Forensic chemistry plays a crucial role in aiding law enforcement investigations by applying analytical techniques for the analysis of evidence. While bloodstains are frequently encountered at crime scenes, distinguishing between peripheral and menstrual bloodstains presents a challenge. This is due to their similar appearance post-drying. Raman spectroscopy has emerged as a promising technique capable of discriminating between the two types of bloodstains, offering invaluable probative information. Moreover, estimating the time since deposition (TSD) of bloodstains aids in crime scene reconstruction and prioritizing what evidence to collect. Despite extensive research focusing on TSD estimations, primarily in peripheral bloodstains, a crucial gap exists in determining the TSD of menstrual bloodstains. This study demonstrates how Raman spectroscopy effectively analyzes biological samples like menstrual blood, showing similar aging patterns to those of peripheral blood and provides proof-of-concept models for determining the TSD of menstrual blood. While this work shows promising results for creating a universal model for bloodstain age determination, further testing with more donors needs to be conducted before the implementation of this method into forensic practice.

The analysis of genetic material may be the only way to identify an unknown person or solve a criminal case. Often, the conditions in which the genetic material was found determine the choice of the analytical method. Hence, it is extremely important to understand the influence of various factors, both external and internal, on genetic material. The review presents information on DNA and RNA persistence, depending on the chemical and physical factors affecting the genetic material integrity. One of the factors taken into account is the time elapsing to genetic material recovery. Temperature can both preserve the genetic material or lead to its rapid degradation. Radiation, aquatic environments, and various types of chemical and physical factors also affect the genetic material quality. The substances used during the forensic process, i.e., for biological trace visualization or maceration, are also discussed. Proper analysis of genetic material degradation can help determine the post-mortem interval (PMI) or time since deposition (TsD), which may play a key role in criminal cases.

Physicochemical property changes observed in a degrading bloodstain can be used to estimate its time since deposition (TSD) and provide a timestamp to the sample\'s age. Many of the time-dependent processes that occur as a bloodstain degrades, such as DNA fragmentation and changes in hemoglobin structure, also exhibit temperature-dependent behaviours. Previous studies have demonstrated that pairing high-resolution automated gel electrophoresis and visible absorbance spectroscopy could be used to quantify the rate of degradation of a bloodstain in relation to time and storage substrate. Our study investigates such trends with an added factor, extreme temperatures. Passive drip stains were stored in either microcentrifuge tubes or on FTA cards at either -20°C, 21°C or 40°C and tested over 11 time points spanning 15 days. For both storage substrates, the wavelength at maximum absorbance for the Soret band and the maximum absorbance of the Alpha band showed a negative trend over time suggesting that spectral shifts are informative for TSD estimates. The ratio of the maximum peak height for DNA fragments lengths of 500-1000 base pairs to 1000-5000 base pairs was the most informative DNA variable in relation to time for both substrates. Cross-validation suggested the appropriate fit of the models with the data and reasonable predictive ability. We integrated both DNA concentration and hemoglobin visible absorbance metrics using principal component analysis (PCA) into a single model. Adding the random effect of the donor to the PCA model accounted for a large portion of the variation as did storage method and temperature. Additionally, canonical correspondence showed that temperature corresponded differently to the response variables for FTA card and microcentrifuge tube samples, suggesting a substrate specific effect. This study confirms that pairing DNA concentration and hemoglobin\'s visible absorbance can provide insight on the effect of different environmental and storage conditions on bloodstain degradation. While the level of uncertainty surrounding TSD estimates still precludes its use in the field, this study provides a valuable framework that improves our understanding of variation surrounding TSD estimates, which will be critical to any eventual application.

The first appearance of ribonucleic acid (RNA) in forensic science research was in 1984 in the study of post-mortem tissues. Since then, many studies have explored the role of gene expression and its potential applications in forensic science. The two main RNA molecules that have been subject to increasing interest in the forensic science community are messenger RNA (mRNA) and microRNA (miRNA). Identification of body fluid type and estimating the time since deposition can be of immense value to criminal investigations. Determining the time since deposition or age of a biological stain can help to indicate either when a crime happened, or whether the biological evidence was deposited before/after a known crime event, in order for samples to be excluded. The research presented here has used reverse transcription quantitative PCR to examine the relative expression ratio (RER) in two types of body fluid-specific markers (saliva and semen), to develop a method to estimate the age of biological stains. mRNA and miRNA markers specific to saliva and semen, along with three reference genes were selected. Biological samples from 20 participants were stored in a dark dry place at room temperature to simulate natural ageing. A series of desired ageing points were set and total RNA was extracted when samples reached each desired point. The degradation behaviour of each RNA marker was analysed, showing that they exhibited unique degradation profiles across a one-year storage interval for saliva and semen samples, where miRNAs and the U6 reference gene were shown to have high stability. The RERs exhibit a non-linear relationship with body fluid stain age and can be considered as a potential method for body fluid stain age estimation, hence the time since deposition.

