BB&T Contributing Editor

SAN JOSE, California – Reflecting its traditional position as a forum for emerging clinical diagnostic technologies, the 38th annual Oak Ridge Conference, sponsored by the American Association for Clinical Chemistry (AACC; Washington), held here in April, continued to provide an intriguing look into the future.

For instance, advances in lateral flow immunoassay technology were described by Michael Natan, PhD, of Oxonica (Mountain View, California). Oxonica has developed a new fluorescence labeling technology called Surface Enhanced Raman Scattering (SERS) nanotags. SERS nanotags consist of gold nanoparticles coated with a layer of fluorescent reporter plus an outer layer of silica. They can be used to enhance a fluorescence signal by a factor of a million-fold or more, and have been used to enhance lateral flow assays such as those manufactured by Meridian Biosciences (Cincinnati) to provide improved performance including four-fold higher sensitivity.

In addition, nanotags loaded with different fluorescent labels can be analyzed simultaneously, allowing multiplex assays to be performed. Oxonica has developed a multiplexed, quantitative lateral flow immunoassay for Flu A, Flu B, and respiratory syncytial virus (RSV) that can be performed using a hand-held reader, and has also developed high-sensitivity no-wash single tube assays for DNA and proteins.

Single-molecule detection was another focus at the conference. Steven Soper, PhD, of the Center for BioModular Multi-Scale Systems (Baton Rouge, Louisiana), discussed diagnostic applications of single molecule detection, including a polymer-based microfluidics system for single-molecule detection now under development. Single-molecule detection assays have certain advantages compared to conventional assays, such as digital counting of reporter molecules leading to high sensitivity, and streamlined processing since some steps normally required in conventional multi-step assays can be eliminated.

Detection of single or small numbers of molecules also can be performed without the need to resort to technologies such as polymerase chain reaction (PCR). Development of single molecule detection technology started in the early 1990s, and until recently the technology was too costly for clinical diagnostic applications.

By 2005, however, system component cost had dropped to around $5,000, making the technology potentially feasible for use in the clinical lab. Companies now marketing single-molecule detection systems for research use include 454 Life Sciences, a subsidiary of CuraGen (both Branford, Connecticut), Helicos BioSciences (Cambridge, Massachusetts), and US Genomics (Woburn, Massachusetts), which now markets the Trilogy 2020 single-molecule analyzer. A key application focus at present is high-speed nucleic acid sequencing.

The use of microfluidics technology in single-molecule analyzers is proving to be an important advance, since it provides a means to automate sample processing while reducing contamination problems at greatly reduced cost compared to existing molecular diagnostic systems.

Microfluidic systems also have the capability to work with very small sample volumes, and can be highly compact, enabling their use in point-of-care testing applications. As discussed by Soper, the cost for a microfluidics chip has now dropped to about $25, and 100 assays can be performed with each chip, making the cost per assay as low as 25 cents. Soper is developing systems using stacked microfluidic chips and specially treated polymers that can be used for nucleic acid extraction to construct low-cost, mass-producible integrated systems for single-molecule analysis.

Singulex (St. Louis) also is developing single-molecule detection technology and described a new assay for cardiac troponin I that uses a 384-well ELISA plate and the Zeptx Digital Molecule Counting System. The assay employs a sandwich format with a fluorescently tagged detection antibody that is chemically released after a wash step. An aliquot from each well in the plate is then pumped through the Zeptx analyzer via capillary flow, and single antibody molecules are counted via laser-induced fluorescence. The assay precision is 10% at a level of 4 picogram/ ml, and sensitivity is 1 picogram/ml, 10-fold higher than that of the cardiac troponin I assay available on the Centaur analyzer from Bayer Diagnostics (Tarrytown, New York).

An initial clinical evaluation compared the Singulex assay to the Centaur assay in 15 patients admitted with chest pain who subsequently were confirmed to have acute myocardial infarction. Testing of the admission sample from each patient with the Centaur assay did not give a positive indication of AMI, whereas the Singulex assay was positive in eight of 15 cases. The troponin I concentration was positive on all subsequent serial samples from the patients on the Centaur, establishing the diagnosis of AMI. The evaluation showed that the Singulex assay detected 53% more cases of acute myocardial infarction than the Centaur when the admission sample was tested.

Microarrays and lab-on-a-chip tech

Microarrays represent another new technology with applications in clinical diagnostics with the potential to revolutionize the way in which testing is performed. In the keynote address at the Oak Ridge Conference, Robert Lipshutz, PhD, of Affymetrix (Santa Clara, California), the leading supplier of microarray devices for biochemical analysis, discussed the latest developments in microarray technology and clinical applications.

Affymetrix currently manufactures chips having up to 6.5 million features, but is developing a new fabrication technology that could allow chips with 160 million features to be produced, and also will allow use of new types of chemistries to expand the diagnostic applications of microarrays.

Affymetrix microarrays already have been commercialized for clinical diagnostics via a partnership with Roche Diagnostics (Indianapolis) to market the CYP450 AmpliChip, a device used to perform a pharmacogenetic test for variations in the CYP2D6 and CYP2C19 genes that affect metabolism of an estimated 25% of all prescription drugs.

