Determination of time since deposition of bloodstains through NIR and UV-Vis spectroscopy - A critical comparison.

Time elapsed since bloodstain deposition is a crucial aspect in forensic investigations, where non-destructive spectroscopic methods play a pivotal role. While extensive research has been conducted by UV-Vis spectroscopy, showcasing its utility in specific cases, there is still a paucity of studies based on NIR spectroscopy, which has the potential to overcome the limitations of the UV-Vis-based methods. To compensate for this disequilibrium, the present study aimed to evaluate the NIR applicability for estimating the age of forensic bloodstains and develop a performance comparison with UV-Vis spectroscopy methods. Capillary blood was sampled and subjected to a 16-day aging, during which it was repeatedly analyzed using both spectroscopic methods. Subsequently, chemometric analysis was applied to process the spectral data and independently assess the methods' performance. Classical preprocessing transforms (i.e., Savitzky-Golay derivatives and SNV transform) were used together with more targeted strategies, such as class centering, whose benefit was highlighted by PCA. Lastly, PLS regression models were computed to evaluate the effectiveness of both spectroscopic methods in estimating the time elapsed since blood trace deposition. Comparable root mean square errors in prediction (RMSEP) - 40 and 55 h for UV-Vis and NIR spectroscopy, respectively - were observed for both techniques, featuring an improvement with respect to the existing literature for NIR spectroscopy. Data fusion strategies for a multi-instrumental platform were also explored, evaluating advantages and disadvantages of low-level and mid-level approaches. The results indicated that NIR spectroscopy integrated with adequate chemometric strategies deserves increased appreciation in forensic bloodstain dating.

Visual identification for species and sex derived from bloodstain based on phosphate-mediated isothermal amplification colorimetric system.

Species and sex confirmation of the biological specimen play a crucial role in crime investigation. However, the specimen found in the scene is always trace quantity, which is hard to be analyzed by current methods. Moreover, the time-consuming DNA extraction, sophisticated apparatus, and complex data processing make it difficult to satisfy the demand of speediness and convenience for point-of-care tests. In this study, we first exhibit a phosphate-based visual system for field-based species and sex identification derived from trace bloodstain. By introducing phosphate ion-based colorimetry into loop-mediated isothermal amplification (LAMP) for result interpretation, not only the bloodstain can be directly submitted to mitochondrial variant amplification owing to the enhanced amplification efficiency by pyrophosphate ion hydrolyzation, but also the colorimetric signal can be recognized by the naked eye for result output within 30 min through molybdophosphate generation. Aerosol contamination, the major conflict of LAMP, has been solved once and for all by integrating uracil-DNA glycosylase into this system that still holds on a constant temperature. As a demonstration, cytochrome b and Y-chromosomal amelogenin are employed to identify species and sex respectively, which has achieved a highly sensitive and specific distinguishability under a strong interferential background. Accurate results can be obtained from both the simulative degraded and dated specimen, which indicates that this novel system may serve as a promising tool in forensic practice.

Characterization of insect stains produced by Dermestes maculatus De Greer (Coleoptera: Dermestidae) resulting from interactions with human blood.

Insect stains produced by adult Dermestes maculatus were characterized during interactions with human blood. Beetles were offered wet or dried blood positioned on ceramic tiles under laboratory conditions. Despite a life history strategy geared toward consumption of dried food stuffs, adult beetles interacted with wet blood more frequently than dried and produced more insect stains after ingesting wet blood. Most (> 95%) of the insect stains produced were the result of fecal elimination. These stains varied in morphologies but were consistently tan/light, black/grey, or red in color; were round to amorphous in shape; and frequently possessed tails. Tailed stains typically were tadpole-shaped or long and tapering from the stain body, yielding Ltl/Lb ratios greater than 1. Tails were the result of beetle locomotion while defecating. Human blood was detected in defecatory stains when using ABA Hematrace® lateral flow assays. When beetles interacted with dried blood, the bloodstains were most often modified due to physical disruption rather than feeding activity. This yielded flaking or dislodgement of the original stains. Within a forensic context, it is unknown whether D. maculatus interacts with any type of bloodstains at a crime scene.

Enhancing forensic investigations: Identifying bloodstains on various substrates through ATR-FTIR spectroscopy combined with machine learning algorithms.

