Six participants (referred to as Participants 1–6 hereafter) were recruited as donors for all three experiments. Two of the selected participants were a high (Participant 2) and low shedder (Participant 4), based on previous Diamond™ Dye (DD) testing (unpublished). Two of the participants were male (2, 6) and four were female with ages that ranged from 18 to 65-years-old. Of the six participants, half were right-handed (3, 4, 5) and half were left-handed (1, 2, 6). Additional participant factors (age, gender, living arrangements, known skin conditions, etc.) are described in Supplementary Data 2.
Handwashing and activityTo minimize the impact of pre-testing behaviour and activity, participants were instructed to wash their hands for 15–20 s with Softsoap© Antibacterial liquid soap (Colgate-Palmolive Company, Manhattan, NY, USA) before drying their hands with paper towel, all while being observed. All sample deposits were made 15 min after handwashing.
Further, for this study, a “clean” and a “dirty” hands conditions were used to assess the impact of activity on shedder status. In the clean hands condition, participants were instructed to not touch anything for 15 min post-handwashing to assess the intrinsic amount of DNA or cells accumulated during that time period. To ensure compliance, participants were supervised during the wait period. In the dirty hands condition, participants were instructed to go back to their normal, daily activities with few exceptions. Participants were instructed not to eat or drink, actively touch other people, wear gloves, or re-wash or sanitize their hands. Participants were also kept from touching shared laboratory surfaces including door handles throughout the 15-min wait period. Surveys were collected at each deposit to ascertain activity information.
ReplicationWith the exception of Participant 1, all samples were collected over the same five-month period, with most participants depositing one sample per week. Participant 1 had a full set of samples (36 samples over Tests 1, 2, and 3) collected over the same five-month period, as well as an additional four samples (two clean and two dirty) collected on glass slides over a period of four weeks, approximately 12 months after they made their first deposits.
Selected shedder testsThe three shedder tests selected (DNA quantification and profiling of a handprint on a glass plate, DNA quantification and profiling of a grip mark on a plastic conical tube, and cell scoring of a Diamond™ Dye-stained fingermark) were replicated from the literature with slight modifications [11, 15, 18] (see Supplementary Data 3 for details of the three original tests and the subsequent modifications used in this study).
Swabbing of glass plates and tubesA two-swab technique (wet and dry) using 155C Copan© swabs (Interpath, Victoria, Australia) was used across all relevant tests.
Test 1: Deposition of a handprint on a glass plateSample depositFifteen minutes post-handwashing, participants were asked to place their dominant hand on a cleaned glass plate (140 × 220 mm; 4 mm thickness) for 10 s, with their fingers and thumb together, applying firm pressure but no friction, as adapted from Goray & van Oorschot [15] (See Supplementary Data 3 for detailed experimental conditions used). The side of the glass plates on which the deposit was made were swabbed in their entirety. Both the clean and dirty conditions were conducted in triplicate for each participant. Each of the deposits were made on a different day.
DNA analysisFor Tests 1 and 2 (Section 2.3), DNA was extracted from both swabs together with the DNA IQ system (Promega, Madison, WI, USA) (end volume 60 µL), quantified using the Quantifiler® Trio DNA Quantification Kit on an ABIPRISM® 7500 (Life Technologies, Carlsbad, CA, USA), and amplified using PowerPlex® 21 (Promega, Madison, WI, USA) (maximum template input of 0.5 ng in a maximum volume of 15 µl, 25 µl total volume, 30 cycles). PCR products were then separated on a 3500 Genetic Analyser (Life Technologies, Carlsbad, CA, USA) and analysed using GeneMapper® ID-X software (v1.6, Life Technologies, Carlsbad, CA, USA) with a detection threshold of 175 RFU. Note, all samples were submitted to amplification, independent of detected DNA concentration.
Result interpretationThe minimum number of contributors (MNC) was determined for each profile. The total number of alleles, allelic peak heights, and mixture proportions was used to inform the number of contributors. The homozygosity threshold was set at of 2,000 RFU. The total amount of DNA was calculated by multiplying the DNA quantification result (in ng/μL) by the volume of an extract (60 μL). This total DNA amount was divided into donor and non-donor contributions using the mixture proportions generated by STRmix™ (v2.9, ESR, New Zealand and FSSA, South Australia).
