Defense Date
2024
Document Type
Directed Research Project
First Advisor
Susan A. Greenspoon, PhD
Second Advisor
Christopher Ehrhardt, PhD
Third Advisor
Catherine Connon, PhD
Abstract
Knowledge of the relative window of time that evidentiary biological material was deposited at a crime scene, referred to as the time since deposition (TSD), would help investigators determine which evidentiary items are most probative and assist with scene reconstruction. One commonly encountered body fluid at a crime scene is blood. Various methods for estimating TSD of bloodstains have been developed, but none to date have been validated for use in the forensic laboratory. Recently, flow cytometry measurements of cellular autofluorescence and size/structure have been investigated as a signature for TSD of saliva and touch epidermal cells. This study assessed whether these signatures could be used to determine TSD of whole blood samples. First, blood samples from two donors (treated with EDTA as an anti-coagulant) were deposited in triplicate onto microscope slides, air dried and maintained at ambient conditions, and collected after designated time periods ranging from T=0 days to 180 days. The first stain was analyzed directly using flow cytometry, the second stain was analyzed using flow cytometry after lysis of the red blood cells (RBCs), and the third stain underwent DNA degradation analysis at the T=0-, 90-, and 180-day time points. To investigate the potential influence of EDTA on autofluorescence, a second time series of fresh whole blood consisting of five intervals over a 90-day period was created by having donors prick their fingers with sterile lancets before deposition on a microscope slide and analyzed using the same workflow.
Results showed that there is no clear difference in autofluorescence of the large cell population over time for whole blood samples containing EDTA, and no DNA degradation was observed in the same time frames. However, detectable differences in green autofluorescence intensity between blood and other biological source types were observed in the T=0-, 7- and 14-day time points. A statistically significant increase in median autofluorescence intensity (p < 0.001) was observed for fresh whole blood over 90 days, with the most notable increase in fluorescence occurring over the first seven days (~8100rfu at T=0 days compared to ~61000rfu at T=7 days). Peak emission wavelengths correspond with those reported in the literature for blood components associated with hemoglobin oxidation. Efforts to create a white blood cell (WBC) fraction by lysing RBCs were unsuccessful; the proportion of RBCs in the sample remained greater than 90% post-lysis.
Lastly, differences were observed across TSDs for a subpopulation of cells in the fresh whole blood samples. Specifically, the number of cells with an autofluorescence intensity greater than 104 rfu increased with time. The proportion of cells within this subpopulation and the sample TSD showed a strong positive correlation (Pearson’s r = 0.93) from T=0 to 7 days, after which it plateaus. Applying a similar method to samples containing EDTA, linear regression analysis reveals a positive correlation between the proportion of cells with fluorescence intensities greater than 103 rfu and TSD through 180 days (r = 0.89). From these findings, there is evidence that the presence of EDTA can affect cellular autofluorescence and, consequently, a TSD estimate from the sample.
Rights
© The Author(s)
Is Part Of
VCU Master of Science in Forensic Science Directed Research Projects
Date of Submission
4-30-2024