The forensic analysis of bloodstains on various substrates plays a crucial role in criminal investigations. This study presents a novel approach for analyzing bloodstains using Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with machine learning. ATR-FTIR offers non-destructive and non-invasive advantages, requiring minimal sample preparation. By detecting specific chemical bonds in blood components, it enables the differentiation of various body fluids. However, the subjective interpretation of the spectra poses challenges in distinguishing different fluids. To address this, we employ machine learning techniques. Machine learning is extensively used in chemometrics to analyze chemical data, build models, and extract useful information. This includes both unsupervised learning and supervised learning methods, which provide objective characterization and differentiation. The focus of this study was to identify human and porcine blood on substrates using ATR-FTIR spectroscopy. The substrates included paper, plastic, cloth, and wood. Data preprocessing was performed using Principal Component Analysis (PCA) to reduce dimensionality and analyze latent variables. Subsequently, six machine learning algorithms were used to build classification models and compare their performance. These algorithms comprise Partial Least Squares Discriminant Analysis (PLS-DA), Decision Trees (DT), Logistic Regression (LR), Naive Bayes Classifier (NBC), Support Vector Machine (SVM), and Neural Network (NN). The results indicate that the PCA-NN model provides the optimal solution on most substrates. Although ATR-FTIR spectroscopy combined with machine learning effectively identifies bloodstains on substrates, the performance of different identification models still varies based on the type of substrate. The integration of these disciplines enables researchers to harness the power of data-driven approaches for solving complex forensic problems. The objective differentiation of bloodstains using machine learning holds significant implications for criminal investigations. This technique offers a non-destructive, simple, selective, and rapid approach for forensic analysis, thereby assisting forensic scientists and investigators in determining crucial evidence related to bloodstains.

Establishment of a random forest regression model to estimate the age of bloodstains based on temporal colorimetric analysis.

Bloodstain age estimation is important in forensic science. Although several studies have used spectroscopy to estimate bloodstain ages, this method has not yet been practically applied due to the need for expensive equipment and low reproducibility. Thus, we aimed to develop a bloodstain age estimation model that can be easily performed using a spectrophotometric colorimeter. First, bloodstains were prepared by placing blood obtained from five healthy volunteers on a plastic plate. The bloodstains were kept on conditions with various brightness and temperatures. Then, each bloodstain was dissolved in saline every 24 h to a final concentration of 1%, measured with a spectrophotometric colorimeter, and subjected to machine learning to generate a random forest regression (RFR) model, and finally, the prediction accuracy of the bloodstain age was verified. We also elucidated the mechanism of the color changes utilizing aminoguanidine, which is an inhibitor of Maillard reaction. Finally, we measured the time-dependent color changes of the blood fluids obtained from healthy volunteers and examined if the method could be potentially applied to estimate postmortem interval (PMI). Our results showed that the RFR model estimated the bloodstain age with no substantial assessment, and it was applicable to bloodstains, regardless of the brightness or temperature. The color changes were affected by the addition of aminoguanidine. Furthermore, the method could be applied to blood fluids, suggesting its potential usefulness for PMI estimation. Considering its feasibility, the present method could potentially be introduced to practical forensic sciences in the near future.

Quantitative PCR analysis of bloodstains of different ages.

An accurate method to estimate the age of a stain or the time since deposition (TsD) would represent an important tool in police investigations for evaluating the true relevance of a stain. In this study, two laboratories reproduced an mRNA-based method for TsD estimation published by another group. The qPCR-based assay includes four transcripts (B2M, LGALS2, CLC, and S100A12) and showed preferential degradation of the 5' end over the 3' end. In this study, the blood-specific marker ALAS2 was added to examine whether it would show the same degradation pattern. Based on our qPCR data several elastic net models with different penalty combinations were created, using training data from the two laboratories separately and combined. Each model was then used to estimate the age of bloodstains from two independent test sets each laboratory had prepared. The elastic net model built on both datasets with training samples up to 320 days old displayed the best prediction performance across all test samples (MAD=18.9 days). There was a substantial difference in the prediction performance for the two laboratories: Restricting TsD to up to 100 days for test data, one laboratory obtained an MAD of 2.0 days when trained on its own data, whereas the other laboratory obtained an MAD of 15 days.

Breaking with trends in forensic dating: A likelihood ratio-based comparison approach.

Further steps toward understanding the time-related information contained within bloodstains found at the crime scene are rightly considered a top priority in forensic science. Contrary to widely held assumptions, the reason for the delayed exploitation of bloodstains dating methods in practice is not the lack of suitable analytical techniques for monitoring degradation processes. The problem lies in the variability of the environmental and circumstantial conditions, playing a vital role in the degradation kinetics of blood deposits. The present article demonstrates the possibility of breaking with current approaches based on absolute age estimations to finally answer time-centered questions in real forensic scenarios. The proposed novel framework for situating forensic traces in time is based on the likelihood ratio assessment of the (dis)similarity between the evidence decomposition and sets of reference materials obtained through supervised aging. In such a strategy, every dating procedure is constructed on a case-by-case basis to fit examined blood traces, thereby limiting the adverse influence of external factors on the validity of age estimations and providing a way for future crime scene implementation.

