testing for as of yet non-established diagnostic and prognostic markers. Developing the knowledge, expertise, and reasonable regulatory pathways to manage these new technologies, without stifling innovation or unnecessarily delaying diagnostic test availability (or novel therapies that they may accompany), will be a balancing act that the agency will face irrespective of candidates or parties. IVD-specific issues and policies that likely will be in the forefront will be FDA’s ever-increasing requests for more data and longer follow-up in establishing the clinical truth of any diagnostic marker and ensuring that physicians and patients understand the true implications of any IVD test result, especially for genetic/genomic markers and directed drug therapies.—Jonathan S. Kahan, JD, partner, Hogan Lovells (Washington, DC)

I will be closely following any legislation related to FDA’s reauthorization of the user fee act, including any provisions that may be related to the reform of the premarket review program-PMA or 510(k)-for IVDs, as well as legislation related to the potential for increased FDA oversight over diagnostic tests developed by a clinical laboratory. So far, around ten bills have been introduced in the House of Representatives. I’m sure many more are to come as the user fee statute is scheduled to sunset next year if it is not reauthorized.
—Michele Schoonmaker, PhD, vice president, government affairs, Cepheid (Sunnyvale, CA)

I will personally be following the Congressional focus on changes to the FDA regulatory approach for diagnostics, as well as Medicare payment changes that might impact those products. Right now, there are a lot of good ideas being batted around on Capital Hill and in the IVD industry. They each take a somewhat different approach to the same basic problem: delays associated with introducing important new diagnostics into the marketplace, and the irrational differences in the regulatory schemes that apply to diagnostics developed by IVD manufacturers and laboratories. Some of these seek fundamental changes in the whole regulatory paradigm to bring us, for example, closer to the European model, while others are more of simple refinements to the existing FDA process. Some of these proposals seek to harmonize the regulatory scheme between IVD manufacturers and laboratories by up-regulating laboratories, while others seek to achieve that harmonization by moderating the regulatory requirements on IVD manufacturers.—Bradley M. Thompson, JD, attorney, Epstein Becker Green (Washington, DC)

IVD Technology's sister publications, MDDI and MX, have launched a microsite called "Medtech Issues in the 2012 Election Year," which will cover and provide the latest news and information on policy issues that affect the medical device and IVD industries.

Figure 1. (a) Polycarbonate microfluidic cassette for bacteria detection in saliva. The cassette integrates a two-stage lysis process, solid-phase nucleic acid isolation, PCR with haptenized primers for post-amplification labeling, incubation for labeling of the PCR product with up-converting phosphor reporters, and blotting on a lateral-flow strip for detection of PCR product as a positive test result. The cassette features conduits, chambers (including PCR), an embedded porous silica membrane, and sites for externally-cooled phase change valves. The phase change valves and PCR chambers are heated/cooled with external thermoelectric elements. (b) Microfluidic cassettes mounted in molded plastic cartridges for interrogation of a lateral-flow strip in a commercial UpLINK laser scanner/reader instrument.

The potential of point-of-care (POC) diagnostics technology to benefit healthcare is widely recognized.^1-6 POC tests enable a distributed delivery of medical diagnostics as an alternative to the traditional reliance on centralized laboratories and hospitals. POC devices facilitate individualized medicine and home testing, and more immediate diagnostics at doctors’ offices, clinics, and hospital bedsides. POC tests can speed up the time-critical acquisition of data needed by emergency medical technicians and other first responders, and can be adapted for the detection of bioterrorism agents and surveillance of epidemics.
Perhaps the greatest impact of POC tests will be in developing nations by bringing affordable diagnostics to resource-poor settings and supporting more-effective eradication efforts for the numerous infectious diseases that still burden much of the world. This article reports on a family of POC devices that were developed in a collaborative effort by several universities with the aim of providing practical, simple-to-use, low-cost immunoassays and molecular diagnostics tests for infectious diseases. This technology features fully integrated sample-to-report devices that accept raw, untreated clinical specimen (e.g., blood, saliva, or urine) and provide a readily interpreted diagnostic test result in less than an hour.
First-generation POC devices include lateral-flow strip immunoassays for home pregnancy and drugs-of-abuse tests, as well as tests for antigens associated with contaminating bacteria in food. Molecular diagnostics (i.e., nucleic acid-based testing) provide even greater sensitivity and specificity compared to immunoassays, plus increased capabilities for analysis. For instance, molecular diagnostics can discriminate between strains and types of bacteria and viruses based on sequence-specific detection of pathogen nucleic acids. Genetic testing and RNA expression profiling are also feasible. However, implementation of molecular diagnostics in a simple, low-cost, easy-to-use POC format is considerably more challenging and remains elusive for many applications.
Nucleic acid-based tests (NATs) generally involve the sequential steps of sample lysis, nucleic acid extraction and isolation, concentration of the molecular target (i.e., sample volume reduction), and enzymatic amplification with pathogen-specific oligo primers. Detection of an amplification product is a test-positive indicator for the presence of bacteria, virus, or parasite in clinical specimens such as blood, saliva, or urine, while the absence of an amplification product is a test negative.
 

Figure 2. A schematic of the supporting instrumentation for a microfluidic cassette including buffer and reagent reservoirs, programmable syringe pumps, thermoelectric elements for actuating phase-change ice valves and heating/cooling of PCR chamber, controlled power supplies, and a computer.

The most common method of amplification is polymerase chain reaction (PCR), which amplifies a pathogen-specific DNA sequence until it reaches a level that can be detected by fluorescence measurements or imaging. Amplification can be preceded by reverse-transcription for sequence-specific detection of RNAs as well. Multiplexed tests that can assay a panel of RNAs or proteins are important for cancer screening and monitoring in which an expression profile is needed for an informative diagnosis. Currently, there is considerable interest in low-cost POC molecular diagnostics for HIV, TB, dengue fever, malaria parasites, SARS virus, E. coli strains causing food poisoning, and avian influenza.

Microfluidics Enables POC Molecular Diagnostics
 

Figure 3. (a) Lateral-flow strip schematic. (b) Conjugation of nucleic acid amplification primers for detection with a lateral-flow strip.

Many POC medical diagnostics systems are based on processing the sample in an inexpensive, single-use (disposable), credit-card-sized plastic microfluidic cassette or chip. The cassette is operated with a companion instrument that provides fluid actuation, flow control, temperature regulation, and sensors for detection. Ideally, the test would utilize untreated, crude clinical specimens such as whole blood, saliva, or urine collected by a syringe or sponge-tipped wand, without the need to pre-process the sample before testing. The test would provide an easily interpreted report within a time frame of 30-60 minutes. All reagents and buffers would be pre-stored in the cassette so that the user merely adds sample at the time of test. The sample and all reagents would be retained on the cassette to facilitate safe and secure disposal. The device would be operated by untrained personnel.
 

Figure 4. A pouch cassette for lateral-flow immunoassays used in a consecutive flow format with separate sample loading, wash, and labeling steps.

Depending on the type of test, results may be qualitative with a yes/no answer for the presence of a pathogen, or alternatively, more sophisticated tests would quantitate the pathogen in the sample (e.g., virus particles per milliliter of sample). Multiplexing capabilities (i.e., detection of several pathogens in a single test, such as simultaneously testing for both HIV and TB) are often desirable since there are many opportunistic infections that are coincident with HIV. These objectives are rather demanding considering the current technology. However, besides perhaps cost, most of them appear feasible as near-term, realistic goals for the next generation of POC devices.
The complicated sample processing for NATs, compared to simpler blood chemistry tests and immunoassays, can be achieved with a microfluidic lab-on-a-chip device. Such a device is comprised of a miniaturized fluidic network of reaction and incubation chambers, mixers, filters, membranes, valves, fluid actuators, conduits, and various interconnections, all formed in a plastic substrate or chip. In general, microfluidic formats offer many potential advantages including automated operation, lower cost per test, faster test times, reduced reagent consumption and waste, more protection from contamination, and potential improved performance with respect to reproducibility, sensitivity, and control.

Technology and Performance Issues
In the past ten or more years, the feasibility of lab-on-a-chip microfluidics components for numerous biotechnology and clinical processing and analysis functions has been well established. Such functions include immunoassays, fractionation and enrichment of biological samples containing cells and viruses, cell and virus lysis, nucleic acid extraction and purification, sequence-specific enzymatic amplification of nucleic acids (e.g., PCR), and various optical, electrical, and electrochemical nucleic acid detection methods.
 

Figure 5. A spring-wound timer system for actuating pouch cassettes. The schematic shows a rotating disc with detent that applies force on actuating balls transmitted to pouches. Position of detent and the balls serves to program the pouch activation as the disc turns sequences the pouch activation.

However, the seamless integration of such functions in a single chip that realizes a fully integrated sample-to-readout diagnostics test is an active area of research, and there are few reports of complete microfluidic molecular diagnostics systems.^5,7-9 To date, there is still no dominant technology platform for commercial-scale realization of molecular diagnostics. Ongoing research and development strive for better engineering solutions in flow control, fluid actuation, mixing, active heating and cooling, reagent delivery, manufacturability, cost effectiveness, reliability and reproducibility, and improved interfacing with instrumentation for control, data acquisition, and communications.
 

Figure 6. A fully automated instrument for pouch cassette actuation and laser scanning/reading of a lateral-flow strip. A sample is loaded on the cassette, and the cassette is placed on the drawer and inserted into the instrument. Electromechanical solenoid actuators depress the pouches according to a control program. A laser scans the nitrocellulose lateral-flow strip that uses up-converting phosphors as reporters. A photodetector records the optical signal from the test and control lines on the lateral-flow strip and sends the data to a computer.

