BBI Contributing Editor

BALTIMORE Clinical diagnostics has historically been one of the most technology-driven segments of the medical device market, challenging competitors to continue investing to develop next-generation products for both central laboratory testing as well as decentralized point-of-care testing. Suppliers of clinical diagnostic products generated worldwide sales of about $26 billion in 2004, in a market forecast to grow at 7% per year through 2009. The leading forum for premiering new diagnostic technologies in the clinical diagnostics industry is the Oak Ridge Conference, organized by the American Association for Clinical Chemistry (Washington) and now in its 37th year. At this year's conference, held here in mid-April, a wide range of technologies with important applications in clinical diagnostics were described, including ultra-high sensitivity detection technologies, technologies for single-molecule detection, lab-on-a-chip systems, point-of-care (POC) testing technologies, non-invasive diagnostics, and advances in bioinformatics. While not all of the emerging technologies discussed at the conference will reach the market, some are likely to play a role in advancing the capability of the lab, physicians, and patients themselves to diagnose and monitor disease.

Pushing the sensitivity envelope

A major focus of development highlighted at the Oak Ridge Conference was advances in detection sensitivity for diagnostic assays. Table 1 below describes high-sensitivity detection technologies discussed at the conference. The keynote speaker at this year's conference, Dr. Joseph Lakowicz of the University of Maryland, Baltimore, described advances in fluorescence detection that have the potential to allow direct measurement of low concentration analytes such as nucleic acid targets and proteins without requiring a separation step, i.e., via direct detection. The technology is based on metallic nanostructures (gold and silver colloids) that are engineered to modify and control the emission of attached fluorescent labels, including the ability to control the direction and wavelength of the emitted light. The combination of metallic particles with fluorescent molecules results in the generation of plasmons upon exposure to an exciting light beam and, via a phenomenon called Radiative Decay Engineering, enhancement of the level of fluorescence emission by potentially up to a million-fold, although at present the level of enhancement is considerably less at five-fold to 100-fold. In addition, the sensitivity of the fluorophore to photobleaching is reduced.

One application is direct sequencing of nucleic acids. A model assay system has been developed using the nanostructure fluorescence technology consisting of DNA attached to a microbead. The DNA is sequentially digested by an exonuclease, and the released bases flow through a microcavity with a nanoparticle coating on its walls. As the DNA bases come into close proximity to the coating, their natural fluorescence is enhanced via a fluorescence resonance energy transfer mechanism, allowing detection and identification of individual bases as they traverse the microcavity.

Another application allows highly sensitive homogeneous detection of DNA using labeled oligomers that bind to the target sequence and capture probes bound to metallic nanoparticles on a quartz surface. A 10-fold enhancement of fluorescence is obtained upon hybridization compared to that observed for labeled oligos in solution, providing a mechanism to directly detect hybridization in real time. Another variant of the nanoparticle enhancement technology relies upon an effect termed Surface Plasmon Coupled Emission (SPCE). By appropriate nano-engineering of a metallic surface, the emission of fluorescent labels near the surface not only can be enhanced, but also the direction and wavelength of emission can be controlled. That effect not only allows more emitted light to be captured and detected, but also can increase discrimination against background, enhancing sensitivity.

One application is for highly sensitive detection for microarray-based assays, in which nanocuvettes are formed in an array comprised of silver posts having nanometer dimensions. The posts are sufficiently small that they can entrap single target molecules, allowing a 10-fold enhancement of fluorescence when the target molecule resides in the nanocuvette. The resulting assay structures thus can provide simultaneous detection of thousands of individual molecules.

Another novel detection technology with potential applications in high-sensitivity analysis was described by Matthew Cooper of Akubio (Cambridge, UK). Akubio was founded in late 2002 to develop and commercialize a label-free detection technology based on acoustics. The technology has potential applications in a wide range of clinical assays, including detection of infectious agents such as viruses and bacteria, cardiac risk marker testing, allergy diagnosis and tests for a number of other low-level analytes in a compact desk-top analyzer, the RAPid Alpha O.2. A low-power hand-held analyzer is under development that will provide multi-analyte capabilities. Acoustic detection technology is based on measurement of changes in the acoustic resonance of piezoelectric quartz crystals as molecules bind to the surface.

While quartz crystal resonator microbalance sensors have been used for biosensing applications for many years, Akubio's RAP technology provides significant enhancements over prior-generation acoustic sensors, including multi-channel analysis, use of microfluidics for sample manipulation, multi-mode high-frequency analysis and automation. Assays can be completed in less than 10 minutes, and the sensing crystal can be reused multiple times. Multiple analytes can be detected on a single sensor. Prototype assays developed for the Akubio analyzer include a myoglobin test, a CRP assay and assays for a number of infectious agents, such as listeria, chlamydia, salmonella and African Swine Fever. A key feature of the technology is the lack of a need for a label in the assay.

