Figure 1 Experimental workflow to assess DNA recovery and STR profiling outcomes from bloodstains deposited onto silver chloride treated fabric. Multiple Sampling and extraction approaches were evaluated prior to quantification and STR analysis.

A schematic overview of the study design is shown in Figure 1. Bloodstains were deposited onto the silver chloride treated fabric and stored for 48 hours prior to sampling. DNA was recovered using multiple sampling approaches (Swabs, Minitapes and direct cutting). The samples were then processed using three extraction methods (Casework Direct, DNA IQ™ and QIAmp DNA Investigator). Samples were quantified by qPCR (Powerplex® ESI 17 fast) and subjected to STR profiling using capillary electrophoresis. DNA yield and profile quality were assessed and compared between methods.

Figure 2 Average DNA yield (pg) obtained from each sampling and extraction approach. The data shown is silver chloride treated samples (a) and positive control samples (b). Error bars represent the standard deviation (n=5).

The highest concentration of DNA after qPCR was from cuttings and Casework Direct, but this combination of methods also displayed the largest variability as indicated by the standard deviation. Tapings yielded the least DNA across all extraction methods; this pattern is observable in both the silver chloride clothing and the control samples. Taped samples that were then extracted using QIAmp were consistently below the optimum PCR input for STR profiling of 500pg³ indicating that these samples may be susceptible to reduced profile quality. In contrast, all other sampling and extraction combinations typically produced yields exceeding 500pg.

Quantification Cycle (Cq) values were similar between the control samples and the treated fabrics ranging from 19.5-21 for every sampling and extraction method combination. The low Cq values indicate a high amount of DNA in the sample. The Cq values suggest a high abundance of amplifiable DNA with no evidence of substantial qPCR inhibition.

Figure 3 Comparison of average peak heights (RFU) between silver chloride treated samples (a) and untreated control samples (b). Error bars represent the standard deviation where n=5

Average peak heights show that swabbed Casework Direct samples produced the greatest peak heights and taped QIAmp samples produced the lowest. Casework Direct samples exhibited the most varied results across swabs and cuttings. Tapings produced lower average peak heights across all extraction methods for silver chloride treated samples and control samples.

Greater variation in peak height was observed between sampling methods than between DNA IQ™ and QIAmp extraction methods. DNA IQ™ and QIAmp generally produced comparable average peak heights and variability across all sampling approaches.

Overall, higher peak heights were generally associated with the sampling and extraction combinations that produced the greatest DNA yields.

Figure 4 Example STR graph showing smaller peaks in the red and yellow dye channels. This sample was recovered with direct cutting and processed with Casework Direct.

Visual inspection of the peaks indicated that peaks in the yellow and red dye channels were generally lower than those in the blue and green channels, particularly within the ≤140bp region on the profile (Figure 4)

To investigate this further, peak heights and individual loci within this region were explored.

Figure 5 compares average peak heights for D3S1358 (blue dye channel) and TH01 (yellow dye channel), two loci located within the ≤140bp region of the STR profile.

Figure 5 Average peak heights (RFU) of silver chloride treated samples at low molecular weight. Loci shown are D3S1358 (Blue) and TH01 (Yellow). Error bars represent the standard deviation where n=5.

Similar patterns were observed at D16S539 and D8S1179 (data not shown). Trends observed in the silver chloride treated samples were also present in untreated controls.

Choice of sampling method generally impacted STR performance more than extraction method. Swabs and cuttings consistently outperformed taping for DNA recovery across both the silver chloride samples and the untreated control samples. Swabs and cuttings consistently produced larger peak heights than tape sampling, particularly at low molecular weight loci such as TH01 and D8S1179.

Tapes were consistently shown to be ineffective for dried bloodstain sampling. The reduced performance was observed across all extraction methods (QIAmp in particular), suggesting that the sampling method itself has a greater influence on DNA recovery and profile quality than the extraction chemistry employed.

These findings support the use of direct cuttings and swab-based recovery methods for bloodstains deposited onto fabric substrates. For forensic practitioners, careful consideration of sampling strategy may be more important than the selection of extraction workflow when processing similar evidence types.

Visual inspection of the STR profiles identified consistently reduced peak heights in the red and yellow dye channels, particularly at loci within the ≤140bp region. Crucially, this pattern was reproducible across multiple samples and was also observed in the untreated control samples. The matching trends in both sample sets suggest that it is unlikely that these effects are due to the silver chloride treatment. However, the consistent occurrence of the effect at specific dye channels and loci indicates that the observed reductions are not random.

The systematic nature of the peak height reductions is consistent with preferential amplification effects where factors such as primer performance, dye chemistry or competition for PCR reagents cause certain loci to amplify less efficiently⁵ Importantly, preferential amplification usually results in consistent, locus-specific effects rather than sporadic reduced peak heights.

The generally sufficient DNA yields and low Cq values suggest that the observed peak imbalance is unlikely to be attributable solely to DNA loss during recovery or extraction. Instead, the reproducible nature of the effects may indicate that factors within the STR amplification process contributed to the reduced peak heights.

This study highlights several areas requiring further investigation. Future work should examine additional antimicrobial technologies, including quaternary ammonium compounds, silver nanoparticles and other metal-ion based treatments to determine whether the observed effects are specific to silver chloride treated fabrics. Alternative STR amplification kits should also be evaluated to investigate whether the observed dye specific and locus specific peak height reductions are assay dependent. Finally, studies using more appropriately matched control fabrics and a wider range of textile compositions would help to isolate the effects of antimicrobial treatments from underlying fabric characteristics.