Electrical characterization of leukocyte activation for monitoring sepsis progression using dielectrophoresis

This thesis describes the development of a rapid dielectrophoretic characterization tool for monitoring leukocyte activation, and its application for monitoring sepsis progression. Sepsis is an uncontrolled activation of the immune system that causes an excessive inflammatory response or an impaired immunity. The unpredictable immune status makes the immunotherapy treatment difficult. One crucial aspect of sepsis is leukocyte activation, which plays an important role in the attacking the pathogens, stimulating the tissues, and resulting in organ function failures. Monitoring leukocyte activation in sepsis may provide real-time indicators for diagnosis, prognosis, and guiding therapy. To measure leukocyte activation, we used Iso-dielectric Separation (IDS) and dielectrohporetic spring (DEP spring) method which can rapid characterize of thousands of cells to provide statistically meaningful results. To adapt to measuring septic blood with clumpy and sticky leukocytes, the doublesided electrodes was developed and characterized for higher throughput and anti-fouling measurement. The human neutrophils were successfully isolated and activated with chemicals and their electrical properties were measured across many conditions and compared to the non-activated ones. We found repeatable and consistent electrical changes using both IDS and DEP spring method: an increase in effective conductivity of activated neutrophils at higher frequencies (above 5MHz). We then developed an electrical model and an experiment pipeline of inhibiting neutrophil functions to hypothesize the underlying mechanism. Using the blood samples from a cecal-ligation and puncture (CLP) model of sepsis in mice, we estimated the number of activated leukocytes by looking into the difference in electrical properties at 10 MHz. We found that the activated leukocyte percentage correlated with the neutrophil activation percentage obtained from flow cytometry, indicating potential use for monitoring sepsis progression. We designed a multi-parametric time series experiment to understand the prognostic value of leukocyte activation and the role of leukocyte activation in sepsis at a system level. We found that the neutrophil activation percentage of non-survivors were significantly larger than survivors at 24 hours after the CLP procedure. We also found the neutrophil activation percentage positively correlates with inflammatory cytokine interleukin-6 and interleukin-10. Finally, we initiated a pilot study of monitoring neutrophil activation in critically-ill human patients with suspected sepsis. We found, with a limited dataset, the effective conductivity of neutrophils in critically-ill human patients is higher than the healthy control. To show the feasibility of becoming an point-of-care bedside device, the sample preparation of red blood cell removal was integrated in for rapid test that can profile >1,000 leukocytes within 15 minutes from sample to result, providing a simple assay to monitor leukocyte activation in sepsis progression. Thesis Supervisor: Joel Voldman Title: Professor of Electrical Engineering and Computer Science