Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Clinical microfluidics for neutrophil genomics and proteomics

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Summary of microfluidic device characterization.
Figure 2: Genomic and protemic characterization of neutrophil lysates.
Figure 3: Summary of RNA extractions from cell lysates collected at six different clinical sites.
Figure 4: Summary of the microarray results for a subset of the clinical samples from Figure 3 .

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Nathan, C. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6, 173–182 (2006).

    Article  CAS  Google Scholar 

  2. Cassatella, M.A., Gasperini, S. & Russo, M.P. Cytokine expression and release by neutrophils. Ann. NY Acad. Sci. 832, 233–242 (1997).

    Article  CAS  Google Scholar 

  3. 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).

    CAS  PubMed  Google Scholar 

  4. Burczynski, M.E. & Dorner, A.J. Transcriptional profiling of peripheral blood cells in clinical pharmacogenomic studies. Pharmacogenomics 7, 187–202 (2006).

    Article  CAS  Google Scholar 

  5. Calvano, S.E. et al. A network-based analysis of systemic inflammation in humans. Nature 437, 1032–1037 (2005).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Nauseef, W.M. Isolation of human neutrophils from venous blood. Methods Mol. Biol. 412, 15–20 (2007).

    Article  Google Scholar 

  8. 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).

    Article  Google Scholar 

  9. Cassatella, M.A. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 16, 21–26 (1995).

    Article  CAS  Google Scholar 

  10. 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).

    PubMed  Google Scholar 

  11. Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 1235–1239 (2007).

    Article  CAS  Google Scholar 

  12. Lyons, P.A. et al. Microarray analysis of human leucocyte subsets: the advantages of positive selection and rapid purification. BMC Genomics 8, 64 (2007).

    Article  Google Scholar 

  13. Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7, 3 (2006).

    Article  Google Scholar 

  14. 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).

    CAS  PubMed  Google Scholar 

  15. Pelletier, M. et al. Evidence for a cross-talk between human neutrophils and TH17 cells. Blood 115, 335–343 (2009).

    Article  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. 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).

    CAS  PubMed  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. Kotz, K., Cheng, X. & Toner, M. Cell capture using a microfluidic device. J. Vis. Exp. published online, doi:10.3791/320 (1 October 2007).

  21. Covert, M.W., Leung, T.H., Gaston, J.E. & Baltimore, D. Achieving stability of lipopolysaccharide-induced NF-κB activation. Science 309, 1854–1857 (2005).

    Article  CAS  Google Scholar 

  22. Theilgaard-Mönch, K. et al. The transcriptional program of terminal granulocytic differentiation. Blood 105, 1785–1796 (2005).

    Article  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. Zhang, X. et al. Gene expression in mature neutrophils: early responses to inflammatory stimuli. J. Leukoc. Biol. 75, 358–372 (2004).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. Ong, S.E. & Mann, M. Mass spectrometry–based proteomics turns quantitative. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  Google Scholar 

  29. de Godoy, L.M. et al. Comprehensive mass-spectrometry–based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).

    Article  CAS  Google Scholar 

  30. Zhang, X. et al. Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin. Electrophoresis 27, 1659–1668 (2006).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. Meheus, L.A. et al. Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages. J. Immunol. 151, 1535–1547 (1993).

    CAS  PubMed  Google Scholar 

  33. Kotz, K., Cheng, X. & Toner, M. PDMS device fabrication and surface modification. J. Vis. Exp. published online, doi:10.3791/319 (1 October 2007).

  34. 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).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. Polpitiya, A.D. et al. DAnTE: a statistical tool for quantitative analysis of -omics data. Bioinformatics 24, 1556–1558 (2008).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Consortia

Contributions

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.

Corresponding authors

Correspondence to Kenneth T Kotz or Mehmet Toner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2205

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research