In this study, we investigated the potential of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy combined with advanced chemometrics for species identification of bloodstains similar to evidence obtained from real crime scenes. Two partial least squares-discriminant analysis classification models (a human-mammal-domestic fowl trilateral model and a species-specific model) were established. The models demonstrated complete separation among the three classes (human, mammal, and domestic fowl) and distinguished six species (human, rat, rabbit, dog, chicken, and duck). Validation was subsequently conducted to evaluate the robustness of these two models, which resulted in 100 and 94.2% accuracy; even human bloodstains placed in an outdoor environment for up to 107 days were successfully identified. Additionally, all bloodstains were positively identified as blood using the squared Euclidean cosine method by comparing the spectra with those of non-blood substances that had a similar appearance or easily produced false positives. These results demonstrate that ATR-FTIR spectroscopy combined with chemometrics can be a powerful tool for species identification of bloodstains.

The time since deposition (TSD) of a bloodstain is a valuable piece of evidence for forensic scientists to determine the time at which a crime took place. The objective of this study was to determine whether attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy could be used to estimate the TSD of a bloodstain in a relatively early period (from 0 min to the time required for the bloodstain to dry out). For this purpose, we used ATR-FTIR to study the variation in absorbance at certain wavelengths as rat and human blood sample dried out. The absorbance at 3308/cm (A3308) was found to have a close correlation with the TSD during this time period, and the changes in A3308 during the drying of rat and human blood drops under the same controlled conditions showed similar results. The current study indicates that ATR-FTIR spectroscopy has potential as a tool for estimating TSD at early time periods of blood deposition.

Knowing the time since deposition (TSD) of an evidentiary bloodstain is highly desired in forensics, yet it can be extremely complicated to accurately determine in practice. Although there have been numerous attempts to solve this problem using a variety of different techniques, currently, no established, well-accepted method exists. Here, a Raman spectroscopic approach was developed for determining the age of bloodstains up to 1 week old. Raman spectroscopy, along with two-dimensional correlation spectroscopy (2D CoS) and statistical modeling, was used to analyze fresh bloodstains at ten time points under ambient conditions. The 2D CoS results indicate a high correlation between several Raman bands and the age of a bloodstain. A regression model was built to provide quantitative predictions of the TSD, with cross-validated root mean squared error and R (2) values of 0.13 and 0.97, respectively. It was determined that a \"new\" (1 h) bloodstain could be easily distinguished from older bloodstains, which is very important for forensic science in helping to establish the relevant association of multiple bloodstains. Additionally, all bloodstains were confirmatively identified as blood by comparing the experimentally measured spectra to multidimensional body fluid spectroscopic signatures of blood, saliva, semen, sweat, and vaginal fluid. These results demonstrate that Raman spectroscopy can be used as a nondestructive analytical tool for discriminating between bloodstains on the scale of hours to days. This approach shows promise for immediate practical use in the field to predict the TSD with a high degree of accuracy. Graphical Abstract Bloodstain aging over time illustrating naturally ocurring processes.

When a firearm has been disposed of in a body of water and becomes corroded, its appearance is altered and determining a time-since-immersion may be of import to the investigation. Therefore, in this study, the corrosion and mass loss of four handgun slides over a period of 180days were examined. Solid-state characterization of the metals and their corrosion products via SEM/EDX and powder X-ray Diffraction (pXRD) was performed. The pXRDs were analyzed against the NIST Powder Diffraction Database to determine the crystalline phases. Filings from the SS416 standard, Llama and Ruger handgun slide predominantly consisted of iron alloys. After 180-days in solution, pXRD indicated that the adherent corrosion products consisted of 1) γ-FeOOH and 2) iron oxide (Fe3O4 or Fe2O3). Additionally, pXRD analysis indicated that the adherent corrosion products of the SS416 standard also consisted of CrO3. Metal filings from the Raven and Jennings handgun slides were a mixture of iron-nickel-zinc and EDX and pXRD analyses of the corrosion products, when submersed in deionized water, indicated that the products consisted of: 1) γ-FeOOH, 2) iron oxide (Fe3O4 or Fe2O3), and 3) ZnFe2O4 or ZnO; where the Jennings adherent rust contained ZnFe2O4 and the Raven adherent rust contained ZnO. Further, pXRD of the corrosion products from these alloys, when submersed in 25 PSU (Practical Salinity Unit) solution, indicated that the products consisted of: 1) ZnO, 2) Zn(OH)2, 3) α-Ni(OH)2, and 4) NaCl. The data thus indicated that both metal composition and the presence of chloride ions had significant impacts on rates and products of corrosion and suggest that the presence of Cl(-) changes not only the rate of corrosion, but also the corroding species itself. While mechanisms and rates of the chloride driven corrosion processes offer explanations as to the different oxides and hydroxides observed between immersion conditions, they do not offer an explanation for the differences observed between handguns. Therefore, utilizing a general approach where surface area coverage of corrosion products is the sole consideration is not sufficient to determine time-since-immersion. Attempts to determine a time-since-immersion would require a priori knowledge of the mechanism of corrosion for a given metal mixture within a specified environment. The results described herein give indications as to the possible corrosion mechanism driving the process in high and low Cl(-) environments and show the necessity of including the metal composition, rust composition and ion concentration in any models that attempt to elucidate the time-since-immersion of handguns for forensic applications.