Master of multiplexing

A German biotech center wanted to strengthen its capabilities in food safety and plant pathogen detection. The Mayo Clinic was looking for a collaborator to develop new genetic testing services for identifying blood disorders. A clinical laboratory needed a more efficient tool for screening potential organ donors.

In developing the FlexMAP 3D instrument, the biggest challenge for electrical engineer Wayne Roth’s team was matching the system’s optics to the microsphere dyes. A prime tool in optics design: the Coherent ModeMaster beam analyzer, which helps calibrate the laser so it focuses on a precise spot size on a bead. Photo by Dan Bryant.

While there’s no shortage of competitors pursuing the multibillion-dollar bioassay market, the 13-year-old Texas firm has distinguished itself by developing instruments that substantially increase the throughput of test results, a major concern for customers such as high-volume clinical labs and pharmaceutical companies competing to bring new drugs to market. The company’s success is evident in its $75 million in sales in 2007, an increase of 42% over the previous year.

Using a multiplexing approach, the company’s latest analyzer—the FlexMAP 3D—can simultaneously test for up to 500 analytes in a single sample, five times as many as the most advanced Luminex system now on the market. FlexMAP 3D is now being readied for FDA 510(k) clearance (the process through which the FDA approves medical devices for market), with a commercial launch anticipated for early 2009.

“In a clinical environment,” said John Carrano, PhD, VP of R&D at Luminex, “physicians want to look at several different factors in diagnosing patients, and our technology allows them to examine many biological targets or analytes simultaneously, rapidly, and accurately. Likewise, in life sciences research and drug discovery, the ability to survey many different targets at once substantially improves the efficiencies of research.”

Delivering those benefits requires a design and test effort that embraces a multitude of technologies in a high-performance flow cytometry instrument: fluidics, microspheres, optics, electronics, mechanics, and software.

The Luminex stable of analyzers, including the Luminex 200 now on the market and the upcoming FlexMAP 3D, are based on the company’s patented xMAP technology. While competitive flow cytometers combine different sizes and color intensities to identify microspheres used in the systems, the xMAP technology relies on microspheres with a uniform 5.6-micron diameter and uses a proprietary dying process to color-code these fluorescent beads into 100 distinct sets. Each bead set can be coated with a reagent designed for a particular bioassay, allowing the analyzer to detect specific analytes when the beads are mixed with a biological sample.

The Luminex multiplexing system features color-coded beads, called microspheres (right), that can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes. Courtesy of Luminex.

High-speed digital signal processors (DSPs), together with system software, classify each microsphere based on its spectral address. Assay results, which typically measure a sample’s fluorescent intensity, are transformed mathematically to depict analyte concentration and are displayed on the system’s workstation screen.

In a typical bioassay, a sample passes through a cuvette surrounded by fast moving sheath fluid that accelerates and separates the microspheres. A red laser excites the fluorescent dye mixture in each microsphere, while a green laser excites a fluorophore reporter tag bound to the surface of the microsphere. Courtesy of Luminex.

Customers have expressed appreciation for the throughput potential of xMAP. “We’re anxious to explore ways to adapt Luminex technology for our business,” said Neal Apple, VP of the Food Safety and Laboratory Service Network of Tyson Foods, which recently signed a collaborative agreement with Luminex. “We believe it will give us the flexibility to gather more testing data faster and develop and validate rapid testing options not currently available commercially.”

The engineers faced with the task of testing the Luminex system must target the individual subsystems, as well as the overall performance of the entire system. It all starts with the tiny polystyrene microspheres, or beads, that form the basic building blocks of the Luminex systems.

Don Chandler, senior director of Chemistry R&D, uses a Coulter particle-size analyzer to ensure that microspheres maintain their uniform size after being dyed. Photo by Dan Bryant.

To ensure uniformity in size and surface characteristics, Luminex evaluates every batch of untreated beads it produces with electron microscopes. After the beads are dyed, test staff employ Coulter particle-size analyzers to ensure that the dyes did not alter the uniform size of beads. The instrument accomplishes this by measuring impedance changes when a particle passes between two electrodes.