U.S. Genomics (Woburn, Massachusetts) has developed a new instrument, the Trilogy 2020, which employs multiple lasers and confocal microscope detection to allow single molecule detection of DNA, RNA and protein. The analyzer is presently marketed only for life science research applications, but additional applications under development include biowarfare agent detection, being developed in collaboration with the Department of Homeland Security (Washington), profiling of RNA expression in human tissues and cancer-related gene expression assays.

In the past, most gene expression analysis in cancer was performed using the Northern blot assay, a complex method having a turnaround time of up to two days. In contrast, the Trilogy analyzer can perform the same analysis at 1,000-fold higher sensitivity with a turnaround time of less than four hours, using only one microgram of DNA. The use of four-channel optics and coincident detection in a focused-flow configuration provides the reduction in fluorescence background and spurious signals from unbound probes needed in order to achieve reliable homogenous single-molecule detection with high throughput. For analysis of a human RNA mixture, the read time is only 20 seconds. A unique feature of the system is its ability to analyze small RNA oligomers down to 20 bases in length. Such targets cannot easily be amplified for analysis by polymerase chain reaction (PCR). In addition, protein targets can be analyzed. A demonstration assay for follicle-stimulating hormone in serum has been developed with sensitivity similar to that for ELISA but requiring only a 1 mL sample, vs. 200 mL for ELISA, and that provides considerably more rapid turnaround time.

Another application under development, of particular interest for applications in oncology, is an assay to determine phosphorylation of epidermal growth factor receptor (EGFR), now one of the most important molecular targets for new-generation cancer therapies. Yet another unique application is mapping of nucleic acid molecules to obtain a rapid signature that is representative of the molecular genotype. Nucleic acid molecules are first conveyed through a microfluidic array that uncoils them into a linear configuration, and then are analyzed in a flow-through mode in the Trilogy system at a rate of 30 megabases per second, with a resolution of 3 kilobases. The analysis generates a fingerprint, similar to a barcode, which can, for example, be used to identify pathogens. U.S. Bioscience is developing the technology in collaboration with the Department of Homeland Security for detection of bioterrorism agents.

Genisphere (Hatfield, Pennsylvania), in partnership with Molecular Probes (Eugene, Oregon), a leading supplier of fluorescent labeling reagents, is developing a single-molecule DNA analyzer using 3DNA dendrimer labels and a flow cytometry platform. The Genisphere dendrimers are multi-layered polymeric structures that contain multiple fluorescent molecules, providing the signal amplification needed for single-molecule detection. The assay consists of a capture probe attached to a bead, which hybridizes to the target, and a reporter probe with the dendrimer label attached. The assay has been used for the analysis of PCR products at levels 1000 times lower than for conventional detection methods, and may in some applications have sufficient sensitivity and specificity to eliminate the need to perform PCR in some types of samples if DNA capture efficiency can be improved by about 20-fold.

Another sensitive nucleic assay method has been developed by NuGEN Technologies (San Carlos, California). As discussed by Nurith Kurn, PhD, at the Oak Ridge Conference, NuGEN has developed the Ovation System, which employs an isothermal linear amplification method for RNA targets. The amplification technology, Single Primer Isothermal Amplification (SPIA), uses chimeric DNA/RNA primers, and has been used both for nucleic acid analysis and protein detection. A variant of the technique, Ribo-SPIA, can be used for amplification of mRNA targets, allowing gene expression profiling to be performed on samples containing as little as one nanogram of RNA.

One application under development by NuGEN is identification of specific tissues including brain, uterus, muscle and liver via analysis of RNA transcripts, which could have important applications in assessment of cancer metastasis. The technology also has applications in high-throughput gene analysis, where it can be used to rapidly amplify targets for microarray detection. Target amplification by a factor of 10 to 100 million-fold can be performed in about one hour.

Mass spectrometry (MS) is another technology for high-sensitivity detection that is being used by an increasing number of research groups for applications in proteomics to identify molecular markers useful in oncology, genetic testing and disease screening. Gary Siuzdak, of Scripps Research Institute (La Jolla, California), described a new technology using nanowires and porous silicon as substrates for mass spectrometry analysis that addresses one of the key limitations of existing MS methods, namely their relatively low throughput as compared to existing methods employed for protein analysis in the clinical lab such as immunoassay.

