ALL Metrics
-
Views
-
Downloads
Get PDF
Get XML
Cite
Export
Track
Data Note
Revised

Flow cytometry dataset for cells collected from touched surfaces

[version 2; peer review: 2 approved]
Previously titled: Flow cytometry analysis of epithelial cell populations from touch samples using the BD Influx flow cytometry platform
PUBLISHED 07 Oct 2016
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

This article is included in the Data: Use and Reuse collection.

Abstract

‘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.

Keywords

forensic science, flow cytometry, epithelial cell, touch mixtures

Revised 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.

See the authors' detailed response to the review by Peter K Rogan
See the authors' detailed response to the review by Dieter Deforce

Introduction

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.

Methods

Dataset 1.Touch epithelial samples.
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.

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).

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.

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.

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.

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.

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.

Data availability

F1000Research: Dataset 1. Touch epithelial samples, 10.5256/f1000research.8338.d137992 (Kwon et al., 2016).

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 23 Mar 2016
Comment
Author details Author details
Competing interests
Grant information
Copyright
Download
 
Export To
metrics
Views Downloads
F1000Research - -
PubMed Central
Data from PMC are received and updated monthly.
- -
Citations
CITE
how to cite this article
Kwon YJ, Stanciu CE, Philpott MK and Ehrhardt CJ. Flow cytometry dataset for cells collected from touched surfaces [version 2; peer review: 2 approved]. F1000Research 2016, 5:390 (https://doi.org/10.12688/f1000research.8338.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
track
receive updates on this article
Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
VERSION 2
PUBLISHED 07 Oct 2016
Revised
Views
15
Cite
Reviewer Report 28 Dec 2016
Dieter Deforce, Laboratory for Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium 
Approved
VIEWS 15
The authors have provided additional data making ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Deforce D. Reviewer Report For: Flow cytometry dataset for cells collected from touched surfaces [version 2; peer review: 2 approved]. F1000Research 2016, 5:390 (https://doi.org/10.5256/f1000research.10476.r16849)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Views
23
Cite
Reviewer Report 07 Oct 2016
Peter K Rogan, Dept of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada 
Approved
VIEWS 23
The authors have addressed my comments with additional data. The analysis ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Rogan PK. Reviewer Report For: Flow cytometry dataset for cells collected from touched surfaces [version 2; peer review: 2 approved]. F1000Research 2016, 5:390 (https://doi.org/10.5256/f1000research.10476.r16850)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
Version 1
VERSION 1
PUBLISHED 23 Mar 2016
Views
38
Cite
Reviewer Report 26 Apr 2016
Peter K Rogan, Dept of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada 
Not Approved
VIEWS 38
The authors describe a dataset used for flow cytometric analysis of sloughed epithelial cells from a set of 6 individuals. It is not at all clear why these data are different from those reported in their copublished research note (http://f1000research.com/articles/5-180/v1) ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Rogan PK. Reviewer Report For: Flow cytometry dataset for cells collected from touched surfaces [version 2; peer review: 2 approved]. F1000Research 2016, 5:390 (https://doi.org/10.5256/f1000research.8964.r13496)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 07 Oct 2016
    Christopher Ehrhardt, Department of Forensic Science, Virginia Commonwealth University, Richmond, USA
    07 Oct 2016
    Author Response
    We agree that the differences between this manuscript and another publication currently in review at F1000 research could have been clearer. The cited manuscript (http://f1000research.com/articles/5-180/v1) was a preliminary survey of ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 07 Oct 2016
    Christopher Ehrhardt, Department of Forensic Science, Virginia Commonwealth University, Richmond, USA
    07 Oct 2016
    Author Response
    We agree that the differences between this manuscript and another publication currently in review at F1000 research could have been clearer. The cited manuscript (http://f1000research.com/articles/5-180/v1) was a preliminary survey of ... Continue reading
Views
24
Cite
Reviewer Report 19 Apr 2016
Dieter Deforce, Laboratory for Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium 
Approved with Reservations
VIEWS 24
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 ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Deforce D. Reviewer Report For: Flow cytometry dataset for cells collected from touched surfaces [version 2; peer review: 2 approved]. F1000Research 2016, 5:390 (https://doi.org/10.5256/f1000research.8964.r13031)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 07 Oct 2016
    Christopher Ehrhardt, Department of Forensic Science, Virginia Commonwealth University, Richmond, USA
    07 Oct 2016
    Author Response
    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 ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 07 Oct 2016
    Christopher Ehrhardt, Department of Forensic Science, Virginia Commonwealth University, Richmond, USA
    07 Oct 2016
    Author Response
    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 ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 23 Mar 2016
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
Sign In
If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password.

The email address should be the one you originally registered with F1000.

Email address not valid, please try again

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.

Code not correct, please try again
Email us for further assistance.
Server error, please try again.