Defense Date
2025
Document Type
Directed Research Project
First Advisor
Christopher Ehrhardt
Second Advisor
Susan Greenspoon
Third Advisor
Catherine Connon
Abstract
Determination of tissue source and the time at which a biological material is transferred from its source to another surface, known as the time-since-deposition (TSD), would assist forensic analysts by providing crucial context for DNA profiles generated from biological evidence. Many methods for cell identification have been explored using biomolecule markers such as mRNA, miRNA, and differential DNA methylation. Forensic laboratories have also developed methods for TSD estimation that focus on degradation of biomolecules. Biomolecules such as mRNA or miRNA degrade at different rates due to some being more stable than others. The ratio of stable degrading biomolecules, therefore, can be related to TSD. However, these methods have not been validated for use in a forensic science laboratory due to high false positive rates, limited application to different biological fluids, and the destructive nature of many existing methods. Flow cytometry is an analytical technique that is used to measure autofluorescence and structural properties to characterize cells in a non-destructive and high-throughput manner. Past studies have utilized flow cytometry to identify tissue source and determine TSD; however, these signatures were always tested in separate experimental efforts on different sets of samples which may not be feasible to implement in the operational workflow of a forensic laboratory.
The goal of the research project reported herein was to develop a single laboratory technique for determining TSD and source tissue for an unknown biological sample. Previous experiments focused on blood samples that ranged in concentration from neat to 1:1,000 dilution and varied in the TSDs between 1 day and six months. To test whether this technique could also be used in mixed tissue samples, blood/saliva mixtures from ten donors were deposited in duplicates onto microscope slides and air dried at ambient conditions. The mixture ratios were prepared as follows: 1:1, 1:5, 1:10, 1:50, and 1:100, with blood as the minor and saliva as the major contributor and collected in intervals between T = 0 days and T = 90 days. Samples were eluted analyzed using a flow cytometer equipped with 488nm excitation laser. After each sample was processed on the flow cytometer, the flow through was collected to undergo DNA analysis.
Results showed that both blood and saliva cells had distinct fluorescence profiles. Blood cells consistently fluoresced at a magnitude of 103 and higher in red and yellow fluorescence channels. Saliva cell consistently fluoresced at a magnitude of 103 and lower in red and yellow fluorescence channels. The observed trends in blood and saliva mixtures, not consistent with trends previously recognized in these fluids independently, did not show a clear TSD signature. Actual cell counts and ratios did not match the theoretical cell counts and ratios that are expected from the body fluids used in this study.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
5-12-2025