Clinical Microfluidics for Neutrophil Genomics and Proteomics

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Nature medicine (Impact Factor: 27.36). 09/2010; 16(9):1042-7. DOI: 10.1038/nm.2205
Source: PubMed


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.

Download full-text


Available from: Alan E Rosenbach, Oct 02, 2015
37 Reads
  • Source
    • "As seen in our previous meta-analysis of mouse microarray data, the different leukocyte lineages can be defined by sets of coregulated genes. The largest leukocyte-enriched cluster is Cluster 2, a set of genes almost exclusively expressed in neutrophils purified using a novel isolation methodology [46]. This involved an affinity capture of the cells using anti-CD66B antibodies, and stimulation with either bacterial lipopolysaccharide (LPS), or granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interferon (IFNγ). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The specialisation of mammalian cells in time and space requires genes associated with specific pathways and functions to be co-ordinately expressed. Here we have combined a large number of publically available microarray datasets derived from human primary cells and analysed large correlation graphs of these data. Using the network analysis tool BioLayout Express3D we identify robust co-associations of genes expressed in a wide variety of cell lineages. We discuss the biological significance of a number of these associations, in particular the coexpression of key transcription factors with the genes that they are likely to control. We consider the regulation of genes in human primary cells and specifically in the human mononuclear phagocyte system. Of particular note is the fact that these data do not support the identity of putative markers of antigen-presenting dendritic cells, nor classification of M1 and M2 activation states, a current subject of debate within immunological field. We have provided this data resource on the BioGPS web site ( and on (
    BMC Genomics 09/2013; 14(1):632. DOI:10.1186/1471-2164-14-632 · 3.99 Impact Factor
  • Source
    • "Collectively, these studies demonstrate the feasibility of subclassifying patients with septic shock, in a clinically relevant manner, based on the expression patterns of a discrete set of genes having relevance to sepsis biology. The availability of clinical microfluidics [58] and digital mRNA measurement technology [59] may allow for clinical feasibility of measuring the 100 class-defining genes in a timely manner that is suitable to direct patient care or for clinical trial stratification. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the past decade several investigators have applied microarray technology and related bioinformatic approaches to clinical sepsis and septic shock, thus allowing for an assessment of how, or if, this branch of genomic medicine has meaningfully impacted the field of sepsis research. The ability to simultaneously and efficiently measure the steady-state mRNA abundance of thousands of transcripts from a given tissue source (that is, 'transcriptomics') has provided an unprecedented opportunity to gain a broader, genome-level 'picture' of complex and heterogeneous clinical syndromes such as sepsis. A trancriptomic approach to sepsis and septic shock is technically challenging on multiple levels, but nonetheless modest, tangible advances are being realized. These include a genome-level understanding of the complexity of sepsis and septic shock, identification of novel candidate pathways and targets for potential intervention, discovery of novel, candidate diagnostic and stratification biomarkers, and the ability to stratify patients into clinically relevant, expression-based subclasses. The challenges moving forward include robust validation studies, standardization of technical approaches, standardization and further development of analytical algorithms, and large-scale collaborations.
    Critical care (London, England) 02/2012; 16(1):204. DOI:10.1186/cc10537 · 4.48 Impact Factor
  • Source
    • "The potential problem with these studies is that isolation may affect gene expression (Holmes et al, 2009; Kotz et al, 2010; Lee et al, 2010; Shim et al, 2010). This may be solved by microfluiditic devices that use cell-specific antibodies that isolate specific cell types at the bed side (Kotz et al, 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Whole genome expression microarrays can be used to study gene expression in blood, which comes in part from leukocytes, immature platelets, and red blood cells. Since these cells are important in the pathogenesis of stroke, RNA provides an index of these cellular responses to stroke. Our studies in rats have shown specific gene expression changes 24 hours after ischemic stroke, hemorrhage, status epilepticus, hypoxia, hypoglycemia, global ischemia, and following brief focal ischemia that simulated transient ischemic attacks in humans. Human studies show gene expression changes following ischemic stroke. These gene profiles predict a second cohort with >90% sensitivity and specificity. Gene profiles for ischemic stroke caused by large-vessel atherosclerosis and cardioembolism have been described that predict a second cohort with >85% sensitivity and specificity. Atherosclerotic genes were associated with clotting, platelets, and monocytes, and cardioembolic genes were associated with inflammation, infection, and neutrophils. These gene profiles predicted the cause of stroke in 58% of cryptogenic patients. These studies will provide diagnostic, prognostic, and therapeutic markers, and will advance our understanding of stroke in humans. New techniques to measure all coding and noncoding RNAs along with alternatively spliced transcripts will markedly advance molecular studies of human stroke.
    Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 07/2011; 31(7):1513-31. DOI:10.1038/jcbfm.2011.45 · 5.41 Impact Factor
Show more