Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples

Interpretation of touch DNA mixtures poses a significant challenge for forensic caseworking laboratories. Front end techniques that facilitate separation of contributor cell populations before DNA extraction are a way to circumvent this problem. The goal of this study was to survey intrinsic fluorescence of epidermal cells collected from touch surfaces and investigate whether this property could potentially be used to discriminate between contributor cell populations in a biological mixture. Analysis of red autofluorescence (650-670nm) showed that some contributors could be distinguished on this basis. Variation was also observed between autofluorescence profiles of epidermal cell populations from a single contributor sampled on different days. This dataset suggests that red autofluorescence may be a useful marker for identifying distinct cell populations in some mixtures. Future efforts should continue to investigate the extrinsic or intrinsic factors contributing to this signature, and to identify additional biomarkers that could complement this system.


Introduction
The difficulties associated with interpreting complex DNA mixtures are well known in the forensic community, and are becoming more prevalent with the sharp increase in 'touch' or trace samples among forensic laboratories' caseloads 1 . Differentiating cell populations from individual contributors in a biological mixture before DNA analysis is a potential way to overcome this issue. While strategies exist to selectively label cell populations from distinct contributors based on their immunochemistry and then physically isolate cells from the mixture prior to DNA profiling 2-4 , there is a dearth of studies demonstrating cell separation techniques on touch samples. This is likely due to the fact that cell populations in these samples mostly, if not entirely, consist of fully differentiated keratinocytes which have limited reactivity to common molecular probes used to target surface antigens 5,6 .
An alternative approach is to avoid the need for probe binding by harnessing the intrinsic fluorescence of compounds found in or on epidermal cells. Here we report on our analysis of autofluorescence in the red region of the spectrum (650-670nm) of epidermal cells collected from surfaces touched by seven different individuals across multiple days, and the implications this may have for processing complex biological mixtures in forensic casework.

Methods
Touch samples were collected from seven volunteers using the following protocol which was approved by the VCU-IRB (#HM20000454_CR). Volunteers rubbed a sterile polypropylene conical tube (P/N 229421; Celltreat Scientific) for five minutes using their entire hand (i.e., palm and fingers). Cells were collected from the surface with six sterile pre-wetted swabs (P/N 22037924; Fisher Scientific) followed by two dry swabs. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 10 mL of ultrapure water (18.2 MΩ•cm). The entire solution was then passed through a 100 µm filter mesh prior to flow cytometry. Flow cytometry analysis of eluted cells was performed on the BD FACSCanto™ II Analyzer (Becton Dickinson) equipped with 488 nm and 633 nm lasers and a 660/20 nm detector filter. Channel voltages were set as follows: Forward Scatter (FSC, 150V), Side Scatter (SSC, 200V) and Allophycocyanin (APC, 250V). FSC and SSC channels were used to gate intact corneocytes for subsequent autofluorescence analysis. Gating of cell populations and generation of histogram profiles for each contributor was performed using FCS Express 4.0 Flow Research Edition (De Novo Software, Inc.).   Table 1). Most experiments resulted in one or more contributor cell population(s) whose fluorescence profile(s) could be distinguished from the others collected that day, such that a fluorescence intensity gate could be designed that would be expected to capture that contributor's cells to the exclusion of (or minimal contribution of) cells from other contributors. However, significant and/or complete overlap was observed between many donor pairs (e.g., A42-B17 in Figure 1a; I66-S07 in Figure 1d). Sometimes, overlap of fluorescence distributions was such that gating could potentially separate the contributors into two or more groups (e.g. Figure 1d: A42, B17, I66, R12 and S07 in one group; D02 and J16 in another group). All contributors from the final experiment exhibited overlapping fluorescence histograms ( Figure 1e).

