Flow cytometry dataset for cells collected from touched surfaces

‘Touch’ or trace cell mixtures submitted as evidence are a significant problem for forensic laboratories as they can render resulting genetic profiles difficult or even impossible to interpret. Optical signatures that distinguish epidermal cell populations from different contributors could facilitate the physical separation of mixture components prior to genetic analysis, and potentially the downstream production of single source profiles and/or simplified mixtures. This dataset comprises the results from antibody hybridization surveys using Human Leukocyte Antigen (HLA) and Cytokeratin (CK) probes, as well as surveys of optical properties of deposited cells, including forward scatter (FSC), side scatter (SSC), and fluorescence emissions in the Allophycocyanin (APC) channel. All analyses were performed on “touch” samples deposited by several different contributors on multiple days to assess inter- and intra-contributor variability.

Flow cytometry has proven a viable approach for differentiating cell populations in many types of uncompromised (i.e.nondegraded) 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.

Methods
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 (18.2MΩ•cm).

Antibody hybridization
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.
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.

Flow cytometry
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.

Amendments from Version 1
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.
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.

Dataset content
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.
We also agree with the reviewer's second point that there may be significant variability in the level of autofluorescence exhibited by touch samples.This in turn may be influenced by a variety of factors such as presence or absence of exogenous compounds, and/or intrinsic biological characteristics of the cells themselves.Understanding this variability, particularly for samples derived from the same contributor or for aged/degraded samples, is necessary to assess the potential utility of flow cytometry-based cell separation techniques such as FACS for downstream DNA profiling of separated cell populations derived from 'touch' biological mixtures.
We would note that although differences in cell yield change the of cells (Y axis) number fluorescing in the red portion of the spectrum at a given RFU value (X axis), it is nonetheless possible to develop a sense of how the average of red autofluorescence exhibited by cells intensity collected from different individuals, or from the same individual on different days, varies by comparing histograms, regardless of cell yield.We agree that an individual's total cellular contribution to a biological mixture will ultimately be an important factor in whether that individual's DNA profile can be successfully generated from a sorted mixture.However, any kind of minimum cell count threshold would be inextricably linked to the sensitivity/efficiency of downstream DNA typing methodologies used on sorted cell populations, and are beyond the scope of this Data Note.
No competing interests were disclosed.

Dieter Deforce
Laboratory for Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium The article suggests "touch samples" however the data set contains no data on forensic relevant touch samples.The six samples were from volunteers rubbing their entire hand.These are "fresh" cells and might not show the same flow characteristics as cells left behind on an object after a touch contact and having "aged" on the object.The dataset provided is as it is not really relevant to the forensic field.
In addition I think that 6 samples is maybe a too limited number for this kind of studies.
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
Author Response 29 Sep 2016 , Virginia Commonwealth University, USA

Christopher Ehrhardt
We agree with the reviewer that there is an important distinction between 'fresh' biological samples

F1000Research
We agree with the reviewer that there is an important distinction between 'fresh' biological samples and ones that are aged and/or degraded since the latter is more likely to be encountered in forensic caseworking samples.Uncompromised samples can nonetheless provide an important foundation for future studies that explicitly examine the effects of aging/degradation on optical signatures identified by the initial study.We also note that there are forensic scenarios where fresh touch samples may be collected and analyzed for an investigation.For example, a firearm may be discarded by a suspect and immediately collected during pursuit by law enforcement.
However, to increase the forensic relevance of this dataset the new version of the manuscript includes samples that were aged between 12 hours and seven days before collection, as well as samples that were collected from forensically relevant substrates such as replica firearms and knife handles.
For the updated version of the manuscript we have also expanded the dataset to include 33 different contributors.
No competing interests were disclosed.Competing Interests: