Results and discussion

Spray device and application optimisation

The number of cells outside of the deposit circle (due to displacement during DD application) was estimated for each of the three spraying methods and the distances of application, by taking images and counting the number of cells outside of the deposit circle. Due to the spread, and in order to encompass all the cells displaced during DD application, the number of images taken per deposit ranged between 10 and 13 (n = 693).

The number of cells outside of the circle ranged between 9 and 140 (av. 51) for single spray with perfume bottle, between 16 and 76 (av. 41) for double spray with perfume bottle and 5 to 193 (av. 45) after the use of the continuous spray device (Table 1). Significantly more cells were found outside of the deposit areas when using single spray perfume device compared to the continuous device (p < 0.05). Fewer cells were found outside deposit after two sprays than continuous device application, however, these differences were not significant. The distance of the device to the deposit area was significant for all spray devices tested (p < 0.05). In general, as the distance from deposit to the device increased, the number of cells displaced outside the deposit decreased. The smallest cell displacement was observed with the continuous spray device and the greatest distance of 20 cm (Table 1).

Distance | Repeat | Perfume bottleSprayed once | Perfume bottleSprayed twice | Voilamart air compressorSprayed continuously
3 cm | 1 | 58 | 31 | 193
 | 2 | 140 | 51 | 107
 | 3 | 86 | 38 | 85
6 cm | 1 | 55 | 36 | 75
 | 2 | 52 | 38 | 42
 | 3 | 58 | 41 | 45
9 cm | 1 | 93 | 76 | 19
 | 2 | 51 | 51 | 31
 | 3 | 63 | 65 | 39
12 cm | 1 | 27 | 34 | 36
 | 2 | 35 | 19 | 28
 | 3 | 43 | 48 | 27
15 cm | 1 | 48 | 31 | 18
 | 2 | 32 | 49 | 16
 | 3 | 21 | 16 | 25
20 cm | 1 | 35 | 20 | 5
 | 2 | 15 | 47 | 10
 | 3 | 9 | 41 | 12

The analysis of spray patterns and cell movements are required in consideration of the potential cell losses associated with each method and the impact it may have on trace DNA recovery. When minute amounts of DNA are present on a surface or an exhibit, collection maximisation is of importance and any losses should be prevented. Assessing the spraying distances, not surprisingly, at close proximity the force of the dye dispersion resulted in the greatest displacement of the cells, as most evident from the results of 3–12 cm distances (Table 1). Looking at the continuous spray device exclusively, significantly more (p ≤ 0.05) cellular displacement was observed at 3 cm distance compared to the other distances, likely due to the high working pressure of 15–50 PSI during device application [30]. The possibility of displacement of biological material on a substrate by air pressure has previously been reported [31]. As the distance between the test devices and the deposit surface increased past 12 cm, cell displacement decreased gradually with the smallest cell displacement noted for the Voilamart device (av. 14 cells for distances of 15–20 cm). Based on these results, the continuous spray device used at the distance of 20 cm represents the best method (of those tested) for cell staining on small target areas during item examination. This is especially to avoid: (a) loss of cells from small items, and (b) displacement of the cells from the deposit areas to areas and surfaces where they did not belong for larger items; resulting in loss of cells or greater sampling areas (and more background DNA collected). The latter point (b) is especially relevant to situations where the location of biological material may be of significance such as during of activity level considerations.

The observed data for single spray with perfume bottle revealed an inverse relationship between tested distance and cell fluorescence intensity. This was also noted, to a lesser degree, with the Voilamart device. As the distance increased, the fluorescence signal diminished, likely attributable to the broader dispersion of the dye over a larger area. This expanded dye distribution resulted in a lower concentration of dye per unit area, consequently reducing the overall fluorescence coverage and intensity detected from the cells. Nevertheless, the introduction of a second spray application markedly enhanced visualization; both with the two sprays of the perfume bottle and the continuous device. This improvement can be attributed to the deposition of an increased concentration of dye per unit area, which effectively increased the overall fluorescent signal and improved the clarity of the cellular imaging.

A notable issue observed with the perfume bottle methods was the inconsistent distribution of the dye. This irregularity manifested as large droplets and non-uniform spray patterns, resulting in uneven fluorescence intensities across the surface. In contrast, the Voilamart application method largely mitigated these problems, producing a more uniform dye distribution and, consequently, a more consistent fluorescence pattern. Our results concur with those of Young et al. [32], who previously compared 15 spray devices and demonstrated that a continuous spray device reduced negative effects such as large droplets and uneven and low intensity during visualising. Based on the results, an application of the dye with the continuous spray device at 20 cm distance is recommended, particularly when treating expansive surface areas. This approach ensures more optimal coverage and enhances the overall effectiveness of the fluorescent visualisation.

