|Send to printer »|
Feature Articles : Apr 15, 2011 ( )
Flow Cytometry Stakes Out New Territory
Reduction in Systems' Complexity Opens Up Diverse Applications for Reliable Technology!--h2>
Flow cytometers are becoming the instrument of choice in a growing number of applications, including marine biology, cancer biology, drug screening, cell-cycle analysis, intracellular analysis, rare event detection, immunophenotyping, and stem cell research. Increasingly firms are developing benchtop systems that can be operated with minimal training while delivering enhanced capabilities, which further increase their utility.
Flow cytometry gathers data not only on cell numbers, but also on such cellular parameters as size, complexity, phenotype, health, and functionality. It has also entered the clinical laboratory for diagnostics and determining patient response to drug regimen. “Flow cytometry is a fundamentally powerful tool,” emphasizes J. Clark Mason, senior director for global marketing for BD Biosciences.
“Getting a more granular perspective of what’s going on in cells is a key driver,” in the growth of flow cytometry applications, according to Jason Whalley, market manager for flow cytometry at EMD Millipore. Ascertaining cause and effect of various actions, not just on a single cell but upon entire populations, relates directly to systems biology and therefore, increases the potential utility of flow cytometry.
“Combining immune phenotyping with the molecular world is the major topic we are working on,” according to Wolfgang Mann, Ph.D., group manager for the single-cell product line at Beckman Coulter. “It’s a very new idea.”
Dr. Mann is using flow cytometry to detect and characterize circulating tumor cells in the bloodstream. “Circulating tumor cells are a rare species of cells. They are present at a rate of a few cells per milliliter. The problem is to pick the few cells that can be expressed in human phenotyping, so you need a high-speed method to screen the cells and find the rare ones. Flow cytometry is the method of choice to isolate them.
“The challenge of isolating rare cells could be applied to other fields,” Dr. Mann insists, suggesting stem cell analysis, as stem cells seem to be related to tumor cells and tumorigenesis. Another application could be the analysis of fetal cells circulating in material blood. “If you want to study cell populations, like human B cells or other homogenous cell populations, one should use a flow sorter, like the Astrios, to prepare these cells, in high quality, for molecular analysis.
“In the past, there was a gap between flow cytometers expertise and people working in genetics. This gap is being closed.”
“Many applications have been around a long time, but were not fully exploited because of the complexity of flow cytometry systems,” says William Gutierrez, director of marketing for Blue Ocean Biomedical, which recently received ISO 13485: 2003 manufacturing certification, paving the way for commercialization of its load & go™ flow cytometers. “Flow cytometry manufacturers generally haven’t taken the lead in integrating and automating the applications. They leave it to customers,” resulting in assays that aren’t standardized. “We hope to be a catalyst in that area.
“Our objective is to take repetitive testing in both research and clinical environments and offer fully automated solutions to the user.” Blue Ocean’s first assay is a lymphocyte subset panel assay for immune monitoring, and assays are in development for stem cell counting for bone marrow transplants, leukocyte counting for blood banks, and patient transplant monitoring. A hematology test kit to reduce the need for WBC differentials is also being developed.
“We’re working with our customers to develop and simplify assays, and to customize some of theirs. The CR Tools package lets Blue Ocean work with customers to create custom load & go assays.”
“Flow cytometers are going into smaller labs now,” Mike Olszowy, business leader for flow cytometer systems at Life Technologies emphasizes, noting their transformation from very expensive, difficult to use, room-sized systems to affordable, benchtop apparatus that can be operated with minimal training.
Whalley agrees. “The expansion in the research market is at the entry level, centered around more intuitive units that make it applicable to individual labs.” In that environment, ease of use is paramount. “In general, an entry-level system with six color detection and eight parameters meets the needs of about 85 percent of users.”
“The bottleneck today is data analysis.Therefore, EMD Millipore developed a complete solution based around the easyCyte flow cytometer, InCyte software, and the FlowCellect family of assays. Those assays are currently focused around cell health. There is also great emphasis on cell signaling, immunology, and stem cell assays, he reports. The InCyte software includes heat map functions and analyzes 96-well plates and six parameters in minutes.
