Defense Date
2026
Document Type
Directed Research Project
Department
Forensic Science
First Advisor
Christopher Ehrhardt
Second Advisor
Sarah Williams
Third Advisor
Memory Dalton
Abstract
Several front-end cell characterization methods have been explored to improve the analysis of mixture samples by identifying and/or separating contributor cell populations before DNA profiling. Front-end methods such as flow cytometry accomplish this by removing cellular material from the collection swab non-destructively, creating two biological fractions: material released into solution and material retained within the swab. While previous studies have shown that sufficient cellular material can be collected for flow cytometry characterizations in this way, little is known about the relative abundance of DNA in each fraction and its biological context (e.g., proportion of extracellular DNA (exDNA) vs intracellular DNA). This can determine which fractions are ideal for front-end separation and which can be directly subjected to DNA profiling.
The goal of this study was to survey the quantity and types of DNA in biological fractions created by removing cells from the collection swab before flow cytometry. Three different tissue types were tested: blood, saliva, and touch/trace epidermal deposits. A total of 14 individuals were used to create 10 replicate samples for each tissue. Following deposition, samples were processed into three fractions: cell-associated DNA (i.e., ‘cell pellet’), cell-free DNA, and DNA retained on the collection swab. Each fraction underwent DNA extraction and quantification. DNA yields were compared across fractions and tissue types.
Results showed that tissue type was the primary factor influencing both total DNA yield (p = 0.004) and DNA distribution across fractions. Blood and saliva yielded higher overall quantities of DNA (120ng average DNA yield for both) compared to touch samples (6ng average DNA yield). Evidence of a difference between tissue types was strong between blood and touch samples (p = 0.009) and moderate between saliva and touch samples (p = 0.011), while no evidence of a difference was observed between blood and saliva samples (p = 0.995). However, DNA was not equally distributed across fractions. Blood and saliva retained a larger proportion of DNA on the swab (74% average for blood and 40.5% average for saliva, p = 0.002) than touch samples, while the latter had a larger proportion of biological material released into solution as exDNA (29% average, p = 0.021). DNA yield in the cell pellet fraction also varied between the tissue types (p = 0.005).
Overall, these results indicate that the cell-associated fraction for blood and saliva samples is suitable for front-end separation, while the remaining fractions have enough genetic material to generate an informative DNA profile. While DNA yield in the cell pellet of touch/trace samples was extremely limited, the abundance of exDNA, as well as material retained in the swab, suggests that DNA profiles may still be recovered from the non-cellular fractions after front-end separation workflows. Ultimately, this can help casework procedures by utilizing the cell pellet, which may comprise a small percentage of the DNA distributed. The retained swab can be subjected to downstream DNA profiling with minimal consumption, while the supernatant can be preserved as a source of exDNA to improve DNA profile recovery, partocularly for low-template samples.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
7-9-2026