Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples.

The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface - one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x10 (3) and ~1x10 (5)) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence.

Introduction

‘Touch’ or trace Dn class="Chemical">NA samples represenpan>t a signpan>ificanpan>t portionpan> of evidenpan>ce submitted to forenpan>sic caseworkinpan>g laboratories. Understanpan>dinpan>g the mechanpan>isms of Dpan> class="Chemical">NA transfer through touch and developing methods to maximize the level of DNA recovery from contact surfaces is a continuing priority for the forensic science community [1]. Historically, the quantity of DNA found in a contact sample was thought to be primarily based on the number of cells that people shed naturally from the outermost layer of skin [2]. This concept continues to be perpetuated in the forensic community and analysts still testify to this effect [3– 5].

However, recent studies have shown that touch samples can also contain ‘cell-free’ or extracellular nucleic acids (referred to as Cn class="Chemical">NAs, eDpan> class="Chemical">NA, or cfDNA; in contrast to intracellular DNA or iDNA) that could be derived from a variety of sources such as sweat and oil secretions [6– 10]. Additionally, it has been suggested that small amounts of saliva can be transferred through touch which may be a source of both cell-free DNA and intracellular DNA (via nucleated buccal cells) to a contact sample [11].

Although there are many possible sources of genetic material in touch evidence, the proportion of cellular and extracellular components is currently unclear. A recent survey of casework samples reported that more than 70% of contact samples contained extracellular Dn class="Chemical">NA, which oftenpan> provided anpan> added value to the short tanpan>dem repan> class="Chemical">peat (STR) profile generated from the pelleted cellular material [6]. The study also found that the relative proportion of extracellular DNA to the total amount of DNA in each sample varied considerably.

In addition to understanding their relative contributions to contact samples, the forensic community would also benefit from determining whether different factors affect the deposition and n class="Chemical">persistenpan>ce of epidermal cells anpan>d extracellular Dpan> class="Chemical">NA on touched surfaces. Addressing these issues has important implications for optimizing DNA collection techniques as well as developing alternative analytical strategies for processing caseworking samples (e.g., 12).

Therefore, the goal of this study was to investigate the relative contributions of extracellular and intracellular Dn class="Chemical">NA anpan>d their relationpan>ship to the quanpan>tity of cells recovered from touch samples unpan>der conpan>trolled conpan>ditionpan>s, anpan>d assess how the tranpan>sfer anpan>d recovery of each typan> class="Chemical">pe of biological material may be influenced by particular actions of the individual contributor. To accomplish this, we used flow cytometry for precise and non-destructive measurements of touch samples that were simultaneously processed using standard caseworking techniques for DNA analyses.

Methods

Sample collection

For initial imaging studies, two individuals were asked to hold a sterile conical tube (P/n class="Chemical">N: 229421; Celltreat Scienpan>tific) inpan> onpan>e hanpan>d for five minpan>utes. Samples were collected from the tube surface with onpan>e sterile, pre-wetted swab (P/pan> class="Chemical">N: 22037924; Fisher Scientific) followed by one dry swab. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 1 mL of Sterile DNAse-Free, Protease-Free Water (P/N: BP24701; Fisher Scientific). All procedures for participant solicitation and consent for human subject research were approved by the VCU-Institutional Review Board (ID# HM20000454_CR).

For exn class="Chemical">perimenpan>ts inpan>volvinpan>g comparisonpan>s of cell anpan>d Dpan> class="Chemical">NA yields between washed and unwashed hands, two sets of two samples (one tube in each hand) were collected from eight individuals using the protocol described above: the first set of 16 was collected before hand washing, and the second after washing hands with soap and water for 20 seconds and air drying. A 20μL aliquot of each 1mL cell solution was used in subsequent flow studies (including cell enumeration), and the remaining 980μL was used for DNA studies.

Another 20 samples were collected without any control for hand washing from these eight n class="Species">donors, alonpan>g with three additionpan>al pan> class="Species">donors, using the protocol described above. The entirety of each of these samples was processed for DNA.