The EasyCyte single sample series was introduced last fall. This family of flow cytometers eliminates the need for sheath fluid, thus letting researchers use less sample and generate less waste. For a machine running 8 hours, that means less than 80 mL of waste versus 8 L, Whalley says.
BD, which commercialized the industry’s first cell sorter in 1973, is addressing both extremes of the markets, Mason says. New product announcements and improvements to the existing platforms will be made later this spring. At one end of the spectrum, BD is developing instruments for individual researchers that Mason characterizes as “less high touch, more personal instruments that are affordable for individual researchers.”
Designed for convenience, these user-friendly models utilize new hardware and software approaches. “A number of them are in beta testing now.”
At the other end, Mason says, “many flow cytometers run in core labs with hundreds of users and multiple applications. They want an instrument they can configure to respond to current and future needs, with the lowest possible background and the highest possible sensitivity.” To meet the needs of these experts, Mason says, “we collaborate with key researchers and opinion leaders, and have a special order program to precisely meet their needs.”
For example, the BD FACSAria™ III Cell Sorting System with six lasers was introduced last summer and included upgrade pathways for the existing user base.
BD’s Special Order Group shipped the first nine-laser, user-operated flow cytometer last November. Called the BD LSR-II Flow Cytometer, it is being used by the Centenary Institute in Australia to detect and analyze rare regulatory T cells. As Advanced Cytometry Facility academic director professor Nick King said in a news release at the time, “The extra lasers on the LSR-II Flow Cytometer will give researchers a greater range of labels to analyze cells so they will only have to run one sample.”
That allows more accurate and more direct measurements, reduces the need to infer relationships, and uses less material than previous flow cytometers. Users report getting more information in a single analysis than was possible before using multiple analyses that required 10 times the amount of material.
BD Biosciences’ multifaceted approach to flow cytometer design mirrors that of the broader industry, which is simultaneously improving ease of use, increasing capacity, and improving the technology itself.
“Although lasers may be considered the powerhouse of the instrument,” Mason says, flow cytometer manufacturers are also making improvements in fluidics, electronics, and signal detection. The just-released FACSAria III Cell Sorting System, for example, uses what Mason refers to as “the next-generation gel-coupled cuvette flow cell” to ensure that the lasers are focused precisely and generate maximum signal. Its optical system also features enhanced sensitivity and resolution to identify dim cell populations, side populations and DNA cell cycle analyses.
Life Technologies’ Attune™ system takes a different approach, using acoustic particle focusing to keep cells of interest in the center of the laser beam, thus producing more accurate reads than the hydrodynamic focusing approach that historically has been used. Hydrodynamic focusing allowed cells to disperse throughout the cylinder, so they were subject to varying intensities of laser light, depending upon where in its beam they were. Therefore, the signaling was imprecise.
“Attune is up to 16 times faster than conventional hydrodynamic focusing,” Olszowy adds. “In cerebral spinal fluid, for example, cells are very dilute and run in hours using hydrodynamic focusing versus minutes with acoustic focusing.” The system has a variable flow rate of 25 to 1,000 µL per minute and can run up to 20,000 cells per second.
The acoustic system isn’t required to use heavy sheathing fluid, which also lets this system use longer transit times and higher throughput, permitting better interrogation of each cell in a sample and the analysis of more cells. It also uses less fluid and has the capability of moving stained cells into clean buffer areas, thus eliminating the need for washing.
Flow cytometry is becoming increasingly powerful as the technologies surrounding it gain power, Olszowy says. Now it can analyze up to 26 parameters simultaneously, separating substances into distinctly fine populations.
“Researchers may realize how simple these systems are becoming,” Gutierrez adds. Newer devices are performing multiple interrogations and producing a high volume of data more efficiently and at a lower cost. Consequently, many more scientists will be able to take advantage of this dynamic technology.
© 2016 Genetic Engineering & Biotechnology News, All Rights Reserved