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Abstract
Determining the time since deposition (TSD) of bloodstains is important to establish a timeline and contextualize physical evidence in forensic investigations, while DNA profiling addresses questions of identity and source attribution. Traditionally treated as separate processes, this study integrates TSD estimation into routine DNA profiling by analyzing typically discarded cell lysate (eluates) from spin-column-based DNA extractions. Fluorescence spectroscopy was used to analyze eluates from bloodstains deposited up to 23 months (99 weeks). Two excitation-emission matrices (EEMs) were acquired for each sample and deconvoluted using parallel factor analysis (PARAFAC) to identify individual fluorophores. We identified tryptophan and noted a time-dependent decrease in its corresponding fluorescence. An increase in fluorescence was observed between 400-500 nm as TSD progressed, attributed to an increase in fluorescent oxidation products (FOX) and advanced glycation end products (AGEs). Different chemometric models were then used to estimate TSD from EEM fluorescence data. Boruta feature selection coupled with random forest regression outperformed all other models and achieved high accuracy, with an R2 of 0.993 and root mean square error of prediction (RMSEP) of 2.83 weeks for the full 99-week period, and an R2 of 0.987 and RMSEP of 2.06 weeks for the 1-year timeframe. Comparisons were also made between anticoagulant-free (AC-free) and anticoagulant-treated (AC-treated) bloodstains deposited up to 3 months. We noted differences in fluorescence based on AC treatment, with AC-free blood exhibiting higher FOX and lower AGE fluorescence than AC-treated blood. A temperature study was also performed using bloodstains deposited at -18 ºC, 4 ºC and 21 ºC, with the strongest FOX and AGE fluorescence recorded at 21 ºC. Our findings demonstrate the effectiveness and feasibility of integrating TSD estimation into routine forensic DNA analyses while maintaining high prediction accuracies.
Supplementary materials
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Supplementary materials
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File contains Table S1 and six supplementary figures
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Table S2
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Table S2. Tentative identifications made from spectra acquired in positive mode for each sample during HPLC-MS analysis.
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Table S3
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Table S3. Tentative identifications made from spectra acquired in negative mode for each sample during HPLC-MS analysis.
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Table S4
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Table S4. Tentative identifications made from composite sample spectra acquired in positive mode during HPLC-MS analysis.
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Table S5
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Table S5. Tentative identifications made from composite sample spectra acquired in negative mode during HPLC-MS analysis.
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Figure 1
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Fig 1. Schematic of the DNA extraction protocol section where the eluate (i.e., cell lysate) was collected and preserved for fluorescence analysis. Created with BioRender.com.
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Figure 2
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Fig. 2. EEM1 (250-500/280-750 nm) and EEM2 (350-600/400-750 nm) plotted side-by-side for each timepoint. Two technical replicates are shown for each EEM and timepoint; replicates with greatest and weakest fluorescence were selected to highlight the variation between samples at the same TSD. Note that the full range of EEM1 is not shown due to lack of fluorescence in the 400-500/550-750 nm range.
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Figure 3
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Fig. 3. Excitation and emission loadings produced by the four-component TSD PARAFAC model, normalized to its maximum excitation and emission intensity.
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Figure 4
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Fig. 4. PARAFAC scores of each component in every sample. Sample names begin with timepoint, followed by biological replicate and technical replicate.
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Figure 5
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Fig. 5. Predicted TSD from Boruta + RFR models plotted against actual TSD, in weeks, for A) TSDs up to 99 weeks and B) TSDs up to 52 weeks. Training and testing datasets are depicted in orange and black, respectively. The dashed blue line represents a perfect fit.
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Figure 6
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Fig. 6. EEM 1 and EEM 2 plotted side-by-side for each timepoint. Two technical replicates are shown for each EEM, timepoint and AC treatment; replicates with greatest and weakest fluorescence were selected to highlight the variation between samples at the same TSD and AC treatment. Samples with AC are denoted as T1/3/4 AC.
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Figure 7
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Fig. 7. EEM 1 and EEM 2 plotted side-by-side for samples deposited 19 (T11) and 23 months (T12). Two technical replicates are shown for each EEM, timepoint and storage temperature; replicates with greatest and weakest fluorescence were selected to highlight the variation between samples at the same TSD and storage temperature.
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Figure. S1
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Fig. S1. Split-half analysis of the four-component PARAFAC model.
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Figure S2
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Fig. S2. EEM of each component in the validated four-component PARAFAC model. Fluorescence intensity is normalized to the maximum fluorescence of each component.
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Figure S3
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Fig. S3. Normalized PARAFAC scores for each component across all samples in the TSD study. For example, component 3 accounts for approximately 75 % of the fluorescence in T0 samples.
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Figure S4
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Fig. S4. Excitation and emission loadings from the four-component AC-treatment PARAFAC model built using EEM2 fluorescence data. Loadings are normalized to their maximum intensities.
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Figure S5
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Fig. S5. Score of each component in every sample used to build the AC-treatment PARAFAC model. Sample names begin with timepoint, followed by biological replicate and technical replicate. Samples in orange were treated with AC. Orange lines were added to delineate samples according to TSD and AC-treatment.
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Figure S6
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Fig. S6. Residual fluorescence from the four-component AC-treatment PARAFAC model. Sample names remain the same as in the barplot shown in Fig. S5. Samples in orange are AC-treated.
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Table 2
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Table 2. List of commonly identified compounds in positive and negative mode HPLC-MS analysis. The 10 red boxes in each mode represent the most observed fluorophores or aromatic amino acid derivatives. The five white boxes in each mode indicate the most common non-fluorescent molecules. Superscript symbols represent alternative identifications: *L-leucine, †L-isoleucine, ‡4-(3-pyridyl)-butanoate.
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Graphical Abstract
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Graphical Abstract - Created with BioRender.com
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