Touch cell persistence on different surfaces over time

The ability to visualise stained cellular material on the six surfaces commonly encountered at crime scenes over a period of one year was investigated using the cell counts at each different time point (Fig. 3, Supplementary data 1). The persistence of the cells was also assessed during two re-spraying events at 6 and 12 months (Sect. 3.3). At initial time point, on average (across all 5 replicates), 122, 354, and 148 cells were detected on the flat, non-porous surfaces: glass, plastic and aluminium, respectively; and an average of 77, 76 and 72 cells were detected on the rough, porous surfaces: melamine, leather and cotton, respectively (Supplementary data 1 and 2). The textured/porous substrates, in general, had lower number of cells visualised compared to those that were flat and non-porous. This may be due to fewer cells being deposited as a consequence of a lesser area of contact (i.e., only contact with the raised surface areas and not the grooves) and/or because the cells settled into the grooves or lower layers of the substrates and were not accessible to DD staining.

Fig. 3: Percentage of visualised cells (average of 5 deposits) remaining at each time point until 6 months (before being resprayed) in hours

Post-staining, there was an initial enhancement in visualisation up to 24 h that was substrate-specific (see Supplementary data 1 and 2). This gradual improvement in visualisation suggests a time-dependent process of dye penetration into the cells and subsequent DNA binding. The observed trend implies that the dye molecules slowly diffuse through cellular membranes and progressively associate with nuclear DNA, resulting in an incremental increase in fluorescent signal strength for these substances. These results suggest that if the precise number of cells on the surface is critical, for example, during shedder testing, it may be pertinent to delay cell counting to 30 min post-staining, depending on the substrate on which the deposit resides.

First losses in visualisation were noted, for some of the replicates, starting at 15 min post-staining (Fig. 3 and Supplementary data 1 and 2). After initial losses, for most of the substates, cell visualisation stabilised for a period of time ranging from few days to a month and then progressive decline in cell visualisation was noted until the re-spraying at six months’ time point. Prior to respraying, 5% to 32% of the initial deposits could still be visualised for the tested substates.

Overall, the greatest to lowest cell losses (at six months before respraying) were noted in the following order for the tested substrates: melamine, cotton, leather, aluminium, plastic and glass. For every substrate, cell visualisation declined significantly between initial time point and six months (p < 0.05). Further, while for some substrates these losses were gradual, for example, on plastic substrate 51% of cells were lost by the 3- months’ time point, for cotton 88% of the cells were lost. Observed trends in cell losses between substrate types may be influenced by factors such as surface topology, chemical interactions between cells and the substrate, and the presence of potential inhibitory or damaging compounds within the surface composition. For instance, when certain materials are subjected to contact forces exceeding approximately 20% of the substrate’s hardness, asperities may form, which can be either permanent or transient [33]. Such asperities can result in uneven surfaces and reduced contact areas for cell deposits as well as more shear- induced cell losses. The comparatively smooth surfaces of the glass, melamine, aluminium and plastic in this study may have thus allowed for better persistence once deposited. It should be noted that small fluctuations in cell counts between adjacent time points may also reflect inherent variability associated with image acquisition and cell counting.

Re-application of Diamond Dye™ to enhance visualisation for targeted sampling

All deposits were visualised prior and post DD re-spraying at the two time points (6 and 12 months) across all substrates. The purpose of the respraying was to test if the losses noted during cell counts are associated with true cell loss or is an outcome of the diminished action of the initial application of DD. Only two timeframes were tested to limit potential impacts of reapplication of DD on the persistence of cells on a substrate. Averaging across all substrates (Supplementary data 1), re-spraying increased cell visualisation by 48% and 14% for the 6 and 12-month time frames respectively (Fig. 4; Supplementary data 2 for further substrate specific details).

Fig. 4: The effect of respraying the substrates on visualisation of cells at 6 months and 12 months estimated based on cell decreases compared to the initial deposits B Before respraying the substrates AAfter respraying the substrates

At six months’ time point, respraying resulted in significant cell increases in visualisation of cellular material for all substates (p < 0.05). These increases were again observed for all substates at 12 months respraying, but was only significantly so for glass, plastic and leather (p < 0.05). These results suggests that cells experience fluorescence decay over time and are responsive to the reapplication of dye in cases where an item needs re-examination several months after the initial analysis and DD application. However, the respraying process itself may result in some cell losses that may need to be weighed against the need to visualise the cells that may have lost fluorescence at these later time points.

Overall, it appears that glass and leather resulted in the lowest cell persistence over the 12-month period compared to plastic, melamine, aluminium and cotton. Interestingly, glass and melamine had the smallest decrease in persistence from 6- to 12-months, indicating that for these surfaces most losses happen in the first 6 month. In contrast, for all other substrates major losses (of approximately half of the deposited cells) were observed between first respraying at 6 months and the second respraying at 12 months.

The persistence observed in the current study is similar to that of several other studies [8, 13, 14, 16, 26]. Lee et al. [14] measured persistence of blood and cultured human keratinocytes by counting alleles and using 7 different substrates in 3 different environments. While the findings of Lee et al. [14] cannot be strictly compared to the results here, due to the use of DNA profiling (rather than cell counts and quantification results) and the differences in biological materials investigated (blood and cultured keratinocytes rather than touch DNA), similar trends were noted. In their study Lee et al. [14] noted that in the indoor conditions partial drop out was observed by 8 weeks and by week 48 all substrates showed drop out and the larger losses were seen in synthetic leather, glass and plastic (compared to cotton/polyester, paper and aluminium). This is comparable to this study where leather showed lowest persistence at the six-month time point.

Similarly, Arsenault et al. [16] assessed persistence of trout cellular DNA, cell-free mouse DNA and mixture of both on various surfaces under differing conditions, including exposures to natural light, dark and high humidity environments. Sampling was conducted at 27 timepoints to track the decline in DNA quantities. Among the tested parameters, substrates stored in the dark most closely matched our study design. Again, the study comparisons are limited by differences in biological material (cellular trout DNA and cell free mouse DNA vs. human touch DNA) and processing techniques (trout DNA quantification vs. human cell counts and qualification). Notably on surfaces such as cotton, wood, carboard and paper, cell free DNA was undetectable after 6 months while on plastic, laminate and aluminium this DNA was still detectable at 12 months. In the same study, cellular DNA was detected on all substrates after 12 months. In contrast, in this study the highest amounts of DNA detected were with cotton and plastic substrates and the lowest with glass, leather and melamine (see Sect. 3.4 for more details). The possible discrepancies between the studies could be due to the use of trout and mouse cells and DNA compared to the human touch DNA utilised in this study.

Cell counts and quantification results comparisons

The average DNA quantities recovered after 12 months from glass, plastic, melamine, aluminium, cotton and leather were 0.004 ng, 0.013 ng, 0.008 ng, 0.010 ng, 0.032 ng and 0.008 ng respectively (n = 5 per substrate; Supplementary data 1). When compared with the average cell counts for these substrates (av. of 60, 152, 36, 67, 16, and 8 cells, respectively), the recovered DNA quantities were markedly lower than expected, i.e.: approximately 99%, 99%, 96%, 98%, 67% and 83% less, assuming a typical cellular DNA content of 6 picogram per cell.

To further investigate the relationship between cell counts and total DNA amounts recovered, linear regression was performed with DNA quantification yield baseline adjusted (0.005 ng was added to each DNA quantification value, ensuring that no result was zero and facilitating better visualisation and analysis). The resulting data were visualised in a scatter plot comparing cell count to DNA quantity for each substrate and replicate (Fig. 5).

Fig. 5: Scatter plot of cell counts against baseline-adjusted total DNA quantity (ng; +0.005 adjustment) for six substrates (and 5 replicates), with linear regression fit

Analysis found no significant association between cell count and DNA quantity, as evidenced by the virtually horizontal regression line and the minimal R2 value observed among different substrates and replicates. These results indicates that the variation in DNA yield is not explained by cell number, supporting the conclusion that DNA recovery efficiency is largely independent of the quantifiable cell count under these conditions. Based on cell counts, it can be hypothesised that DNA recovery should increase proportionally with cell number; as the number of recovered cells increases, the total amount of DNA is expected to rise accordingly. One possible explanation for the lack of correlation is DNA degradation in touch deposits. It is well established that DNA in touch samples is often degraded, which can substantially diminish quantification results leading to lower DNA yields than would be predicted from cell counts alone [34, 35]. Further, recovery and extraction efficiency and DD’s non-human specific DNA binding can also play a role [18, 21, 22, 35, 36].