Affymetrix now is developing a wide range of new applications of microarray technology for clinical diagnostics. For example, the ability to subclassify leukemias into subtypes that correlate with response to drug therapy has been demonstrated in a collaboration with Haferlach at Ludwig-Maximilians-University (Munich, Germany), an application that is moving ahead into clinical trials. Studies also have been performed using microarray genetic analysis of saliva samples to detect markers of breast cancer, creating the possibility for an oral breast cancer-screening test that could be run in a dental office.

Another collaboration involving bioMerieux (Marcy l’Etoile, France) and ExonHit Therapeutics (Paris) is focused on development of a microarray breast cancer assay. That program is now entering the clinical trial phase, with a study that will enroll 1,000 patients. Lipshutz said the assay may actually be detecting genetic variations in the immune system in response to breast cancer, rather than measuring tumor nucleic acid shed in blood, since the assay is detecting very small tumors that are unlikely to be releasing significant amounts of genetic material.

The key advantage of microarray technology in such applications is its ability to reliably analyze large numbers of single nucleotide polymorphisms (SNPs) at once in a sample, with current versions of the Affymetrix chips capable of testing for up to 50,000 SNPs.

Another application that takes advantage of that feature is a leukocyte genetic profiling assay being developed in collaboration with Contragene. The assay can potentially be used as a remote diagnostic tool to detect a variety of diseases, such as schizophrenia and bipolar disorder.

Microfluidics and lab-on-a-chip technologies are additional examples of the application of microfabrication technologies in clinical diagnostics. Richard Taylor, PhD, director of LabCD programs at Tecan U.S. (West Boxford, Massachusetts), described the development of a centrifugal microfluidics platform called LabCD with applications in rapid enzyme assays, nucleic acid assays, and immunoassays. The LabCD technology uses high-precision injection molding to form microfluidic networks on the surface of a compact disc.

The technology was originally developed by Gamera beginning in 1995, and purchased by Tecan in 2000. So far, $60 million has been invested by Tecan in development of the technology. Microfluidic networks can be fabricated that include valves, metering devices, and sequential reagent delivery devices using a combination of channel diameter and rotational speed of the disc. The technology allows reagent volumes to be reduced ten-fold compared to conventional assay systems, and provides highly precise fluid control with variations in volume of only 1% to 3%.

The LabCD technology already is in use for pharmaceutical development, but new applications under development at Tecan will address molecular diagnostic testing, including PCR-based assays. Clinical applications include a diabetes panel disc that performs tests for glucose, hemoglobin and glycated hemoglobin; and a LabCD metabolic panel. Tecan is developing a new LabCD analyzer that will be priced at under $50,000 and will have the capability to perform colorimetric, fluorescence and chemiluminescence measurements. Cost per test is projected at 30 cents to 40 cents, which should compare favorably to costs for conventional ELISA assays of 70 cents to 80 cents per test.

Another company developing microfluidics technology for clinical diagnostics, TeleChemistry (Turku, Finland), described a Liquid MicroProcessor (LMP) that can replace conventional large-volume chemistry analyzers with mass-produced 3-D microfluidic devices fabricated from electroformed metal alloys. Thermal phase transition inside a microchannel junction is used to create valves. Microscopic solid plugs are automatically formed whenever liquid is present within a channel, and a laser beam is then used to turn selected solid plugs into liquid by heating the outside of the channel. An electroformed bellows dispenser is included to drive liquids through the device, and all operations are digitally controlled.

TeleChemistry is using the LMP as the key component of a stand-alone chemistry analyzer that can be operated remotely and has the capacity to run 20,000 tests on a single reagent pack including the 20 to 40 most frequently ordered chemistry tests. Conventional chemistry assay methodologies are employed, but required reagent volume is one-tenth that of existing mainframe chemistry analyzers. The system accepts whole blood samples, and includes automatic quality control features.

The company plans to place the system in sites that have no or limited access to central laboratory testing services, allowing physicians and nurses to have direct access to blood analysis without the need to establish a laboratory.

Agilent Technologies (Santa Clara, California) is using microfluidics technology in its 2100 Bioanalyzer to characterize the size distribution of high-density lipoprotein (HDL) particles in blood. The analyzer can perform an HDL particle size distribution analysis in less than three minutes using 1 ml of serum, and runs 12 samples plus a calibrator on each microfluidic chip. An initial clinical evaluation of the benchtop analyzer showed good repeatability and acceptable correlation with existing methods (Lipoprint). HDL particle distribution analysis is used in assessment of coronary artery disease risk.

Advances in molecular diagnostics

New technologies for the molecular diagnostics laboratory also were introduced at the conference. Eric Olivares, PhD, of Invitrogen (Carlsbad, California) discussed the EasyPlex ChargeSwitch gDNA plates for use in genomic DNA purification, amplification, and detection in a single well. The ChargeSwitch technology uses a novel resin molecule that allows nucleic acids to be manipulated by varying pH.

Target DNA molecules can be captured on a well of an EasyPlex plate by use of a buffer with an appropriate pH. The pH then is altered to release the target molecules, and PCR amplification can be performed. Amplified product can then be extracted from each well by simple aspiration, eliminating the need for centrifugation or filtration. A model assay for lentivirus requires 30 minutes from sample addition to initiation of PCR.

Other applications under development include HLA typing and detection of additional viruses. The EasyPlex technology, however, is expected to have broad applicability for rapid, simple processing of samples for nucleic acid testing, and in many cases allows pre-processing steps to be eliminated. A future application under investigation is to combine cell isolation technology from the company’s Dynal division with ChargeSwitch technology to enable nucleic acid testing in specific cell populations.