1. IntroductionIn court, expert witnesses are often asked to address questions that concern the activity which led to the deposition of a defendant’s DNA on an item or surface. In particular, if the source of the DNA is considered to be from biological material deposited after skin contact, it is common that the defendant has an alternative explanation as to how his DNA was deposited which often involves indirect transfer of DNA. According to the recommendations of the International Society of Forensic Genetics (ISFG) [1Gill P. Hicks T. Butler J.M. Connolly E. Gusmao L. Kokshoorn B. Morling N. van Oorschot R.A.H. Parson W. Prinz M. Schneider P.M. Sijen T. Taylor D. DNA commission of the International society for forensic genetics: assessing the value of forensic biological evidence - guidelines highlighting the importance of propositions: Part I: evaluation of DNA profiling comparisons given (sub-) source propositions., 2Gill P. Hicks T. Butler J.M. Connolly E. Gusmao L. Kokshoorn B. Morling N. van Oorschot R.A.H. Parson W. Prinz M. Schneider P.M. Sijen T. Taylor D. DNA commission of the International society for forensic genetics: assessing the value of forensic biological evidence - guidelines highlighting the importance of propositions. Part II: Evaluation of biological traces considering activity level propositions.], evaluation of the DNA findings should be reported using a likelihood ratio (LR) approach based on case specific propositions. The scientist assesses the value of the evidence of two alternate propositions. Activity level is addressed by Bayesian networks, a graphical tool to evaluate complex probabilities [2Gill P. Hicks T. Butler J.M. Connolly E. Gusmao L. Kokshoorn B. Morling N. van Oorschot R.A.H. Parson W. Prinz M. Schneider P.M. Sijen T. Taylor D. DNA commission of the International society for forensic genetics: assessing the value of forensic biological evidence - guidelines highlighting the importance of propositions. Part II: Evaluation of biological traces considering activity level propositions., 3Taylor D. Biedermann A. Hicks T. Champod C. A template for constructing Bayesian networks in forensic biology cases when considering activity level propositions., 4Bayesian networks for evaluating forensic DNA profiling evidence: a review and guide to literature.]. As the probabilities involve DNA transfer, persistence and recovery, an individuals’ shedder status may have an impact on the outcome. Fonneløp et al. [[5]Fonnelop A.E. Ramse M. Egeland T. Gill P. The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario.], found that the shedder status of an individual influenced the transfer rate of DNA to a T-shirt in a simulated attack scenario. There was a significantly higher amount of DNA deposited by high shedders compared to low shedders; indirect transferred DNA was more likely to be detected on the low shedder’s T-shirt compared to the T-shirt of the high shedders.It has been well-known for decades that a full DNA profile can be obtained from an item after skin contact, along with the possibility to detect indirect transferred DNA [6van Oorschot R.A. Jones M.K. DNA fingerprints from fingerprints., 7Ladd C. Adamowicz M.S. Bourke M.T. Scherczinger C.A. Lee H.C. A systematic analysis of secondary DNA transfer., 8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces.]. Furthermore, different studies on the topic have shown that there are inter- and intra-individual differences in the propensity to deposit DNA [8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces., 9The tendency of individuals to transfer DNA to handled items., 10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer.], and that the time of deposition since hand wash, post 15 min, did not have a clear effect [11Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders., 12Kanokwongnuwut P. Kirkbride P. Linacre A. Speed of accumulation of DNA in a fingermark, Australian.]. It is not completely understood why individuals have different tendencies to deposit DNA. A review by Burill et al. [[13]Burrill J. Daniel B. Frascione N. A review of trace “Touch DNA” deposits: variability factors and an exploration of cellular composition.] concluded that self-cellular components may consist of shed corneocytes from hands, DNA residues in fragmented cells, shed nucleated cells from hands, cells transferred from elsewhere on the body and cell-free DNA. Other studies have shown that the presence of sebum and sweat or other body fluids, like saliva and blood, can promote DNA transfer [14van den Berge M. Ozcanhan G. Zijlstra S. Lindenbergh A. Sijen T. Prevalence of human cell material: DNA and RNA profiling of public and private objects and after activity scenarios., 15Lacerenza D. Aneli S. Omedei M. Gino S. Pasino S. Berchialla P. Robino C. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.]. Hence, the presence of transferred nucleated cells or cell-free DNA on the hands may occur because of habits: for instance touching the face or forehead, could affect a person’s shedder status. Gender and age have also been reported to be a dependent factor as several studies have concluded that males have a higher propensity of depositing DNA than females [10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer., 11Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders., 15Lacerenza D. Aneli S. Omedei M. Gino S. Pasino S. Berchialla P. Robino C. A molecular exploration of human DNA/RNA co-extracted from the palmar surface of the hands and fingers.], and that children aged 1–10 years old deposit a higher amount of DNA compared to older individuals; elderly individuals over 60 years old deposited less DNA compared to younger adults [10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer., 16Poetsch M. Bajanowski T. Kamphausen T. Influence of an individual’s age on the amount and interpretability of DNA left on touched items.]. Also, skin diseases that increase the keratinocyte turnover may result in a higher deposition of DNA; Kamphausen et al. [[17]Kamphausen T. Schadendorf D. von Wurmb-Schwark N. Bajanowski T. Poetsch M. Good shedder or bad shedder--the influence of skin diseases on forensic DNA analysis from epithelial abrasions.], showed that patients with atopic dermatitis or psoriasis had a higher propensity of depositing DNA when handprints were examined, compared to healthy individuals.There have been several attempts to develop a method to categorize a person’s shedder status. Studies have addressed this by asking participants to hold a plastic tube followed by DNA analysis of the handprint on the tube [8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces., 9The tendency of individuals to transfer DNA to handled items., 10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer.]. Participants were categorized into “good” and “poor shedders” [8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces., 9The tendency of individuals to transfer DNA to handled items.] or into “good”, “intermediate” and “poor shedders” [[10]Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer.] based on the number of donor’s alleles detected. Fonneløp et al. [[5]Fonnelop A.E. Ramse M. Egeland T. Gill P. The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario.], adopted a similar approach but determined shedder classification into “high” and “low shedders” by combining the DNA quantity and the STR results. Kanokwongnuwut et al. [[11]Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders.] published a different approach where they assessed shedder status by counting the number of cells in a fingerprint by using Diamond™ nucleic acid dye and fluorescence microscope combined with direct PCR of the fingermark. They observed a good association between the number of detected cells and the strength of the DNA result (total rfu value or number of detected alleles) in fingerprints in two separate studies [11Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders., 12Kanokwongnuwut P. Kirkbride P. Linacre A. Speed of accumulation of DNA in a fingermark, Australian.], where the participants were categorised as “heavy”, “intermediate” or “light shedders” based on the number of cells detected by microscopy [[11]Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders.].The aim of this study was to find a quick, reliable and reproducible method to establish a person’s shedder status, which is applicable in both research and casework when transfer related questions are considered. We compared the cell count method (CC method) to the classical handheld tube method (HH method). For the CC method we used Diamond™ nucleic acid dye which is a fluorescent dye that binds to the grooves of the DNA molecule [], in both cell-free and nuclear DNA; however cell-free DNA is unlikely to be detected by microscopy using 220X or less magnification [12Kanokwongnuwut P. Kirkbride P. Linacre A. Speed of accumulation of DNA in a fingermark, Australian., 19Kanokwongnuwut P. Martin B. Taylor D. Kirkbride K.P. Linacre A. How many cells are required for successful DNA profiling?.]. The number of cells that appear as bright spots in a fluorescence microscope, within a fingerprint, was counted. The HH method was followed by direct PCR analysis after swabbing the tubes. A handprint was considered to be representative for casework as the question of shedder status is often raised when evaluating the evidence of DNA transfer to a touched item. Furthermore, the aims were to:1)

