With multiplexing, one slide can hold a thousand samples, each tested on a microarray that produces multiple results. Add cost-efficiencies (such as reduced consumable use), automation, clinically useful results, and high sensitivity and specificity, and multiplexing and microarrays are poised to become a significant methodology for clinical laboratories. New products are permitting clinical labs to expand their testing menus and increase volume.

Nanogen’s NanoChip 400

Shirley Williams, PhD, a scientist at Children’s Hospital of Orange County, in Orange, Calif, has noted some of these benefits since the installation of the NanoChip 400 from Nanogen Inc, San Diego, a little over 1 year ago.

“The machine permits a higher throughput with less hands-on time required,” Williams says. “We are now trying to complete large population studies, which weren’t possible with a manual system due to the need for too much pipetting and transfer of samples.”

Features she has found particularly helpful include automated loading and unloading, onboard dilution, and the ability to work with both 96-well and 384-well plates.

“You can complete a PCR reaction and transfer the entire PCR plate from the PCR machine directly into the NC400,” Williams says. “There is no pipetting or dilution, so once you are done with the PCR and program the computer, you’re done.”

Less hands-on time has allowed the laboratory to increase its testing menu and volume, although the hospital runs primarily home brews. The NC400 is used in the hematology thrombosis lab, and the tests ordered look for hematological markers. Because the patients are pediatric, home brews are easier to use than available assays whose tests must be chosen selectively to accommodate pediatrics.

The NC400 makes testing even easier, according to Williams. Her biggest challenge is optimizing the PCR phase (which does not involve the NC400) to be sure there is no preferential amplification of any one target.

The NC400 automated system incorporates mutation/polymorphism detection, pharmacogenomic testing, and pathogen sequence detection. The NanoChip electronic microarray cartridge runs multiple samples with multiple targets and can be reused until all test sites have been utilized. Automated post-PCR, software-driven reagents, sample dispensing, and bar-code reading reduce manual requirements, decreasing error and increasing laboratorian time.

Nanogen submitted the device, along with an assay for cystic fibrosis carrier and newborn screening, for 510(k) clearance in April .

Seegene’s Seeplex

A new test from Seegene, Rockville, Md, can also save a lot of time (and money): The Seeplex 18-Plex Respiratory Test is capable of detecting 18 different respiratory infections in one tube. The new diagnostic test kit is designed to assist physicians with cost-effective active surveillance and treatment of respiratory illnesses. By identifying the cause of a respiratory illness more quickly, the appropriate treatment can be administered sooner, potentially improving the patient’s outcome.

Multiplexing Stem Cells

Multiplexing and microarrays are playing a large role in drug discovery, and thanks to work by researchers at Rensselaer Polytechnic Institute in Troy, NY, they may also play a larger role in stem cell research. Two separate teams have developed tools to rapidly test stem cell response to different drugs and/or genes, as well as create a supply of healthy, viable stem cells. Microarrays permit the analysis of significant amounts of material on one slide.

The team led by Jonathan Dordick, the Howard P Isermann professor of chemical and biological engineering; Tiago Fernandez, a visiting doctoral student; and Joaquim MS Cabral, a professor from the Instituto Superior Téchnico-Lisbon, has developed technology to test millions of stem cells for drug response. Dordick aims to contribute to research on the elimination of stem cells that are destined to become cancerous. The group prepared up to 1,000 drops as small as 20 nanoliters on a chemically modified slide to permit various forms of cell-based screening.

The second team, led by professor of chemical and biological engineering Ravi Kane and Rensselaer doctoral student Randolph Ashton, focuses on the genes impacting stem cell development. Working with neural cells, the team developed a method to test clonal stem cell populations for desired behavior, such as normal development. The goal is to isolate specific genetic sequences that guide the development of stem cells and permit the creation of medical treatments that will use these correct sequences in programmed cells to produce normal development.

—RD

The new test detects 11 respiratory RNA viruses, two DNA viruses, and five pneumonia bacteria using one multiplex PCR in one tube. Samples accepted include nasopharyngeal aspirates, nasopharyngeal swabs, and bronchoalveolar lavage. Capillary electrophoresis permits the automated detection of pathogens on agarose gel; an auto-sequencer can also be used. A small amount of the target molecule can be detected, often within 5 hours.

Seeplex technology is based on the company’s proprietary dual priming oligonucleotide (DPO) primer system, which optimizes PCR. The technology links two separate priming regions with a polydeoxyinosine linker, which serves to separate the two regions. One acts as the “stabilizer,” preferentially binding to the template DNA and initiating “stable annealing.” The other selectively binds to a target site and determines “target-specific extension,” thereby earning the label “determiner.”

This architecture allows high sensitivity, high specificity, and test reproducibility. Advantages include multidetection, internal quality control, reduced carryover risk, and cost efficiencies. Economies are realized from rapid turnarounds, the elimination of cross-reactivity, and the value of 18 tests at a price similar to that for one.