Microscopic imaging

In order to separate intact cells from debris and cellular fragments for imaging purposes, after passing cell susn class="Chemical">pensionpan>s through a 100 µm mesh filter, Fluorescenpan>ce-Activated Cell Sortinpan>g (FACS) was pan> class="Chemical">performed on the BD FACSAria™ Ilu (Becton Dickinson) flow cytometer using a 488 ηm Coherent solid-state laser. Channel voltages were set as follows: FSC, 50V; SSC, 200V. Events falling into gate “K” (see Figure 1) were sorted into a new tube, then imaged using the Amnis ® Imagestream X MK II Software (EMD Millipore) by activating the Bright Field channel. Pictures were analyzed and exported with the IDEAS ® Software v6.1 (EMD Millipore).

Figure 1.

Optical characterization of a touch sample.

Cell events fall into two distinct populations along the Forward Scatter (FSC) and Side Scatter (SSC) axes: intact cells (‘K’) and cell debris (‘D’). Right insets show images of individual events within the K population. Scale bar=7 µm.

Flow cytometry analysis and cell enumeration

Cell susn class="Chemical">pensions were passed through a 100 µm mesh filter prior to flow cytometry analysis on the BD FACSCanto™ II analyzer (Becton Dickinson) using 488 ηm and 633 ηm lasers. The channel voltages were set as follows: FSC, 150V; SSC, 200V; n class="Chemical">FITC, 335V; PE, 233V; PE-Cy5, 300V; PE-Cy7, 400V; and APC, 250V. Data acquisition was performed using the FACSDIVA Software v8.0.1 (Becton Dickinson) and analyzed using FCS Express 4.0 (DeNovo).

In order to precisely quantify the cells in our samples during flow analysis, we spiked our cell solutions with a known concentration of 123 eBeads (01-1234-42; Affymetrix eBioscience), fluorescently-labeled microparticle standards that are 7 µm and easily distinguishable from our target cell population both in size and fluorescence (n class="Chemical">FITC, pan> class="Chemical">PE, and APC channels). The ratio of cells to beads was then used to determine the concentration of cells in the sample through the following formula:

The concentration of cells in the 20µL aliquot was then used to estimate the total number of cells present in the entire volume of eluent (1mL) recovered from the collection swabs. Flow cytometry analysis of bead standards was conducted on events detected within the ‘K’ gate for each sample. The procedures for detecting and differentiating eBeads from target cells followed the manufacturer's suggested protocol ( http://www.ebioscience.com/media/pdf/tds/01/01-1234.pdf).

Isolation and extraction of extracellular and cell pellet DNA

Once an aliquot was removed for cell quantification, the remaining cell susn class="Chemical">pensionpan> was tranpan>sferred to a new 2mL collectionpan> tube anpan>d cenpan>trifuged at 10,000 xg for five minpan>utes at room tempan> class="Chemical">perature. The supernatant was added to a pre-washed Amicon filter (UFC210024; EMD Millipore). The remaining pellet was washed with 500 µL of sterile water twice, each time adding the supernatant to the Amicon filter. The combined supernatant fraction was then centrifuged at 3,220 ×g for 30 minutes, followed by a wash step in 2 mL 1xTE Buffer (P/N 50-843-203; Teknova). The DNA was collected by inverting the filter and centrifuging at 1,000 xg for 2 minutes at room temperature. The final volume of the eluted retentate was approximately 20 µl.