The average RFU of the donor alleles was calculated by dividing the donor’s total RFU contributions by the total number of alleles detected, excluding Amelogenin. STRmix™-generated mixture proportions were further used to assign major and minor contributors, as applicable, where the major contributor deposited over 70% of the total DNA in a mixed sample. Where a major donor couldn’t be established, majority and minority contributors were defined. DNA profiles were deconvoluted in STRmix™ and compared to reference profiles from the participants, investigator, and other users of the shared laboratory space to identify relevant contributors.
Shedder categorisationGoray & van Oorschot [15] described five shedder status allocations where participants were evaluated in terms of total DNA, number of alleles, and relative fluorescence units (RFU) per deposit. Additional factors considered included mixture proportions, as well as a participant’s tendency to be the major contributor within a mixture. Ultimately, participants could be described as low, low-intermediate, intermediate, intermediate-high, or high shedders. For the purposes of this study, the exact results described in Goray & van Oorschot [15] were used to generate the cut-off ranges used for categorisation. We recognise that the ranges described were based off data collected from 10 participants and a larger sample size may have resulted in different cut-off values, as is the case with most investigations into shedder categorisation. Participants were placed into one of the five shedder categories (Table 1) based on thresholds met in at least two of the following three categories: total DNA, number of alleles, and average RFU. Additionally, to be classified as an intermediate-high or high shedder, participants had to always be the major contributor in a mixture, contributing 70% or more of the DNA collected in each sample deposited. Goray and van Oorschot’s [15] categorisation method was applied to DNA quantity and quality data obtained from samples generated using both Test 1 and Test 2 deposition methodologies.
Table 1 Shedder status criteria as used by Goray & van Oorschot [15]Test 2: Deposition of a grip mark on a plastic conical tubeSample deposit and DNA analysisFifteen minutes post-handwashing, participants were instructed to grip a cleaned plastic conical tube (Greiner centrifuge tube, 50 mL, 30 × 115 mm) with firm pressure for 10 s using their dominant hand, as adapted from Fonneløp et al. [18]. Participants were asked to ensure that their entire hand spanned the body of the tube and that no part touched the cap. The body of each plastic tube was swabbed, while the caps remained unsampled. Both the clean and dirty conditions were conducted in triplicate for each participant. Each of the deposits were made on a different day. The DNA analysis of these samples was the same as for the Test 1 samples described in the "DNA Analysis" Section.
Shedder categorisationFonneløp et al. [18] used a binary system of classification, which only recognised individuals as high or poor shedders (see Supplementary Data 3 for more detail). Participants were classified as high shedders if at least two of their three deposits produced a higher than average (for all tested participants) DNA quantification result and a high-quality profile with 12 or more full donor loci. All other participants were categorised as poor shedders. The average thresholds were determined using data generated by our six test participants. The following modification was adopted in this study: Fonneløp’s analysis utilised PowerPlex(R) 17, which analyses alleles at a total of 17 loci, including Amelogenin, where 12/17 complete loci represents approximately 71% coverage. The present study utilised PowerPlex(R) 21, assessing 21 loci inclusive of Amelogenin. To account for the increased number of typed loci, a similar 71% cut-off was applied, and a minimum of 15 full donor loci was required for a high shedder status designation. The Test 2 categorisation method was applied to quantity and quality data obtained from samples generated after applying Test 1 and Test 2 deposition methodologies.
An additional “modified Fonneløp et al.” categorisation method, as first described in Goray et al. [39], was applied to the cell counts generated in Test 3. Cell scores were averaged for all participants in the clean and dirty conditions and participants were deemed high shedders if at least two of their three deposits produced a higher than average number of cells for said condition. This allowed for the binary categorisation of Test 3 results and subsequent comparison to Tests 1 and 2 results.
Test 3: Deposition of a thumbprint on a glass slideSample depositAs the thumb was not involved in the tube gripping manoeuvre (as per instructions to participants), Test 3 was conducted immediately after Test 2. Participants were asked to place their dominant thumb on a cleaned DNA-free glass slide with medium pressure for 15 s, as adapted from Kanokwongnuwut et al. [11] (see Supplementary Data 3 for more detail). Both the clean and dirty conditions were run in triplicate for each participant. Each of the deposits were made on a different day.
