Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.
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Touch samples were obtained pursuant to VCU-IRB approved protocol ID no. HM20000454_CR3. Volunteers were asked to rub a sterile polypropylene conical tube (P/N 229421; Celltreat Scientific) using their palm and fingers for 5 min. Cells were collected from the surface with sterile pre-wetted swabs (P/N 22037924; Fisher Scientific) followed by dry swabs. A total of six wet swabs and two dry swabs were used to sample the entire tube surface. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 s in 10 mL of ultrapure water (18.2 MΩ cm). The entire solution was then passed through a 100-μm filter mesh prior to antibody hybridization, conventional flow cytometry, and imaging flow cytometry (IFC). Separate aliquots of the resulting cell solution were used for each analysis method.
For studies of exogenous influences on autofluorescence in touch samples, each donor handled a specific material prior to depositing cells on a conical tube. These materials included purple nitrile gloves (Precision brand, powder-free, P/N PCS775), plant material, and conical tubes marked with Sharpie marker ink. Prior to handling these materials, donors washed their hands with antibacterial soap under running water for 15 s and then allowed them to air dry. For nitrile glove experiments, donors wore a nitrile glove on their right hand, leaving the left (control) hand bare, and proceeded to grip/handle various items with their gloved hand for 5 min to simulate normal activity (e.g., pipette, door handle, tools). The glove was then removed and the contributor held a conical tube in each hand for 5 min. For experiments involving plant material, subjects handled individual leaves of kale or collard greens using only their right hand for 5 min (left hand was not used and served as a control cell population). The handling procedure involved lifting/tossing leaves with fingers and palmar surface and tearing individual leaves into smaller pieces, approximating how this material might be handled during food preparation. Subsequently, donors rinsed their hands with water for approximately 5 s (to remove pieces of plant material) and allowed their hands to air dry before depositing touch samples by holding a conical tube in each hand for 5 min. For marker ink experiments, each donor held a conical tube that had been marked with a black or green marker in his/her right hand for 5 min before depositing touch samples by holding an unmarked conical tube in each hand for 5 min. For each of these experiments, cells were collected from the surface of each tube and eluted into solution as described above. Separate aliquots of the resultant cell solutions were used for flow cytometry analysis and IFC.
Amnis imaging of individual flow cytometry events from the fluorescent cell population shown in Fig. 4e (red histogram). Each event was visualized in two different microscopic settings: Brightfield (left image in gray) and APC channel fluorescence (right image in black background). The X-axis of each frame is 20 μm
Ultimately, our observations regarding variations in red fluorescence in touch deposits suggest that there will be some touch cell mixtures that are more susceptible to being separated into individual components (or at least broken down into less complex cell mixtures) based upon this characteristic than others. Because flow cytometry is non-destructive, evidence samples could potentially be screened for favorable fluorescence distributions. A mixed cell sample that exhibits two or more peaks (e.g., Fig. 6c) on a fluorescence histogram may be a more promising candidate for cell separation than one that exhibits a unimodal fluorescence distribution (e.g., Fig. S3c, see ESM), as the latter suggests a high degree of overlap between contributor cell populations. Our preliminary results appear to bear out this proposition (compare Table 1 and ESM Table S1), but further research is required to develop a standardized set of screening criteria.
Finally, because a significant portion of the genetic material in many touch samples may be unavoidably extracellular, characterizing the chemical and physical relationship between cell-free DNA and the surface of intact epidermal cells may be an important area of future research. If extracellular DNA associates with epidermal cells, as it has been observed to do in other cell types [34], flow cytometry protocols could potentially be optimized to maintain surface-bound DNA through the cell sorting process. If it emerges that extracellular DNA is not bound to epidermal cells at the time of transfer, or is not bound in a way that can be maintained through the sorting process, this DNA source can be separately collected for typing (e.g., the workflow described in [20]).
Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data.
Descriptions of marker expression profiles for particular cell populations in flow cytometry often use terms like positive-negative, or bright-dim, to qualify the amount of target present. Since BD FACS CAP is a standard platform for screening a wide range of cell types, and antibody concentrations were not optimized for these particular PMBC samples, results are reported as the percentage of cells above the isotype gate rather than positive or negative. Followup studies, including single color titrations and competition experiments, are needed to definitively show that a marker is present. Markers that have been previously characterized using BD FACS CAP with 90% of the cells above the isotype threshold are usually confirmed as positive using titration and competition experiments, while staining in markers with 10% of cells above the isotype threshold is often the result of nonspecific binding (data not shown). Note that these percentages refer to the fraction of cells above the isotype threshold, but this does not necessarily imply heterogeneous staining in multiple populations. 2ff7e9595c
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