BB&T Contributing Editor

SAN JOSE, California — Diagnostics, including laboratory-based and point-of-care (POC) in vitro diagnostics as well as diagnostic imaging, is a dynamic segment of the medical device market, driven both by new developments in genomics and personalized medicine and also by advances in analytical technologies including miniaturization and microfluidics. Although new developments have been slow to become commercialized for clinical use, there is a large emerging base of technologies that will provide the platforms for next-generation diagnostic devices and imaging modalities.

The Oak Ridge Conference — organiz-ed by the American Association for Clinical Chemistry (AACC, Washington), and now in its 40th year — provides an important opportunity for presentation of emerging diagnostic technologies, including a recent expansion in scope to include advances in diagnostic imaging in response to an evolving integration of diagnostic modalities.

The newest developments in genetic testing and disease risk assessment were de-scribed at the Oak Ridge Conference, in-cluding approaches based on proteomics that provide an added dimension for disease characterization with increased clinical relevance.

But perhaps one of the most vibrant areas of this year's conference was the focus on new point-of-care (POC) tests, because this is where the diagnostics sector is seeing the most rapid expansion and the most obvious potential for new profits. This is especially true since POC testing addresses so many segments of the global market, including developing countries that can increase spending on lower-cost systems.

POC in new generations

As shown in Table 1 below, the market for POC IVD testing products is estimated at $12.6 billion for 2007 and includes products for hospital-based testing, physician's office testing and home/self-testing. The global market has expanded at 10%-11% a year over the past two years as measured in U.S. dollars, although at a somewhat lower rate on a currency-adjusted basis.

Leading suppliers of POC testing products — primarily the top manufacturers of whole blood glucose testing products, due to the large size of that segment — include Roche Diagnostics (Indianapolis), Lifescan/J&J (Warren, New Jersey), the Abbott Diabetes Care unit of Abbott Laboratories (Abbott Park, Illinois), and Bayer Diabetes Care, a unit of Bayer AG (Leverkusen, Germany).

Another key player in the POC testing market is Inverness Medical Innovations (Waltham, Massachusetts), which has acquired numerous suppliers of POC testing products over the past several months to become the dominant supplier in the POC segment, excluding whole blood glucose, with 2007 pro-forma sales of $1.1 billion.

New developments in the POC segment, however, are often coming not from market leaders but from emerging companies in the sector.

A new POC testing system targeted initially at POC infectious disease testing in developing countries was described by Paul Yager, PhD, of the Department of Bioengineering at the University of Washington (Seattle) at the conference.

Yager's team is developing a system called the DxBox, a compact microfluidics-based analyzer being developed in partnership with Micronics (Redmond, Washington). Micronics is a leading company in the development of microfluidics technology for near patient diagnostic testing, and it has created a large-scale lamination technology for fabrication of microfluidic devices.

An initial application for the DxBox analyzer employs surface plasmon resonance imaging for detection of drugs of abuse in saliva, targeted for use by law enforcement agencies.

Another application is a POC testing device for detection of multiple pathogens, under development in partnership with the Bill & Melinda Gates Foundation (gatesfoundation.org). The system will be configured as a compact battery-powered device that will employ both immunoassay and nucleic acid testing technologies, enabling infections to be detected at an early stage before an immune response occurs as well as at later stages of infection (e.g., 4 to 5 days post-exposure).

The projected turnaround time for immunoassays is less than 10 minutes, while nucleic acid assays require about 22 minutes, including a PCR step, and cost is projected to be low enough to allow purchase by developing countries. The system is targeted for market introduction in 2010.

POC measurement of blood glucose

A new system for POC measurement of blood glucose, an application that accounts for about two-thirds of the POC testing products market, was described at the conference by Alastair Hodges, PhD, of Universal Biosensors (Victoria, Australia). The system employs a thin-layer cell design with opposing electrodes and tri-pulse electrochemical detection, enabling a test time of about five seconds using a 0.4 L blood sample.

A key advantage of the system is the ability of the sensor to eliminate interference from a variety of sources including hematocrit, temperature, or electrochemically active components such as anti-oxidants that are common sources of error for existing blood glucose sensors. Studies using whole blood have shown no bias due to hematocrit or temperature over a wide range of glucose levels (15-600 mg/dL).

