For years, flow cytometry has represented a valuable instrument to examine single cells, but the analysis of large numbers of samples is still technically and computationally challenging. Bruce S. Edwards, Ph.D., research professor of pathology and director of the shared flow cytometry resource, together with Larry A. Sklar, Ph.D., Regents professor of pathology and director of the center for molecular discovery at the University of New Mexico, and other collaborators, recently developed HyperCyt®, a high-throughput flow cytometry platform that creates temporal gaps in the collection process by introducing air bubbles between distinct samples, before delivering them to the flow cytometer.
Instead of collecting and saving each data point individually during the analysis, HyperCyt continuously collects data for the whole plate and stores the information as a single file. This approach eliminates delays that are otherwise caused when separately collecting data for each sample of large datasets.
“This system provides a new way to analyze the data. The technology that we developed decreased the time requirement about 20 fold, and we can now analyze 40 samples in a minute,” Dr. Edwards said. “We can routinely sample an entire 384-well plate without stopping and only save the data at the end.”
By using this approach, Dr. Edwards and collaborators have developed a high-throughput phenotypic assay to screen for small molecules that induce intracellular granularity, which is one of the known markers of cellular apoptosis, and from over 24,000 compounds that were screened they identified 95 molecules of interest.
“The biggest challenge will be to develop software that is able to handle the huge volume of data generated during the screening process,” predicted Dr. Edwards. “In some experiments, each well of a 384-well plate performs up to seven assays, and each of those sets of data can tell us about 10,000–50,000 cells, which represents a lot of data to analyze and archive, and this information overload is the real challenge of the future.”
The University of New Mexico flow-cytometry screening program was recently selected into the Molecular Libraries Probe Production Centers Network, a nationwide consortium of nine small molecule screening centers that proposes to use small chemical compounds to understand different cellular processes and identify new therapeutic targets.
“Our groups and some others have been proposing to use much more sophisticated classification tools to separate signals in a more sophisticated way,” said J. Paul Robinson, Ph.D., professor of immunopharmacology and biomedical engineering and director of the Purdue University cytometry laboratories.
At the Boston meeting, Dr. Robinson emphasized the need to develop more advanced hardware and software applications to facilitate high-throughput assays such as the ones needed for drug screening and functional studies, which often require an additional level of sophistication. “The analytical tools that we have are not adequate for the needs. I think this is a fundamental area that needs change, and I think it will be changed.”
One of the recent advances has been the development of multispectral flow cytometry, which provides several advantages over existing approaches. “Another thing that I think will happen over the next few years is an approach toward multispectral analysis,” Dr. Robinson noted. “I don’t think multispectral instruments will replace current systems, I think they represent a different tool to supplement existing technologies. By using hyperspectral fingerprinting, we will be able to identify certain aspects of cells that are more difficult to extract.”
An important application of flow cytometry is to monitor CD4 lymphocytes, the primary targets of HIV, during the course of the infection. CD4 levels are informative about the stage of the infection in seropositive individuals, help monitor the response to antiretroviral therapy, and recently were also shown to predict the risk to develop malignant tumors during the course of the infection.
While the level of CD4 lymphocytes needs to be regularly monitored during therapy, ideally once every three to four months, in many locations worldwide this is difficult or impossible to accomplish due to high costs. It has been estimated that only approximately 2–3% of the approximately 35 million HIV-positive individuals from developing countries receive adequate treatment.
The Cytometry for Life Program, founded in 2006 by Dr. Robinson and his colleague Gary Durack, intends to construct robust, user-friendly instruments that would reduce costs and make individual CD4 tests affordable in underserved regions worldwide.
To bring attention to the importance of this issue, Dr. Robinson recently climbed the world’s tallest mountain. “It was easier to climb Mount Everest than to solve this problem,” he said. “We need an instrument that is easy to use and has low costs. The only solution is the ultimate solution. And the ultimate solution might well be something ridiculously simple and extremely cheap.”