BB&T Contributing Editor

BALTIMORE – The Oak Ridge Conference, an annual event organized by the American Association for Clinical Chemistry (AACC, Washington), is the leading venue in the clinical diagnostics industry for presentation of emerging technologies which will shape diagnostic testing in the future. This year's conference, held here in April, highlighted developments in the two most rapidly growing segments of the market, point-of-care (POC) testing and molecular diagnostics.

Those two segments combined now account for about 45% of the global in vitro diagnostics market, and are expected to comprise an even larger percentage of the total in the future. New POC testing technologies for use in cardiovascular diagnosis; cancer diagnosis and therapy monitoring; and infectious disease testing were described at the conference that promise improved performance compared to existing methods, while advances in DNA sequencing technology are making low cost, rapid molecular analysis an increasingly viable prospect, including the advent of the $1,000 and perhaps the $100 genome.

Diagnostic imaging technology also was highlighted at the conference, with a focus on molecular imaging and its relation to laboratory diagnostics through the use of biomarkers.

Microfluidics and lab-on-a-chip technologies form the basis for many of the new testing platforms now in development. Those technologies enable advanced tests to be implemented at the point of care, allowing tasks such as sample preparation which add complexity to laboratory-based testing to be carried out on-chip without user intervention.

In addition, laboratory-based tests that may not be economically feasible if implemented with conventional technologies may become viable in lab-on-a-chip formats. For example, newborn screening test panels are becoming increasingly complex, with the number of individual tests in recommended screening panels numbering in the dozens and additional markers being added.

David Millington, PhD, of Duke University (Durham, North Carolina) described newborn screening tests for Lysosomal Storage Diseases (LSDs) ,which have become more important due to the increased use of LSD therapies such as bone marrow transplants and enzyme replacement therapy.

Millington is collaborating with Advanced Liquid Logic (Morrisville, North Carolina) to develop microfluid chip assays which enable multiple LSD markers to be analyzed at low cost and, perhaps more importantly, using very small sample volumes. Millington said that such testing might not be feasible in the public health laboratory environment of today without employing lab-on-a-chip technology.

POC next generation

Point-of-care testing technologies occupied a majority of the agenda for the Oak Ridge Conference. POC testing products comprise a significant portion of the total IVD market. As shown in Table 1, the market is estimated at $13.3 billion worldwide in 2008, with about two-thirds of the total attributable to whole blood glucose self-testing products. Growth versus 2007 was 8.7%.

The POC testing products market consists of multiple segments spanning bedside testing in the hospital, physician's office testing, rapid testing in the outpatient clinic and emergency department, testing in a variety of non-hospital institutional settings such as skilled nursing facilities, and self-testing. Whole blood glucose self-testing remains the largest segment of the market by far, but other segments such as POC coagulation monitoring are growing rapidly.

A new generation of POC testing products is under development based on microfluidics and chip-based assays that could drive additional growth in the market by expanding the number of sites performing tests as well as the types of tests performed. Many of the new platforms address testing in low-resource settings, an application that has attracted funding from sources such as Program for Appropriate Technology in Health (PATH, Seattle, Washington).

An example of a new POC testing system under development by researchers from the PATH group in partnership with TwistDX Ltd. (Cambridgeshire, UK) was described at the conference by David Boyle of PATH. The system is designed to perform molecular diagnostic assays such as infectious disease tests at the point of care and in field settings.

The technology employed in the TwistDX system, Recombinase Polymerase Amplification or RPA, is an isothermal nucleic acid amplification technology that can provide results in as little as 15 minutes using a kinetic assay format. The system employs lyophilized reagents which can be stored at room temperature, an important feature for diagnostic systems designed for use in low resource settings, and requires only the addition of sample and buffer to perform a test. A portable, battery-operated analyzer has been developed that uses individual test strips to perform each test.

The first test to be developed is an HIV-1 proviral DNA assay which gives results in less than 20 minutes and is particularly well-suited for testing of infants. The existing version of the test has a sensitivity of 38 copies, but the RPA technology is capable of single-copy detection for DNA targets. The company anticipates that a completely disposable version of the technology will be developed in the near future, making it well-suited for field use.

Another research team from PATH led by Bernhard Weigl is developing a non-instrumented cartridge-based test system for POC infectious disease screening based on loop-mediated isothermal amplification (LAMP) technology. The initial application under development is a malaria assay with single-genome sensitivity and a one-hour maximum turnaround time. A unique feature is use of an exothermal chemical reaction to generate the heat required to bring the cartridge to the temperature needed for amplification, avoiding the need for electrical heating and the associated instrumentation.