A new development in non-invasive measurement of blood glucose was reported by Yasuhiro Yamakoshi of yu.sys Corporation (Kanazawa, Japan) and Chiba University (Chiba, Japan). The yu.sys technology is based on the method of Pulse Glucometry, which involves interrogation of tissue with near infrared radiation and collection of a large number of transmittance spectra.

The technique analyzes the cardiac-related pulsatile changes in transmitted radiation through the fingertip, sampling at a rate of 100 spectra per second, to extract a signal related to the level of arterial blood constituents including blood glucose. A key challenge for such non-invasive measurement methods is development of calibration techniques that ensure accuracy over a wide range of patient types and measurement conditions.

Yu.sys has systematically studied a number of multivariate calibration methods, and has found two, Artificial Neural Networks and Support Vector Machines Regression (SVMR), that provide the most accurate results.

Studies in 27 healthy volunteers have shown that Pulse Glucometry combined with SVMR calibration provides accurate glucose measurements (92% of measurements in region A and 8% in region B of the Clarke Error Grid). However, a considerable amount of additional evaluation in patients as well as development of a small, portable device suitable for patient use remains to be completed before the technology is ready for clinical use.

A non-contact POC method for dynamic evaluation of thrombosis was described by Francesco Viola and others from Hemosonics (Charlottesville, Virginia). The method is based on sonorheometry, which uses acoustic radiation force to produce small, localized displacements within the blood, and monitoring of returned echoes to determine the viscoelastic properties of the blood.

Studies with human blood samples have shown the method to be accurate, with a precision of about 5% achieved with fresh blood, and significant (25%) increases in clotting time have been observed in samples from patients with deep venous thrombosis while a large decrease (46%) is observed for hemophiliac patients.

Targeting toxins, infectious agents

A new system for high-sensitivity detection of toxins and infectious agents, the Compact Bead Array Sensor System (cBASS), is under development by researchers at the U.S. Naval Research Laboratory (Washington).

As discussed by Shawn Malvaney at the conference, the cBASS technology employs Fluidic Force Discrimination (FFD) which relies on both lateral and torque forces applied to micron-size beads captured on an avidin-coated silicon nitride surface when subjected to laminar fluid flow. FFD allows highly efficient removal of unbound beads and non-specifically bound material from the substrate, leaving only the specifically-bound beads on the surface which bind the target analyte, creating a sandwich-assay format. The beads can then be counted either by magnetic sensing or optical imaging to achieve detection sensitivity in the attomolar range.

A further enhancement of the technology which produces a 1,000-fold sensitivity increase involves a semi-homogeneous version of the assay in which analyte-specific beads are pre-mixed with the sample, providing efficient target capture in solution, followed by capture and separation on the sensor substrate via FFD.

Applications demonstrated so far for the cBASS system include detection of staphylococcal enterotoxin B at a level of 50 femtograms per ml in clinical samples, and high-sensitivity detection of ricin toxoid in serum and whole blood.

The technology can also be applied to detection of nucleic acids, as exemplified by multiplexed DNA hybridization assays for genomic bacterial DNA without the need for PCR amplification. Sample types that have been analyzed successfully using the cBASS system include serum, plasma, whole blood (at a 10-fold dilution), saliva, urine and feces extracts.

A 30-minute assay for detection of fecal mitochrondial DNA in wastewater has been demonstrated. Sensitivity can be traded off for assay turnaround time to achieve attomolar sensitivity in as little as 10 minutes.

Royal Philips Electronics (Eindhoven, The Netherlands) also is developing a portable biosensor-based system for POC testing applications, including roadside drugs-of-abuse testing and protein analysis. The Philips system uses magnetic nanoparticles as labels to achieve low background along with the ability to concentrate and wash in a miniaturized assay format.

Using magnetic sensing

Rather than employing fluid forces for separation as used in the cBASS system, the Philips analyzer uses magnetic forces to remove unbound particles, while the nanoparticles that bind specifically to the analyte are concentrated on the sensor surface. Detection is achieved via current wires embedded in the sensor substrate along with Giant Magnetic Resistance Elements (GFRs) that enable sensing of the particles bound to the surface.