A group at the University of Pennsylvania, in collaboration with New York University College of Dentistry, Lehigh University, and Leiden University Medical Center, and with the support of the National Institute of Dental and Craniofacial Research at the National Institutes of Health, has developed a family of POC microfluidic devices for detecting pathogens.9-19 The initial focus was testing for HIV antigens, anti-HIV antibodies, and HIV RNA in oral fluid samples. This multi-target detection capability provides more informative and confirmatory diagnostics with regard to the stage of the disease, its infectivity, the host immune response, quantitative viral load, and the effect of anti-viral therapy.
The current generation of HIV POC tests are used for screening and require a subsequent confirmatory test before a diagnosis is made. The HIV POC tests developed at the University of Pennsylvania combine antibody screening with a parallel nucleic acid/antigen confirmatory test in less than one hour. Subjects who test positive can be immediately assigned to a treatment program. The technology is generic and can be readily adapted to other pathogens (e.g., malaria, TB) or other types of samples (e.g., blood, urine, environmental samples). In the future, combination POC tests (e.g., antigen, antibody, DNA and/or RNA) may also be useful in discriminating between vaccinated and infected or exposed subjects when such need may arise.
 

Figure 7. (a) A pouch-based molecular diagnostics cassette for sample lysis, nucleic acid isolation by solid-phase extraction with a porous silica membrane as the binding phase, PCR, and detection of labeled PCR products on a lateral-flow strip. All buffers are contained in pouch reservoirs, and PCR components are pre-stored as paraffin-encapsulated, dried form inside the PCR chamber. (b) A photo of the cassette.

As mentioned above, a system architecture that is common to most microfluidic medical diagnostics systems is based on processing the sample in a plastic microfluidic cassette or chip. Ideally, the cassette would have no or few moving parts, could be mass produced at low cost, and as such would require a companion instrument for controlled heating and cooling, fluid delivery and actuation, flow control, and supporting electronics and optics for analyte detection and data processing/archiving, with options for GPS and communications. There is much interest in exploiting smart cell phones to perform the detection, control, and wireless communications functions of a POC system. There is also interest in non-instrumented cassettes that can function alone without any supporting processing, similar to commercial lateral-flow strip tests.^19,20

Implementing Microfluidics Diagnostics
In the POC systems developed at the University of Pennsylvania, the microfluidic cassette network integrates sample metering, lysis, solid-phase nucleic acid extraction, enzymatic amplification with pathogen-specific primers, and detection options using either real-time in situ fluorescence measurements of the amplification reaction (e.g., real-time PCR or loop-mediated amplification [LAMP]), or a lateral-flow strip for detecting the labeled amplification product. The solid-phase extraction is based on various nucleic acid binding phases such as porous silica glass fibers, cellulose (e.g., Whatman FTA), or nanoporous aluminum oxide membranes.
 

Figure 8. An oral fluid sample collector device with a Porex membrane. Also shown are various stages of insertion and elution in the cassette sample introduction slot, as tested with a red dye as the sample.

Solid-phase extraction is crucial for removing substances that may inhibit or otherwise reduce the sensitivity of the enzymatic amplification reaction. The solid-phase extraction step also serves to concentrate the target analyte since the lysate volume loaded on the membrane is much greater than the volume of membrane with bound nucleic acid, and much greater than the volume of nucleic acid eluted from the membrane into the amplification reactor. A concentration step is important since the volume of the sample must often be large (100-1000 microliters) in order to ensure a sufficient amount of sparse analytes, while the volume of the enzymatic amplification reaction should be small (about 10 µl) to conserve expensive enzymes and facilitate precise temperature control.
The cassettes were made out of polycarbonate or acrylic, and prototyped with a CNC mill or CO₂ laser cutting machine. Channel dimensions are on the order of 0.1 mm, and no coatings or surface treatments are required. The chips are compatible with high-volume, low-cost production by injection molding or stamping. Sealing the cassettes with pre-loaded, lyophilized reagents can be done with solvent bonding, adhesives, or ultrasonic welding. But thermal bonding methods that are often used to assemble microfluidics devices were avoided in order to assure compatibility with pre-loaded reagents.

Nucleic Acid-Based Tests on a Chip
The first-generation of nucleic acid test chips was a direct implementation of processes based on the commercial silica membrane spin column kits that are widely used for benchtop nucleic acid isolation (see Figures 1 and 2).⁹ The sample is shuttled between different chambers for lysis, solid-phase extraction using a porous silica membrane as the NA binding phase, PCR, conjugation of PCR amplicon with labels, and blotting of the labeled PCR product on a lateral-flow strip. A raw clinical specimen is collected with a sponge-tipped collector, and 100-500 µl of sample is injected into the cassette through an inlet port. The sample is lysed within a two-step process by incubation with a combination of proteinase K, detergents, and chaotropic salts.

Figure 9. A schematic of a pouch-based chip for combined lysis, nucleic acid extraction, and amplification (PCR or LAMP) in a single chamber, with pre-loaded, encapsulated reagents. This version of the chip uses a lateral-flow strip for detection of the amplicon. The cassette can be further simplified by measuring real-time fluorescence from the amplification chamber, eliminating the need for the lateral-flow strip but adding more complexity to the companion instrument.

To isolate the nucleic acids, the lysate is filtered through a porous silica membrane embedded in the flow path of the microfluidic circuit. The chaotrope lysing agent promotes binding of the nucleic acids to the silica. The silica membrane is washed with ethanol solutions to remove proteins and other lysis debris, and the membrane-bound isolated NA is eluted from the membrane and mixed with PCR reagents. The mixture of PCR cocktail and isolated nucleic acid fills a PCR chamber that is sealed by inlet and outlet valves, and temperature cycled by an external thermoelectric element built into the instrument stage upon which the chip is docked. The PCR primers are conjugated with dioxygenin (DIG) and biotin (see Figure 3). The PCR product is labeled by incubation with avidin-coated up-converting phosphor particles (UCPs) and blotted on a nitrocellulose lateral-flow strip mounted on the cassette.
The labeled amplicon migrates along the lateral-flow strip and binds to anti-DIG antibody that is formed as a test line to capture the UCP-labeled PCR product. The lateral-flow strip is interrogated by a commercial laser scanner instrument (UPLink) that is specifically designed for reading lateral-flow strips that use up-converting phosphors as reporters. Up-converting phosphors are more sensitive since the up-conversion process is excited by an infrared light source, and the long wavelength infrared background autofluorescence can be filtered from the green light emission of the UCP particles. This chip requires numerous interconnections with external programmable syringe pumps to provide fluidic power and controlled delivery of reagents and buffers to the chip, numerous valves for flow control and chamber-sealing, and computer control of thermoelectric elements for heating/cooling of the ice valves and PCR reactor (see Figure 2).

Streamlining Microfluidic Molecular Diagnostics
Although the chip performed well as a PCR-based molecular diagnostics test, it was overly complex for point-of-care applications. The group at the University of Pennsylvania completely rethought this approach using the following design criteria: greater simplified flow control, elimination of all interconnections between the chip and the outside world (excluding sample introduction), and pre-storage of all buffers and reagents on the cassette during manufacturing. The cassette would be made at high volumes but for a low cost, and would be available in several versions with varying degrees of sophistication for different types of users, including a self-contained cassette with chemical heating and visual endpoint detection and no need for any companion instrumentation.

Figure 10. Simplified molecular diagnostics chips: a) a cassette integrating FTA isolation of the nucleic acids and a LAMP reactor; b) measuring real-time fluorescence with a commercial ESE reader; and c) a recently-developed chip that uses an exothermal chemical reaction to heat the LAMP reaction.

To this end, a series of pouch-based chips were developed (see Figure 4). These cassettes feature pouches (i.e., chambers with flexible covers) that serve as reservoirs for buffers (about 100 µl) or are air-filled such that when they are squeezed, they propel a fluid charge through the microfluidic circuit.¹⁰ Similar structures can be adapted as diaphragm valves for flow control and gate-valve sealing of chambers and flow paths. A timed sequence of pouch manipulations executes a flow control program for a specified sample processing protocol. In the simplest version of the chip, the pouches are finger actuated by the user. In a mechanized but non-electrical version, the chip is coupled to a palm-sized timer device with a wind-up clock-spring that drives reciprocating balls to deflect the pouches (see Figure 5).¹¹ A fully automated version features a microcontroller, solenoids to actuate the pouches, thermoelectric heating/cooling, and optical excitation and detection (see Figure 6).
Pouch-based chips for molecular diagnostics were also developed (see Figure 7).¹² These chips perform the same processing steps as the chip shown in Figure 1. Combined with pre-storage of PCR components, the pouches yield a considerable simplification in both the design and operation of the chip. For the basic molecular diagnostics protocol, the sample is introduced into the cassette, mixed with chaotropic lysing agents, and filtered through a porous silica membrane. The chaotrope promotes binding of nucleic acids to the porous silica. Pouches are actuated to wash the membrane, and the nucleic acid is eluted into a temperature-controlled amplification chamber for PCR. The operation of the cassette has been further simplified by pre-storing lyophilized reagents in the amplification chamber.
The PCR cocktail (i.e., polymerase, primers, nucleotides, intercalating fluorescent dyes, and buffer salts) was dried in the amplification chamber and encapsulated with paraffin wax. The PCR chamber is downstream from the silica membrane, and the flow-through of the silica membrane during sample loading and washes exits through the PCR chamber. The dried reagents are protected from premature dissolution by the paraffin film. As the isolated nucleic acid is eluted from the membrane into the PCR chamber, the chamber is sealed and thermal cycled by a thermoelectric element, whereupon the PCR components are released and reconstituted in the correct proportions. This chip is compatible with real-time fluorescence measurements using a miniature fluorescent reader that is built into either the portable instrument, which the chip mates with, or a nitrocellulose lateral-flow strip mounted on the chip as described earlier for immunoassay chips.
The pouch-based chips also include a slotted input port for sample introduction with an oral fluid collector (see Figure 8). The single-use disposable saliva collector is affixed with a Porex (porous polyethylene) disc that adsorbs oral fluid when swirled around in the mouth. Loaded with sample, the collector disc is inserted into a slotted opening in the chip, and a pouch containing buffer is depressed to flow buffer through the porous disc and elute the oral fluid into a sample metering chamber. For blood specimens, a screw-lock introduction port or septum can accommodate connection to a sample collection syringe. In another option, a lance for finger pricking and wicking of blood into the cassette by capillary action could also be molded into the cassette.
Further simplification of the chip can be accomplished by combining the lysis, NA isolation, and PCR steps in a single chamber (see Figure 9).^13-15 The raw sample is filtered through a porous cellulose FTA filter paper membrane that is impregnated with lysis agents. The filter paper is mounted in the amplification chamber along with paraffin-encapsulated PCR reagents. The nucleic acid bound to the FTA membrane is washed with ethanol solution, but unlike the devices described above, the nucleic acid is not eluted from the membrane. Instead, the membrane-bound nucleic acid serves as the template for amplification: after the wash step, the PCR chamber containing the membrane is filled with water, sealed, and thermally cycled. The PCR product is detected in situ by real-time fluorescence, or alternatively, a lateral-flow strip is used to detect the PCR product as previously described.
More recent versions of the chip have realized still more simplification by using an isothermal (about 65° C) amplification method called LAMP in place of PCR, thus obviating the need for temperature cycling and by merging the lysis, nucleic acid isolation, and isothermal amplification steps into a single chamber on the chip, as previously described (see Figure 10).¹³ A self-contained, user-triggered, exothermic chemical reaction that is fueled by reagents pre-stored on the chip has also been developed to incubate the amplification reaction at 60-65° C for 30-60 minutes, thus avoiding the need for any external electrical heating, using the same chemistry of disposable hand warmers.¹⁸