The use of tailored surfaces for desorption of samples for MS analysis increases throughput dramatically as compared to solution-based MS analysis, allowing up to 50,000 analyses per day performed, and also allows assessment of molecular affinity via the use of molecular tailoring of the surfaces. Sensitivity also is improved, to 800 yactomoles (800 x 10-24 moles). Another unique feature of the technology is its ability to perform separations as part of the desorption process, since the tailored nanowire surface functions as a chromatography column that can be designed to have specific release characteristics. Existing applications of mass spectrometry in the clinical lab include neonatal screening for protein markers of genetic disorders such as phenylketonuria as well as screening for heavy metal exposure, but the advances described by Suizdak could make it practical to use the technology for a much wider range of applications, including analysis of oncogene expression in tumor specimens, high-throughput screening for drug discovery, and assessment of proteomic profiles of patients for more precise cancer staging.

Chad Mirkin, PhD, of Northwestern University (Evanston, Illinois), described technology developed by Nanosphere (Northbrook, Illinois) for high-sensitivity analysis. Nanosphere is a venture-backed life sciences company founded to commercialize technology based on nanoparticles coated with oligonucleotides. The technology has the potential to provide sensitivity equivalent to PCR amplification for some applications, and extends such sensitivity to encompass protein as well as nucleic acid analysis. The company has commercialized the technology for research applications in the Verigene system, which provides rapid detection of specific nucleic acid sequences by analysis of DNA melting curves via changes in light scattering.

The most recent innovation is Biobarcodes, a technology licensed exclusively by Nanosphere, which provides highly sensitive detection of proteins in biological matrices. A model prostate-specific antigen (PSA) assay has been developed using magnetic particle separation for post-surgical screening in prostate cancer patients. Another potential application that takes advantage of the assay's 30-attomolar sensitivity is a breast cancer screening test. According to Mirkin, PSA has been shown to occur in women at very low levels in early stage breast cancer.

Another assay targeted for development by Nanosphere is a test for ADDL in cerebrospinal fluid, and perhaps in blood, for detection of Alzheimer's disease. While ADDL has yet to be shown to be a valid marker for Alzheimer's disease, the Nanosphere technology has been shown to have sufficient sensitivity to detect the marker at clinically relevant levels. Other applications described by Mirkin include assays for inhibin (a putative marker of ovarian cancer), p24, Troponin I, tau protein and prions, as well as a DNA assay for anthrax that avoids the need for pcr amplification. That application also demonstrates the ability of the platform to perform assays for both DNA and proteins.

New sensor technologies for high-sensitivity analysis were also described at the conference. Adam Heller, PhD, of the University of Texas (Austin), discussed electrochemical sensors using wired enzyme technology. The base technology was commercialized by a company founded by Heller, TheraSense (Alameda, California), now a unit of Abbott Laboratories (Abbott Park, Illinois), in a new glucose sensor that provided a 500-fold sensitivity improvement over existing sensors and allowed measurements to be performed using a 0.3 uL blood sample, enabling sampling from sites such as the forearm rather than the fingertip.

Heller said he believes the technology has much wider applicability, however. Using a cross-linked hydrogel as the reaction matrix, ambient oxygen as a substrate, and a bilirubin oxidase enzyme label, Heller has demonstrated the capability to detect 300 DNA copies in a 5 uL droplet using a $100 hand-held instrument. A nucleic acid assay to detect Shigella at one femtomolar concentration has been developed for the U.S. Army. Another application in nucleic acid analysis is detection of mutations in the BRCA gene, for identification of women with a genetic predisposition to breast cancer. Heller also believes the sensor technology can be applied to immunoassay detection, and estimates a sensitivity of 3 pg/ml can be achi-eved, representing an order of magnitude improvement over existing immunoassays.

Lab-on-a-Chip systems for POC testing

A number of companies described lab-on-a-chip technologies at the Oak Ridge Conference that may allow a wider range of tests to be performed at the point of care. Meso Scale Diagnostics (Gaithersburg, Maryland) has developed a multi-analyte test strip and cartridge employing electrochemiluminesence detection that can be read in an imaging device, the Sector Imager Reader, and provides 0.1 pg/uL sensitivity for protein detection using a 25 uL serum sample. Target assays include FSH, LH, TSH and hCG, as well as cardiac markers, using whole blood or plasma samples. The system is capable of performing up to 100,000 assays per hour. The company plans to develop a POC version that will satisfy the requirements for CLIA-waived tests.

