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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Nathan, C. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6, 173–182 (2006).
Cassatella, M.A., Gasperini, S. & Russo, M.P. Cytokine expression and release by neutrophils. Ann. NY Acad. Sci. 832, 233–242 (1997).
McDonald, P.P., Bald, A. & Cassatella, M.A. Activation of the NF-κB pathway by inflammatory stimuli in human neutrophils. Blood 89, 3421–3433 (1997).
Burczynski, M.E. & Dorner, A.J. Transcriptional profiling of peripheral blood cells in clinical pharmacogenomic studies. Pharmacogenomics 7, 187–202 (2006).
Calvano, S.E. et al. A network-based analysis of systemic inflammation in humans. Nature 437, 1032–1037 (2005).
Laudanski, K. et al. Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways. Proc. Natl. Acad. Sci. USA 103, 15564–15569 (2006).
Nauseef, W.M. Isolation of human neutrophils from venous blood. Methods Mol. Biol. 412, 15–20 (2007).
Elghetany, M.T. & Davis, B.H. Impact of preanalytical variables on granulocytic surface antigen expression: a review. Cytometry B Clin. Cytom. 65, 1–5 (2005).
Cassatella, M.A. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 16, 21–26 (1995).
Cheng, X. et al. A microchip approach for practical label-free CD4+ T-cell counting of HIV-infected subjects in resource-poor settings. J. Acquir. Immune Defic. Syndr. 45, 257–261 (2007).
Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 1235–1239 (2007).
Lyons, P.A. et al. Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification. BMC Genomics 8, 64 (2007).
Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7, 3 (2006).
DeForge, L.E., Kenney, J.S., Jones, M.L., Warren, J.S. & Remick, D.G. Biphasic production of IL-8 in lipopolysaccharide (LPS)-stimulated human whole blood. J. Immunol. 148, 2133–2141 (1992).
Pelletier, M. et al. Evidence for a cross-talk between human neutrophils and TH17 cells. Blood 115, 335–343 (2009).
De, A.K. et al. Selective activation of peripheral blood T cell subsets by endotoxin infusion in healthy human subjects corresponds to differential chemokine activation. J. Immunol. 175, 6155–6162 (2005).
Gosselin, E.J., Wardwell, K., Rigby, W.F. & Guyre, P.M. Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-γ and IL-3. J. Immunol. 151, 1482–1490 (1993).
Cobb, J.P. et al. Application of genome-wide expression analysis to human health and disease. Proc. Natl. Acad. Sci. USA 102, 4801–4806 (2005).
Singh, R. et al. Microarray-based comparison of three amplification methods for nanogram amounts of total RNA. Am. J. Physiol. Cell Physiol. 288, C1179–C1189 (2005).
Kotz, K., Cheng, X. & Toner, M. Cell capture using a microfluidic device. J. Vis. Exp. published online, doi:10.3791/320 (1 October 2007).
Covert, M.W., Leung, T.H., Gaston, J.E. & Baltimore, D. Achieving stability of lipopolysaccharide-induced NF-κB activation. Science 309, 1854–1857 (2005).
Theilgaard-Mönch, K. et al. The transcriptional program of terminal granulocytic differentiation. Blood 105, 1785–1796 (2005).
Kobayashi, S.D., Voyich, J.M., Braughton, K.R. & DeLeo, F.R. Down-regulation of proinflammatory capacity during apoptosis in human polymorphonuclear leukocytes. J. Immunol. 170, 3357–3368 (2003).
Fessler, M.B., Malcolm, K.C., Duncan, M.W. & Worthen, G.S. A genomic and proteomic analysis of activation of the human neutrophil by lipopolysaccharide and its mediation by p38 mitogen-activated protein kinase. J. Biol. Chem. 277, 31291–31302 (2002).
Zhang, X. et al. Gene expression in mature neutrophils: early responses to inflammatory stimuli. J. Leukoc. Biol. 75, 358–372 (2004).
Malcolm, K.C., Arndt, P.G., Manos, E.J., Jones, D.A. & Worthen, G.S. Microarray analysis of lipopolysaccharide-treated human neutrophils. Am. J. Physiol. Lung Cell. Mol. Physiol. 284, L663–L670 (2003).
Kobayashi, S.D., Voyich, J.M., Whitney, A.R. & DeLeo, F.R. Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor. J. Leukoc. Biol. 78, 1408–1418 (2005).
Ong, S.E. & Mann, M. Mass spectrometry–based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).
de Godoy, L.M. et al. Comprehensive mass-spectrometry–based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).
Zhang, X. et al. Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin. Electrophoresis 27, 1659–1668 (2006).
Berger, T. et al. Lipocalin 2–deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 103, 1834–1839 (2006).
Meheus, L.A. et al. Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages. J. Immunol. 151, 1535–1547 (1993).
Kotz, K., Cheng, X. & Toner, M. PDMS device fabrication and surface modification. J. Vis. Exp. published online, doi:10.3791/319 (1 October 2007).
Li, C. & Wong, W.H. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl. Acad. Sci. USA 98, 31–36 (2001).
Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121 (2001).
Zimmer, J.S., Monroe, M.E., Qian, W.J. & Smith, R.D. Advances in proteomics data analysis and display using an accurate mass and time tag approach. Mass Spectrom. Rev. 25, 450–482 (2006).
Qian, W.J., Jacobs, J.M., Liu, T., Camp, D.G. II & Smith, R.D. Advances and challenges in liquid chromatography–mass spectrometry–based proteomics profiling for clinical applications. Mol. Cell. Proteomics 5, 1727–1744 (2006).
Petyuk, V.A. et al. Spatial mapping of protein abundances in the mouse brain by voxelation integrated with high-throughput liquid chromatography–mass spectrometry. Genome Res. 17, 328–336 (2007).
Polpitiya, A.D. et al. DAnTE: a statistical tool for quantitative analysis of -omics data. Bioinformatics 24, 1556–1558 (2008).
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.
The authors declare no competing financial interests.
Supplementary Figures 1–3, Supplementary Tables 1–2,4,6-7,9-12 and Supplementary Methods (PDF 857 kb)
Significantly perturbed genes and proteins following ex vivo stimulation of whole blood by LPS or GM+I. (PDF 1485 kb)
Gene expression across all subjects for the genes in Figure 2f comparing microfluidic isolation with Ficoll-dextran isolation (PDF 607 kb)
Significantly perturbed genes after severe trauma injury (PDF 5588 kb)
About this article
Semiquantitative Proteomics Enables Mapping of Murine Neutrophil Dynamics following Lethal Influenza Virus Infection
The Journal of Immunology (2019)
Scientific Reports (2019)
A Dean-flow-coupled interfacial viscoelastic fluid for microparticle separation applied in a cell smear method
The Analyst (2019)
Proteome Analysis of Human Neutrophil Granulocytes From Patients With Monogenic Disease Using Data-independent Acquisition
Molecular & Cellular Proteomics (2019)
In-Depth, Proteomic Analysis of Nasal Secretions from Patients With Chronic Rhinosinusitis and Nasal Polyps
Allergy, Asthma & Immunology Research (2019)