Another rapid molecular diagnostic system which is targeted at POC testing applications is being developed by Lynntech (College Station, Texas). The Lynntech analyzer employs real-time polymerase chain reaction (PCR) technology, and uses a novel closed-loop convective thermal cycler that is not only compact but which can also amplify targets much more rapidly than conventional thermocyclers. The convective cycler does not require heating and cooling of individual wells, but instead relies upon flow patterns produced by thermal gradients to move reactants between constant temperature zones. The theoretical cycle time is 42 seconds, indicating that a typical 30-cycle amplification could be performed in less than 30 minutes, although at present 40 minutes are required for a 15-cycle amplification.

Lynntech also is developing a sample preparation module employing magnetic particle separation to enable automation of the entire testing process. Applications under development include rapid detection of gram-negative and gram-positive bacteria in platelets and detection of influenza A and other viral targets in less than one hour.

Another rapid molecular diagnostics analyzer for use in POC settings was described at Oak Ridge by Robert Jenison of Great Basin Scientific (Salt Lake City). The company is developing a rapid, low-cost molecular diagnostics system which employs a compact cartridge, the PORTRAIT cartridge, which integrates all steps of the assay in a single device. Read-out of the test result is performed with a compact benchtop analyzer employing an imaging device based on cell-phone camera technology.

The system is targeted at applications in infectious disease detection in low-resource settings, physician offices, and small hospitals and clinics. Assays are being developed for methicillin-resistant staphylococcus aureus (MRSA), Group B streptococcus, and human papilloma virus. Reagent cost is $0.01 per test.

The technology uses dextran polymer-based enzymatic signal amplification, rather than target amplification as is employed in PCR analysis, which results in a simple, rapid, and low-cost assay format. Typical turnaround time for a test is 27 minutes, including a five-minute extraction step. The PORTRAIT Dx system has exhibited high sensitivity in clinical evaluations, exceeding that of conventional pcr assays for MRSA detection.

A chip format also is in development that can provide up to 100-fold higher sensitivity. In a pilot clinical study of MRSA detection, 100% concordance with reference methods has been achieved. Great Basin, which closed a $3.9 million financing in October 2008, plans to introduce the product by late 2009.

Another rapid, low-cost DNA detection system is under development by X-Bar Diagnostic Systems (Mendenhall, Pennsylvania). The system is designed for use in diverse settings and includes an All-in-One test processing station and DNA analysis via PCR and fluorescence detection.

Immunoassay systems in the spotlight

A number of new immunoassay systems for use at the point of care were described at the Oak Ridge Conference. As shown in Table 2, applications include cardiac marker testing, infectious disease screening and diagnosis, hormone analysis, and cancer screening and diagnosis.

Key characteristics of technologies which are well-suited for use at the point of care include compact size of the test system, extending to non-instrumented tests; rapid turnaround time; low cost; sensitivity and precision approximating that available in the central lab; room temperature reagent/test cartridge storage; small blood sample volume; and ease of use.

Examples of POC immunoassay systems that succeeded in penetrating the market include the Triage from Biosite/Inverness Medical (Waltham, Massachusetts), which performs cardiac marker and D-dimer assays; and the i-STAT System from Abbott Diagnostics (Abbott Park, Illinois), with a wide menu including cardiac marker immunoassays as well as chemistry tests.

The new systems under development described in Table 2 are targeted at expanding the range of immunoassays that can be performed at the point of care, and at improving the performance of POC tests. The number of sites at which POC testing is performed is continuing to expand in the U.S., as well as outside the U.S.

As shown in Table 3, growth has been particularly rapid in testing sites in ambulatory surgery centers, home health agencies, pharmacies, mobile laboratories and ambulances. The Physician's Office Laboratory (POL) segment also is continuing to expand, adding the largest number of new testing sites of any segment between 2005 and 2008. The statistics do not include self-testing, which is continuing to increase in terms of the number of individuals performing tests as the prevalence of diabetes grows and more patients adopt coagulation self-testing.

Microfluidics technology is playing a key role in many of the POC systems now under development. The 4CastChip technology, originally developed by Amic (Uppsala, Sweden) and acquired by Ortho Clinical Diagnostics (Raritan, New Jersey) in June 2008, is an example of a microfluidic technology that is showing promise for POC immunoassay testing. As described by Ib Mendel-Hartvig of Amic/OCD at the Oak Ridge Conference, the 4CastChip uses a micropillar structure which is formed by silicon micromachining, in concert with fabrication technology developed for manufacture of compact discs.

