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Microfluidic device allows collection, analysis of hard-to-handle immune cells

Ability to gently collect neutrophils may improve understanding of immune response to serious injury

A team led by Massachusetts General Hospital (MGH) scientists has developed a new microfluidic tool for quickly and accurately isolating neutrophils – the most abundant type of white blood cell – from small blood samples, an accomplishment that could provide information essential to better understanding the immune system's response to traumatic injury. The system, described in a Nature Medicine paper that received advance online release, also can be adapted to isolate almost any type of cell.

"Neutrophils are currently garnering a lot of interest from researchers and clinicians, but collecting and processing them has been a real challenge," says Kenneth Kotz, PhD, of the MGH Center for Engineering in Medicine, lead author of the study. "This tool will allow a new range of studies and diagnostics based on cell-specific genomic and proteomic signatures."

Part of the body's first-line defense against injury or infection, neutrophils were long thought to play fairly simple roles, such as releasing antimicrobial proteins and ingesting pathogens. But recent studies find their actions to be more complex and critical to both chronic and acute inflammation, particularly the activation of the immune system in response to injury.

Studying patterns of gene expression and protein synthesis in neutrophils could reveal essential information about the immune response, but gathering the cells for analysis has been challenging. Standard isolation procedures take more than two hours and require relatively large blood samples. Neutrophils also are sensitive to handling and easily become activated, changing the molecular patterns of interest, and they contain very small amounts of messenger RNA, which is required for studies of gene expression.

Building on their experience developing silicon-chip-based devices that capture CD4 T cells for HIV diagnosis or isolate circulating tumor cells, Kotz's team developed a system that gathers a neutrophil-rich sample from microliter-sized blood samples in less than 5 minutes, reducing the risk of disturbing cells in the process. To meet the requirements for speed and precision, the researchers completely redesigned the geometry, antibody-based coating and other aspects of the cell-capture module at the heart of the device. The samples collected were successful in revealing differences in gene and protein activity relevant to the cells' activation status.

While the laboratory tests were encouraging, samples from critically injured patients need to be handled and processed in real-world clinical environments. Through the efforts of study co-author Lyle Moldawer, PhD, of the University of Florida College of Medicine, the devices were tested at six additional sites participating in a major national study of the immune response to injury. Analyzing samples from 26 patients with serious burns or other traumatic injuries revealed complex gene expression patterns that shifted during the 28 days after injury, probably reflecting complex interactions between various immune system components.

Ronald Tompkins, MD, ScD - chief of the MGH Burns Service, a study co-author and principal investigator of the "Inflammation and the Host Response to Injury" initiative - says, "This technology has been widely implemented in our 'Glue Grant Program,' with a major impact. The ability to capture specific cells in a routine clinical environment rapidly and accurately offers a possible change in the paradigm of normal clinical diagnostics." The Nature Medicine study is part of the National-Institute-of-Health-funded, large-scale collaborative research program and involved its seven clinical sites and seventeen academic institutes across the United States.

Kotz says, "Until now, it's been logistically impossible to study neutrophils to the extent we have in this paper." He notes that their analysis of neutrophil samples from trauma patients is the largest such investigation to date and adds, "This technology – which is much faster and gentler than current approaches to isolating cells – can be scaled and modified to capture just about any cell type, and we're working to apply it to other cell-based assays."

Mehmet Toner, PhD, director of the BioMEMS Resource Center in the MGH Center for Engineering in Medicine, is senior author of the Nature Medicine article. In addition to Tompkins and Moldawer, primary co-authors are Aman Russom, Alan Rosenbach, Jeremy Goverman, Shawn Fagan and Daniel Irimia, MGH; Wenzong Xiao, Weihong Xu, Julie Wilhelmy, Michael Mindrinos, and Ronald Davis, Stanford Genome Technology Center; Carol Miller-Graziano, Asit De and Paul Bankey, University of Rochester School of Medicine; Wei-Jun Qian, Brianne Petritis, David Camp, and Richard Smith, Pacific Northwest National Laboratory; Elizabeth Warner, University of Florida College of Medicine; and Bernard Brownstein, Washington University of St. Louis.

Massachusetts General Hospital, established in 1811, is the original and largest teaching hospital of Harvard Medical School. The MGH conducts the largest hospital-based research program in the United States, with an annual research budget of more than $600 million and major research centers in AIDS, cardiovascular research, cancer, computational and integrative biology, cutaneous biology, human genetics, medical imaging, neurodegenerative disorders, regenerative medicine, systems biology, transplantation biology and photomedicine.

Sue McGreevey | EurekAlert!
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