In addition, the Luminex system itself is used to evaluate the potential assay performance of beads as well as dye uniformity and accuracy. “Every lot of beads is tested on the analyzer to make sure the targeting is accurate,” said Chandler. “In the Luminex 200, for example, there are 100 targets per each sample well, and we need to make sure that we are targeting the center of each of these 100 regions.”

What QC looks for in such tests is a tight grouping of fluorescent signatures from the microspheres in a given region. In the Luminex 200, this appears as a two-dimensional grid or “bead map.” For example, one internal dye in a bead might have a fluorescent intensity of 1000, while the second dye might have an intensity of 500. This results in a dot at the coordinates (1000, 500) on the bead map. Every time a bead comes through the analyzer, another dot is recorded. A third fluorescent signature is simultaneously measured—this one associated with reporter molecules on the surface of the microsphere.

The development of the FlexMAP 3D system posed new test challenges for the Luminex team. To facilitate the increased multiplexing associated with the new instrument, each bead incorporates a third internal dye as well as the reporter dye. This third dye, which creates a “third dimension” in the classification bead map, is what allows the instrument to analyze up to 500 targets.

“We had to find an additional dye that we could excite using the same two lasers as before,” recalled Chandler. “There is a lot of crosstalk between dyes, which absorb, emit, and share energy between each other. As you add more dyes, the interactions are a significant factor in choosing the final dye set.”

A key tool here was fluorescent spectrometry, which helped the R&D staff eliminate dyes whose energy transfer was not compatible with the design. “Some dyes that would seem to be ideal based on wavelength weren’t very efficient in energy absorption and emitting, so they were ruled out,” noted Chandler.

Engineers in the company’s QC department also use spectrometers to inspect new dyes in manufacturing, and they employ spectrophotometers (for both Fourier-transform infrared spectroscopy and ultraviolet-visible spectroscopy) to assess beads and reagents used in the bead manufacturing process. In addition, they turn to high-pressure liquid chromatography to test dye purity.

At Luminex, the development of bead chemistry goes hand in hand with the design of the system hardware that analyzes the beads. “Moving from 100 beadsets to 500 for the FlexMAP 3D required a lot of back and forth between the instrument and chemistry teams,” said Chandler. “We had to make sure that the hardware engineers could capture the light from the dyes we were proposing.”

The Luminex systems feature an open architecture suitable for many applications, noted John Carrano, VP of R&D.

To address this optics design, the hardware engineers needed spectrophotometers to measure the spectra of the dyes and to precisely measure the blocking performance and edges of the optical filters. “Besides picking the right spectra to transmit to your detector, almost more important is what wavelengths you want to block from the detector,” explained Roth.

Equally crucial are the tests on the system’s lasers. Engineers in R&D and QC use Coherent ModeMaster beam analyzers to measure the M² rating, or beam quality factor, of lasers that the company buys from suppliers. The same instrument also helps the engineers calibrate the laser so it focuses on the precise spot size on a bead that is targeted for illumination.

Other test challenges stem from changes in FlexMAP 3D’s design. For example, the R&D team developed a patented air-pressure-driven fluid-delivery system that compensates for variations in fluid viscosity. To ensure that beads move at the required constant speed through the cuvette, the engineers installed a variable electronic pressure regulator and microprocessor, which automatically measure the sheath temperature and adjust the air pressure to compensate for viscosity change.

In developing that design, the engineers put the entire instrument in an environmental chamber, changing temperature and measuring flow rates. “This helped us create the pressure profile we need to apply across the temperature range the system is exposed to,” said Roth.

During the environmental test, the rate of fluid flow through the cuvette was measured in real time, using a digital oscilloscope connected to the red and green photodetectors. The time that a bead takes to transmit between the two separated laser spots is measured in microseconds.

In fact, said Roth, digital oscilloscopes are “probably the most important tool we have in this company as far as testing.” Scopes come into play in applications ranging from adjusting analyzer optics to measuring the voltage that detectors produce upon capturing the reflected light from the microspheres.

For example, oscilloscopes assist engineers in achieving optimal alignment of the laser beams, which is important for ensuring consistent results during assays. Said Roth: “We hook up the oscilloscope to the optical detectors and adjust the lasers and lenses to get the consistency we need from one bead pulse to the next, as seen on the oscilloscope screen.”