Slide stainingSlides were stained with 10 μL of a 20-fold dilution of the Diamond™ Nucleic Acid Dye (Promega, Madison, WI, USA) stock (10,000x). The dye was left to dry for 30 min before slides were visualized under a Dinolite fluorescent digital microscope (Dino-Lite Australia) equipped with an emission filter of 510 nm and a blue LED excitation light source (480 nm).
Cell scoringCells were manually scored by counting the number of “bright spots” or cells in three frames, each 1 mm2 in size, at 220 × magnification. Each of these frames was thought to represent a different “high-density” area of the thumbprint, as first conducted by Kanokwongnuwut (personal communication). The cell counts within these frames were averaged to produce an overall cell count per deposit.
Shedder categorisationKanokwongnuwut et al. [11] recorded their 11 donors as being either high, intermediate, or low shedders, albeit recognising that shedder status is best represented by a continuum. While the original study utilised RFU rather than cell scores for the purposes of categorisation, cell ranges were extracted from the figure titled “Illustrating the amount of cell nuclei deposited at four different time points” [11], where deposits of 1–15 cells/mm2 were made by low shedders, deposits of 16–30 cells/mm2 were made by intermediate shedders and deposits of more than 30 cells/mm2 were made by high shedders. Although these ranges may not have been intentioned for use by the authors, these values have been the basis of subsequent shedder studies [12, 34]. As such, these ranges were used for the purposes of designation in Test 3. A correlation between cell scores and RFU generated (R2 = 0.92175) was found for 9/11 participants in Kanokwongnuwut et al. [11], with the outliers being two intermediate shedders.
Evaluation of complete fingermarksTo further evaluate different shedder methods and the effects of categorisation criteria, the fingermarks generated in Test 3 were overlayed with a 5 mm2 grid and images of every square with at least 50% cell coverage, regardless of density, was taken at 50 × magnification, as modified from Kaesler et al. [34]. ImageJ (LOCI, University of Wisconsin) was used to perform automated cell counts on each of these 5 mm2 frames. To accurately compare these results to the Test 3 designations, a modified Kanokwongnuwut et al. [11] method was used. Three 5 mm2 frames of high cell density were selected and the generated cell counts were averaged and divided by 25 to account for differences in grid size used (5 mm2 versus 1 mm2 in Test 3). Two other methods of classification were applied, including modified Fonneløp et al. [18] (as described in the ''Shedder Categorisation'' Section) and an in-house, “sample-adjusted” method. In this sample-adjusted method, for each condition, all participant replicate results were ranked from lowest to highest. These results were then divided into equal thirds, each of which represented the accepted ranges for a low, intermediate, and high shedder, respectively. In the instance that a specific value spanned two categories, it was conservatively estimated to be the upper limit of the lower ranked shedder category. A total of 665 squares were counted over the entire study. The resulting shedder designations are presented in Table 7.
Shedder categorisation terminologyThe terminology used to describe similar shedding patterns vary between publications, as some will use good/high or bad/poor/low shedder terms interchangeably (see Supplementary Data 1). In this article, we used high, intermediate, and low shedder terms.
Quality controls and cleaning protocolsTo ensure the glass plates and plastic conical tubes were DNA-free prior to use, they were sprayed in sequence with 1% hypochlorite, 100% ethanol, and distilled water. Both surfaces were dried with paper towel after each solvent was applied and each item was individually stored in plastic bags. Control swabs were taken from 15 random glass plates and plastic conical tubes after cleaning. No quantifiable DNA and no DNA profiles were generated from control samples. Both positive and negative controls were appropriately implemented during DNA processing and produced the expected results.
The glass slides used were cleaned with 100% ethanol. Once deposits were made, the slides were stored in a cupboard until staining to prevent cell degradation due to environment [40]. Negative control slides (10) were identically cleaned, stored, and stained on random sampling dates. No cells were observed on these controls.
Statistical analysisGiven the continuous nature of the variables assessed, a Mann–Whitney U-test was used to analyse the differences in DNA deposits (ng) made between Tests 1 and 2, as well as between the clean and dirty hands conditions (p < 0.05 significance). Inter-person variation was also assessed for each test conducted. All statistical analyses were performed in SPSS Statistics (IBM, v29.0.1.0).
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