RESUMO
Severe burns result in T lymphocyte specific immunologic changes. In addition to decreased levels of circulating lymphocytes, changes in cytokine secretion and receptor expression also take place. Our finer understanding of the inflammatory response has led to the development of immune-targeted therapeutics, requiring specialized gene-expression monitoring. The emerging field of bio-micro-electromechanical systems can be used to isolate highly pure T lymphocytes in a clinically relevant and timely manner for downstream genomic analysis. Blood samples from healthy volunteers and burn-injured patients were introduced into microfluidic devices developed in our laboratory. Utilizing cell-affinity chromatography for positive selection of T lymphocytes, the devices served as a platform for RNA extraction and downstream cytokine analysis via quantitative real-time polymerase chain reaction (PCR). From a 0.5-mL whole blood sample, the microfluidic devices captured highly pure T lymphocytes from healthy volunteers and burn-injured patients. Cell capture was of sufficient quantity, and extracted RNA was of sufficient quality, for evaluating the gene expression of cytokines: interferon-gamma, interleukin-2, interleukin-4, and interleukin-10. Microfluidics is a useful tool in processing blood from burn-injured patients. Though in its very early stages of development, cell-specific information obtained by this platform/technology will likely be an important component of near-patient molecular diagnostics and personalized medicine.
Assuntos
Queimaduras/genética , Queimaduras/imunologia , Perfilação da Expressão Gênica , Inflamação/genética , Inflamação/imunologia , Microfluídica/métodos , Linfócitos T/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Queimaduras/complicações , Separação Celular , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Inflamação/complicações , Masculino , Microfluídica/instrumentação , Pessoa de Meia-Idade , RNA/genética , RNA/normas , Linfócitos T/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Adulto JovemRESUMO
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.