Investigate the association between the number of cells detected within a fingerprint by microscopy and the strength of the DNA result from handheld tubes using 20 participants in duplicate. The purpose was to evaluate if the CC method, on its own, could be used to classify a person’s shedder status.

2)

Compare the results from the HH method followed by direct PCR for 20 participants at three different time points, minimum 1 h post-handwashing, and

3)

To classify the 20 participants in three categories defined as low, medium and high shedders.

2. Methods2.1 Ethical declaration

This study was approved by the Data protection officer (DPO) at Oslo University Hospital prior to initiating this project (reference 20/17/479). The project was carried out according to the approved procedures and protocols, and all participants have given informed consent.

2.2 Pre-studiesSeveral small pilot studies were carried out prior to this study to find a method that is sensitive and reproducible, but not too labour intensive. We tested 1) the cell count method (CC method) with Diamond nucleic dye and direct PCR of fingerprints, 2) different DNA sampling methods and 3) CC method combined with handheld tube method (HH method) and direct PCR of handprints (see Appendix A, Supplementary material, for details).

From different pre-studies, we concluded that the handheld tube method combined with direct PCR worked well. In addition, we consider a handprint to be representative for casework as the question of transfer often rises in context of a touched item (i.e. a contact that involves the whole hand rather than a finger).

2.3 Sample collection

Twenty volunteers (5 male and 15 female) were asked to supply two fingerprints and three handprints on microscope slides and plastic tubes respectively; collected minimum 1 h post handwashing. Participants were asked to carry out normal activities between washing their hands and depositing finger-/handprints, but to refrain from using hand cream, hand disinfection and gloves.

All slides and plastic tubes were pre-cleaned with ethanol wipes. Negative controls (blanks) were collected from two pre-clean tubes. One allele was detected at one locus in both samples: the negative controls were considered to be clear. Fingerprints were collected by placing the index finger of the dominant hand on a microscope slide (Independent Forensics, SPERM HYLITER™ Microscope Slides 1 × 11 mm), applying medium pressure, for approximately 3–5 s, within the designated ring of 11 mm diameter. Directly after depositing the fingerprint, the handprint of the dominant hand was collected by grasping a 15 mL plastic tube (vwr™, Centrifuge tube high Performance), also applying medium pressure, for 10 s. The DNA sample was collected immediately by swabbing the entire body of the plastic tube (not the lid) by using a moistened cotton swab (mwe, Tubed Sterile Dryswab™, MW1041). The swab was placed in an evidence paper bag until analysed. The sampling was repeated twice, but fingerprints were only collected on the first two occasions. A total of 40 fingerprints and 60 handprints/ DNA samples were collected. The sampling for each individual was carried out on different days, or two on the same day with intervals of at least 3 h between each set.

2.4 Diamond dye staining and microscopyThe microscope slides were stained with Diamond™ Nucleic Acid Dye (Promega). Thirty µl of 20X Diamond dye solution (1 µl DD 10 000 X stock solutions added to 500 µl of 70% ethanol) was added to each slide, and left to dry. As a control of background dye a blank slide was also included in the experiment, and no clear bright sports were observed. The slides were studied under a Zeiss Axio fluorescence microscope at 100X magnification with blue excitation light (475 nm) and green emission filter (540 nm). The approximate number of fluorescent cells (bright spots/flakes) within the 11 mm ring was counted and recorded. Fig. 1 shows an example of Diamond dye stained cells from one of the participant’s fingerprint.

Fig. 1Photo of fluorescence cells in a fingerprint. The white arrows represent 100 µm. The photo is taken with Zen 2.6 software using Alexa Fluor 430 filter. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article).

2.5 DNA analysis

The samples were analysed by direct PCR. The tips of the cotton swabs were placed into 0.2 mL PCR tubes. Samples were amplified using the PowerPlex®Fusion 6 C System (Promega) as recommended by the manufacturer (25 µl reaction volume, 29 amplification cycles). Amplification was carried out using a Veriti® 96-Well Thermal Cycler (Applied Biosystems®). Samples were injected onto the Applied Biosystems 3500xl Genetic Analyzer at 1.2 kV for 24 s. The results were analysed using the GeneMapper® ID-X Software version 1.6 (Applied Biosystems®) and the limit of detection (LOD) for alleles was set to 100 rfu. No stochastic threshold value was applied for the homozygote alleles. The DNA profile was compared to the reference profile from the donor, and the number of alleles, loci and the total (summed) rfu value of the donor’s allele was recorded, including amelogenin. Homozygote alleles were counted as two. The Y-chromosome STRs were disregarded in this experiment.

The total rfu value served as a quantification variable. Samples with more than one unknown allele were defined to be a mixture. The total rfu value representing the donor was adjusted in mixtures by multiplying the rfu value of the entire profile by the mixture proportion of the donor. This was calculated using the software EuroForMix v3.0.4 [[20]Bleka Ø. Storvik G. Gill P. EuroForMix: an open source software based on a continuous model to evaluate STR DNA profiles from a mixture of contributors with artefacts.]. The number of foreign (non-donor) alleles was recorded and their impact as indirect transferred DNA is discussed in Section 3.5.2.6 Shedder status criteria

The participants were divided into high, medium and low shedders according to the strength of the DNA result from the handheld tubes as described in 2.6.1. To compare the two methods, shedder status classification was also defined from the cell count method, but employed the same proportion of individuals in each group as for the handheld tubes.