Additionally, in order to maximize the recovery of cell material and/or Dn class="Chemical">NA, the wet swab tips were placed inpan> a spinpan> basket (P/pan> class="Chemical">N 19597; Investigator Lyse & Spin Basket Kit; Qiagen) immediately following the initial elution, and centrifuged at 10,000 ×g for 5 minutes at room temperature (absent any additional reagents). The resulting liquid eluent that passed through the spin basket was added to the supernatant solution (prior to Amicon filtration) and remaining cell pellet in the spin basket was dissolved in ~50 µl of sterile water, combined with its respective fraction, and subjected to DNA extraction using the following protocol. The cell pellet material was incubated with 500 µL Cell Lysis Buffer (P/N BDB559759; BD Pharmigen) and 10 µL Proteinase K (P/N EO0491; Fisher Scientific) in a 56°C water bath for 17 hours. The sample was then centrifuged at 10,000 ×g for 5 minutes at room temperature. The supernatant was purified with an equal volume of UltraPure Phenol:Chloroform:Isoamyl Alcohol (P/N 15593-031; Life Technologies (25:24:1, v/v)), then 1xTE Buffer, and finally concentrated to a final volume of 20–40 µL using a pre-washed Amicon filter.

DNA quantitation

Dn class="Chemical">NA quantitation was n class="Chemical">performed using the Investigator Quantiplex ® Human Kit (P/N 387016, Qiagen) coupled with the ABI Prism 7500 Sequence Detection System (Applied Biosystems). A 25 µl reaction was used for all samples following manufacturer’s suggested protocol (‘Investigator Quantiplex Handbook’, www.qiagen.com).

Results

Initial characterizations of touch samples with flow cytometry showed two distinct size fractions (‘K’ and ‘D’ populations in Figure 1). Size and morphological information derived from AMn class="Chemical">NIS images of the K fractionpan> from two touch samples revealed that this populationpan> was conpan>sistenpan>t with fully differenpan>tiated keratinpan>ocytes ( i.e., cornpan>eocytes) ~20–40 µm inpan> diameter, while the D fractionpan> was conpan>sistenpan>t with cellular debris/fragmenpan>ts. Other epithelial cell typan> class="Chemical">pes ( e.g., buccal cells >60 µm) were not observed among the AMNIS images captured ( Figure S1, Figure S2).

Cell counts and Dn class="Chemical">NA yields were compared across 31 touch samples genpan>erated from eight differenpan>t individuals that used both dominant and non-dominant hands to hold the substrate. To investigate the effect of hand washing on the transfer of cellular and extracellular componenpan>ts of a touch sample, half of these samples were collected after n class="Species">donors had washed their hands and the other half without immediate hand washing.

An estimated ~5×10 3 to ~1×10 5 cells were recovered from washed hand samples, versus ~1×10 3 to ~8×10 4 cells from unwashed hand samples ( Figure 2; Table S1). Overall, we observed greater transfer of cells in the washed hand samples than the unwashed hand samples (median of 2.5×10 4 cells vs. 8.6×10 3 cells, resn class="Chemical">pectively). Despite the oftenpan> high recovery of cells from touch samples, Dpan> class="Chemical">NA recovery from the cell pellet was consistently low, whether from washed or unwashed hands. DNA was detected in the cell pellet of one unwashed hand sample (0.220 ng) and three washed hands samples (0.049, 0.042, 0.060 ng). No DNA was detected in any of the other cell pellets.

Figure 2.

Cell counts and DNA yields from touch samples from washed and unwashed hands.

For each graph, the Y axis represents the number of “K events” (cells) detected in solution from collection swabs (unwashed hands in a and b; washed hands in c and d), while the X axis represents the number of nanograms of DNA recovered (from supernatant ( a) and cell pellet ( b) of unwashed hands, and from supernatant ( c) and cell pellet ( d) of washed hands).

For each graph, the Y axis represents the number of “K events” (cells) detected in solution from collection swabs (unwashed hands in a and b; washed hands in c and d), while the X axis represents the number of nanograms of Dn class="Chemical">NA recovered (from supan> class="Chemical">pernatant ( a) and cell pellet ( b) of unwashed hands, and from supernatant ( c) and cell pellet ( d) of washed hands).