In addition, Amic has developed a dextran-based chemistry used to couple antibodies to the chip's surface. The microfluidic technology provides precise control over fluid flow rates and patterns, allowing quantitative assays to be performed. The technology enables analysis of whole blood samples, and while at present the required sample volume is 100 uL -200 uL, Mendel-Hartvig said the volume will eventually be reduced to 7 uL by miniaturization of the device.

Recent clinical evaluations of the first assay, an NT-proBNP test with applications in diagnosis and monitoring of heart failure, have shown a high degree of correlation with existing laboratory immunoassays, quoted at 0.97 compared to the Elecsys assay from Roche Diagnostics (Indianapolis), although agreement between the methods is not as good at low levels of NT-proBNP.

Amic also has demonstrated the ability to perform multiple different assays on one chip, such as simultaneous NT-proBNP and CRP assays. Amic/OCD has not yet announced a target date for introduction of the Forecast system. However, the company plans to target the system for POL and Emergency Department testing, and is planning to develop both CLIA-waived and non-waived tests.

Claros Diagnostics (Woburn, Massachusetts) also is developing a microfluidics-based system for POC immunoassay testing. The system consists of a handheld analyzer and individual test cartridges with reagents stored on the cartridge in microcapillary channels. By configuring the channels appropriately, a pre-programmed sequence of reagent delivery is achieved, avoiding the need for complex external pumps.

High sensitivity along with robust detection is achieved by using absorbance measurement combined with precious metal (gold and silver) labels which provide a high level of signal amplification. Up to 10 optical detectors are included in the system, enabling multiple different assays to be performed on a single chip.

Claros is developing both line-powered and battery-powered versions of the system, and has demonstrated good (r = 0.95) correlation with existing laboratory assays such as the Centaur assay from Siemens Medical (Einhoven, Germany) with a prototype PSA test.

Philips Healthcare (Best, the Netherlands), a leading supplier of diagnostic imaging and patient monitoring equipment, is developing a POC IVD system, the Magnotech analyzer, which will have applications in point-of-care immunoassay. The system was first described publicly at the MEDICA exhibition in November of last year, as reported by Biomedical Business & Technology.

The Magnotech system does not employ flow-through microfluidics as do most POC immunoassays, but instead moves magnetic particles through the stationary fluid and concentrates them at the detection surface to perform a separation. By using evanescent wave detection, only the labeled molecules which are bound to the particles are detected. The quoted sensitivity for Troponin I of 1 picomolar (0.02 µg/L) compares favorably to that for existing POC systems such as the Triage (0.05 µg/L) and the RAMP assay from Response Biomedical (Vancouver, British Columbia) of 0.03 µg/L, but is about two-fold less than that of the Roche Elecsys of 0.01 µg/L.

Vivacta (Kent, UK) is developing a POC immunoassay system that employs a unique piezo-optic detection technology to achieve high sensitivity and rapid test time in a compact, low-cost analyzer. The polyvinylidine fluoride piezo film used in the test cartridge responds to thermal and mechanical shocks by generating a voltage. Antibodies are bound to the film surface, and the sample containing the target is then applied to the film.

After the target binds, labeled second antibodies are bound to the target. The film is then exposed to pulsed light from an LED, and the labeled molecules generate heat when the light is absorbed, resulting in a voltage pulse. Bound versus non-bound particles can be reliably distinguished by measuring the time course of the generated pulses. Multiplexed assays are possible, and Vivacta has demonstrated a prototype cartridge capable of performing three assays at once. Cost of the cartridge is about $1. The system is now in the prototype stage, and the company expects to be in pilot production by September.

Another growing segment of the POC testing market involves coagulation and platelet function testing for patients undergoing anti-coagulation and anti-platelet drug therapy. Anti-platelet therapy is being used increasingly for patients with drug-eluting stents to protect against thrombosis, and in patients with cardiovascular disease to prevent stroke and heart attack.

According to data from Accumetrics (San Diego), more than 29 million prescriptions are written annually for Plavix, the most widely used anti-platelet medication other than aspirin, but up to one-third of patients do not respond to the drug, and consequently are not protected against adverse events as expected. Platelet function tests are available both for lab use and for POC use, including the VerifyNow test from Accumetrics, but adoption has been limited so far.

A new platelet activation test under development by Biomedical Diagnostics Institute (Dublin, Ireland) was described at the Oak Ridge conference by Antonio Ricco. The test employs microfluidics technology to measure the propensity of platelets in a patient sample to adhere to a collagen surface coated with Von Willebrand Factor, simulating the action of platelets in vivo. A CMOS camera is used to image platelets as they flow over the surface, tracking migration patterns which differ depending on the state of platelet activation.

