Defense Date
2024
Document Type
Directed Research Project
First Advisor
Sarah Seashols-Williams, PhD
Second Advisor
Tracey Dawson Green, PhD
Third Advisor
Joseph Reiner, PhD
Abstract
DNA interpretation of biological mixtures, a recurrent consequence of sexual assault-type evidence, is arguably one of the most challenging and time-consuming steps in the forensic DNA analysis workflow, often requiring the use of complex back-end mixture deconvolution procedures. This has led the research community to seek out a reliable front-end cell separation method that is capable of isolating different cell types in an attempt to remove biological mixtures entirely. The standard cell separation method used by most laboratories to deconvolute sexual-assault mixtures at the front-end is differential extraction, a method that requires extensive manual processing and does not guarantee single-source STR profiles. An alternative approach is optical trapping, a non-invasive method that utilizes the properties of light to create an optical trap for cell separation and manipulation. The optical trap is created by tightly focusing a laser beam through a high-numerical immersion objective on an inverted microscope, where the gradient force applied by the laser allows for individual cells or cell clusters (e.g., spermatozoa, vaginal epithelial cells, and leukocytes) to become captured and fixed to a high-intensity region; through the use of a joystick, the cells are manipulated in the X, Y, and Z directions and displaced from their original positions. Previous work has demonstrated successful isolation and complete, single-source STR profiles by trapping 15 leukocytes or 40 sperm cells, with minimal carryover from the female epithelial donor when evaluating mock sexual assault evidence. Recent work has focused on optimizing optical trapping in a closed microfluidic device system, which generates equivalent results and has reduced contamination potential. This research continued to optimize the optical trapping procedure and setup in order to demonstrate reliability in obtaining a pair of single-source profiles of both the male and female contributor when trapping 40 spermatozoa and 20 vaginal epithelial cells on a single microfluidic device. After demonstrated success, a series of prepared liquid male-female mixtures were separated using optical trapping and differential extraction, then processed through DNA analysis to compare the separation efficiency across methods. As a result, the sperm fraction samples of both methods received high separation efficiency with an average above 93%. While the optically trapped sperm fractions fell slightly short in comparison to the differentially extracted samples, optical trapping outperformed with respect to the non-sperm fractions with a separation efficiency of 92.27%, compared to 60.97% from differential extraction. Optical trapping continues to show potential to provide superior separation compared to that of differential extraction, and ultimately, could reduce the labor and time required by the analyst in an attempt to eliminate mixtures at the beginning of the DNA analysis workflow.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
4-29-2024