In contrast, consistent differences were observed in eDn class="Chemical">NA recovery from samples genpan>erated from washed versus unpan>washed hanpan>ds. Little to no Dpan> class="Chemical">NA was recovered from the extracellular fraction of touch samples left by donors who had washed their hands, with quantitation values ranging from zero to 0.242 ng ( Figure 2c). In samples from unwashed hands, extracellular DNA recovery varied between zero and 4.646 ng ( Figure 2a). There was no apparent correlation between the number of cells and the quantity of DNA recovered from the samples (either eDNA or cell pellet). Neither could DNA recovery with or without hand washing be correlated to hand dominance, in contrast to findings by others [13].

The additional 20 samples tested for relative quantity of eDn class="Chemical">NA versus inpan>tracellular Dpan> class="Chemical">NA produced results that are consistent with the above findings ( Table 1, compilation of all samples without hand washing (n = 35)). In samples where DNA was detected, the total proportion of eDNA ranged from 84–100% with the majority of the samples at or near 100%.

Table 1.

Proportion of DNA in supernatant and in cell pellet after three washes.

Sample	Extracellular DNA (ng)	DNA in Cell Pellet (ng)	Percentage eDNA
D11	0.607	ND	100
	1.191	0.068	95
	0.603	ND	100
	0.842	ND	100
	2.023	ND	100
E14	2.296	0.037	98
	1.134	0.220	84
	0.504	ND	100
	ND	ND	n/a
E15	2.162	0.284	88
	0.567	ND	100
	4.646	ND	100
	0.940	ND	100
	2.842	ND	100
C81	ND	ND	n/a
	ND	ND	n/a
	0.282	ND	100
D02	ND	ND	n/a
	0.374	ND	100
	ND	ND	n/a
	0.028	ND	100
	ND	ND	n/a
H73	ND	ND	n/a
	ND	ND	n/a
	0.780	ND	100
I66	0.286	ND	100
	1.240	ND	100
	1.110	ND	100
J16	1.804	0.021	99
	1.262	ND	100
Y02	0.058	ND	100
	0.106	ND	100
	0.314	ND	100
K08	ND	ND	n/a
S07	ND	ND	n/a

ND=below the limit of detection, ~1 pg/µl. Samples refer to individual donors. Each row within a single donor shows results from replicate experiments performed on different days.

n class="Chemical">ND=below the limit of detection, ~1 pg/µl. Samples refer to individual n class="Species">donors. Each row within a single donor shows results from replicate experiments performed on different days.

Discussion

Our results contribute to the forensic community’s growing body of knowledge on touch samples. We found that the vast majority (~84–100%) of nuclear Dn class="Chemical">NA recovered from touch samples collected unpan>der the conpan>ditionpan>s described above is extracellular. Amplifiable Dpan> class="Chemical">NA from the pelleted cellular fraction was detected in only eight of the 51 touch samples analyzed ( Figure 2, Table 1).

Although this finding is generally consistent with other recent studies suggesting the significance of extracellular Dn class="Chemical">NA inpan> touch evidenpan>ce [6, 8], the prevalenpan>ce anpan>d proportionpan> of extracellular Dpan> class="Chemical">NA relative to the total DNA yield shown in Table 1 was higher than observed in other studies [6]. It is possible that the multiple wash steps performed on the pelleted cell material for this study removed more eDNA than efforts utilizing a single wash. In a separate analysis of seven replicate samples, we found that additional eDNA was often recovered with additional wash steps, and concurrently, that a clear systematic cell loss at each wash step was not observed—a Student’s t-test on cell counts before and after three wash steps yielded an average p-value of 0.28 with only two of the individual replicates yielding p-values less than 0.01 ( Table S2). This suggests that while some cells may have been unintentionally removed from some cell pellets by our methodology, this phenomenon is unlikely to explain the consistent increased DNA recovery in the supernatant with additional washes across samples.