A typical platelet population is heterogeneous, consisting of a mix of resting and activated states. By characterizing the flow pattern of the platelet population, the state of platelet activation can in principle be determined. The test requires a 150 µL -200 µL whole blood sample, and can be performed in a few minutes.

Ricco said the company is working to reduce the required sample volume, and is also experimenting with additional molecular components such as fibrinogen to coat the collagen surface to more precisely mimic the in-vivo environment. Although at present the system is configured for use in a laboratory environment, Ricco said a POC version is planned. A limitation, however, is that fingerstick sampling, as is typically employed for many POC tests, cannot be used in the BDI test since the platelet activation profile is modified by fingerstick sampling.

New DNA sequencing, proteomics tech

New developments in DNA sequencing and proteomics were highlighted at the Oak Ridge Conference which promise to expand the role of molecular diagnostics in the clinical lab, and to potentially allow molecular testing to be performed in POC settings. Han Cao of BioNanomatrix (Philadelphia) described a new nanochannel array technology which has applications in DNA analysis, including haplotyping, methylation analysis, detection of gene rearrangements, and assessment of DNA damage caused by toxic agents or radiation.

Ultimately, the NanoAnalyzer technology may be capable of high-speed gene sequencing. The technology is based the use of semiconductor fabrication techniques to form fluidic channels with a width of about 100 nanometers in a chip substrate. At that size, only a single DNA molecule can pass through a channel, and passage is possible only if the molecule becomes uncoiled and linearized.

Cao said that previous attempts to linearize single DNA molecules had failed because the channels were not sufficiently narrow. In addition, it is necessary to position a graded series of pillars in front of the channels with successively smaller separations, allowing the DNA to gradually uncoil, reducing the energy barrier to linearization of the molecule. Once individual molecules have been directed into the channels, they can be imaged to identify structural features, and various labeling techniques can be used to perform haplotype analysis or methylation studies.

Ultimately, with sufficiently high image resolution and labeling, it may be possible to perform single-molecular sequencing, according to Cao. Since each chip can contain thousands of channels, and each channel can be analyzed separately, it is possible to perform population studies to obtain clinically useful results.

BioNanomatrix holds patents on nanofluidics technology licensed from Princeton University (Princeton, New Jersey), and is pursuing a variety of potential applications including molecular cytogenetics, biopathogen detection, and epigenetic studies. Analysis of a DNA sample requires 20 seconds, vs. five to 12 hours for alternative methods such as gel electrophoresis. In addition, because intact molecules are analyzed instead of fragments or short sequences as is done with other methods, more accurate sequencing analysis is possible, according Cao said.

The use of advanced DNA sequencing technology for assessment of anti-viral drug resistance and for improved tissue typing was described by Birgitte Simen of 454 Life Sciences (Branford, Connecticut), a unit of Roche Diagnostics. The technique, ultra-deep sequencing, employs pcr amplification as a first step to produce viral sequences of interest with a typical length of 250 base pairs. The amplicons are then attached to beads for separation and purification, followed by a sequencing-by-synthesis step employing the proprietary pyrosequencing technology licensed from Pyrosequencing AB (Stockholm, Sweden) to obtain the detailed sequence. The technique allows rapid determination of characteristics such as codon variants associated with drug resistance.

In the case of HIV drug resistance, conventional PCR analysis is unable to detect some variant sequences which occur at a low level, resulting in the development of resistance in spite of a test result that indicates the patient should respond to the drug. With deep sequencing, low-level variants can be detected reliably.

Simen has found that the deep sequencing method can detect twice as many mutations associated with drug resistance compared to conventional analysis, and the presence of the mutations is associated with failure of therapy in HIV-infected patients undergoing drug therapy. Patients whose HIV strains show more than 20% of the newly identified mutations have an average time to treatment failure of 135 days vs. 706 days for those without any of the mutations. Simen also has used deep sequencing to analyze drug resistance for patients infected with hepatitis B and C virus.

Mathias Uhlen, PhD, of the Royal Institute of Technology (Stockholm, Sweden) discussed recent advances in human proteomics, including a human protein atlas now in development for use in biomarker discovery. Uhlen, along with Dr. Michael Snyder of Yale University (New Haven, Connecticut) and Dr. Peter Hudson of CSIRO (Melbourne, Australia), is a co-chairman of the Human Antibody Initiative, a program of the Human Proteome Organisation (HUPO; Montreal, Quebec).

The program consists of a worldwide network of scientific laboratories which are producing antibodies to human proteins, with a goal of eventually producing antibodies to all proteins in the human proteome. About 20,500 human genes are now believed to exist, according to Uhlen, and at present 14,000 antibodies have been produced and validated within the Human Antibody Initiative to characterize proteins produced by human genes, representing about 70% of the human genome.