2.6.1 Shedder category based on handheld tube method

The participants were divided into high, medium and low shedders according to the strength of the DNA result (total rfu value) and the quality of the DNA profile. “High shedders” were classified when at least two of the three profiles had a total rfu value above the average (53,533 rfu) of all the participants and they provided a high quality profile, i.e. ≥ 20 out of 24 full loci (≥ 83%). “Low shedders” were categorized if all three samples had rfu values below 10,000 rfu and gave partial or negative profiles, i.e. < 20 full loci (< 83%). Remaining participants were categorized as “medium shedders”.

2.6.2 Shedder category based on cell count method

The participants were divided into high, medium and low shedders according to the average number of detected cells and the distribution of mean value of all the participants. In addition, the classification followed the same proportion of individuals in each group as for the handheld tube method.

2.7 Statistical analysis

t-tests were carried out to see if the expected values were the same between groups at 5% significance level. Furthermore, linear regression was used to test the association between the cell count method and handheld tube method, i.e. number of detected cells by microscopy versus strength of the DNA results.

Statistical analyses were performed using Stata 16.1. Figures and plots were made in Stata and R (www.r-project.org).4. DiscussionThe shedder status of an individual may be important to consider in the context of Bayesian networks used to ascribe probabilities of transfer, persistence and recovery of DNA following an alleged assault, for example. The higher the shedder status, the more likely it is that an individual will deposit cells on an item; indirect transfer is also more likely [5Fonnelop A.E. Ramse M. Egeland T. Gill P. The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario., 8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces., 9The tendency of individuals to transfer DNA to handled items., 21van Oorschot R.A.H. Szkuta B. Meakin G.E. Kokshoorn B. Goray M. DNA transfer in forensic science: a review.]. Currently, we are comparing two methods proposed to determine shedder status a) handheld tube (HH) method, and b) fluorescent cell count (CC) method [5Fonnelop A.E. Ramse M. Egeland T. Gill P. The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario., 8Lowe A. Murray C. Whitaker J. Tully G. Gill P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces., 9The tendency of individuals to transfer DNA to handled items., 10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer., 11Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders., 12Kanokwongnuwut P. Kirkbride P. Linacre A. Speed of accumulation of DNA in a fingermark, Australian.]. The latter method is much simpler and faster to use, since DNA analysis is not required. In order to apply either method in casework, it will be necessary to ask a person of interest to provide a sample by one or both of the methods.Several authors have previously inferred that a person’s shedder status is difficult to classify due to intra-individual difference in depositing DNA [9The tendency of individuals to transfer DNA to handled items., 10Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer., 22Samie L. Taroni F. Champod C. Estimating the quantity of transferred DNA in primary and secondary transfers.]. In this paper we have classified a person’s shedder status into high, medium and low by applying the handheld tube method in triplicate, and by combining the total rfu value and the quality of the DNA profile. When the three shedder groups were compared, there was a significant difference in the DNA deposited (total rfu value). The five low shedders were consistent in their propensity of depositing DNA. Likewise, the high shedders had an overall high deposition of DNA. The medium shedders, that defined most of the participants, had a performance between the high and low shedders. However, five of the medium shedders provided one sample that fell within the high shedder category, i.e. rfu value > mean and a good quality DNA profile, and four other participants in the medium shedder group provided one sample within the high shedder category and another sample within the low shedder category (rfu value below 10,000 and negative or partial profile). This demonstrates that the medium shedders have to be considered with care as they may act as a low or a high shedder in a transfer event. A similar study by Manoli et al. [[10]Manoli P. Antoniou A. Bashiardes E. Xenophontos S. Photiades M. Stribley V. Mylona M. Demetriou C. Cariolou M.A. Sex-specific age association with primary DNA transfer.], found that 77% of their participants changed shedder status if replicates were considered separately. Compared to our study, 65% of the participants changed shedder status, and 35% retained their shedder category if repeats were evaluated separately. This emphasizes the challenge of determining a person’s shedder status and the importance of carrying out several trials to take into account for a person’s intra-individual tendency in DNA deposition.Previous work by Kanokwongnuwut et al. [11Kanokwongnuwut P. Martin B. Kirkbride K.P. Linacre A. Shedding light on shedders.,