Conclusion
A microfluidic approach to POC diagnostics has been developed, which enables convenient, low-cost nucleic acid-based testing. The designs exploit simplification that is enabled by reagent lyophilization, temperature-activated encapsulation layers, and chemical heating, along with new isothermal enzymatic amplification and filter-paper-based nucleic acid extraction methods. The devices employ flexible pouches that are built into the chip as liquid storage reservoirs and flow actuators, and can be actuated by finger, non-electrical mechanical devices, or fully-automated companion instruments. There are two detection options: lateral-flow strips and real-time fluorescence that can be implemented with a cell phone camera. A streamlined, efficient, robust POC test device has been achieved without sacrificing the functionality of conventional clinical laboratory diagnostics. This technology can be adapted for a wide range of costs, automation, functionality, and ease-of-use specifications, thus addressing the diverse applications envisioned for POC devices.

References
1. CP Price, A St. John, and JM Hicks, Point of Care Testing, 2nd ed. (Washington DC: AACC Press, 2004).
2. L Gervais,  N de Rooij, and E Delamarche, “Microfluidic Chips for Point-of-Care Immunodiagnostics,” Advanced Materials (2011) (DOI 10.1002/adma.201100464).
3. K Ohno, K Tachikawa, and A Manz, “Microfluidics: Applications for Analytical Purposes in Chemistry and Biochemistry,” Electrophoresis 29 (2008): 4443-4453.
4. J Wang, et al., “A Self-Powered, One-Step Chip for Rapid, Quantitative and Multiplexed Detection of Proteins from Pinpricks of Whole Blood,” Lab on a Chip 10 (2010): 3157-3162.
5. C van Berkel, et al., “Integrated System for Rapid Point of Care (POC) Blood Cell Analysis,” Lab on a Chip 11 (2011): 1429-1255.
6. LY Leo, et al., “Microfluidic Devices for Bioapplications,” Small 7, no. 1 (2011): 12-48.
7. ML Sin, et al., “System Integration: A Major Step Toward Lab on a Chip,” Journal of Biological Engineering 5, no. 1 (2011): 6.
8. RH Liu, et al., “Self-Contained, Fully Integrated Biochip for Sample Preparation, Polymerase Chain Reaction Amplification, and DNA Microarray Detection,” Analytical Chemistry 76, no. 7 (2004): 1824-1831.
9. J Wang, et al., “A Disposable Microfluidic Cassette for DNA Amplification and Detection,” Lab on a Chip 6 (2006): 46-53.
10. X Qiu, et al., “Finger-Actuated, Self-Contained Immunoassay Cassettes,” Biomedical Microdevices 11, no. 6 (2009): 1175-1186.
11. C Liu, et al., “A Timer-Actuated Immunoassay Cassette for Detecting Molecular Markers in Oral Fluids,” Lab on a Chip 9, no. 6 (2009): 768-776.
12. D. Chen, et al., “An Integrated Microfluidic Cassette for Isolation, Amplification, and Detection of Nucleic Acids,” Biomedical Microdevices 12, no. 4 (2010): 705-19.
13. X Qiu, et al., “A Portable, Integrated Analyzer for Microfluidic-Based Molecular Analysis,” Biomedical Microdevices (2011) (DOI 10.1007/s10544-011-9551-5).
14. C Liu, et al., “An Isothermal Amplification Reactor with an Integrated Isolation Membrane for Point-of-Care Detection of Infectious Diseases,” Analyst 136, no. 10 (2011): 2069-2076.
15. BL Ziober, et al., “Lab-on-a-Chip for Oral Cancer Screening and Diagnosis,” Head & Neck 30, no. 1 (2008): 111-121.
16. C Liu, MG Mauk, and HH Bau, “A Disposable, Integrated Loop-Mediated Isothermal Amplification Cassette with Thermally Actuated Valves,” Microfluidics and Nanofluidics (2011) (DOI 10.1007/s10404-011-0788-3).
17. X Qiu, et al., “A Large Volume, Portable, Real-Time PCR Reactor,” Lab on a Chip 10, no. 22 (2010): 3170-3177.
18. C Liu, et al., “A Self-Heating Cartridge for Molecular Diagnostics,” Lab on a Chip (2011) (accepted for publication).
19. JL Osborn, et al., “Microfluidics Without Pumps: Reinventing the T-Sensor and H-Filter in Paper Networks,” Lab on a Chip 10 (2010): 2659-2655.
20. B Weigl, et al., “Towards Non- and Minimally Instrumented, Microfluidic-Based Diagnostic Devices,” Lab on a Chip 8, no. 1 (2008): 999-2014.
 

Michael G. Mauk, PhD, is a researcher in the Mechanical Engineering and Applied Mechanics Department in the School of Engineering and Applied Science at the University of Pennsylvania (Philadelphia). He can be reached at mmauk@seas.upenn.edu.

Chang-chun Liu, PhD, is a research associate, in the Mechanical Engineering and Applied Mechanics Department in the School of Engineering and Applied Science at the University of Pennsylvania (Philadelphia).

Paul Corstjens, PhD, is a research group leader at Leiden University Medical Center (Leiden, The Netherlands).

Haim H. Bau, PhD, is a professor in the Mechanical Engineering and Applied Mechanics Department in the School of Engineering and Applied Science at the University of Pennsylvania (Philadelphia).

Calorimetry is the science of measuring the heat absorbed or released in a chemical reaction or physical transition. It provides a direct physical measurement of what is perhaps the most fundamental property of chemical and biochemical reactions: the change in heat.^1,2 The field of calorimetry dates back to the late 18th century and the Scottish physician Joseph Black, whose work represents some of the early developments of modern thermodynamics.³ The first practical calorimeter was developed by Antoine Lavoisier and Pierre-Simon Laplace and described in a 1783 publication of a method to measure the heat produced by oxidation of various substances.⁴ In this design, the amount of heat exchanged during a reaction was monitored as it flowed into or out of a solution directly surrounding the reaction chamber.
 

Figure 1. A schematic of a differential scanning calorimeter. Sample and reference chambers are heated at a controlled rate under constant pressure (P). The temperature difference between the chambers (delta T) is monitored and kept in thermal balance by heaters controlled by feedback electronics.

Knowledge of the heat capacity of the surrounding material and accurate measurement of its change in temperature permitted direct and precise measurement of the quantity of heat released or absorbed during the reaction or transition. Because design characteristics of calorimeters have improved substantially over the years, improved accuracy in measuring heat changes of chemical reactions have occurred and have facilitated steady advances in the fields of thermodynamics and thermochemistry. These fields have contributed dramatically to the present understanding of chemical compounds and their reactions.

Differential Scanning Calorimetry (DSC)
The differential scanning calorimeter was first developed by E.S. Watson and M.J. O’Neill more than 50 years ago.⁵ This device consists of sample and reference chambers that are heated at a constant rate at a temperature range of interest. A generic schematic of the device is shown in Figure 1. The electrical power output to each chamber is computer-controlled in order to maintain the same temperature in both chambers throughout the analysis. When the test sample undergoes a reaction that produces a change in heat (either exothermic or endothermic), excess heat in the form of electrical power is transferred to or from the sample to maintain the thermal balance between the sample and reference chambers. This electrical power compensation is directly proportional to the excess heat capacity of the solution, and the resulting instrument output is known as a thermogram, which can be plotted as the excess heat capacity versus temperature.

Figure 2. Schematic of an ideal two-state protein denaturation thermogram.