The nature of the samples likely played a role as well, as there may have been more opportunities to n class="Gene">pick up nucleated cells for some casework samples described inpan> other research [6] thanpan> our conpan>trolled conpan>ditionpan>s. The fact that the “typical” or “stanpan>dard” touch sample evades definpan>itionpan> poses a challenpan>ge whenpan> designpan>inpan>g studies to better unpan>derstanpan>d these kinpan>ds of samples. It has beenpan> suggested that saliva, which conpan>tainpan>s buccal cells, may be anpan> importanpan>t ( i.e., Dpan> class="Chemical">NA rich) component of some touch samples [11]. We observed no evidence of such cells – which generally appear larger than corneocytes (>60 µm for buccal cells versus 20–40 µm for corneocytes) – in microscopic surveys of individual cells within two touch samples ( Figure S1, Figure S2). However, this does not preclude the possibility that non epidermal cells were present, since only a portion of the sample was surveyed, and because deformed or fragmented cells from different tissues may be indistinguishable from corneocytes. Future work could explicitly test for the presence of buccal cells in touch samples through, e.g., antibody hybridizations targeting tissue specific surface antigens coupled with flow cytometry.

The mechanism of touching could also affect the proportion of eDn class="Chemical">NA to iDpan> class="Chemical">NA in touch samples; our preliminary data from touch samples deposited by rubbing suggest that this action may result in considerably higher cell pellet yields than samples deposited by holding, perhaps by exposing deeper (i.e., undifferentiated) layers of cells. However, in these preliminary experiments we also observed that the amount of eDNA left by rubbing the substrate was similar to levels of eDNA left by holding. This suggests that the transfer of eDNA may not be as affected by the manner in which a substrate was handled as iDNA transfer.

In any case, our results lend further support to the concept that extracellular Dn class="Chemical">NA is particularly crucial to the anpan>alysis of touch samples. Measures should be explored to exploit this source of inpan>formationpan> to the greatest extenpan>t possible. For sample collectionpan> anpan>d processinpan>g purposes, this may dictate that touch samples be treated differenpan>tly thanpan> other typan> class="Chemical">pes of forensic biological sample. To avoid the significant loss of DNA that may be associated with extraction, it may make sense to process the eDNA-containing supernatant separately via direct amplification; our results suggest that care should be taken to maximize the amount of eDNA washed into the supernatant.

Our finding that the number of cells in touch samples was uncorrelated to the amount of extracellular Dn class="Chemical">NA or the total Dpan> class="Chemical">NA yield suggests that not only is the recoverable DNA primarily extracellular but that it is not immediately derived from the large numbers of epidermal cells that are shed daily. DNA was not detected in the cell pellet of samples that contained more than 100,000 cells, while samples comprised of far fewer cells (~2000) yielded DNA. Our extraction methodology likely had some impact on overall DNA yield [14]; we have found in other experiments that other extraction methodologies (e.g., DNA IQ) resulted in low (<80pg) but quantifiable DNA yields in samples that yielded no DNA after processing with the extraction method utilized here. However, this does not change the fact that a considerable portion of DNA from the touch samples that we analyzed was extracellular, and that the number of cells shed was not a reliable indicator of DNA yield. These results are compatible with previous medical research showing that corneocytes from the outermost epidermal layer (i.e., stratum corneum) have little to no genomic DNA owing to the controlled degradation of intracellular components during differentiation [15].

Accordingly, epidermal cells – even when present in large quantities – may make a fairly insignificant contribution to either intra- or extracellular Dn class="Chemical">NA recovery from touch samples. Conpan>sistenpan>t with recenpan>t studies that founpan>d no evidenpan>ce of fragmenpan>ted Dpan> class="Chemical">NA in the epidermal layers (in contrast to sebaceous cell sources) [10], the majority of extracellular DNA in touch samples is likely derived from alternate sources such as oil and sweat secretions, or saliva [8, 11]. Where intracellular (i.e., cell pellet) DNA levels from touch samples are considerably higher than those observed in this study, a nucleated cell source (i.e., non-epidermal, or more basal epidermal) may be implicated, though certain skin conditions are known to result in the aberrant retention of nuclear DNA in corneocytes [15].

