Abstract
Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.
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Acknowledgements
We thank O. Hurtado, K. Eken, K. Richter and A. Gupta for microfabrication support. We thank C. Vanderburg for help with nucleic acid analysis and the use of the Harvard NeuroDiscovery Center Agilent Bioanalyzer 2100. We thank the University of Florida technical staff (A. Abouhamze, C. Tannahill and R. Ungaro) for managing clinical implementation of the microfluidic devices. K.T.K. was supported by a US National Institutes of Health (NIH) training grant T32 GM-007035-32. These studies were supported by the US NIH Inflammation and the Host Response to Injury Large Scale Collaborative Project, U54 GM-062119, BioMEMS Resource Center P41 EB-002503 and Proteomics Research Resource for Integrative Biology RR018522. The ex vivo stimulation studies and genomics protocol development were partially supported by US National Institutes of Health grants R01-GM-36214 and P01 HG000205, respectively. The proteomics work was performed in the Environmental Molecular Sciences Laboratory, a US Department of Energy Office of Biological and Environmental Research national scientific user facility on the Pacific Northwest National Laboratory (PNNL) campus. PNNL is multiprogram national laboratory operated by Battelle for the Department of Energy under contract number DE-AC05-76RLO 1830.
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K.T.K. performed and analyzed experiments. W. Xiao and W.-J.Q. preformed microarray and proteomics analyses. K.T.K., W. Xu, J.W., M.N.M., W. Xu, A.R., E.A.W., L.L.M., D.I., B.H.B., R.W.D. & M.T. designed genomic experiments. K.T.K., W.-J.Q., D.G.C. II and R.D.S. designed proteomic experiments. J.G., S.P.F., A.E.R. and R.G.T. aided in clinical sample studies at Massachusetts General Hospital. K.T.K., C.M.-G., A.D., L.L.M., W. Xiao, M.N.M., J.W., W.-J.Q., B.O.P., D.G.C. II, A.E.R., P.E.B. and M.T. designed, conducted and analyzed the ex vivo stimulation experiment. K.T.K., C.M.-G., W. Xiao, M.N.M. and L.L.M. wrote the manuscript. All authors contributed to the final editing of the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–3, Supplementary Tables 1–2,4,6-7,9-12 and Supplementary Methods (PDF 857 kb)
Supplementary Table 3
Significantly perturbed genes and proteins following ex vivo stimulation of whole blood by LPS or GM+I. (PDF 1485 kb)
Supplementary Table 5
Gene expression across all subjects for the genes in Figure 2f comparing microfluidic isolation with Ficoll-dextran isolation (PDF 607 kb)
Supplementary Table 8
Significantly perturbed genes after severe trauma injury (PDF 5588 kb)
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Kotz, K., Xiao, W., Miller-Graziano, C. et al. Clinical microfluidics for neutrophil genomics and proteomics. Nat Med 16, 1042–1047 (2010). https://doi.org/10.1038/nm.2205
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DOI: https://doi.org/10.1038/nm.2205
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