Keywords
forensic science, flow cytometry, epithelial cell, touch mixtures
This article is included in the Data: Use and Reuse collection.
forensic science, flow cytometry, epithelial cell, touch mixtures
In the revised manuscript we have expanded the dataset to include more contributors as well as aged samples. Additionally, we have included flow cytometry data from antibody hybridization experiments on touch samples involving Human Leukocyte Antigen and Cytokeratin Probes.
See the authors' detailed response to the review by Peter K Rogan
See the authors' detailed response to the review by Dieter Deforce
Flow cytometry has proven a viable approach for differentiating cell populations in many types of uncompromised (i.e. non-degraded) forensic mixture sample (Dean et al., 2015; Schoell et al., 1999; Verdon et al., 2015). However, application to ‘touch’ or trace epithelial cell mixtures remains a challenge since many cell surface features are lost or obscured during the process of keratinocyte differentiation, leaving few biochemical or structural features in shed corneocytes that vary between individual contributors. Preliminary research has identified specific optical characteristics—namely, red autofluorescence and forward scatter (FSC) and side scatter (SSC) profiles—that may vary between touch samples deposited by different contributors and collected immediately after deposition (Stanciu et al., 2016). For this dataset, we built on these studies by examining optical properties such as these on an additional two flow cytometry platforms (including different settings, such as channel voltages). We also investigated the capacity of touch epithelial cells to bind to two different classes of antibody probes: Human Leukocyte Antigen (HLA), which has been successfully utilized to separate uncompromised mixtures of blood and other bodily fluids (e.g. Dean et al., 2015) and Cytokeratin (CK), which is known to be a dominant component of epidermal cells. This dataset includes samples that were collected and analyzed immediately after deposition (i.e. ‘fresh samples’), as well as samples that were collected up to seven days after deposition.
Touch samples were collected from six volunteers using the following protocol which was approved by the VCU-IRB (#HM20000454_CR). Contributors either rubbed a sterile polypropylene conical tube (P/N 229421; Celltreat Scientific) for five minutes using their entire hand (i.e. palm and fingers), or held a tube in their hands (no rubbing) for five minutes. Cells were then immediately collected from the surface with six sterile pre-wetted swabs (P/N 22037924; Fisher Scientific) followed by two dry swabs or were collected between 12 hours and seven days after deposition.
Cells were also deposited onto a wooden kitchen knife handle and the grip of a resin mold of a handgun by handling each substrate for five minutes. Several contributors donated samples on multiple days to assess intra-contributor variation of epithelial cell populations.
To elute the collected cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 10 mL of ultrapure water (18.2 MΩ∙cm).
For antibody hybridization experiments, three milliliters of cell solution were centrifuged at 5,000×g for five minutes. The supernatant was decanted and the pellet was resuspended in 100 µl PBS buffer and 1 µL of Human Fc Receptor block (Cat# 130-059-901, Miltenyi Biotec) to increase the specificity of antibody binding before reaction with either HLA or CK probes. This suspension was allowed to incubate at room temperature for 10 minutes.
For HLA hybridizations, cells were then incubated with one of three different antibody probes for 30 minutes: mouse anti-human monoclonal antibody (mAb) HLA-ABC-FITC (Cat# 311403, BioLegend), HLA-A02-FITC, (Cat#343303, Biolegend), HLA-B7-FITC, (Cat#sc-53304, Santa Cruz Biotechnology). Cells incubated with anti-mouse IgG2a-FITC (Cat# 343303, BioLegend) for 30 minutes served as the isotype control for this set of hybridizations. Cells were then pelleted and washed once in 1x FACS buffer [PBS supplemented with 2% Fetal Bovine Serum (FBS, Cat# 100-106, Gemini BioProducts) and 10% Sodium Azide (Cat# S2002, Sigma-Aldrich)] and re-suspended in the same FACS buffer solution until flow cytometry analysis.