Because of the high sensitivity of modern microcalorimeters, which can reliably measure heat changes as low as 0.1 microcalories, DSC is the method of choice for thermodynamic studies of protein denaturation, in which thermal-induced unfolding of proteins can be directly measured. A typical DSC thermogram for a simple and ideal protein denaturation reaction is depicted in Figure 2. The excess heat capacity of the reaction is plotted as a function of temperature, producing a curve with an essentially Gaussian shape. In order to simplify analytical procedures, it is often assumed that the melting transition occurs in a two-state manner (i.e., the protein is either fully folded or completely denatured).
The area under the thermogram is the enthalpy (ΔH) of the temperature-induced unfolding reaction. Integration of the curve yields a transition or melting curve from which the fractions of folded and unfolded molecules may be determined. Under a given set of buffer conditions, every protein has a characteristic denaturation thermogram that provides a fundamental thermodynamic signature for that protein. Many proteins have complex structures that can lead to a substantial departure from the ideal two-state behavior. Corresponding thermograms may have complex shapes (e.g., multiple peaks) that may reflect melting of individual structural domains in the tertiary structure. A majority of the most predominant proteins in blood plasma (e.g., albumin, IgG, fibrinogen, transferrin, IgA, α2-macroglobulin, α1-antitrypsin, complement C3, IgM, and haptoglobin) have complex structures with thermograms that deviate from the ideal two-state melting behavior.⁶
DSC thermograms are an extensive property of a protein solution and are directly related to the mass of proteins present. For example, if the weight concentration of a protein is doubled, the calorimetric heat response will also double. Likewise, in a solution containing mixtures of proteins, the relative heat response will correspond to the total mass of proteins present. Fundamentally, this property constitutes the basis for DSC-based diagnostic applications, since thermograms of protein mixtures can be resolved into characteristic melting curves of individual protein components. In a non-interacting mixture, each protein has a characteristic curve shape, melting temperature, and melting enthalpy. Thus, the thermogram observed for the mixture can be represented as the weighted sum of all constituent individual protein thermograms, weighted according to their relative molar mass and concentration.

DSC Applications
DSC’s high sensitivity and ability to evaluate sample purity and material properties have enabled wide applications of the technology in industrial settings, particularly as a quality control instrument. DSC analysis is commonly applied in food science research to help define and control processing parameters, and in the pharmaceutical industry to help characterize drug compounds.^7-9 The use of DSC in medical diagnostics is currently being developed, with promising potential applications for diagnostic analysis of human blood plasma.

The Plasma Proteome
In recent years, the emergence of numerous FDA-approved plasma/serum diagnostic assays has profoundly affected the world of medical diagnosis. Detecting a disturbance in the interrelationships among proteins in blood plasma is increasingly relied on to indicate the presence of infection, inflammation, malnutrition, or autoimmune disease. Human blood plasma is known to contain more than 3,000 unique proteins (the plasma proteome) with 10 proteins contributing 90% of plasma by weight and only 22 proteins comprising 99%. The remaining 1% is a complex mixture of low abundant proteins.^6,10 Disturbances among the major proteins can provide critical information early in the course of a disease, leading to improved patient outcomes and reduced costs for patient care.
Interest in the array of proteins and other biological molecules in plasma/serum have been primarily focused on the low molecular weight components of the serum or plasma proteome, or the peptidome, which has been touted as a “treasure trove of diagnostic information that has largely been ignored.”^11,12 Analytical methods such as mass spectrometry (in particular SELDI methods), protein electrophoresis, and immunochemical assays have made analysis of the peptidome possible. Both electrophoresis and mass spectrometry separate plasma proteins on the basis of size and charge.
In contrast, measuring the thermal properties of proteins present in plasma by DSC is independent from and complementary to these procedures. DSC thermograms of composite protein mixtures such as plasma provide an entirely new approach to analyzing the plasma proteome. DSC measurements of protein concentrations and interactions in human blood plasma have the potential to be powerful clinical tools for detecting, diagnosing, and monitoring diseases and associated pathophysiological processes.^2,6

The Interactome
Many components of the peptidome bind with the more abundant serum proteins, particularly albumin and immunoglobulins. This has led to the concept of the interactome, which for plasma/serum is “comprised of a network of protein-protein and peptide-protein interactions.”¹³ Proponents of the interactome concept believe that in disease states, low-molecular-weight proteins or peptides unique to that disease will increase in concentration in plasma or serum.¹³
Interactions of these biomarkers with the more abundant plasma proteins can alter their denaturation properties, producing characteristic changes of shape in observed thermograms. Interestingly, the original paper that introduced the interactome concept concluded that “the discovery of novel biomarkers in serum/plasma requires new biochemical and analytical approaches, and most importantly, it is clear that no single sample preparation or detection method will suffice if biomarker investigations are to be broadly successful using current technologies.”¹³
DSC assays are sensitive to binding interactions in ways that current electrophoresis and mass spectrometry assays are not. This is because binding interactions between small peptides and larger proteins can result in dramatic changes in thermal transitions, which can be detected by DSC, but result in only small changes in mass or charge, which can be challenging for electrophoresis or mass spectrometry to detect. While the denaturation of very small proteins and biomarkers themselves may not be observed directly by DSC, consequences of their interactions are seen indirectly through alterations of the melting properties of the more abundant proteins, to which the biomarkers bind. With proper calibration, changes in thermograms resulting from binding interactions can be related quantitatively to the biomarkers’ binding strengths.
Because they are sensitive to changes in protein composition both in a non-interacting mixture and as a consequence of interactions with other components, DSC thermograms are promising as a molecular diagnostics and biomarker discovery tool. Recent experiments have shown DSC thermograms to be sensitive to changes in plasma in relation to various pathological conditions.^2,6 Blood plasma thermograms were measured for patients suffering from various clinical diseases and revealed uniquely identifiable patterns associated with each disease state.
Louisville Bioscience Inc. has expanded such research and development efforts into activities aimed at commercializing the Plasma Thermogram technology. Ongoing clinical studies are directed toward characterizing thermograms for several different classes of disease. Thermograms for each disease type and stage are stored in an expanding database that is refined with every sample examined. The database provides a reference to which diagnostic thermograms can be compared and classified. As shown in several examples that follow, these promising results provide a compelling argument for the utility of DSC in clinical diagnostics.

Experimental Methods
In a typical Plasma Thermogram experiment, an aqueous plasma solution (at a concentration of approximately 2 mg/mL) is heated at a constant rate in the calorimeter sample cell alongside an identical reference cell containing only the solvent (buffer). An initial sample preparation step is performed before DSC data collection in order to standardize the sample buffer conditions. Only 100 µl of plasma is required, which allows for potential loss throughout the entire process. However, as little as 50 µl of sample can be used but would be considered the minimum sample volume required.
A major advantage of the DSC instrument is that it can be automated for higher sample throughput. For instance, multiple samples may be loaded into a 96-well plate, stored in a refrigerated compartment, and loaded into the calorimeter cells by a robotic system that also cleans the cells between runs. Calorimetric experiments are made serially with each DSC thermogram collected in approximately two hours. However, throughput of the system may be enhanced by increasing the scan rate or narrowing the range of temperatures scanned.
Initial data processing steps include a necessary baseline correction and thermogram normalization with respect to total protein concentration. Baseline-corrected and normalized thermograms are interpolated over a standardized temperature scale and stored in a database for easy access and future analysis. Statistical and chemometric analytical models have been developed to characterize and classify thermograms according to pattern and sample characteristics.¹⁴ Using these tools, unclassified thermograms can be compared to previously classified and characterized thermograms in the database to evaluate similarity in pattern. Based on this analysis, newly acquired thermograms can be assigned to established categories with associated probabilities or confidence levels.

Diagnostic Applications
The National Institutes of Health report of the Autoimmune Diseases Coordinating Committee (ADCC) states that as many as 24 million people in the United States are afflicted with autoimmune diseases.¹⁵ Diagnosing autoimmune diseases is particularly difficult due to the highly diverse clinical manifestations; symptoms are often not apparent until a disease has reached relatively advanced stages. The ADCC states that “autoimmune diseases present many complex challenges to the clinician. Prominent among these are the difficulties in establishing a diagnosis early in the course of disease and the lack of surrogate markers to monitor therapy and predict clinical outcomes. Thus, new tools are needed to ensure that the most promising experimental approaches will lead to better clinical outcomes.”¹⁵

Figure 3. Autoimmune disease Plasma Thermograms.

Measurements of blood plasma thermograms by DSC may provide an additional method for aiding in the differential diagnosis of autoimmune diseases. Plasma Thermogram assays from patients with the following four commonly misdiagnosed autoimmune diseases have been measured: lupus, rheumatoid arthritis, scleroderma, and myositis. The results showed clear differences between thermograms from healthy control subjects and thermograms from patients suffering from each of these diseases. Figure 3 shows the mean thermograms measured for each of the four disease categories and the mean normal thermogram collected from healthy subjects. These results show the potential utility of DSC technology in this medical area.

Improved Cancer Monitoring
New minimally invasive tools are imperative for improving therapeutic monitoring and early detection of cancer remission and relapse. The current methods for cancer detection and monitoring include surgical biopsy, serological testing, ultrasound, and imaging (x-ray, MRI, CT scan, PET, etc.). Although methods exist for detecting metastatic diseases at advanced stages, many suffer from limitations and drawbacks for early detection of disease relapse.¹⁶ In addition, FDA has recently announced an initiative to reduce unnecessary radiation exposure from medical imaging procedures. A noninvasive blood assay that can discriminate early from more advanced stages of carcinoma would highly benefit oncology diagnostics.

Figure 4. Comparison of Plasma Thermograms from diseased (red) and healthy (green) plasma. Diseased samples were taken from patients diagnosed with cervical cancer (top), melanoma (middle), and lung cancer (bottom).

DSC measurements of blood plasma could advance oncology diagnostics toward this goal. Figure 4 shows mean Plasma Thermogram assays obtained from patients who were diagnosed with cervical cancer, melanoma, and lung cancer. The thermograms for each disease category are significantly different in shape from the healthy control thermograms and from each other. Comparable results have been obtained for various other types of cancer, including ovarian, uterine, and endometrial cancer. These results suggest that DSC thermogram assays may be applicable to cancer detection and diagnosis. Thermograms from patients at different stages of cervical cancer and melanoma have also been measured. As Figure 5 indicates, the Plasma Thermogram assay could monitor disease progression through characteristic curve shapes associated with different stages of disease.

Biomarker Discovery
Applying DSC thermogram analysis to human plasma may also form the basis of a new, powerful, and broadly applicable biomarker discovery platform that is fundamentally different from standard biomarker assays. Differences in the shapes of plasma thermograms have been attributed to interactions of circulating components (e.g., peptides, proteins, metabolites, lipids, fatty acids, etc.) with the most abundant plasma proteins. Both experimental and clinical studies support the theory that in diseased states, concentrations of circulating low molecular weight proteins, peptides, or nucleic acids indicative and unique to a disease become elevated in plasma and other bodily fluids.
 