Uhlen is now using antibodies derived from the program to analyze protein expression in human cancers, including breast, colorectal, prostate, ovarian and liver cancer, with a goal of identifying new, clinically useful cancer biomarkers. The studies employ tissue microarrays derived from cancer patients who have been followed clinically for up to 20 years, enabling a rigorous analysis of protein expression relative to cancer type and stage.

Some new markers have already been identified, which appear to have value in predicting outcome, including a breast cancer marker, HPA001467, which is correlated with a 15% change in survival. Another breast cancer marker, HPA8338, has potential utility in combination with estrogen receptor status as a predictor of response to adjuvant chemotherapy. A marker has also been identified which appears to have utility in colorectal cancer to guide the choice of therapy in patients with Stage II/III disease.

Uhlen has now begun using suspension microbead antibody array analysis employing the xMAP system from Luminex (Austin, Texas) to accelerate the biomarker discovery process, and has expanded the program to include discovery of new markers for kidney disease, including markers for biotoxicity resulting from drug therapy in collaboration with AstraZeneca (London).

New frontiers in molecular imaging

The scope of clinical diagnostics is in the initial stage of a transformation to integrate information from in vitro laboratory tests with information from non-invasive imaging, in particular that information derived from molecular imaging, to provide a more comprehensive and clinically meaningful assessment of disease. Integrated diagnostics has particular advantages for patients, who can have all diagnostic procedures including biopsies and imaging exams performed in one stop at a single, integrated facility.

Integration of radiology and laboratory reports has value for referring physicians who can realize improved efficiency and possibly reduced error rates by having a single report to interpret.

Diagnostic imaging was first included within the scope of the Oak Ridge Conference at the 2008 meeting, and was again incorporated into the topics addressed this year. Some early-stage initiatives to establish integrated radiology and pathology services are already in place in various countries including the U.S., but integration is at an early stage

Cancer diagnostics is an important application for integrated diagnostics, as the field is now termed by suppliers such as Siemens Diagnostics.

One potential application in prostate cancer diagnostics was addressed by Martin Pomper of Johns Hopkins University (Baltimore) at the conference. Pomper has developed a molecular imaging agent which targets prostate-specific membrane antigen (PSMA), a cell membrane protein which is expressed on nearly all prostate cancer cells and on blood vessels in solid tumors.

The agent employs an antibody fragment rather than an intact antibody as is currently used in ProstaScint, a SPECT imaging agent which targets PSMA that is manufactured by Cytogen (Princeton, New Jersey). ProstaScint has been available for about 10 years but is not highly sensitive or specific due to its use of intact whole antibody, according to Pomper. The antibody fragment agent developed by Pomper is expected to provide higher sensitivity and specificity, and is about to enter clinical trials.

Molecular Insight Pharmaceuticals (Cambridge, Massachusetts) has licensed molecular imaging agents developed by Pomper for imaging of metastatic prostate cancer, and its product pipeline includes Trofex, a PSMA imaging agent. Pomper also has developed molecular imaging agents for infectious bacteria and viruses including herpes simplex virus, as well as targeted therapeutic agents for cancer.

Dr. Joshua Rychak of Targeson (Charlottesville, Virginia) discussed new molecular ultrasound imaging agents under development for imaging of inflammatory disease, including vulnerable plaque imaging. Targeson is an angel- and grant-funded venture focusing on development of ultrasound imaging agents.

The Targestar imaging agents employ lipid-encapsulated perfluorocarbon microbubbles which can be ligated with antibodies or other specific binding molecules. The company has received an SBIR grant to develop a targeted ultrasound agent for imaging of tumor angiogenesis which binds to vascular endothelial growth factor R-2.

In addition, an agent for non-invasive ultrasound imaging of blood vessels is being developed which targets P-selectin glycoprotein ligand-1 (PSGL-1) and Vascular Cell Adhesion Molecule (VCAM). PSGL-1 mimetics have been identified which have the unique property of binding to regions of shear stress in blood vessels, which could prove useful in detecting high-shear regions of the vasculature that may be prone to formation of vascular lesions.

The Targeson molecular imaging agents are in pre-clinical development.

Ultimately, integrated diagnostics may enable information from molecular imaging to be combined with biomarker data to provide comprehensive diagnosis and staging of cancer as well as to guide targeted, personalized therapy, or, in the case of cardiovascular diseases, enable more accurate identification of individuals at risk for an adverse event and guide targeted preventative interventions.