Although hand washing resulted in the transfer and subsequent recovery of little to no eDn class="Chemical">NA, we founpan>d that cells were nonpan>etheless tranpan>sferred. Inpan> fact, we observed greater levels of cellular tranpan>sfer amonpan>g washed hanpan>d samples thanpan> unpan>washed hanpan>d samples. It is possible that the act of hanpan>d washinpan>g loosenpan>s or sloughs off cornpan>eocytes, anpan>d that these cells (pan> class="Chemical">perhaps because of their flattened morphology) are more likely to persist through the washing process than eDNA. Regardless of the explanation, an estimated thousands to hundreds of thousands of cells survived the hand washing process to be transferred from the palmar surface by simple touching.

Consistent with Locard’s principle, while these shed corneocytes may not contain sufficient levels of nuclear Dn class="Chemical">NA to genpan>erate a probative STR profile, there is the possibility that other, nonpan>-genpan>etic signpan>atures could be anpan>alyzed, so that the most challenpan>ginpan>g touch samples (i.e. those that conpan>tainpan> little to no Dpan> class="Chemical">NA) may provide forensically relevant information. For example, the average size of individual corneocytes has been shown to vary with source factors such as age, sex, and anatomical region [16– 18], as does the composition of intracellular cytokeratin components [19]. While further research is of course necessary to assess the degree of inter- and intra-individual variance in particular cellular features, determining such source attributes from unknown contributors could potentially provide leads or exclude suspects in specific types of investigations, e.g., sexual assault, molestation. Further, the absence of amplifiable nuclear DNA in corneocytes does not necessarily preclude the presence of sufficient levels of mitochondrial DNA to permit typing. Combining techniques to sort epidermal cells into donor populations (e.g., using factors described above) and typing the mtDNA of those populations is an avenue that warrants further exploration.

Overall, our observations suggest that many traditional explanations of Dn class="Chemical">NA anpan>alysis from touch samples used inpan> expan> class="Chemical">pert testimony – which often seek to explain the quantity and quality of DNA detected (or lack thereof) in terms of an individual’s inherent or circumstantial susceptibility to shed epidermal cells – may need to be modified to reflect fundamental shifts in the forensic community’s understanding of touch evidence. Future research efforts should continue to examine the relationship between the transfer of eDNA, iDNA, and intact corneocytes onto touch surfaces by testing other types of depositional circumstances, e.g., different substrate material or touch samples from multiple donors.

The manuscript “Optical characterization of epidermal cells and their relationship to Dn class="Chemical">NA recovery from touch samples” by Stanpan>ciu CE et al. is focused onpan> the “Touch Dpan> class="Chemical">NA” topic and is aimed at investigating the relative contributions of extracellular and intracellular DNA and their relationship to the quantity of cells recovered from touch samples under controlled conditions. It also assesses how the transfer and recovery of each type of biological material may be influenced by particular actions of the individual contributor.

To this aim, the Authors used flow cytometry for precise and non-destructive measurements of touch samples that were simultaneously processed using standard caseworking techniques for Dn class="Chemical">NA anpan>alyses. The well described anpan>alytical methods anpan>d results show that there was no apparenpan>t correlationpan> betweenpan> the number of cells anpan>d the quanpan>tity of Dpan> class="Chemical">NA recovered from the samples, neither could DNA recovery with or without hand washing be correlated to hand dominance.

According to recent studies, they found that the vast majority (~84–100%) of nuclear Dn class="Chemical">NA recovered from touch samples collected unpan>der the conditions described above is extracellular (i.e. oil and sweat secretions, saliva), and suggest that future work could explicitly test for the presenpan>ce of buccal cells in touch samples through, e.g., antibody hybridizations targeting tissue sn class="Chemical">pecific surface antigens coupled with flow cytometry.