Optical profilometry for forensic bloodstain imaging.

Understanding the physical, chemical and biological changes that occur during the drying of a bloodstain is important in many aspects of forensic science including bloodstain pattern analysis and time since deposition estimation. This research assesses the use of optical profilometry to analyze changes in the surface morphology of degrading bloodstains created using three different volumes (4, 11, and 20 µL) up to 4 weeks after deposition. We analyzed six surface characteristics, including surface average roughness, kurtosis, skewness, maximum height, number of cracks and pits, and height distributions from the topographical scans obtained from bloodstains. Full and partial optical profiles were obtained to examine long-term (minimum of 1.5-h intervals) and short-term (5-min intervals) changes. The majority of the changes in surface characteristics occurred within the first 35 min after bloodstain deposition, in agreement with current research in bloodstain drying. Optical profilometry is a nondestructive and efficient method to obtain surface profiles of bloodstains, and can be integrated easily into additional research workflows including but not limited to time since deposition estimation. Optical profilometry is a non-contact tool to scan bloodstains in ambient conditions Drying phases are observable in small drip bloodstains Significant surface morphology changes occur within 35 min after deposition.

Evaluation of the detectability of different ages of bloodstains on fabrics in different washing conditions and at various wavelengths.

PURPOSE: The detection of bloodstains at crime scenes is extremely useful in forensic investigations. This study aimed to investigate the effects of washing temperature, fabric type, fabric color, and stain age (time from staining to laundering) on the detection and identification of bloodstains on fabrics after washing. MATERIAL AND METHOD: A total of 240 fabrics (4 different colors and 5 different types) were stained with blood and washed in 4 different washing temperatures with 3 different lag times. The evaluations of fabric images were performed using the FLS system (Forenscope-Mobile Multispectral UV-VIS-IR Imaging Systems®) on a total of 1200 images using 5 different wavelengths and filter options. The bloodstained areas of the fabric pieces were then excised, and the hemoglobin presence was analyzed using the SERATEC® HemDirect hemoglobin test. RESULTS: The analyses of laundered samples using the FLS system revealed that the best images were obtained from velvet, cotton fleece, denim, and polyester fabrics, in that order. Except for polyester fabrics, the SERATEC® HemDirect hemoglobin screening test, which was used to detect bloodstains on fabrics, showed positive results after washing at low temperatures (approximately 15 °C and 30 °C). At higher temperatures (60 °C and 90 °C), the SERATEC® HemDirect hemoglobin test yielded negative results. CONCLUSION: The fabric type and color played a crucial role in stain detection using the FLS system on the laundered fabrics. The FLS system and the SERATEC® HemDirect hemoglobin test revealed that stain age had a limited effect on the stain's detectability.

Fatal penetrating iliac wound: A case report with determination of physical activity and time to collapse.

A case of fatal left iliac penetrating wound in a thirty-year-old man, during a fight, was presented. The medical file described a transfixing five-centimeter large wound on the left iliac vein and a puncture wound on the left iliac artery. After performing an autopsy, we concluded that the death was secondary to multivisceral failure, secondary to hemorrhagic shock. The judge investigating the case requested a detailed forensic medical reconstructive opinion focusing on the sequence of events leading to the fatal blow. Our having access to the videorecording and the photographs of the crime scene, added to the testimonies of witnesses proved to be a great help to the investigation. The data from the crime scene video recordings allowed us to estimate the victim's physical activity after the fatal stabbing (running between 20 and 140 meters) and the time when he collapsed (between 32s and 1 min) after the stabbing which is rarely described with methods using Closed-Circuit Television material, especially in cases of fatal vascular injuries. Those data are usually estimated from eyewitnesses' accounts, which remain an unreliable method.

Identity of the numerous bloodstains at the murder scene: molecular identification of fly artifacts and fly species by CO1 analysis.

