Flow cytometry has reached a stage in its evolution where fundamental technical improvements are paying huge rewards.
“We can now go a lot farther, and delve more deeply into fundamental scientific problems,” says Jeannine Holden, M.D., director of scientific affairs for flow cytometry at Beckman Coulter Life Sciences. “We’re reaching the point where signal to noise is not about instruments, but about the biology.”
Multilaser, multidetector systems, combined with improved chemistries and fluorophores, have emerged in rapid succession, enabling scientists to answer scientific questions that were previously unapproachable.
Flow is now capable of analyzing and sorting microparticles, which was previously impractical due to instrument variability and noise. Microparticles are heterogeneous, submicrometer, extracellular vesicles that express antigens specific to their cells of origin. Illnesses as diverse as sepsis, preeclampsia, and heart disease are associated with elevated microparticle populations.
“We’ve known that microparticles were significant, but we previously lacked the tools to study them,” Dr. Holden adds. Beckman Coulter’s AstriosEQ cell sorter, for example, identifies and sorts microparticles.
Flow cytometry has traditionally used cell surface markers to identify and sort cells, but with a few exceptions observing intracellular events, or intra-extracellular interactions, involved cumbersome workflows. Recent advances now allow the simultaneous examination of surface and intracellular events, and in some cases their interactions.
Several vendors offer kits for achieving this. Beckman Coulter’s PerFix reagent kit reduces prep and handling time by about 50%, according to the company. A variation on this theme, PerFix Expose, covers phosphorylated targets, which opens up possibilities for studying cell signaling.
“The ability to study surface and intracellular markers simultaneously, in many thousands of cells responding differently to experimental stimuli, in one sample, has been a breakthrough,” Dr. Holden explains. After sorting, researchers may recover selected populations for further study. “Flow doesn’t provide the detail of microscopy, but it hits a sweet spot for maximizing information from large numbers of cells.”
Vendors of flow cytometry reagents have done well to standardize kits to eliminate dispensing-related variability. Global diagnostics companies, for example, are mindful that assays be consistent across the hall or across the globe.
One way to eliminate reagent dispensing issues entirely is through wells or tubes that contain reagents in dry form out of the box. The sample becomes, in effect, the reconstitution buffer. Through its July 2013 acquisition of ReaMetrix, Beckman Coulter became a leading purveyor of stable, dry, ready-to-use reagents in familiar container formats. “The rigor that’s been the standard in clinical work is entering research as well,” Dr. Holden comments. “The multiparameter aspect of flow no longer represents a multiparameter means of introducing variability.”