Kenneth T. Kotz

Clinical microfluidics for neutrophil genomics and proteomics

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.

Biopolymer system for cell recovery from microfluidic cell capture devices.

Microfluidic systems for affinity-based cell isolation have emerged as a promising approach for the isolation of specific cells from complex matrices (i.e., circulating tumor cells in whole blood). However, these technologies remain limited by the lack of reliable methods for the innocuous recovery of surface captured cells. Here, we present a biofunctional sacrificial hydrogel coating for microfluidic chips that enables the highly efficient release of isolated cells (99% ± 1%) following gel dissolution. This covalently cross-linked alginate biopolymer system is stable in a wide variety of physiologic solutions (including EDTA treated whole blood) and may be rapidly degraded via backbone cleavage with alginate lyase. The capture and release of EpCAM expressing cancer cells using this approach was found to have no significant effect on cell viability or proliferative potential, and recovered cells were demonstrated to be compatible with downstream immunostaining and FISH analysis.

Separation of Leukocytes

Leukocytes (e.g., neutrophils, monocytes and/or lymphocytes) can be captured and separated from blood by removing platelets using a spiral channel, followed by capturing individual leukocyte types in a series of cell capture channels having leukocyte binding moieties. Accordingly, various microfluidic-based cell affinity chromatography methods can be used to separate leukocytes from whole blood.

Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage.

Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b+ cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.

Cell assay using a two-photon-excited europium chelate

We report application of two-photon excitation of europium chelates to immunolabeling of epidermal growth factor receptor (EGFR) cell surface proteins on A431 cancer cells. The europium chelates are excited with two photons of infrared light and emit in the visible. Europium chelates are conjugated to antibodies for EGFR. A431 (human epidermoid carcinoma) cells are labeled with this conjugate and imaged using a multiphoton microscope. To minimize signal loss due to the relatively long-lived Eu3+ emission, the multiphoton microscope is used with scanning laser two-photon excitation and non-scanning detection with a CCD. The chelate labels show very little photobleaching (less than 1% during continuous illumination in the microscope for 20 minutes) and low levels of autofluorescence (less than 1% of the signal from labeled cells). The detection limit of the europium label in the cell assay is better than 100 zeptomoles.

Inertial focusing cytometer with integrated optics for particle characterization

Microfluidic inertial focusing has been shown as a simple and effective method to localize cells and particles within a flow cell for interrogation by an external optical system. To enable portable point of care optical cytometry, however, requires a reduction in the complexity of the large optical systems that are used in standard flow cytometers. Here, we present a new design that incorporates optical waveguides and focusing elements with an inertial focusing flow cell to make a compact robust cytometer capable of enumerating and discriminating beads, cells, and platelets.

Microfluidics for T‐ Lymphocyte Cell Separation and Inflammation Monitoring in Burn Patients

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. Clin Trans Sci 2011; Volume 4: 63–68

Feature issue introduction: biophotonic materials and applications

Biophotonics can be defined as the interplay of light and biological matter. The percolation of new optical technology into the realm of biology has literally shed new light into the inner workings of biological systems. This has revealed new applications for optics in biology. In a parallel trend, biomolecules have also been investigated for their optical applications. Materials are playing a central role in the development of biophotonics. New materials, fabrication methods, and structures are enabling new biosensors, contrast agents, imaging strategies, and assay methods. Similarly, biologic materials themselves can be used in photonic devices. In this context, two open-access, rapid-publication journals from The Optical Society of America, Optical Materials Express and Biomedical Optics Express, will publish a joint feature issue covering advances in biophotonics materials.