Another lab-on-a-chip device was described at the conference by Gyros AB (Uppsala, Sweden). Gyros is partnering with Fujirebio Diagnostics (Malvern, Pennsylvania) in the development of a new device configured as a microfluidic compact disc. The technology has already been commercialized for bioanalytical research. It functions as a complete, integrated centrifugal analyzer and can perform 104 different tests in 24 to 50 minutes using a 200-nanoliter sample. The system incorporates integrated sample metering, microfluidic manipulation of reagents, a laser light source and fluorescence detection. Read time for a single assay is 210 seconds. A model assay for AFP has a 7.8% coefficient of variation for 10 picomolar levels of AFP, and a lower detection limit of 0.15 picomoles if a 100 uL sample volume is employed. The device also can perform kinetic assays by monitoring of the reaction zone of the disc. In addition to AFP, prototype assays have been developed for IL-6 and CEA.

LabNOW (Austin, Texas) described a new microfluidic system coupled with an electrochemical sensor for use in point-of-care and laboratory testing. One version of the system uses microbeads as self-contained reactors. The beads are mass-produced, and each bead can perform one chemistry test. Using an array of beads deposited on a microchip, a multi-analyte test panel can be performed. Readout is accomplished using pH indicator dyes and a neural network image recognition system for analysis of the color pattern. The LabNOW analyzer can perform a battery of tests in 10-30 minutes, with a target cost of $2 per chip. For added sensitivity, epifluorescence detection can be employed, using a miniaturized, low-cost microscope system. A prototype LabNOW chemistry analyzer has been used to perform an assay for CRP in saliva, and exhibited a sensitivity that was 10,000-fold better than that for conventional CRP assays.

New POC testing technologies also were highlighted at the Oak Ridge gathering. The market for POC testing products continues to expand, due to increases in prevalence of certain diseases such as diabetes and cardiovascular disease for which POC testing is widely used. As shown in Table 2 below, the clinical POC testing market includes both major players in the diagnostics industry as well as niche suppliers, with total revenues well in excess of $6 billion worldwide, and most suppliers reporting double-digit growth. Opulus (Philadelphia) described a new quartz crystal microbalance sensor that is designed for use in both lab-based and POC coagulation and sepsis testing. The sepsis assay measures endotoxin as a precursor to sepsis. In its current configuration, the assay requires 90 minutes to perform, but preliminary studies indicate a new two-channel POC analyzer is feasible that will provide significantly reduced test time and require a sample of a few microliters.

Michael Pugia of Bayer Diagnostics (Tarrytown, New York) discussed a new technology platform for hand-held diagnostics based on microfluidics with potential applications in POC immunoassay and chemistry testing. The technology has been developed in partnership with MicroParts (Dortmund, Germany) and uses surface treatment technologies to control fluid movements within a microchip device. A chemistry chip has been developed that can perform 48 glucose assays simultaneously, and a prototype hemoglobin A1c assay has also been demonstrated.

Luis Garcia-Rubio, of the University of South Florida (Tampa, Florida), discussed a biophotonic technology employing Spectral Acquisition Process Detection (SAPD), an ultraviolet-visible spectral analysis technique that can be applied to whole blood, plasma, or serum samples for rapid, low-cost, portable analysis of infectious diseases including malaria, Chlamydia infection, and dengue fever. The technology involves analysis of scattered light at multiple wavelengths and at one or many angles, and correlation of the spectral properties to microorganism size and chemical composition. Up to seven different bacterial strains can be differentiated, and cost per test is estimated at three cents. The analyzer also can be used to detect sickle cell anemia and bacterial contamination of blood products such as platelets at levels of one contaminant particle per one million normal cells. The technology requires no reagents, and current prototypes are already field-portable. In addition, preliminary studies have indicated that the technology may be able to perform non-invasive analysis, i.e., without the need to draw a blood sample.

Joel Dryer of ChemSensing (Northbrook, Illinois) described another non-invasive assay technology employing a colorimetric sensor array. The technology has potential applications in testing for Helicobacter pylori infection, as well as in the detection of ventilator-acquired pneumonia and possibly lung cancer. Existing breath tests for H. pylori infection require ingestion of a radioactive agent and laboratory-based analysis of exhaled air. The ChemSensing assay, in contrast, requires ingestion of non-radioactive urea, and detection of expired ammonia via a color change sensor employing metalloporphyrin dyes. Animal studies have demonstrated 100% sensitivity and specificity for H. pylori detection.

Emerging technologies for disease detection

A number of presenters at the Oak Ridge Conference described new technologies with applications in the diagnosis and management of specific diseases, including cancer, AIDS and tuberculosis infection, and also for applications in monitoring of transplant patients. Lance Liotta, MD, of the National Cancer Institute (Bethesda, Maryland), discussed recent studies using protein microarrays and signal pathway profiling for guidance of individualized therapy in cancer patients. Liotta's studies have focused on children with rhabdomyosarcoma who are likely to fail therapy with conventional drugs. Using laser capture microdissection performed with the Veritas system from Arcturus (Mountain View, California), along with proteomic microarray and cell-signaling network analysis, Liotta has demonstrated the ability to construct molecular signatures from pre-treatment biopsy specimens that separate responders from non-responders.