The company also offers the technology in multiplex diagnostic test kits for HPV genotyping (Seeplex HPV 18-plex Genotyping Test), STDs (Seeplex STD 7-plex Test), and Lamivudine-resistant HBVs (Seeplex HBV Lamivudine-resistant Test). Tests in development include a 39-plex HPV genotyping test; a 23-plex STD test; a 22-test mycobacteria test; a 20-plex blood screening for sepsis; and a 24-plex blood screening for viruses, including hepatitis, HIV, and West Nile.

Luminex’s xMAP

Luminex Corp, Austin, Tex, is also working to expand its multiplex testing offerings. “The challenge in many genetic profiling environments is to provide a higher level of multiplexing,” says David E Smith, PhD, scientific awareness manager at Luminex. The company has an instrument in the final stages of development that will provide 500-plex testing, an improvement over the 100 tests per well currently available. Other improvements, according to Smith, will include greater sensitivity and wider dynamic range.

Smith colleague Keld Sorensen, PhD, director of research and development for Luminex’s bioscience group (and its assay-development arm), estimates there are roughly 40 FDA-approved diagnostic test kits and several hundred research uses currently on the market. Applications are varied and include protein-expression profiling, genetic disease testing, adverse drug reaction determination, immunodiagnostic testing, and home brews.

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Luminex offers a number of tests, as well as access to the xMAP technology upon which the tests are based. The FDA-approved xMAP platform incorporates traditional technologies—such as flow cytometry, microspheres, lasers, digital signal processing, and traditional chemistry—to perform various bioassays. The innovative process allows multiplexing of up to 100 unique assays within a single sample.

“We can look at multiple analytes per well using very straightforward technology—flow cytometry, which everyone in the lab environment is familiar with. Users can assay for just about anything they want. The difference is that rather than looking at cells, we are looking at microspheres,” Sorenson says, citing proteins, receptor ligands, enzymes, and oligonucleotide sequences as test targets. The microspheres are coated with substrates specific to the target molecule(s).

Because the xMAP architecture is open, companies have developed compatible kits and software. Luminex also offers test kits as well as a service dedicated to helping others develop their own assays. Diagnostic kits using xMAP technology include tests for allergies, autoimmune disease, cystic fibrosis, cytochrome p450, HLA, and infectious disease. Research applications include cancer markers, cardiac markers, cellular signaling, cytokines, chemokines and growth factors, endocrine, gene-expression profiling, genotyping, isotyping, matrix metalloproteinases, metabolic markers, neurobiology, and transcription factors/nuclear receptors.

The technology’s flexibility is one of its advantages; another is its ability to both detect and quantitate protein and nucleic acids. “In a clinical environment, you sometimes need a quantitative answer for proteins and nucleic acids. [xMAP] allows researchers to perform integrated systems biology. For instance, when you see a genetic change, it’s important to verify that it results in a change in the protein or whatever you are measuring,” Smith says.

These results can be obtained from small samples, which is particularly beneficial in the pediatric setting. Built-in controls ensure quality with each run; one well can be devoted to quality assurance. Automated instruments add further value. Luminex is working with partners to provide automated systems that are flexible and cost-effective. “We’re working on automation so the laboratorian can put trays in, walk away, and do the assay overnight,” Smith says.

BioTek’s ELx50

The new ELx50 Filter Microplate Washer has been recommended by Luminex for use with the xMAP system.

BioTek Instruments Inc, Winooski, Vt, is one such company. Its new ELx50 Filter Microplate Washer (FMV) has been recommended by Luminex for use with the xMAP system. The system automates filter bottom plate washing, eliminating the need for manual labor.

The system will accept filter pore sizes from 0.45 µm to 1.2 µm and a range of fluid viscosities. Selectable vacuum levels and aspirations times increase effectiveness. Two sets of dispense and aspiration tubes per well provide even more consistency in the 96-well format.

The hardware features a syringe-drive fluid-delivery system, a Dual-Action manifold that washes both 96- and 384-well plates, a unique priming trough built into the removable microplate carrier, optional automatic buffer switching, isolated electronics (away from the fluidic system), and an aerosol shield.

According to Luminex, the recovery of microspheres in the xMAP assay platform remains good even after high vacuum during aspiration and up to six washes. Blotting requirements are minimized or unnecessary, and the filter microplate wells do not dry out from the aspiration.

The only downside to multiplexing and microarrays can be the overwhelming amount of data that results from the tests. “The large amount of data produced can startle the end user, particularly in research cases where tests can produce 9,000+ bits of data,” Sorensen says. But for clinical labs, this problem is minimized with automated data flow into the LIS; and for prolific researchers, the data permits publication of multiple studies.

“If prepared to deal with it, large amounts of data can be a huge advantage,” Smith says. This is a good thing, since multiplexing is expected to take on a bigger role in the lab.

Renee DiIulio is a contributing writer for CLP.