December 1, 2008 (Vol. 28, No. 21)

Flow cytometry has come a long way since it was first conceived by Wolfgang Göhde at the University of Münster in 1968. Originally known as Impulscytophotometrie, it became better known after a name change ten years later. The principle is straightforward, based on the use of a laser beam of light focused on a stream of fluid to detect suspended particles.

The scattering of the light beam by the particles as well as the emission of light by fluorescent tags bound to them are picked up by detectors and the signal analyzed by the computer innards of the cytometer. As the years have passed, the technology has improved dramatically through advances in hardware and software and the development of better fluors including molecules that can be presented to living cells.

“Manufacturers have responded to the needs of researchers by developing faster systems that can measure more variables over shorter time periods,” according to Gillian Bryne, product specialist at Applied Cytometry. The improvements in instrument technology, however, have run way ahead of the software that analyzes the deluge of data pouring out the new, highly advanced cytometers. For example, rare-event analysis such as detecting tumor cells in peripheral blood in ancient cytometers with a maximum acquisition speed of only 1,000–3,000 events per second is slow and tedious.

With frequencies of one stem cell in a population of millions of circulating lymphocytes, the time required to build a sufficient store of data could be prohibitive. New, high-powered machines put this number to shame with their acquisition speed of 50,000 or more events per second. Another trend that Bryne cites is the increasing number of fluors and multiple lasers that can capture as many as 18 parameters per event simultaneously.

Doctors Albert and Vera Donnenberg of the University of Pittsburgh say that Applied Cytometry software, which uses scalable parallel processing to maximize data analysis throughput, makes the analysis of rare events routine. According to Bryne, VenturiOne software allows up to 400 files to be fully studied simultaneously in less than ten seconds. This speed is achieved through the use of patent-pending multiprocessor technology, which forms the basis of Applied Cytometry’s platform.

VenturiOne version 3 also produces enhanced reports, generated from within the software itself. Because of the system’s capability to deal with large numbers of files, the new version of VenturiOne can attack data generated from multiple well formats, Bryne says. As a standard, the new software works in background calculation mode, which means that files are automatically analyzed. This feature considerably speeds up the data analysis process, she adds. When larger data files (>100 megabytes) need to be analyzed, for example in rare-event detection, however, the background mode can be switched off. This allows files to be analyzed sequentially, thereby avoiding any delays in reporting. The rapid performance of the system allows immediate review of alternative models, identifying classifier populations and outcomes, an approach that would not be practical with conventional, slower software packages.

The analytical problems of older systems are exacerbated when multiple fluorochromes are used to address the signal-to-noise ratio problem, which arises when one attempts to distinguish rare events from background. Especially with tumor material, dying cells and normal cells within the tumor can confound the analysis, requiring additional fluorescent parameters to identify and remove these events from the analytical picture Now with the improved software and by increasing the number of fluorochromes used to detect the signal, background noise can be detected and eliminated.

The Donnenbergs extol the efficacy of VenturiOne software in their investigations of cancer-causing stem cells. “Using the old software, we were only able to look at the data in a limited way, and we experienced countless crashes,” Dr. Albert Donnenberg says. “With VenturiOne, we increased our analytical speed by a factor of ten. It was amazing.”

“Today, new desktop computers have two cores, but they are not utilized to their full extent by conventional software,” Tony Burpee, CEO of Applied Cytometry, adds. “This is because the software does not tie the cores together in a fashion that allows them to perform computing tasks simultaneously. Our software does this, which means that it can tremendously accelerate not only biological jobs such as evaluating data generated by labeling with multiple fluorchromes but also other tasks involving massive amounts of data such as drug screening.”

“By the 2000s, the easy-to-understand technology of flow cytometry had not yet translated into an easy-to-use instrument,” states Jack Ball, CCO at Accuri Cytometers. Ball and his colleagues feel that flow cytometry has always been a poor stepsister to the other, more widely accepted instrumentation such as PCR and standard light microscopy. Conventional cytometers have been consigned to core facilities due to their expense, high maintenance costs, and expensive training required for operators.

In 2004 Accuri set out to design an instrument that would overcome the drawbacks of conventional devices by improving on data quality, affordability, and software while reducing complexity. Its solution was the C6 Flow Cytometer® System, which the company believes optimizes and locks together the four interdependent components of fluidics, optics, electronics, and software.

Innovations include the use of peristaltic pumps with pulse dampeners and dynamic feedback to replace conventional syringe pumps or pressurization; a simplified and stable optical design; advanced electronics and intuitive software that does not require training classes, while fitting the needs of both novice and expert.

The practical results of the application of the C6 and CFlow® software were described in a poster presentation given by Clare Rogers, Ph.D., and her colleagues at Accuri Cytometers at the “International Congress of the Society for Analytical Cytology.”

