Diagnostics & Imaging Week Contributing Editor

ST. LOUIS — The Oak Ridge Conference, organized here by the American Association for Clinical Chemistry (AACC, Washington) and now in its 39th year, provides a forum for presentation of the latest developments in clinical diagnostics and assessment of their impact on the market. A key focus of this year’s meeting was point-of-care testing and assay technologies for use in “low-resource” settings — formerly known as third-world countries.

POC testing now comprises about one-third of the clinical diagnostics market worldwide, with sales reaching about $11 billion in 2006 and continuing to grow more rapidly than the overall clinical diagnostics market.

Ove Ohman, PhD, of Amic (Uppsala, Sweden) described recent developments with the company’s Forecast technology, a chip-based microfluidic platform using the 4castchip, an injection-molded plastic device consisting of an array of micropillars which drive the flow of sample and reagent in a controllable manner. The underlying technology is based on fabrication processes and materials used in digital video disks.

The device can be used to perform assays on whole blood samples, and, mainly due to its highly controlled flow properties, provides precise results when used in POC settings. A proprietary coating technology is employed to reduce non-specific binding and facilitate attachment of antibodies to the surface.

Miniaturized, the device contains a number of active elements integrated into the design, such as a blood separation filter, a flow channel, a wicking zone and a unique detection zone using on-chip diffraction grating to more effectively couple light into the device and distribute emitted light to the detector.

POC assays have been developed for cardiac Troponin I (with a sensitivity of 0.03 pg/mL), TSH, CRP and IgE. The precision of the technology is exemplified by the CRP assay, for which the coefficient of variation is quoted at 6%.

The Forecast technology also has applications in the clinical lab, where microfluidics reduces the need for pumps and fluid handling devices.

Another platform employing microfluidics technology for use in POC testing — and in particular for infectious disease diagnostics in low resource settings — was described by William Rodriguez, MD, of Massachusetts General Hospital (Boston).

There is growing demand for diagnostic products for HIV/AIDS, malaria, and tuberculosis in low resource settings, driven in part by organizations such as the Clinton Foundation, which are funding disease-fighting efforts. Fewer than 10% of the 40.4 million people living with HIV infection worldwide have undergone an HIV test, and only 1% of these have access to the tests needed to manage their treatment.

The problem extends beyond public health to impact the economies of such countries, since in some cases projections indicate that up to 25% of the agricultural work force will be lost due to deaths from AIDS. This in turn has created a small but growing demand for easy-to-use, low-cost diagnostics.

Rodriguez described microfluidic devices developed by his group that incorporate on-chip immuno-affinity chromatography for detection and can accurately perform CD4 cell counts using a 3 microliter whole blood sample in under two minutes, without the use of additional reagents, labels, or sample preparation.

A similar approach is being used to develop microfluidic platforms for HIV viral load and tuberculosis testing.

Lumora (Cambridge, UK) is developing a low-cost platform for quantitative molecular diagnostics called Bioluminescent Assay in Real-Time (BART), with POC applications.

BART simplifies nucleic acid testing by using isothermal amplification, pyrophosphate chemistry and luminescence detection, combined with simple hardware, including a constant-temperature block and an optical detector. The method is capable of single-copy detection and is configured in a closed-tube format appropriate for non-laboratory settings. Applications under development include detection of Chlamydia in urine and sepsis detection.

BART also has been developed in a configuration suitable for the central lab, because offering low-cost, simple operation, rapid turnaround and quantitative real-time readout.

Gonzalo Domingo, PhD of PATH (Seattle) discussed another microfluidic platform with applications in diagnostics for low-resource settings. PATH is developing three microfluidic disposable-based diagnostic platforms, including a PCR-based device for detection of bacterial pathogens in diarrhea; a device combining nucleic acid and immunoassay technology for detection of viruses and bacteria associated with measles, dengue, influenza, malaria, typhoid and Rickettsia; and a PCR-based platform for detection of sexually transmitted diseases and vaginal infections including Trichomonas vaginalis, Chlamydia, Neisseria gonorrheoeae, and bacterial markers of vaginosis.

The company’s Lab-on-a-Card device and portable reader, the DxBox, is a POC system that can perform an assay in 30 minutes with performance equivalent to central lab systems.

Other new technologies for use in POC and low resource settings were described by speakers from Wave 80 Biosciences (San Francisco, CA) and Cleveland Biosensors (Brisbane, Australia).

As discussed by Daniel Laser, PhD, Wave 80 is developing a new microfabricated device platform with initial focus on malaria diagnosis, initially being developed for use by the U.S. military, and consequently has a rather high target cost of $10 per test. However, Laser believes that the cost could be reduced 10-fold for high-volume applications in clinical diagnostics.

The device uses an electro-osmotic pump based on technology employed in semiconductor devices, a microarray-based reaction zone with immobilized antibodies specific for malarial pathogens (P. falciparum and P. vivax), and immunogold/immunofluorescence detection.

Test time with a prototype device is about three minutes using a 20uL fingerstick whole blood sample. A key advantage of the Wave 80 technology is the use of the high-pressure electro-osmotic pump, voiding the need to rely on capillary flow dynamics as in most other types of POC microfluidic devices.

Cedric Robillot, PhD, of Cleveland Biosensors, described the Biofiniti system, a handheld immunosensing device based on self-contained microfluidics, ferromagnetic actuation, magnetic capture, and electrochemical detection. The device uses a moving magnetic field coupled to a ferrofluid to pump sample and reagents through a network of microfluidic channels formed in an injection molded cartridge.

Assay sensitivity is enhanced via the use of liposome particles as labels. The liposomes contain an electroactive label that is released during the detection step by lysis, providing a high degree of signal amplification. The first assay to be developed is a test for microcystins, toxic peptides produced by cyanobacteria that can contaminate public water supplies.

The Cleveland Biosensors’ assay has a sensitivity of 0.1 nM, producing a result in 30 minutes compared to three hours typically required for existing methods. Another application under development is a rapid (10 minute) assay for the heart failure marker BNP, offering a sensitivity of 100 pg/mL, the typical threshold for BNP assays now on the market.

Cleveland Biosensors initially is targeting the food and water testing market, but the technology has obvious applications in clinical diagnostics. The company is collaborating with Biosite (San Diego) in its development program.