Beyond their arsenal of bench instruments, Luminex engineers also rely on a long menu of software packages to develop and test the company’s multiplexing instruments. Among the many examples cited by Roth:

• Microsoft Excel is used extensively for calculations, including the timing sequence of a new dual-syringe system that will boost throughput in the FlexMAP 3D system. In this design, one syringe aspirates the sample out of a well in the microtiter plate, while the other syringe dispenses another sample of microspheres through the cuvette.
• MathCAD was called on for numerous tasks, such as modeling the egg-shaped regions that distinguish one bead set from another. This was an essential step for setting up the classification algorithms for the bead sets.
• PSPICE design software helps electrical engineers simulate filters for the system’s analog electronics, while mechanical engineers turn to SolidWorks 3D CAD to model hardware assemblies and the analyzer’s enclosure.
• COSMOSFloWorks computational fluid dynamic software models fluid movement, while COSMOS finite-element analysis software provides thermal analysis of components such as the stainless-steel optics plate on which the lasers rest.
• LabView plays a role in many applications, including the design of a software-based accelerated life test for valves in the fluid-delivery system.
• Homegrown test applications, programmed in C++ and C#, test assemblies such as the positioning system responsible for moving the microtiter plate that contains the bioassay samples. The FlexMAP 3D system will handle both 96-well and 384-well plates, meaning that there could be 384 different patient samples in one well plate for in vitro diagnostics.
• Microsoft Visual Studio Test Edition, Bugzilla, and homegrown software emulators test the system firmware that runs embedded microprocessors, field-programmable gate arrays, and DSPs.

Digital oscilloscopes come into play in applications ranging from adjusting optics to measuring the voltage that detectors produce upon capturing the reflected light from the microspheres. Photo by Dan Bryant.

To perform this integrated testing, the Luminex team had to develop the key performance metrics and then set up a design of experiments on the machine to test those metrics and gather statistics to prove reproducible results. Among the key parameters that the system measures:

• throughput, or the amount of time it takes the system to read a 96-well plate, using a 100-plex-per-well standard;
• dynamic range, including the system’s ability to detect light in multiple light paths with different signals;
• coefficient of variance, a measure of the instrument’s precision;
• classification efficiency, or how well each bead is read by the instrument; and
• assay performance, typically involving known assays with readily recognizable signals.

One measurement that addresses many of these parameters is the dose response curve, which analyzes signals from the reporter dye (Phycoerythrin) coating on beads of the same mixture from an entire microtiter plate. “That kind of test allows you to look at limits of detection, dynamic range, classification efficiency, a certain level of multiplexing, and throughput,” said Carrano. “This is an example of a design of experiments that gives you a solid feel for the true functioning of the instrument.”

Luminex is now setting up its manufacturing line and QC system for the new FlexMAP 3D, according to Oliver Meek, the Luminex VP for Quality Assurance and Regulatory Affairs. Working with R&D, QC engineers developed the battery of tests to be used in manufacturing test and will rely on many of the same procedures and instruments that R&D used in developing the instrument. This includes 100% testing of all major subassemblies. Assembled units also will undergo additional tests, including hipot.

To a large extent, the Luminex multiplexing instrument itself serves as the most important tester. In demo batches, Luminex engineers use the analyzer and its software to test parameters such as classification efficiency and how well each bead is read by the instrument. Courtesy of Luminex.

While the Luminex 200 will continue to be a workhorse, the company expects FlexMAP 3D to open up even more opportunities, ranging from high-volume clinical diagnostic labs that serve doctors and hospitals to tissue typing for transplants and the emerging field of molecular diagnostics. “The FlexMAP 3D is important to the research community,” said Steven Binder, director of Technology Development for Bio-Rad Laboratories’ Clinical Diagnostics Group. “The new design, supporting 500 targets, moves Luminex into a larger space regarding protein discovery.”

R&D VP Carrano points to the company’s open architecture as a key Luminex advantage. “Different users, whether they come from in vitro diagnostics, drug discovery, food safety, or genetic research, can use our platform without any changes. And that puts us in a very good position for continued success.”