Using 24-bit digital-signal processing, the flow cytometer is able to obtain much more accurate representation and processing of fluorescence and scatter signals. Because of the wider dynamic range and greater signal resolution of the C6, there is no need to adjust detector gain or voltage, thereby saving time, precious sample material, and obviating the problem of over- or underamplification of signals, Dr. Rogers explained. Furthermore, the zoom function of the C6 allows the user to identify and precisely focus on populations of interest that might otherwise go unnoticed, as shown by the group’s analysis of rare subpopulations such as monocyte precursors.

Accuri Cytometers says that it designed the C6 flow cytometer to overcome the drawbacks of conventional devices by improving on data quality, affordability, and software while reducing complexity.

Clinical Trials

ACM-Pivotal Global Central Laboratory is a worldwide clinical facility that adopts a focused approach to keep clinical research studies on schedule and budget,” states Andrew Botham, Ph.D., senior scientist for R&D. The company participates in clinical trials across the globe with operations that extend to more than 60 countries. Flow cytometry multiplexing is a major component of the firm’s activities.

Flow cytometry lends itself especially to the monitoring of HIV patients through the analysis of CD4/CD8 ratios. These markers of the T-helper cells and cytotoxic T cells, respectively, are important indicators of the patient’s health status, the progress of the disease, and the effect of various treatment regimes.

Another disease that flow cytometry has been of great value in analyzing is paroxysmal nocturnal hemoglobinuria (PNH). This chronic hemolytic anemia is a rare, acquired condition of unknown etiology. The basis for paroxysmal nocturnal hemoglobinuria lies in a deficiency of the regulatory protein, decay acceleration factor. This protein inhibits the formation of complement C4 convertase and is attached to the cell membrane through a glycophosphotidylinositol (GPI) anchor. Individuals who manifest the condition are deficient in proteins anchored by GPI, which serve as markers for flow cytometry, now the gold standard for diagnosing this disease.

According to Dr. Botham, flow cytometric analysis of cell populations is the most accurate and convenient method for diagnosing and monitoring patients in order to obtain an accurate prognosis.

“Another important area in which flow cytometry has made a major impact is in the assessment of factors such as cytokines, present in serum,” Dr. Botham continues.

“By using fluorescent beads of different sizes conjugated to appropriate antibodies, we can simultaneously quantify up to 20 analytes in a single sample.” Not only is this a rapid and convenient approach, it requires small samples of serum (25 µL in total compared to 100 µL per analyte) or more for a conventional ELISA assay.

The advances in flow cytometry hardware and software have led to a wealth of new applications that can speed basic research and drug development. Companies need to be aware of the range of questions that can now be conveniently investigated with this greatly improved technology. The new options lend themselves especially to monitoring cancer of the blood, HIV infections, and various hereditary conditions. Rapid and non-invasive, flow cytometry will continue to offer an alternative approach to biotechnological investigations.

According to ACM-Pivotal, flow cytometric analysis of cell populations is the most accurate and convenient method for diagnosing and monitoring paroxysmal nocturnal hemoglobinuria patients in order to obain an accurate prognosis.

Rugged Hardware for Use in the Field

The invention of the sheathless fluidics flow cell by Philippe Goix led to the founding of Guava Technologies in 1998 and the introduction of the first truly, easy-to-use benchtop flow cytometer in 2001, according to Paul Wheeler, Ph.D., vp for global sales and marketing.

Guava’s microcapillary flow cytometer comes with assay-specific reagents and software. In addition, Guava systems do not require sheath fluid, and thereby can accommodate smaller sample volumes, Dr. Wheeler adds. This feature translates into less waste, lower daily operating costs, and more convenient set up and maintenance than traditional flow cytometers.

One important area of global healthcare that lends itself to the Guava Technologies flow cytometry platforms is the HIV/AIDS global pandemic.

Dr. Wheeler cites statistics compiled by WHO documenting that almost 65% of new infections occur in highly inaccessible regions of the world such as Africa, where not only adults but children are infected. Adults are monitored for disease progression via their absolute CD4 T-cell counts. Absolute CD4 T-cell counts in children undergo variation with age, however, and development and an accepted indicator for this patient population is the CD4 percentage of total lymphocytes.

Guava Technologies has developed an assay for the analysis of both pediatric and adult HIV samples; the Guava AutoCD4/CD4% Assay, that simultaneously detects both total CD4 and CD4% T-cell counts.

The assay also automatically sets analysis gates, greatly enhancing ease-of-use and reducing operator error. “This auto-gating feature, along with the ability to simultaneously get both assay results, greatly simplifies training and assay costs,” says Dr. Wheeler.

The Guava AutoCD4/CD4% System has been widely evaluated in the clinic on both adult and pediatric samples as documented at the “AIDS Conference” held in Mexico City earlier this year. The Guava AutoCD4/CD4% System demonstrated excellent accuracy and precision when compared with established clinical methods, Dr. Wheeler notes.

“We believe that the accuracy and precision of our latest technology coupled with its simplicity and robustness make it an attractive alternative to traditional methods of T-cell enumeration for both adult and pediatric samples.”

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