Another unique approach under investigation by Liotta is the use of silica or gold nanoparticles to selectively collect protein markers from blood, which can then be subjected to proteomic analysis. That approach can provide selective enrichment of low-level markers. Liotta has found that many protein markers with potential utility in cancer diagnosis are typically not detected in blood samples because they are bound to albumin and often are lost from the specimen during centrifugation when preparing a serum sample for analysis. At least 800 new proteins have been discovered by Liotta's group by analyzing such bound molecules.

Mary Lopez of Perkin Elmer Life & Analytical Sciences (Boston) discussed the use of the protein enrichment technique for early detection of ovarian cancer. Noting that existing biomarkers for ovarian cancer have low sensitivity and/or specificity, Lopez is combining protein enrichment technology with mass spectrometry analysis and bioinformatics to develop an ovarian cancer test with improved sensitivity and specificity. In partnership with Vivascience AG (Hannover, Germany), a subsidiary of Sartorius (also Germany), Lopez has developed Membrane Absorber technology, which uses an open porous membrane for analyte capture and biomarker enrichment.

In addition, bioinformatics software, the BAMF software using a learning rules-based algorithm, has been sourced from Predictive Diagnostics (Vacaville, California), a subsidiary of Large Scale Biology (Madison, Wisconsin). Combining those technologies has resulted in a powerful biomarker-profiling platform, the BioXpression system. Another application being studied by Lopez is a test for Alzheimer's disease using the BioXpression system. A preliminary study of samples from more than 1,000 individuals from 40 religious orders in the U.S., including priests, nuns and brothers, 150 of whom have developed Alzheimer's disease, has allowed development of a biomarker profile that can diagnose the disease with a sensitivity of 94% and a specificity of 89%, based on preliminary results.

Cell-based assays were another area of focus at the Oak Ridge Conference. PortaScience (Moorestown, New Jersey) has developed a whole blood point-of-care test for white blood cell count, the PortaWBC test, which provides a quantitative WBC count in whole blood in the physician's office or in the home. The test uses a 20 uL fingerstick blood sample, requires 10 minutes to perform, and uses a palm-sized digital reader to report the result. A test strip technology is used that consists of a capture membrane and colorimetric enzyme assay reagents to produce a quantitative color change that is proportional to the number of white cells per microliter. Precision studies have demonstrated a CV of 8.6% for low WBC count and 2.8% for normal WBC count. A correlation of 0.91 has been demonstrated vs. a conventional cell counter.

The initial application envisioned for the test is in-home monitoring of patients undergoing chemotherapy. Such a test could improve the efficiency of patient management by allowing patients to verify that their white count is sufficiently high before traveling to the hospital for chemotherapy. Other applications include assessment of cardiac risk, monitoring of antibiotic therapy and monitoring of treatment with drugs such as Neupogen. As shown in Table 3, the estimated market for point-of-care devices to perform blood counts is substantial.

Micronics (Redmond, Washington) described a development-stage microcytometer lab card for use in rapid labeling, counting and sorting of rare blood cells, including cancer cells and stem cells. The device includes a cell injector for laminar flow antibody labeling that greatly accelerates the labeling process, as well as a microfabricated monolayer-sorting slit to speed up cell interrogation by allowing the analysis of a complete monolayer of cells. Using epifluorescence imaging, the system is expected to enable rare cell detection approximately 100 times faster than existing flow cytometry analysis, with a goal of an analysis time of under one hour vs. about 28 hours for existing methods. The system, which includes the MicroFlow benchtop instrument and microcytometer lab cards, will be one of the first products to be introduced by Micronics.

Another cell analysis device, and one that, like the PortaScience device, can be used at the point of care, is being developed by LabNOW. The LabNOW technology not only can be used for protein analysis as described previously, but also has applications in cell analysis, and is being used to develop a POC device for performing CD4 counts, a test that is used to monitor all patients with AIDS. The LabNOW assay uses a microchip that filters a whole blood sample to isolate white blood cells, followed by CD4-specific staining and automated microscopic analysis to product a CD4 cell count. The assay can be performed in 10 minutes at a cost of about $3 vs. $30 to $50 for a typical flow cytometry CD4 assay. An initial evaluation of the test vs. flow cytometry demonstrated a correlation coefficient of 0.98.

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