Figure 5. Comparison of Plasma Thermograms from stages of melanoma (red) with healthy samples (green). Diseased samples were taken from patients diagnosed with minimal (top) and advanced (bottom) stages of melanoma.

If they do indeed interact and bind with one or more of the abundant proteins in a fluid, such substances could prove to be viable biomarkers specific for particular diseases. Because they may alter the melting profile of any of the major proteins, interactions of candidate biomarkers may produce changes in observed thermograms relative to the characteristic normal signature. Plasma Thermogram assays may present a method for discovering or confirming the existence of disease specific biomarkers and understanding their role in disease pathology.

Conclusion
DSC has advanced during the past 200 years into a robust and reproducible physical technique with a multitude of potential applications. This article has presented recent research results that used DSC for the diagnostic characterization of human plasma for the detection, clinical diagnosis, and therapeutic monitoring of various clinical disease states including autoimmune diseases and cancers. Although the results presented in this article in the context of plasma analysis provide a compelling argument for the further development of DSC-based diagnostics, many significant challenges must be addressed before DSC diagnostics can be developed into a mainstream diagnostic tool.
For example, quantitative assessments of metrics such as sensitivity and specificity that define diagnostic performance will require the collection and analysis of many more samples, with disease incidence and prevalence within the population being critical parameters of the sample sets. Direct comparisons with gold-standard industry diagnostic techniques have yet to be established, which include clinically defined protein markers that are detected using antibody technology or physical-chemical techniques such as equilibrium dialysis, electrophoresis, and ultrafiltration. Ultimately, in order to gain market adoption, results of DSC diagnostics must be demonstrated to be comparable to more established, conventional techniques. The results presented in this article provide confidence that these comparisons are likely to yield favorable results. In addition, high-throughput capabilities of DSC must be fully demonstrated to achieve practical efficiency of the technique.
Promising additional applications beyond clinical diagnostics include biomarker discovery, and absorption, distribution, metabolism, excretion, and toxicity screening in which sensitive, robust, and reproducible screening of small molecule and protein interactions can provide valuable insights for drug discovery and development processes. For clinical diagnostic applications, further validation of DSC will be required through additional clinical studies and quantitative characterization of thermograms and their behaviors in the presence of small molecules or other potential therapeutic agents. Although many challenges remain, with strong results in future development trials, DSC could emerge as a central player in medical research and clinical diagnostics.

Conflict of interest statement: JBC, ASB, DJF, GPB, and NCG are coinventors on patent applications describing the DSC Plasma Thermogram technology. Louisville Bioscience Inc. holds an exclusive license from the Univ. of Louisville for the DSC Plasma Thermogram technology.

References
1. B Wunderlich, Thermal Analysis (New York: Academic Press, 1990), 137–140.
2. NC Garbett, et al., “Interrogation of the Plasma Proteome with Differential Scanning Calorimetry,” Clinical Chemistry 53, no. 11 (2007): 2012-2014.
3. KJ Laider, The World of Physical Chemistry (Oxford University Press, 1993).
4. AC Buchholz and DA Schoeller, “Is a Calorie a Calorie?,” American Journal of Clinical Nutrition 79, no. 5 (2004): 899S–906S.
5. ES Watson and MJ O’Neill, “Differential Microcalorimeter,” U.S. Patent 3,263,484, August 2, 1966.
6. NC Garbett, et al., “Calorimetry Outside the Box: A New Window into the Plasma Proteome,” Journal of Biophysics 94, no. 4 (2008): 1377-1383.
7. JA Dean, The Analytical Chemistry Handbook (New York: McGraw Hill Inc., 1995), 15.1–15.5.
8. E Pungor, A Practical Guide to Instrumental Analysis (Boca Raton, FL: 1995), 181–191.
9. DA Skoog, FJ Holler, and T Nieman, Principles of Instrumental Analysis, 5th ed. (New York: 1998), 805–808.
10. S Surinova, et al., “On the Development of Plasma Protein Biomarkers,” Journal of Proteome Research 10, no. 1 (2010): 5-16.
11. LA Liotta and EF Petricoin, “Serum Peptidome for Cancer Detection: Spinning Biologic Trash into Diagnostic Gold,” The Journal of Clinical Investigation 11, no. 1 (2006): 26-30.
12. LA Liotta, M Ferrari, and E Petricoin, “Clinical Proteomics: Written in Blood,” Nature 425, no. 6961 (2003): 905.
13. M Zhou, et al., “An Investigation into the Human Serum Interactome,” Electrophoresis 25, no. 9 (2004): 1289-1298.
14. DJ Fish, et al., “Statistical Analysis of Plasma Thermograms Measured by Differential Scanning Calorimetry,” Biophysical Chemistry 152, no. 1-3 (2010): 184-90.
15. The Autoimmune Diseases Coordinating Committee, “Progress in Autoimmune Diseases Research,” Report to Congress, U.S. Department of Health and Human Services, National Institutes of Health, NIH Publication No. 05-5140, March 2005.
16. JD Wulfkuhle, LA Liotta, and EF Petricoin, “Proteomic Applications for the Early Detection of Cancer,” Nature Reviews Cancer 4 (2003): 267-75.

Mark A. Wisniewski, MBA, is the Chief Operating Officer at Louisville Bioscience Inc.

Nichola C. Garbett, PhD, is Instructor of Medicine and Biophysical Core Facility Manager at James Graham Brown Cancer Ctr. at the Univ. of Louisville and a founder and director of Laboratory Operations of Louisville Bioscience Inc. She can be reached at nichola.garbett@louisville.edu.

Daniel J. Fish, PhD, is Vice President for Theoretical Development at Louisville Bioscience Inc. He can be reached at dfish@lbidx.com.

Greg P. Brewood, PhD, is Vice President for Product Development at Louisville Bioscience Inc. He can be reached at gbrewood@lbidx.com.

James J. Miller, PhD, is Professor of Pathology and Laboratory Medicine at the Univ. of Louisville and the Director of Clinical Chemistry and Toxicology at the Univ. of Louisville Hospital Laboratory. He is a principal advisor to Louisville Bioscience Inc. He can be reached at jmiller@louisville.edu.

Jonathan B. Chaires, PhD, is a Professor in the Dept. of Medicine at the Univ. of Louisville Health Sciences Center, a Senior Scientist in the James Graham Brown Cancer Center, and a founder of Louisville Bioscience Inc. He can be reached at j.chaires@louisville.edu.

Albert S. Benight, PhD, is the founder and President of Louisville Bioscience Inc. He can be reached at abenight@lbidx.com.
Mark A. Wisniewski, MBA, is the Chief Operating Officer at Louisville Bioscience Inc.

Thermo Scientific Nunc Immobilizer Streptavidin and Nunc Immobilizer Amino surfaces.

The stability of all components in an IVD kit reflects its ability to maintain consistent, and therefore accurate, performance over time. Unlike commonly measured attributes, stability is problematic to assess prior to experimental use, and as such, there is a great onus on IVD manufacturers to ensure that any stability claims are substantiated. In today’s culture of mass manufacturing, extending stability to ensure that shelf-life does not expire prior to shipping is becoming increasingly important.
Furthermore, guaranteed stability is perceived as a value added component, specifically in international markets or areas where storage can be problematic. Such guaranteed stability increases the product’s usability, regardless of shipping time and any storage issues, including power failures or temperature deviations in a cold storage environment. In addition, once the IVD kits are shipped, they likely will undergo further storage before use, not necessarily at optimal temperatures. By extending the usable shelf-life of such products, concerns over stability are alleviated, and end users can continue to work with a significant level of assurance and confidence that resulting data are accurate.
Shelf-life is commonly assessed using two different types of stability testing: real-time and accelerated. In real-time stability testing, the IVD kit components are stored under recommended conditions and monitored until viability is lost. In accelerated stability testing, a product is stored in an environment in which conditions such as temperature and humidity are higher than recommended. Degradation at the recommended storage conditions can therefore be predicted using known relationships between the acceleration factor and the degradation rate. As the most commonly used acceleration factor, temperature has a strong relationship with the Arrhenius equation, which is used to determine an estimation of the degradation rate.

Solid Capture Surfaces
As an integral component of any diagnostic kit, solid capture surfaces (e.g., microwell plates) are used to bind effectively the component of interest. In order to maximize usability and consequently obtain consistent and reliable results, these surfaces ideally need to remain effective over long periods of time. In order to increase the shelf life of diagnostic immunoassay kits and provide the required level of quality assurance, plate activity should remain constant with storage at room temperature. This eliminates the need for long-term storage in cooling facilities and makes the immunoassay techniques more accessible to laboratories where cooling facilities may not exist.
For diagnostic purposes, the solid surfaces of the plates are coated with a capture molecule before being used in a screening set-up. Capture proteins are fixed to the surface and used to detect specific biomarkers from serum or urine, or an antibody response to infection. The surface coating can consist of a range of different-sized molecules, from small peptides to large antibody molecules, the activity of which can be maintained during storage using a variety of stabilization methods. For example, previous methods of increasing reagent stability include freeze-drying, which preserves the protein-coated surfaces that are unstable in aqueous solutions.1 Frozen samples are preserved through the removal of water, which blocks several degradation pathways.2-5 However, the freeze-drying process itself can lead to protein degradation and subsequent loss.6-8 These risks are significantly reduced by adding a stabilization agent prior to freeze-drying in order to provide a reliable method to simplify transport and storage, and extend shelf-life.
This article assesses the stability of a range of different stabilizers to increase shelf-life, thus maximizing usability and improving cost-efficiency. Such assessment is demonstrated via accelerated aging by which capture molecules of different sizes can maintain their full activity for at least four years, when attached to an amino binding or streptavidin immobilization surface.9