Crime scenes may contain insect artifacts as well as samples of human origin. While the presence of insects can be important evidence in forensic medicine and forensic entomology, the insect artifacts sometimes interfere with the interpretation of bloodstain pattern analysis (BPA) which can be critical for accurate crime reconstruction. Fly artifacts are especially complicated to distinguish from true bloodstains. Indeed, we encountered a murder scene with numerous bloodstains inconsistent with the cause of death and had trouble interpreting them. The morphological method has been developed to distinguish them, but this method has to rely on the analyst's experience and opinion. This study aims not only to distinguish fly artifacts from true bloodstains but also to identify fly species by detecting fly DNA in small amounts of bloodstains at the scenes. Melt curve analysis of real-time PCR (qPCR) targeting cytochrome c oxidase subunit 1 (CO1) of mitochondrial DNA (mtDNA) was able to detect fly DNA in bloodstains from a murder scene. The fly DNA was sequenced from the qPCR product, and the fly species were identified by BLAST search. Fluorescence-labeled specific primers for four species of necrophagous flies were designed based on the sequences of the CO1 region, and differences in the length of the amplification products were used to identify fly species from trace amounts of fly DNA in the artifacts.

Proteomic Difference Analysis of Whole Blood and Bloodstains / 法医学杂志

OBJECTIVES@#To study the changes of protein levels in peripheral blood after it dried.@*METHODS@#The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed.@*RESULTS@#A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains.@*CONCLUSIONS@#The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Proteomic Difference Analysis of Whole Blood and Bloodstains.

OBJECTIVES: To study the changes of protein levels in peripheral blood after it dried. METHODS: The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed. RESULTS: A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains. CONCLUSIONS: The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Can scene bloodstains be used to quantify drug concentration at the moment of injury?

Blood is the primary material for quantitative toxicological analysis and interpretation of the results. However, in some cases like hit-and-run, it may be collected after a long time since the incident, resulting in the complete elimination or a significant decrease in the concentrations of potentially present drugs. If the suspect was injured and bloodstains were revealed, they can become the best or sometimes, the only material to be used to prove driving under the influence of drugs. In this context, the aim of this work was to develop a simple procedure that would allow for the estimation of drug concentrations in forensic bloodstains of unknown volume. In this work endogenous amino acids: valine and leucine were used to determine the unknown volume of blood from which the stain was formed. In order to isolate the analytes from bloodstains on different surfaces (plastic and cotton) the elution mixture consisted of an acetonitrile: water (60:40) and sodium chloride (0.9%) was used. The developed protocol was tested on 32 authentic forensic samples (mostly cases of people driving under the influence of amphetamine). The results of bloodstains analyses were compared with the results of whole blood analyses. The accuracy was in the range from -87.7 to +471.0%. However, for most of the cases, more accurate results were obtained. The differences between amphetamine values estimated from bloodstains and determined from whole blood were analysed using the Bland-Altman difference plots that revealed significant agreement. The variables concentrations estimated from bloodstains and concentrations calculated from whole blood were found to be strongly correlated. The Pearson correlation coefficients were: for plastic r(29) = 0.85 and for cotton r(25) = 0.91. This is the first work in which quantitative analyses of drugs in bloodstains have been attempted. A special protocol was developed for this purpose. The conducted research indicates that estimation of the concentration of drugs in bloodstains from the blood of an unknown volume is possible. The developed protocol has been extensively tested on authentic forensic samples and the obtained results were successfully compared with drug concentrations determined in whole blood. Nevertheless, the proposed method of estimating drug concentrations in bloodstains is characterized by many disadvantages, and the determined values should be treated with great caution, at best as estimates.

Direct STR profiling from laundered bloodstains: an investigation of different factors of laundering.

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90 °C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide.

The effect of reactive dyeing of fabric on the morphology of passive bloodstains.

The majority of fabrics at crime scenes have been coloured in some way. The effect of such treatments on resultant bloodstains has not been considered. In this work, horse blood was dropped onto reactively dyed calico fabrics (100% cotton, plain woven) with three different masses of 91 g m-², 171 g m-² and 243 g m-² and the results compared to previous work on the not-coloured calico fabric. Five impact velocities were used from 1.7 ms-1 to 5.4 ms-1. The use of reactive dye increased the thickness (from 0.38 - 0.56 mm to 0.39 - 0.6 mm) and mass per unit area (from 85.1 - 224.6 g/m² to 91 - 243 g/m²) of the calico fabrics. The reactively dyed fabrics had larger bloodstains (e.g. lightest calico 41.2 - 78.6 mm²) compares to the not-coloured fabrics (e.g. lightest calico 21.4 - 67.5 mm²) across all three mass per unit areas. The dyeing of the fabrics altered the intra-yarn spaces to a more optimum size for wicking blood, increasing the ease with which the blood could wick along the yarns in the dyed calico. The amount of wicking varied depending on individual variations within the fabrics and yarns. More variation in dry bloodstain area was seen among dyed calico specimens than for the not-coloured fabric. The amount of wicking which was seen on the dyed calico meant there was no correlation between dry bloodstain area and impact velocity, a correlation which was seen on the medium and heavy not-coloured calico in the previous work.

