New Systems Focus on Throughput

The growing volume and complexity of genomic research requires higher-throughput, higher-density techniques that can simultaneously capture multiple data points with greater sensitivity at a lower cost per point. Rare variant research, validation studies and biomarker discovery are among the applications that will directly benefit. Such advancements are also applicable to the development and application of molecular diagnostic tools. Among the companies advancing the throughput and volume of their technologies for such applications are Roche Applied Science, Luminex and Invitrogen. New product introductions from each company will enable the companies to enter new markets, as well as provide improved sensitivity at a lower cost.

Quantitative real-time PCR (qPCR) systems for life science research have progressed in recent years to encompass higher-throughput solutions and multiplexing strategies. To address higher-throughput applications and increase sensitivity via smaller testing volumes, Roche Applied Sciences last month launched the LightCycler 1536 Real-Time PCR System. Based on Roche’s LightCycler 480 Platform, the system employs a 1536 Multiwell Plate developed by IT-IS International for reaction volumes of 0.5–2 µL. The system can process 1536 data points in less than 50 minutes, according to Roche. “We find users for the LightCycler 1536 System in the high-throughput facilities of pharmaceutical companies, selected molecular diagnostic companies and their respective service providers. Also the green biotech companies have throughputs that require the highest possible throughput,” stated Robertus van Miltenburg, global marketing director for Roche Diagnostics GmbH qPCR & NAP Systems. Mr. van Miltenburg told IBO that gene expression profiling is the primary application for the LightCycler 1536. “Studies involving several thousands of samples are increasing and require a high-throughput workflow, while at the same time reducing the price per data point generated. At the same time, we also identified the need for more targets to be analyzed per sample, e.g., for gene expression profiling.”

For quantitating gene expression, qPCR is considered the gold standard. As Mr. van Miltenburg explained, “for this purpose, arrays are not the optimal solution because of missing design flexibility and insufficient dynamic range in detecting different expression levels. Customers appreciate the flexible design for number of targets and number of samples according to their need, without too many restrictions put upon them by the choice of format.” Other applications for the 1536 LightCycler include genotyping and copy number determination.

Compared to other high-throughput PCR platforms on the market, such as BioTrove’s glass array–based system or Fluidigm’s microfluidic format, the 1536 Lightcycler is more flexible, according to Mr. van Miltenburg. “Several pipetting robots are capable of mixing the required number of samples with the required number of assays. Other formats require either a fixed ratio of targets per sample, or even require the pre-loading of the device by the supplier. Another benefit of the use of a microtiter plate is the relative robustness. It is less vulnerable than a microfluid system.” Flexibility also extends to the amount of starting material. “Because the LightCycler 1536 System works in volumes up to 2 µl, the amount of starting material can be relatively high, leading to sensitivities that are almost the same as achieved in 384-well plates.”

Another technology making the leap to higher throughput and higher volume is the xMAP platform. Luminex announced the full commercial launch of the FLEXMAP 3D system in June. The system provides the ability to multiplex up to 500 analytes per well, compared to 100 analytes per well using the Luminex 100 or 200 systems. To increase throughput, the FLEXMAP 3D can run 384-well plates as well as 96-well plates and employs dual sample fluidics. The read time for a 384-well plate with 2,500 beads per well measuring 500 analytes per well is two hours and 15 minutes, or 85,000 tests per hour, according to the company.

Asked which end-user markets the FLEXMAP 3D targets, Darin Leigh, vice president for Sales and Marketing at Luminex, told IBO: “We have seen a lot of interest from pharmaceutical and academic customers, and this has also manifested itself in many of the first orders for FLEXMAP 3D going to these types of customers. We see a good fit for the system in both the biomarker discovery and high-throughput screening segments, where customers have identified a number of signatures from their vast array analysis and now want to run anywhere from 10–500 plex assays to validate these markers.”

The system will also need the needs of current customers. “We also see a number of current Luminex users that either have a need for a higher throughput or have reached their capacity with 100 plex on the Luminex 200. Many of those customers will ‘upgrade’ to a FLEXMAP 3D for the increased throughput and enhanced performance with regard to sensitivity and dynamic range.” Assays used on the Luminex 200 can be transferred to the FLEXMAP 3D.

Currently, in the life science research market, Luminex technology is primarily used for protein assays. The higher-density, higher-throughput capabilities of the FLEXMAP could change this, as it better positions Luminex to enter new markets. As President and CEO Patrick Balthrop recently said on a quarterly conference call, “we are evaluating a variety of strategic options that will allow us to use that platform to penetrate life science research in nucleic acids.” Mr. Leigh cites gene expression, SNP genotyping and high-throughput screening as possible applications. The system also fits with the company’s growing business in the molecular diagnostics field, both through its own and partners’ assays. “We also have a number of initiatives internally that will result in new assays and methodologies being delivered to the market in Q3 and Q4 of 2009. By the start of 2010, we should have a comprehensive offering in the molecular research space in particular,” said Mr. Leigh.

Such applications also benefit from the unique aspects of xMAP technology. “One of the main differences is the ability to be flexible with your analytes (through addition or subtraction). This allows you to change assay content very easily as you start to focus on narrowing your range of biomarkers you wish to validate,” explained Mr. Leigh. “The fact that xMAP is proven for both nucleic acid and protein applications is also something that is fairly unique about our technology.” A higher-volume system also delivers costs savings. “Our customers in this market segment are also more and more focused on cost per data point. This could mean the ability to get more results out of a smaller sample now that we are able to go up to 500 plex and use 384-plates.” The list price for the FLEXMAP 3D is $150,000.

One company that Luminex can expect to compete against in the high-throughput Human Leukocyte Antigen (HLA) research market is Invitrogen, a division of Life Technologies. (HLA testing products using the Luminex platform are offered by One Lambda and Gen-Probe, formerly Tepnel.) Invitrogen’s Prodigy System, introduced in June, is a microarray-based solution for immunogenetic testing, such as HLA. Designed for ease of use and high throughput, the Prodigy provides automated hybridization, detection and analysis. Invitrogen states that the system can process 290 genotypes in nine hours. Like xMAP technology, the system will eventually provide analysis of both DNA and protein assays. “The instrument currently performs DNA assays and we will launch a protein assay at the end of 2009,” said Beth Button, head of Marketing for Transplantation Diagnostics at Life Technologies.

The system can analyze up to 12 eight-well Prodigy Typing Strips. Each well contains a glass microarray with Sequence-Specific Oligonucleotides (SSO). As Ms. Button explained: “Loci-specific oligonucleotide probes are conjugated to the surface of a large glass slide. This glass slide is then dissected into individual chips. The individual chips are then fixed into the bottom of an eight-well strip. The size and colorimetric principles currently provide real estate for 500 density.” The density allows for high-volume testing and the addition of newly discovered alleles. “Future generations will enhance the chemistry and increase density at least two fold.” The system’s list price is $98,500. Ms. Button told IBO, “the assay prices will vary depending on application, but the current HLA product averages $35 per loci per sample.”

A high-throughput, automated system is especially important for immunogenetic testing, according to Ms. Button. “Specifically, with the major histocompatibility complex (MHC), multiple loci testing is required and this region is highly polymorphic. This results in 5–10 tests per individual sample and volumes quickly accumulate.” HLA testing is conducted for organ transplants and disease association. “In addition, bone marrow registries worldwide test in high volumes with limited funding. A walk-away, fully automated, high-throughput system is essential to drive efficiencies in registry laboratories.”

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