Accelerated Aging
In order to ensure experimental integrity is maintained, plates are often coated to modify surface properties and increase ligand binding affinity. With the ability to improve significantly enzyme immunoassay sensitivity, having a plate with low well-to-well variability and strong adsorption is crucial and minimizes the loss of any material during the repeated washing steps.10 Such plate types are widely used for immobilizing captured antibodies, since they provide a generic surface that maintains molecular activity.11 To demonstrate the suitability of plates with amino and streptavidin binding molecules as solid surfaces for diagnostic kits, different plate types (e.g., the Thermo Scientific Nunc Immobilizer Streptavidin and Nunc Immobilizer Amino surfaces) were coated with a typical capture molecule, and their activity were measured.
Three different capture molecules were adapted for covalent binding of NH2- or SH-groups to the Immobilizer Amino surface, or were biotinylated for attachment to the Immobilizer Streptavidin surface: C-peptide from human insulin (3 kDa), human chorionic gonadotropin hormone (hCG) (37 kDa), or rabbit anti-goat immunoglobulin (Ig) (170 kDA).To assess the long-term stability of the coated surfaces, different commercially available stabilizers were compared to a coated, unstabilized surface. Since storage at 4º C is considered non-aging and room temperature storage is considered real-time aging, all surfaces were stored at 50º C. Doing so simulates accelerated aging, in which one day at 50º C equals 6.5 days at room temperature and enables stabilization experiments to be performed during a shorter timeframe.9

Materials and Methods
Amino binding strips were coated with either C-peptide (Bachman H2470), hCG (Sigma CG5), or polyclonal rabbit anti-goat Ig (Dako 2Z0228). C-peptide (1 mg/ml) was diluted in 0.1 M sodium phosphate buffer (pH 8.0) to a final concentration of 600 ng/ml, and 100 µl was added to the well. hCG (65 µg/ml) was diluted in 0.1 M sodium phosphate buffer (pH 8.0) to a final concentration of 400 ng/ml, and 100 µl was added to the well. Polyclonal rabbit anti-goat Ig (7.1 mg/ml) was diluted in 0.1 M sodium phosphate buffer (pH 8.0) to a final concentration of 10 µg/ml, and 100 µl was added to the well. The strips were incubated for two hours and agitated at 300 revolutions per minute (rpm), followed by three washes with 350 µl/well of 0.15 M phosphate buffered saline.
C-peptide and hCG were biotinylated using a commercially available biotinylation kit (EZ-Link Sulfo-NHS-LC-Biotinylation kit by Pierce), in accordance with the manufacturer’s guidelines. Streptavidin strips were prewashed three times with 350 µl/well of 0.15 M phosphate buffered saline and PBS with 0.05% Tween 20 (pH 7.2). They were coated with either 300 ng/ml of biotinylated C-peptide, 200 ng/ml of biotinylated hCG, or 5 µg/ml of biotinylated polyclonal rabbit anti-goat Ig, all of which were diluted in PBS-Tween. Each well was coated with 100 µl of capture molecule for three hours and gently agitated at 100 rpm at room temperature. The strips were washed three times with 350 µl/well of 0.15 M phosphate buffered saline (pH 7.2).
The stabilization process was performed by adding 200 µl/well of either of the following: starting block blocking buffer by Thermo Fisher Scientific; 0.15 M (5% w/v) sucrose by Acros; coating stabilizer and blocking buffer diluted 1:1 with water by BioDesign International; or luria broth (LB) medium by Sigma for one hour with gentle agitation (100 rpm). The strips were aspirated and dried at 37˚ C for 30 minutes. After this stabilization, the strips were packed in alubags with desiccants and stored at 4° C, room temperature, or 50° C accordingly until the protocol began.
To assess the effectiveness of the stabilization protocol, the surfaces were tested and their conditions were recorded after two weeks, one month, two months, four months, six months, and eight months of storage at the specified temperatures. Uncoated strips were also freshly prepared as baseline reference values for normalization by using the coating process described above. The stabilized strips were washed three times with 350 µl/well of PBS-Tween prior adding the antibody solutions:

•     The C-peptide-coated strips were incubated for three hours with a mixture of HRP-conjugated polyclonal mouse anti-human C-peptide Ig 1:64000 and monoclonal mouse anti-human C-peptide 1:400.
•     The hCG-coated strips were initially incubated for ninety minutes with polyclonal rabbit anti-hCG Ig 1:400. They were subsequently incubated for an additional ninety minutes with a mixture of HRP-conjugated polyclonal swine anti-rabbit IgG 1:2000 and polyclonal swine anti-rabbit IgG 1:1000.
•     The rabbit anti-goat Ig-coated strips were incubated for three hours with a mixture of HRP-conjugated polyclonal swine anti-rabbit IgG 1:2000 and a polyclonal swine anti-rabbit IgG 1:1000.

The substrate reactions were initiated by adding 100 µl/well of O-phenylenediamine dihydrochloride (OPD/H2O2). All incubation steps were performed using 100 µl/well of substrate at room temperature, gentle agitation at 100 rpm. Reactions were terminated after five minutes by adding 100 µl/well of 2N sulfuric acid. Absorbance was consequently measured at 492 nm.
Linear regression was used to evaluate the change of activity level due to aging. An α value greater than or equal to 0 is considered no change in aging, and an α value less than 0 is considered a decrease in activity level due to aging. A two-way analysis of variance was used to evaluate the effect of stabilization compared to the unstabilized assays, with a significance level of p less than 0.05 (n=9).

Results
In this study of accelerated aging, amino-binding and streptavidin surfaces were coated with a variety of different capture molecules and stored at 50° C. All six model assays demonstrated sustained activity at eight months after coating, which is equivalent to four years of storage at room temperature (see Figures 1 and 2).
 

Figure 1 (left). Determined activity for the immobilizer amino surface coated with C-peptide (A), hCG (B), and rabbit anti-goat Ig (C). The coated surfaces were stored as unstabilized or stabilized with either a coating stabilizer, LB medium, blocking buffer, or sucrose, and tested after storage at 50 degrees Celsius for 1, 2, 4, 6, and 8 months, corresponding to 1/2, 1, 2, 3, and 4 years' storage at room temperature (normalized data, n=7).

Figure 2 (right). Determined activity for the immobilizer streptavidin surface coated with biotinylated C-peptide (A), hCG (B), and rabbit anti-goat Ig (C), unstabilized or stabilized with either a coating stabilizer, LB medium, blocking buffer, or sucrose, after storage at 50 degrees Celsius for 1, 2, 4, 6, and 8 months, corresponding to 1/2, 1, 2, 3, and 4 years' storage at room temperature (normalized data, n=7).

In the majority of assays, activity levels were constant, and the relative absorbance was high compared to the unstabilized surfaces. The greatest decrease in measured activity was found in the biotinylated C-peptide assay stabilized with blocking buffer (see Figure 2a). The decrease was calculated to be 24.7% by linear regression. However, as an inherently variable structure, C-peptide is a problematic model to assess for stability, and there may be some inherent background noise which is not accounted for here.

Amino-Binding Surfaces
C-Peptide. As shown in Figure 1a, the C-peptide coated and unstabilized amino-binding surface maintained an activity level of approximately 70% for up to four years. Coated surfaces which are stabilized with either LB-medium or sucrose maintained an activity level of approximately 90% throughout the testing period, while surfaces stabilized using blocking- or coating-buffer showed a decrease in activity. LB medium or sucrose demonstrated a stabilization effect, as the activity of these surfaces was significantly higher than the unstabilized surface.
hCG. An activity level of greater than 100 % was achieved after four years of aging using the hCG-coated amino-binding surface in combination with LB medium or sucrose. This indicates that hCG is interacting with the stabilizing buffer to cause non-specific binding of the poyclonal antibody. This combined with minimal degradation of hCG shows that the effect of using LB medium or sucrose is significant compared to the unstabilized surface and the surfaces treated with a coating stabilizer, or blocking buffer, which demonstrated a significant decrease in stability between the ½- and four-year time points. However, the coating stabilizer resulted in an activity level of approximately 90% after four years of aging (see Figure 1b).
Polyclonal Rabbit Anti-Goat Ig. When stabilized with sucrose, Ig demonstrated a steady activity level of greater than 90% after four years of aging. Surfaces treated with coating stabilizer and blocking buffer both demonstrated a steady activity level of greater than 80 % after four years of aging. Unstabilized Ig lost activity immediately following coating, and after four years of aging, the activity levels were down to 25% (see Figure 1c). For this model assay, all stabilization methods resulted in a significant increase in activity compared to the unstabilized surface.
Discussion. Despite the size of the capture molecule, all stabilization methods had a positive effect on the molecules immobilized onto the amino-binding surface. Favorable results were obtained on the C-peptide- and hCG-coated plates with both sucrose and LB medium. Maintained stability and activity levels demonstrated that by using these methods, plates can be stored for up to four years. Data show that sucrose has the greatest stabilizing effect for the polyclonal rabbit anti-goat Ig-coated plate, while LB medium provided optimal stabilization for the hCG-coated plate.

Streptavidin Surfaces
C-Peptide. When C-peptide was immobilized to the streptavidin surface, no significant differences between an unstabilized surface and stabilized surfaces were observed (see Figure 2a).
hCG. As shown in Figure 2b, a high degree of stability is reached when hCG was immobilized onto the streptavidin surface. The activity level was maintained close to 1.0. Stabilization with sucrose or a coating stabilizer had a long-term effect. Unstabilized and LB medium set-ups have also shown activity levels between 80% and 90%, which is accurately maintained during four years of aging. Surfaces treated with sucrose and a coating stabilizer showed significant stabilization compared to the unstabilized surface and surfaces treated with a blocking buffer and LB medium.
Polyclonal Rabbit Anti-Goat Ig. When Ig immobilized on the streptavidin surface was treated with a coating stabilizer, activity levels are maintained at 95% for up to four years of real-time aging. Sucrose demonstrated a long-term stabilizing effect, with the ability to maintain an activity level of 90%. The activity level of unstabilized molecules decreased to 50%, and to a lesser extent, the coated surfaces stabilized with LB medium or a blocking buffer also decreased. As with the amino-binding surface, all stabilization methods resulted in a significant improvement in activity compared to the unstabilized surface.
Discussion. The stabilizers did not have any effect on the activity of the C-peptide. For hCG, the coating stabilizer and sucrose have improved the retention of activity over four years. However, the LB medium has shown no effect, while the blocking buffer appears to accelerate a loss of activity. Coating stabilizers provide the best stabilization for Ig, followed by sucrose, the blocking buffer, and finally the LB medium. Therefore, when using the most suitable stabilizers for each assay, data have shown that coated surfaces can be stored at room temperature for up to four years with a consistent and stable level of activity.