For CK hybridization experiments, cells were incubated with either cytokeratin probe ‘AE1’ (recognizes CKs 10, 14, 15, 16 and 19; Cat# 14-9001-80, Affymetrix eBioscience) or ‘AE3’ (recognizes CKs 1, 2, 3, 4, 5, 6, 7, 8), Cat# 14-900-80, Affymetrix eBioscience) for 30 minutes followed by reaction with a secondary antibody, anti-mouse IgG1-APC (Cat# 17-4015-80, Affymetrix eBioscience). We used anti-mouse IgG1-APC (Cat#17-4714-42, Affymetrix eBioscience) to create the isotype control for these experiments, incubating for 30 minutes. As before, cells were washed once and then resuspended in 1xFACS buffer prior to analysis.
Cell solutions—both those treated with antibody probe and those that were not—were passed through 100 µm filter mesh prior to flow cytometry. Flow cytometric analysis of cells was performed on three different platforms: BD FACSCanto™ II Analyzer, BD FACSAria™ II High-Speed Cell Sorter, and BD Influx™ Cell Sorter (all from Becton Dickinson and Company, Franklin Lakes, NJ, USA). The FACSCanto and FACSAria platforms were equipped with 488nm and 633nm lasers. The Influx cell sorter was equipped with 488 561, and 640nm lasers.
For HLA and CK studies, flow cytometry analysis was performed on the BD FACSCanto™ II Analyzer. Channel voltages were set as follows: Forward Scatter (FSC, 150V), Side Scatter (SSC, 200V), Alexa Fluor 488 (FITC, 335V), Phycoerythrin (PE, 233V; PE-Cy5, 300V; PE-Cy7, 400V), and Allophycocyanin (APC, 250V).
Autofluorescence studies of touch samples were performed on one of two BD FACSAria™ II flow cytometers, or on the BD Influx Cell Sorter (additionally, analysis of unstained samples conducted on the FACSCanto, at the settings described above, are the equivalent of autofluorescence studies). Channel voltages for the FACSAria were set as follows: FSC, 200V; SSC, 475V; and APC, 400V. Channel voltages for the BD Influx were set to the following: FSC, 17.5V; SSC, 16V; and APC, 74.6V.
Flow cytometry source data for all samples are provided in Flow Cytometry Standard (.fcs) format files. Source data files are organized into three different repositories labeled, “HLA Hybridization”, “Cytokeratin Hybridization”, and “Autofluorescence”. Flow cytometry data collected on aged touch samples is contained within the Autofluorescence data folder. File names are labeled with the anonymized sample ID number, date of analysis, and the instrument platform used. Source data files are also labeled by the specific probe used where applicable (e.g., AE1, HLA-A02, etc...) as well as by the voltage setting when different from those described in the above Methods.
F1000Research: Dataset 1. Touch epithelial samples, 10.5256/f1000research.8338.d137992 (Kwon et al., 2016).
CE conceived the study. CE, CS, and YK designed the experiments. CS and YK carried out the research. CE and KP prepared the first draft of the manuscript.
This project was funded by the National Institute of Justice Award number 2013-DN-BX-K033 (PI: Ehrhardt). Flow cytometry analyses were performed at the University of Virginia Flow Cytometry Facility which is supported through the University of Virginia Cancer Center National Cancer Institute P30-CA044579-23 Center Grant.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Views | Downloads | |
---|---|---|
F1000Research | - | - |
PubMed Central
Data from PMC are received and updated monthly.
|
- | - |
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | ||
---|---|---|
1 | 2 | |
Version 2 (revision) 07 Oct 16 |
read | read |
Version 1 23 Mar 16 |
read | read |
Click here to access the data.
Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly.
Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
Sign up for content alerts and receive a weekly or monthly email with all newly published articles
Already registered? Sign in
The email address should be the one you originally registered with F1000.
You registered with F1000 via Google, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Google account password, please click here.
You registered with F1000 via Facebook, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Facebook account password, please click here.
If your email address is registered with us, we will email you instructions to reset your password.
If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance.
Comments on this article Comments (0)