Sepsis is an uncontrolled response to infection. Severe sepsis is associated with
organ dysfunction, and has mortality rate of 30-50%. Identification of severity of sepsis
and prediction on mortality is crucial in making clinical decisions. Recently, cell-free
DNA (cfDNA) in blood was found to have high discriminative power in predicting ICU
mortality in patients with severe sepsis. In an analysis of 80 severely septic patients, the
mean cfDNA level in survivors (1.16±0.13μg/ml) was similar to that of healthy
volunteers (0.93±0.76μg/ml), while that of non-survivors (4.65±0.48μg/ml) was notably
higher. Therefore, rapid quantification of cfDNA concentration in blood will enable
physicians to quickly predict mortality of sepsis and decide on treatment.
Current methods for quantification of cfDNA involve multiple steps including
centrifugation, DNA-extraction from plasma, and its quantification either through
spectroscopic methods or quantitative PCR. The whole process is time consuming, thus is
not suitable for immediate bedside assessment. To solve the problems, a microfluidic
device is designed and fabricated in this thesis, which is potential for cfDNA
quantification directly using blood in 5 minutes. The goal is to use this device for
distinguishing survivors or healthy donors from non-survivors in patients with severe
sepsis. The two-layer device consists of a sample channel (top) and an accumulation
channel (bottom) that intersect each other. The accumulation channel is preloaded with
1% agarose gel, and the blood containing cfDNA and intercalating fluorescent dye is
loaded in the sample channel. Fluorescently labeled DNA is able to be trapped and
concentrated at the intersection using a DC electric field, and fluorescent intensity of the
accumulated DNA is representative of its concentration in the blood. The simulated
electric field in the sample channel reveals that both the magnitude and the gradient of
electric field reach their maximum values at the intersection. Force analysis shows that
DNA was driven into the gel by the dominate electrophoretic force, while red blood cells
moved away from the gel due to a strong dielectrophoretic force.
In this thesis, 4 types of samples have been used to characterize the performance
of the device. It showed that DNA was efficiently accumulated at the intersection, and the
fluorescent intensity could be measured using a fluorescent microscope. Samples from
healthy donors were able to be distinguished from that of severely septic patients in 5
minutes. However, better resolution was needed for differentiating various cfDNA
concentrations in patient samples. The discussion on the effect of applied voltage showed
that 9V is an optimized setting compared with 3V and 15V. In addition, it has been
proved that the fluorescent reagent could be immobilized in the device and the sample
preparation could be absolutely eliminated. In summary, the device proposed in this thesis is capable of distinguishing
severely septic patients from healthy donors using clinical plasma in 5 minutes, and is
potential to be applied in clinical blood samples. It has low cost, and is ready to be
developed into a fully functioned system. This tool can be a valuable addition to the ICU
to rapidly assess the severity of sepsis for informed decision making. / Thesis / Master of Applied Science (MASc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/16562 |
| Date | 06 1900 |
| Creators | Yang, Jun |
| Contributors | Selvaganapathy, P. Ravi, Biomedical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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