Conclusion
This accelerated shelf-life study has demonstrated that capture molecules can be immobilized onto amino-binding or streptavidin surfaces. They can subsequently be detected after accelerated storage at 50° C for eight months. By effectively stabilizing the interaction between the amino-binding or streptavidin surface and the capture molecule, degradation is not an issue when stored at room temperature.
 

Table 1. Recommended stabilizers and their degree of effect when used with Nunc Immobilizer Amino and Nunc Immobilizer Streptavidin surfaces. -: No effect, +: either long-term stabilizing effect or effect on activity level, ++: both effect on activity level and long-term stabilizing effect.

For several capture molecules, introducing a stabilizer has a positive effect on long-term stability (see Table I). Where stabilization is necessary to maintain activity, stabilization can be achieved using the appropriate combination of capture molecule and immobilization surface. Data obtained in this study allows specific stabilizers to be recommended. For amino surfaces coated with C-peptide or hCG, LB medium or sucrose should be used. Amino plates coated with polyclonal rabbit anti-goat Ig should be stabilized with a coating stabilizer. For biotinylated surfaces coated with C-peptide, inconclusive results prevent specific recommendations to be made, and further investigation is required. With hCG and polyclonal rabbit anti-goat Ig, optimal stabilization can be achieved with coating stabilizer or sucrose.
In general terms, the data have shown that coated surfaces can be stored at room temperature for up to four years, while maintaining a stable level of activity. However, not all combinations of capture molecules and stabilization methods show a stable level of activity throughout the testing period. Retention of activity can be enhanced through the use of a stabilization buffer to maintain the integrity of tertiary protein structures on the surface of the plate. As a result, after coating with the optimal stabilization buffer, immobilizing plates are suitable for inclusion in diagnostic kits, with a shelf-life of up to four years at room temperature.

References
1. E Nilsson and A Larsson, “Stability of Chicken IgY Antibodies Freeze-Dried in the Presence of Lactose, Sucrose and Threalose,” The Journal of Poultry Science 44 (2007): 58-62.
2. MC Manning, K Patel, and RT Borchardt, “Stability of Protein Pharmaceuticals,” Pharmaceutical Research 6 (1989): 903-918.
3. JL Cleland, MF Powell, and Shire SJ, “The Development of Stable Protein Formulations: A Close Look at Protein Aggregation, Deamidation, and Oxidation,” Critical Reviews in Therapeutic Drug Carrier Systems 10 (1993): 307–377.
4. BS Chang and NL Fischer, “Development of an Efficient Single-Step Freeze-Drying Cycle for Protein Formulations,” Pharmaceutical Research 12 (1995): 831–837.
5. SJ Prestrelski, KA Pikal, and T Arakawa, “Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried-State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy,” Pharmaceutical Research 12 (1995): 1250–1259.
6. SD Allison, et al., “Hydrogen Bonding Between Sugar and Protein is Responsible for Inhibition of Dehydration-Induced Protein Unfolding,” Archives of Biochemistry and Biophysics 365 (1999): 289–298.
7. CC Hsu, et al., ”Surface Denaturation at Solid-Void Interface: A Possible Pathway by which Opalscent Particulates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures,” Pharmaceutical Research 12 (1995): 69–77.
8. MZ Zhang, et al., ”A New Strategy for Enhancing Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor,” Pharmaceutical Research 12 (1995): 1447–1452.
9. ASTM International Designation: F 1980-02, “Standard Guide for Accelerated Aging of Sterile Medical Device Packages.”
10. L Tiefenauer and D Bodmer, “Antibody Coating Using Various Avidin-Biotin Complexes Employed to an Enzyme Immunoassay for Estradiol,” Analytical and Bioanalytical Chemistry 330 (1988): 342.
11. L Valimaa, et al., “A High Capacity Streptavidin-Coated Microtitration Plate,” Bioconjugate Chemistry 14, no. 1 (2003):103-111.
 

Lena Brandt Larsen was formerly a senior research scientist at Thermo Fisher Scientific, Laboratory Specialty Products, with expertise in immunology.

Tina Kristensen Marwood, PhD, is a research manager at Thermo Fisher Scientific, Laboratory Specialiy Products. She can be reached at tina.marwood@thermofisher.com.

Thomas Anderson is a senior technical adviser. at Thermo Fisher Scientific, Laboratory Specialty Products, managing technical support for the business unit. He can be reached at thomas.andersen@thermofisher.com.

Companion diagnostics are emerging as a key part of personalized medicine. Particularly in oncology, patients are being better served by drugs for which patients are selected via in-vitro diagnostic tests.
The field is promising but nascent: so far, the potential of companion diagnostics is greater than the number of drug-and-diagnostic products that is actually commercially available.
To find out why this is the case, and to learn more about the future of companion diagnostics, IVD Technology editor Richard Park spoke with Walter Koch, PhD, vice president and head of global research for Roche Molecular Systems.

IVD Technology: How would you characterize the current state of companion diagnostics for personalized medicine?
Walter Koch: A metaphor comes to my mind: cherry trees at the beginning of the spring season. I think the field is blossoming. We’re seeing the first flowers on the tree, but there are a lot of buds waiting to explode.
That is how I envision companion diagnostics right now. We have a few examples already in the marketplace. Just recently, Zelboraf and the cobas 4800 BRAF test is the first time in at least ten years that the FDA has approved a diagnostic together with a therapeutic. The field is nascent but coming on strong.

You sound optimistic. Are there other examples of products or companies that give you that impression of blossoming within the field of companion diagnostics?
Yes, specifically in the field of oncology, but not limited to that area. The state of the science has reached a point where specific aberrations in cancer cells can be targeted at a level of a therapeutic. But you need to know that that aberration is present to target it, and that’s where this model is playing out quite a bit. In fact, within a couple of weeks of our approval, the anaplastic lymphoma kinase, or ALK, inhibitor Xalkori was approved, and it also requires a diagnostic to identify the three to four percent of lung-cancer patients whose cancer is driven by an ALK fusion gene. Just within a few weeks of each other, there are two examples where this type of approach is really coming to fruition.

What are the current challenges that remain when IVD manufacturers develop companion diagnostics for personalized medicine?
The biggest challenge is the somewhat disparate development processes and timelines for diagnostics and therapeutics. In the BRAF mutation test/Zelboraf example, starting back in 2005 or 2006, we planned in preclinical stages of the therapeutic development and developed a prototype assay. We got that assay ready for Phase 1 and 1B studies so that we could obtain an investigational device exemption.
In the case of Zelboraf, there was a very strong indication that the presence of the BRAF mutation was associated with response of melanoma patients, something you rarely see with a 30-patient safety study. Importantly, it also gives the diagnostic manufacturer time to lock down the assay, the instrument platform, and the software in time for the pivotal trials, the Phase 2 and then the Phase 3 efficacy trials.
Thanks to a great collaboration, in the case of Zelboraf and our test, we managed to have both the therapeutic and the drug approved on the same day.
On the other hand, a less desirable scenario is that thedrug enters the market without any selection of patients. An example of that is the KRAS mutation test for colorectal cancer patients, where for several years now we have known that colorectal cancer patients with KRAS mutations do not respond to monoclonal antibodies to EGFR.It’s the standard of care , but there is no FDA-approved test because of the conundrum of trying to register a test that had not been used in the clinical trial. So the earlier we are engaged, the better.

How can IVD manufacturers overcome challenges such as these when developing companion diagnostics for personalized medicine?
Early engagement and involvement in the co-development project planning. In the Roche Zelboraf example, we have actually had a diagnostics team member embedded within the pharma development team. That is the ideal situation.

What sort of challenges are involved in overcoming differences in culture between the diagnostics and pharmaceutical businesses? Is culture a factor as the two groups work on a major project like this?
The first time a diagnostics group tells a pharma team what is involved in developing a companion diagnostic, jaws do drop. They are surprised at the level of rigor that is required from the FDA.
So when you tell pharma colleagues the timelines, the multiple lots that have to be made in the manufacturing scenario, that have to then be tested in multiple sites by multiple users on multiple days to validate performance, they are a bit surprised.
It is an education process as much as it is a cultural difference. I believe we understand much better now, because of our several years of experience.

What companion diagnostics products does Roche currently have on the market?
The cobas 4800 BRAF mutation test was approved in the United States and also has CE mark approval. The cobas KRAS mutation test also has CE mark approval. Ventana has the newly approved Inform HER2 Dual ISH assay to select breast cancer patients who may be eligible for treatment with Herceptin.
Ventana also has estrogen receptor and progesterone receptor immunohistochemistry tests that are used for breast cancer patients to determine if they will receive hormonal therapy.

What companion diagnostics development projects is Roche currently involved in and what is the status of those projects?
I would say across the entire Roche organization there are well over 100 codevelopment and personalized healthcare projects across all therapeutic areas.
Every drug development team considers a biomarker strategy. If the hypothesis is sound, and there are early preclinical data that suggest a certain level of evidence that it should go forward, we work on a potential companion diagnostic.
I don’t personally believe that every drug is going to have a companion diagnostic, but it is at least considered in every program.  

Among the many projects that Roche is involved in, what clinical area or disease state is most represented?
I believe oncology is clearly the area where we have the greatest presence and where there’s also the greatest amount of activity. Oncology is where we see the most intense activity, but I certainly know of several in virology as well, and in the neurology area.