Results and discussion
Cell populations from J16 and D02 showed a great deal of disparity in fluorescence intensity in the first experiment, such that overlap between these populations was minimal ( Figure 1a). There was somewhat less distinction -and thus more overlap -observed between the same contributors during a second replicate ( Figure 1c); during a third, overlap between the two populations was substantial ( Figure 1d). As these results suggest, fluorescence intensity values for cell populations derived from any given contributor varied in distribution across replicate experiments on different days. Figure 1f shows overlayed histograms for J16 cell populations; mean fluorescence intensity values ranged from 589 to 2606 relative fluorescence units (RFUs) across five sampling days ( Table 1).
The underlying cause of red autofluorescence in these epidermal cell samples is currently unclear. Cells deposited through touch are likely primarily derived from the outermost epidermal layer (stratum corneum) which can contain a number of fluorescent compounds including tryptophan and tyrosine 7,8 , melanin, keratins, NADH and flavins 9 , lipofuscins 10 , and porphyrins and porphyrin precursors 11,12 . However, many of the corresponding emission maxima for these molecules occur at shorter wavelengths than what was examined in this study (e.g., amino acids, keratin, NADH, all have maxima below 550nm 9 ). Porphyrin molecules exhibit emission maxima between 630-680nm 11 . Their abundance within the epidermis may be influenced by bacteria on the skin that produce porphyrin molecules with similar fluorescence emission profiles 13 . Exogenous sources such as plasticides 14 or other biological compounds (e.g., chlorophyll 15 ) may also produce fluorescence, and could potentially be transferred to donors' hands and subsequently to the tube surface (with cells) through touch or contact.
Regardless of the ultimate source for the observed differences in cell population fluorescence, this initial data set indicates that autofluorescence may be a useful marker for distinguishing between cell populations in a mixture. The non-destructive nature of flow analysis and the fact that autofluorescence monitoring does not require special reagents beyond those maintained in any laboratory (e.g. no probes required) are advantages when considering their potential front-end use in forensic analyses.  Data is organized according to the histogram overlays shown in Figure 1. Mean (arithmetic) and median values are in relative fluorescent units (RFUs).
2 Flow cytometry cell 'events' correspond to populations within FSC and SSC gates that select for intact epidermal cells.
The variation across multiple samples from the same donor suggests that the level of autofluorescence is likely not a unique or identifying feature for a particular individual. However, to be of use in separating components of a biological mixture, a feature need not be unique; it simply needs to be distinctive among the contributors to that particular mixture. The ability to separate out even one contributor (or to separate a mixture of four contributors into two mixtures of two) may render the remaining mixture more interpretable in downstream DNA analysis. Further, the possibility that some combination of endogenous and/or exogenous factors could impart distinct optical properties to contributor cell populations in a particular mixture sample warrants further exploration.
Future efforts will continue to focus on isolating the molecule(s) responsible for fluorescent differences in touch epidermal cells through a combination of targeted immunofluorescent assays, chemical characterizations, and complex spectral analysis of autofluorescent profiles. Additionally, we are working on using optical signatures such as these to facilitate physical isolation of epidermal cell populations using flow cytometry-based strategies such as fluorescent activated cell sorting (FACS) for the purposes of generating single source genetic profiles from touch mixtures. Although previous work suggests that analyzing DNA profiles directly from isolated epidermal cells may be a challenge due to the prevalence of extracellular or 'cell-free' DNA in touch samples 16 , the sheer quantity of cells that may be recovered from these sample types (up to ~1×10 5 , 16 ) may help to overcome such obstacles.

Data availability
F1000Research: Dataset 1. Flow cytometry source data for individual contributors, 10.5256/f1000research.8036.d113749 17 Author contributions CE conceived the study. CE, CS, KP, EB, and YK designed the experiments. CS, EB, YK carried out the research. KP assisted with data analysis and provided expertise in the area of forensic casework. CE, CS, and KP prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.

Competing interests
No competing interests were disclosed.

Grant information
This project was funded by the National Institute of Justice Award number 2013-DN-BX-K033 (PI: Ehrhardt). Flow cytometry services in support of the project were provided by the VCU Massey Cancer Center, supported in part with funding from NIH-NCI P30CA016059.
I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1.

2.
3. Separation of cellular genetic material is central to forensic DNA analysis. Separation stages include:

Open Peer Review
Cells. Labs cells out of mixed cellular populations based on physical characteristics. For separate example, differential extraction can enrich sperm in one cell fraction relative to epithelial cells in another fraction. The red autofluorescence described in the paper occurs at this front-end stage.
DNA. Extraction procedures nucleic acids from other molecules. Such procedures separate include organic, non-organic, chelex, FTA or silica methods.
Loci. PCR short DNA regions from the rest of the genome by amplifying (i.e., purifying) separates these specific regions relative to background sequence.
Fragments. Fluorescent electrophoresis DNA fragments by size, identifying length separates polymorphism alleles and determining their relative quantity.
Genotype. Computer of electrophoretic data can determine the genotypes of each separation contributor to a DNA mixture . Back-end methods that do not separate contributors have limited applicability for resolving complex DNA mixtures. This paper focuses on the first step, front-end separation of mixed epithelial cell populations. The authors investigate a novel physical assay -red autofluorescence in the 650-670 nm range. Their preliminary results demonstrate effective separation, with some cell populations clearly distinguished from others based on fluorescent frequency.
There are some limitations to the current procedure. The partial autofluorescence separation is incomplete, and can vary for the same subject between samplings. However, as the authors note, "the ability to separate out even one contributor [subset] may render the remaining mixture more interpretable in downstream DNA analysis." This ability, coupled with automatic fluorescent activated cell sorting (FACS) and automatic DNA mixture data analysis (e.g., TrueAllele® computing), could be quite powerful.
Joint Bayesian analysis of multiple STR samplings of low-level DNA mixtures can recover considerable identification information . The autofluorescence method reported here could produce analogous data by 1 2 3 between sampling and analysis. It would be of benefit to repeat this study with older samples, perhaps placed onto a swab and dried for a week before analysis, compared to pristine cells collected via a glass bead method without the need for swabs at all.
The cells collected and analysed herein may not all be borne of the individual who touched the item -how controlled were the volunteers before touching? Did they have to wash their hands? Did they have contact with others or other work environments? The only way to assess this fully would be to examine the profiles from these samples post-analysis.
Following this, the samples may not actually yield sufficient DNA, as rightly pointed out in the discussion. It seems a shame that these samples were not separated and profiled to examine this further.
Overall, the publication of this type of data should be encouraged, and hopefully more thorough experiments of this kind will follow on from this work. No competing interests were disclosed. Competing Interests: 1