Philips researchers are collaborating with Future Diagnostics (Wijchen, The Netherlands) to develop a POC assay for parathyroid hormone, and with Cozart Biosciences (Oxfordshire, UK) for development of a morphine assay. The Future Diagnostics assay measures PTH at clinically relevant levels with a turnaround time of 12 minutes using a 10 L sample.

Philips also has developed a model assay for cardiac Troponin I in buffer with a turnaround time of about four minutes, though when translated to a whole blood test the performance may differ. The Philips magnetic nanoparticle sensor technology can also be used for rapid (three-minute) high-sensitivity detection of DNA amplicons produced in a PCR assay.

Magnetic sensing is also being used in a new system under development by T2 Biosystems (Cambridge, Massachusetts).

As discussed at the conference by Lee Josephson of the Center for Molecular Imaging Research of Massachusetts General Hospital (Boston) and a co-founder of T2, The T2 system uses magnetic nanoparticles as labels in a variety of assay formats that generally employ aggregation to detect an analyte.

The T2 relaxation rate, also known as the spin-spin relaxation rate, for a sample containing magnetic nanoparticles depends on the state of particle aggregation, enabling a sensitive, non-contact approach for detecting aggregation in response to binding of antibody-labeled particles to a target analyte, or for detection of other types of agglutination reactions. In effect, the magnetic nanoparticles act as magnetic relaxation switches (MRSw).

The MRSw method is insensitive to the optical characteristics of a sample, making it useful in clinical diagnostics where specimen quality can be an issue. It also can be used to measure a diverse range of analytes including DNA, RNA, proteins, viruses, enzymes and small molecules, such as glucose. In a model system, detection sensitivity using multiple enhancement techniques is 0.14 picomolar.

Josephson also described an in vivo glucose sensor employing MRSw technology which consists of a reservoir containing glucose-labeled magnetic particles and a glucose-binding protein (Concalvin A) covered with a semi-permeable membrane.

Glucose from the external medium enters the reservoir through the membrane and competes with glucose attached to the nanoparticles for binding to ConA, changing the state of aggregation and producing a T2 change in proportion to the external glucose concentration.

The concept can be expanded to measurement of a number of different analytes, including monitoring of levels of the cancer market beta-hCG as demonstrated in a model sensor developed by Michael Cima at the Massachusetts Institute of Technology (Boston).

Nanogen in POC sector

Nanogen (San Diego, California) is another company with a position in POC diagnostics, having acquired or sourced a portfolio of POC cardiac marker and drugs-of-abuse testing products from Spectral Diagnostics (Toronto, Ontario) and LifeSign (Somerset, New Jersey).

The company currently is developing a third-generation POC testing platform that employs fluorescence detection and lateral flow assay technology in partnership with the U.S. Centers for Disease Control and Prevention (Atlanta) and HX Diagnostics (Emeryville, California).

The Nanogen system consists of a portable reader and a single-use test device and uses synthetic nucleotide polymers (pRNA) as high-affinity binding agents along with fluorescent microbead labels for detection. A serum assay for NT-proBNP has been developed that can produce results in 15 minutes with a 1 pg/mL quantitation limit.

Additionally, a multi-analyte influenza assay (Type A, Type B, H1/H3 and H5) is about to enter clinical trials that has a 100-fold higher sensitivity than existing lateral flow assays from suppliers such as Quidel (San Diego) and BD (Franklin Lakes, New Jersey), and produces results in under 30 minutes.

Another POC testing system, designed for use in pathogen detection in developing countries, was described at the conference by Bernhard Weigl, PhD of PATH (Seattle, Washington). The ICS Strip Test is a new non-instrumented assay platform that combines the sensitivity of nucleic acid amplification with the simplicity of a visually read test strip.

A disposable handheld device is used which is manually manipulated to accomplish the various assay steps, including actuation of an exothermic heating pad along with phase change materials to perform cell lysis and isothermal autocirculation-based amplification. An initial application under development is a loop-mediated amplification (LAMP) assay for Plasmodium falciparum, the causative agent of malaria.

PATH, a non-profit research organization, reports that it is searching for partners to assist in commercialization of the assay for low-resource settings.