VCU AAFS Poster.pdf
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, would assist forensic analysts by providing crucial context for DNA profiles generated from biological evidence. Many methods for cell identification have been explored with mostly a focus on biomolecule markers such as mRNA and miRNA. Forensic laboratories have also developed methods for TSD estimation that focus on degradation of biomolecules. 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.
Therefore, the goal of this research was to develop a single laboratory technique for determining TSD and source tissue for an unknown biological sample. Initial experiments focused on blood samples that ranged in concentration from neat to 1:1000 and varied in had TSDs between 1 day and six months. Blood samples from three donors were deposited in quadruplicates (two separate stains per slide) onto microscope slides and air dried at ambient conditions. One set of stains was used as a reference sample. The other set of stains was used to perform red blood cell (RBC) lysis to analyze white blood cells (WBCs). To separate the WBCs from the whole blood sample, red blood cells were lysed using Ammonium-Chloride-Potassium (ACK) Lysing Buffer prior to analysis on the flow cytometer. A second method that did not utilize a WBC separation technique was also used by creating a gate based on events that were measured after running whole blood samples and data found in literature to separate WBCs from RBCs and platelets present in the whole blood sample. To test whether this technique could also be used in mixed tissue samples, blood/saliva mixtures from six donors were deposited in duplicates onto microscope slides and air dried at ambient conditions. After the designated time periods ranging from T = 0 days to T = 120 days, samples were collected using a sterile cotton swab moistened with deionized water and air-dried. Sampled were filtered through a 100 µm mesh filter and analyzed using a flow cytometer equipped with 488nm excitation laser.
Results showed that autofluorescence profiles of blood cell populations showed incremental changes within the first two weeks, e.g., median autofluorescence in wavelengths between 535nm and 600nm increased from ~2200 RFU at one day to ~2500 RFU at 14 days. Samples that had been aged for more than two months showed a 40% decrease in median autofluorescence (~400RFU) which was consistent across each contributor tested. Results also showed that forward scatter (FSC) and side scatter (SSC) profiles collected from the same flow cytometry dataset were able to differentiate blood cells from saliva cells. Autofluorescence in wavelengths between 650nm and 700nm was capable of further discriminating between the two cell sources. These measurements were successful in detecting the presence of blood cells in a sample even when saliva was present in ratios between 1:5 and 5:1. The observed trends, consistent with that previously recognized in other biological samples, show promise for the development and implementation of flow cytometry as a method for time-since-deposition determination and cell identification in forensic laboratories.