Is developing a companion diagnostic for a particular drug something that is always taken into consideration and discussed at the beginning of the drug-development process?
Absolutely. Regardless of how early it is, once a drug development team has a molecule that looks like it might have the right kind of properties to hit a target based on cell culture data, in vitro systems, and so on, the team starts to contemplate whether there would be a biomarker for that. That is probably the best way to deliver effective medicines.
No drug works for everyone. If you’ve got a headache, there are some people that respond better to aspirin, while some respond better to acetaminophen and others to ibuprofen. We don’t always know why, but it clearly reflects differences in humans that manifest in many other ways as well. There are drugs that don’t provide much benefit for a very significant number of people, and if you could avoid treating them with something that doesn’t work, not only are you sparing them unsuccessfully treating their malady, but also potential side effects as well.
It makes sense from every perspective that you would want to try to develop a companion diagnostic test if a good predictive biomarker exists.

Roche’s COO, Daniel O’Day, was recently quoted as saying, “Efforts by government to curb healthcare spending will be beneficial to growth in companion diagnostics for personalized medicine.” Do you agree?
I agree. It is a logical extension that with the aging demographics of the planet, and every healthcare system in the world under some duress to deliver quality healthcare with a finite amount of resources, anything you can do to improve the efficacy of how you deliver healthcare is a good thing. You can quantify it in a variety of different ways.
If we can make new therapeutic agents more efficacious and focus more on the patients who will benefit versus those who will not, by using companion diagnostics, that is simply a dollars-and-cents equation.
Diagnostics can increasingly improve the value of the therapeutics not only to the individual patients, which is the first thing we care about, but also to the healthcare systems and the payers.
It is clear to see that there are many opportunities for diagnostics to contribute to improving outcomes with medicines and better healthcare delivery. And we certainly plan to be a part of that, with oncology leading the way.

FDA recently released its draft guidance on companion diagnostics. Do you think the document adequately addresses the various regulatory issues and challenges IVD manufacturers encounter when developing companion diagnostics for personalized medicine?
It is good that the agency is thinking about this and that they’re soliciting feedback from industry and professional organizations.

What is currently the level of clinical use of companion diagnostics by doctors, physicians, and other clinicians?
There is a lot going on, some of which is actually standard of care, mainly for oncology. For breast cancer patients there are three biomarkers that are going to be evaluated: ER, PR, and HER2, which covers about seventy-five percent of breast cancer patients who, based on test results, are now going to be candidates to receive a targeted therapy-either an aromatase inhibitor, or tamoxifen, or Herceptin.
In colorectal cancer, a patient with metastatic disease who is a candidate for the EGFR monoclonal antibody therapies will get a KRAS test today, despite the fact that there’s not an FDA-approved test. In the United States, Europe, and throughout the world, this is the standard of care. For melanoma, patients will be tested for the presence of the BRAF V600 mutation.

So from what you understand, have physicians, doctors, and clinicians accepted the usage of companion diagnostics as part of the standard of care, without much resistance?
When a companion diagnostic has been validated in a rigorous clinical trial, there appears to be rapid uptake.

As long as it’s validated, they essentially are on board.
Yes. And particularly if it ends up in the professional guidelines, clinicians will do it, because if you don’t, you’re really not practicing modern medicine. Those professional guidelines are a critical element.

What has been the reception to and usage companion diagnostics outside the United States?
I don’t think there is any fundamental difference. There are differences in regulatory paths for getting into the market. Zelboraf is not yet approved in Europe, but it is approved in the United States.
Interestingly, it is a bit easier for us to get diagnostics approved in Europe. The CE-mark process, with some exceptions, is a self-certification process. But that is the only fundamental difference. Drug and diagnostic development companies are global.

What are the future prospects for companion diagnostics?
I think they are really, really good. I believe we’ll see many more examples in the coming years where diagnostic tests are developed together clinically in the same clinical trial with the therapeutics, because it enables the personalized medicine approach and can specifically improve the outcomes for patients over the one-size-fits-all approach.
The continued focus on companion diagnostics and personalized medicine is good for the industry. It’s good for patients. It’s good for healthcare systems and payers.

What future challenges will IVD manufacturers encounter when developing companion diagnostics for personalized medicine?
From where I sit in Research, science and technology are moving at such an extremely rapid pace. And with ever-decreasing costs, there are daily announcements of new biologically relevant biomarker discoveries. In academic centers that are on the cutting edge, we’re seeing some of those discoveries converted into laboratory-developed tests very rapidly, with the results used long before they’ve gone through the more typical rigor of a randomized clinical trial that would be required for an FDA approval.
Once the FDA has approved a new therapeutic with a specific clinically validated companion IVD, there may already be several laboratory-developed tests on the market, and the clinical laboratories will market their tests for use with that therapeutic, even though that use is not supported by clinical data, nor has it been subject to the FDA’s rigorous review of safety and effectiveness.  
We believe there needs to be a more consistent, risk-based regulation of diagnostic tests regardless of the originator, and that without it, patient safety may suffer.

Do you have any final comments on the state of personalized medicine and companion diagnostics?
It is exciting and exhilarating for those of us who are able to participate in this new way of developing medicines. And I am convinced we’re making a fundamental difference for patients.
We’ve had patients come to our site and tell us what a difference our tests made for them and how their disease was managed. People who had been diagnosed with malignant melanoma and, frankly, didn’t have any treatment options left, have a new lease on life with Zelboraf following BRAF testing-based eligibility.
So it’s very gratifying to see that we can help patients. It is very exciting to be a part of this.

Walter H. Koch, PhD is vice president and head of global research for Roche Molecular Systems. He has held this position since 2005. As a member of the Executive Leadership Team, he sits on the Life Cycle and Business Development committees and chairs the Research Portfolio Committee. He can be reached via Jacqueline Wallach at jacqueline.wallach@roche.com.

Back in late July of this year, the Institute of Medicine (IOM) recommended that FDA replace the 510(k) medical-device clearance process with something brand new. (Under current regulation, to obtain 510(k) clearance, a manufacturer must demonstrate that the device is substantially equivalent to a device that was in legal commercial distribution in the United States before May 28, 1976, or to a device that has been determined by FDA to be substantially equivalent.) The committee stated that FDA’s resources would be better used in the development of a new framework using both premarket clearance and improved postmarket surveillance of device performance to provide reasonable assurance of the safety and effectiveness of Class II devices throughout the duration of their use. “The 510(k) process lacks the legal basis to be a reliable premarket screen of the safety and effectiveness of moderate-risk Class II devices and cannot be transformed into one,” the IOM committee concluded in its report.

510(k): Imperfect, but worth defending? Since its release, IOM’s report, titled “Medical Devices and the Public’s Health: The FDA 510(k) Clearance Process at 35 Years,” has garnered much attention and discussion among industry and those who monitor it, and the recommendation to abandon 510(k) has been met with some derision.
In a letter sent to FDA’s Division of Dockets Management on September 30, the Advanced Medical Technology Association (AdvaMed), wrote that while it commends the IOM committee’s efforts and believes that the report “contains a number of valuable observations and recommendations,” including instituting an internal quality-assurance program in CDRH, AdvaMed nevertheless “strongly disagrees with [IOM’s] central recommendation to abandon the current 510(k) program and replace it at some unknown date with an untried, unproven, and unspecified new legal structure.”
In an interview with IVD Technology in October, AdvaMed’s executive vice president of technology and regulatory affairs, Janet Trunzo, echoed that statement: “We were surprised by that recommendation. The IOM opened up some opportunities to submit any kind of information [related to 510(k)]; they were very willing to accept any kind of commentary and information. They made an information-gathering effort. We were just surprised that the key recommendation after all that work was that we should scrap the 510(k) program and that FDA should not expend any more of its resources on making improvements to it.” She said she could not speculate on how the IOM arrived at that recommendation.
Glen Freiberg, RAC, a longtime regulatory, clinical, and quality consultant for the medical device and diagnostic industry, believes that the current 510(k) process could “work quite well to meet everyone’s objectives” with improved management. As an example, he points to FDA’s recent draft guidance for infusion pumps. “Infusion pumps traditionally went through the 510(k) system with bench testing and without clinical trials,” he says. “If you take a look at the recall database, you will be amazed to learn how many pump vendors have had recalls, for a variety of reasons.” He says that while the new draft guidance for infusion pumps “is not perfect,” it does show how FDA can flexibly address safety and effectiveness under the current 510(k) paradigm. In short, this draft guidance is evidence in support of keeping and working with 510(k).

Disagreement on how to keep unsafe products off the market. Reliance on substantial equivalence to already-cleared products or products on the market prior to 1975 cannot assure that devices reaching the market today are safe and effective, the IOM committee concluded. The majority of the devices used as the basis for comparison were never reviewed for safety or effectiveness, according to IOM. This does not mean that they or the devices that followed them are unsafe, IOM adds, and the continued use of many of these products in clinical practice provides a level of confidence in their safety and effectiveness. But 510(k) clearance does not determine a device to be safe or effective, the report asserts.
Freiberg responds, “To date, stakeholders have been looking in the wrong direction. Specifically, everyone is looking at the regulations and intent of 510(k) without having conducted an effective system management assessment.” He contends that most medical device safety and effectiveness problems that come up result not from issues within 510(k) itself but from manufacturing and quality problems at individual production sites. “A more appropriate approach,” he suggests, would be for CDRH to recognize that the issues raised in recalls should be compliance-oriented rather than clearance-oriented. “That’s not to say there is no role for clinical data in traditional device submissions,” he adds, “but the authority and resources for this aspect of the historical device problems should be delegated to the field-inspection system via improved compliance-policy guides to the investigators.”
Freiberg summarizes this way: “FDA should be defending the 510(k) substantial equivalence notification procedure for what it is-demonstration of equivalence-while concurrently boosting enforcement of quality systems via inspection and enforcement rather than via the review process.”