They also highlighted the n class="Chemical">peculiarities, anpan>d conpan>sequenpan>tly the anpan>alytical challenpan>ges, of touch samples, thus suggestinpan>g these samples to be treated differenpan>tly thanpan> other typan> class="Chemical">pes of forensic biological sample as for sample collection and processing purposes; e.g. to avoid the significant loss of DNA that may be associated with extraction, it may make sense to process the eDNA-containing supernatant separately via direct amplification.

An interesting and innovative element represented in the manuscript is the possibility that non-genetic signatures could be analyzed, so that the most challenging touch samples (i.e. those that contain little to no Dn class="Chemical">NA) may provide forenpan>sically relevanpan>t inpan>formationpan>. To this regard, it is reported that “… the average size of inpan>dividual cornpan>eocytes has beenpan> shownpan> to vary with source factors such as age, sex, anpan>d anpan>atomical regionpan>, as does the compositionpan> of inpan>tracellular cytokeratinpan> componpan>enpan>ts … combinpan>inpan>g technpan>iques to sort epidermal cells inpan>to pan> class="Species">donor populations (e.g., using factors described above) and typing the mtDNA of those populations is an avenue that warrants further exploration …”.

Finally, as an interested researcher in the field of “Touch Dn class="Chemical">NA”, I appreciate the efforts of the Authors in preparing this interesting manuscript, that is well writtenpan> and follows a logical structure. I just have a minor suggestion to improve the manuscript, that is to include some STR profiles in order to show the quality of the DNA recovered from the analyzed samples.

I have read this submission. I believe that I have an appropriate level of exn class="Chemical">pertise to confirm that it is of an acceptable scienpan>tific standard.

The manuscript ‘’Optical characterization of epidermal cells and their relationship to Dn class="Chemical">NA recovery from touch samples’’ by Stanpan>ciu CE et al. describes the genpan>etic (nuclear Dpan> class="Chemical">NA) and biological components (Cells and Cell debris) in samples created by touch or contact with a surface, both in controlled and non controlled situations. The question to understand and characterize the components of touch sample is of paramount importance in forensic science research. The manuscript attempts to decipher the mechanism of DNA transfer in touch samples and suggests methods to maximize the recovery of DNA from the touch samples.

On the cellular components side, the FACS coupled with microscopy technique was employed to explore the number and identity of  the cells in touch samples. The cells in touch samples were found to be consistent with the keratinocyte morphology and size. This work also shows that after washing hands there is tendency to shed more cells in touch samples but the increase in the cell number is not correlated with the increase in Dn class="Chemical">NA yield, rather there was no or very little Dpan> class="Chemical">NA recovered after washing hands. Thus, this study provides evidence that there is no or very little DNA associated with the cellular component of the touch samples. It makes a strong case for the next important question to ask. How we can differentiate keratinocytes from different individuals?

As the authors suggests that other biological information still may be present on the keratinocytes, it on class="Chemical">pens up the possibilities of a new field for characterizationpan> of keratinpan>ocytes from touch samples. The manpan>uscript’s conpan>clusionpan> that the source of majority of Dpan> class="Chemical">NA in touch samples comes from extracellular components, not from the cellular components, is in well agreement of previous studies but the proportion of extracellular DNA is found to be higher in this study than the earlier reports. The manuscript is well written and presents the data in a logical way. Overall, the manuscript adds further knowledge to the body of knowledge existing in this field and I recommend this manuscript for indexation.

However I have following minor suggestions/comments to improve the manuscript.

n class="Chemical">No profiles, convenpan>tional or LCn class="Chemical">N, are shown to show the quality of DNA recovered.

It will be interesting to explore the contributions (both of Dn class="Chemical">NA as well as Cells componenpan>ts) made by normal flora of n class="Species">human skin.

‘’....flow cytometer using a 488 ηm Coherent solid-state laser.... ‘’ in this line and elsewhere the nanometer symbol should be written as ‘nm’.

I have read this submission. I believe that I have an appropriate level of exn class="Chemical">pertise to confirm that it is of an acceptable scienpan>tific standard.