The use of fly artifacts in a crime scene: Is there any application for forensic toxicology?

Fly artifacts (FA) are bloodstains resulting from insect activity at a crime scene, usually by feeding on human blood. Whether these artifactual stains might be useful for forensic toxicological investigations in cases of absence of conventional and unconventional matrices, for example, in cases concealment of the body or of extensive putrefaction, has not yet been investigated. The purpose of this study is to understand if FA trace evidence permits toxicological analysis when traditional matrices are not available. To this aim, FA experimentally produced by Calliphora vomitoria feeding on human blood of a cocaine and heroin user were collected from absorptive and non-absorptive material. FA material was analyzed by a new simple and fast LC-MS/MS method. Results were evaluated in terms of presence of the drug and relative amount of the detected molecules. From a qualitative point of view, the analysis of FA revealed all the substances originally detected in post-mortem blood in both cases. The ratios of cocaine/benzoylecgonine, codeine/morphine, and 6-monoacetylmorphine/morphine recovered in FA from cotton-textile materials and from non-absorptive surfaces were consistent with data resulted from original post-mortem blood. The preliminary study herein reported demonstrated that FA are extremely informative in case of cocaine and heroin users and merit further research in order to be applied in real caseworks.

Finding blood in the dark: A comparison of infrared imaging devices for the detection of bloodstains on dark fabrics based on their resolution.

The use of infrared (IR) light to locate bloodstains on dark fabric is a search technique that is employed in forensic examinations in a number of organisations worldwide. IR is used to complement existing, established visual white light search techniques. There exist a variety of commercially available products that can be purchased for this purpose as well as the option of using IR-converted standard DSLR (Digital Single Lens Reflex) cameras. In this study, a number of IR systems with contrasting resolutions were explored and their performance was assessed on a variety of bloodstain types and fabrics in comparison with white light. The systems ranged from low-budget, low resolution options, such as portable webcams, to vision-industry standard, high resolution, purpose-built cameras for more detailed blood searching of suitable items in the laboratory. Blood spatter, transfer bloodstains, dilute bloodstains, blood mixed with other body fluids and environmental contaminants were among the samples tested on eight different dark fabric types under IR conditions to assess the impact of the resolution differences. All IR systems were able to locate bloodstains, with significantly more bloodstains being found with IR compared to white light. The higher resolution systems were able to locate significantly more bloodstains than the systems with the lower resolution. The webcams were able to locate many of the larger areas of bloodstaining but performed less well in terms of locating smaller bloodstains and dilute blood. False positives such as mud, make-up and brown sauce were detected under IR but were readily discriminated under white light and with presumptive chemical tests. The balance between the ability to locate bloodstains based on system resolution and practicality and possible efficiency gains is discussed.

Differential responses of adult Calliphora vicina to dry bloodstains on porous versus non-porous surface materials.

Necrophagous flies are presumed to feed on wet and dried blood at crime scenes, but no empirical information exists detailing fly interactions with dried bloodstains. In the present study, the foraging behavior of adult Calliphora vicina was characterized during interactions with dried bloodstains formed on a variety of porous, and non-porous materials that are commonly encountered in a household. Continuous digital recording and image analysis were used to monitor fly interactions with dried bloodstains and to determine mechanisms of stain modification. Flies displayed differential responses to bloodstains based on the porosity and topography of the surface material. For instance, blood that was not tightly adhered to the materials was flaked or dislodged by fly activity and was not consumed by the flies. On other non-porous surfaces, most stains were consumed following moistening by regurgitation. Feeding activity on such bloodstains frequently yielded partial, perimeter and skeletal stains. In contrast, adult flies rarely mechanically altered dried blood on porous fabrics and there was no evidence of modification due to feeding. Feeding avoidance behavior was observed due to tactile inhibition with blood dried on rough, uneven surfaces like cotton and denim.

Short and Long Time Bloodstains Age Determination by Colorimetric Analysis: A Pilot Study.

Bloodstains found at crime scenes represent a crucial source of information for investigative purposes. However, in forensic practice, no technique is currently used to estimate the time from deposition of bloodstains. This preliminary study focuses on the age estimation of bloodstains by exploiting the color variations over time due to the oxidation of the blood. For this purpose, we used a colorimetric methodology in order to easily obtain objective, univocal and reproducible results. We developed two bloodstain age prediction algorithms: a short-term and a long-term useful model for the first 24h and 60 days, respectively. Both models showed high levels of classification accuracy, particularly for the long-term model. Although a small-scale study, these results improve the potential application of colorimetric analysis in the time-line reconstruction of violent criminal events.