Various body fluids such as blood, semen, vaginal secretions, and saliva are frequently encountered at crime scene. In cases of sexual assault, semen stains are one of the most reliable evidence of biological origin. In this study, our objective was to develop a method for estimating the time since deposition of semen stains on five different fabric types using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy, with a focus on a time frame of up to 8 weeks. Semen samples from six different volunteers were dripped onto five distinct fabric materials, and ATR-FTIR measurements were obtained at 17 different time points. Principal component analysis (PCA) and partial least squares (PLS) methods were employed to differentiate semen stains on various fabric samples and estimate the age of semen stains. Models constructed using PCA and PLSR achieved high R2 values and low root-mean-square error (RMSE). While the performance varies depending on fabric types, it was observed that age estimation of semen stains can be made within following intervals: 0.39-0.76 days for 0-7 day range, 2.59-3.38 days for the 1-8 week range, and 3.98-8.1 days for the 0-56 day range. This study demonstrates the effectiveness of using ATR-FTIR spectroscopy in combination with chemometrics to estimate the age of human semen stains on various fabric types based on time-dependent spectral changes.

BACKGROUND: Blood and semen stains are the most common biological stains encountered at crime scenes. The washing of biological stains is a common application that perpetrators use to spoil the crime scene. With a structured experiment approach, this study aims to investigate the effects of washing with various chemicals on the ATR-FTIR detection of blood and semen stains on cotton.
METHODS: On cotton pieces, a total of 78 blood and 78 semen stains were applied, and each group of six stains was immersed or mechanically cleaned in water, 40% methanol, 5% sodium hypochlorite solution, 5% hypochlorous acid solution, 5 g/L soap dissolved pure water, and 5 g/L dishwashing detergent dissolved water. ATR-FTIR spectra gathered from all stains and analyzed with chemometric tools.
CONCLUSIONS: According to performance parameters of developed models, PLS-DA is a powerful tool for discrimination of washing chemical for both washed blood and semen stains. Results from this study show that FTIR is promising for use in detecting blood and semen stains that have become invisible to the naked eye due to washing of the findings.
CONCLUSIONS: Our approach allows blood and semen to be detected on cotton pieces using FTIR combined with chemometrics, even though it is not visible to the naked eye. Washing chemicals also can be distinguished via FTIR spectra of stains.

Semen stain is one of the most important biological evidence at sexual crime scenes. Age estimation of human semen stains plays an important role in forensic work, and it is rarely studied due to lack of well-established methods. In this study, the technique called attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) coupled with advanced chemometric methods was employed to determine the age of semen stains on three different substrates: glass slides, tissues and fabric made of regenerated cellulose fibres up to 6 d. Partial least squares regression (PLSR) was used in conjunction with spectral analysis for age estimation, and the results generated high R 2 values (cross-validation: 0.81, external validation: 0.74) but a narrow margin of error for root mean square error (RMSE) (RMSE of cross-validation: 0.77 d, RMSE of prediction: 1.02 d). Additionally, our results indicated the robustness of PLSR model was not weaken by the influence of different substrates in this study. Our results indicate that ATR-FTIR, combined with chemometric methods, shows great potential as a convenient and efficient tool for age estimation of semen stains. Moreover, the method could be applied to routine forensic investigations in the future.

Semen stains are the most important biological evidence when identifying the aggressor in sexual assault cases. Current detecting assays of semen stains species identification were not confirmative enough. In this study, we investigated the potential of species identification of semen stains by using attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy combined with advanced chemometrics methods. The effect of substrates types and time since deposition (TSD) were considered in the study. A partial least squares-discriminant analysis (PLS-DA) classification model was established which demonstrated complete separation between human and other species (rabbit, dog, boar, bull, and ram). Validation was conducted which showed prediction abilities with 100% accuracy. Additionally, we found species identification could be achieved without sperm cells which proved ability of spectroscopic methods detecting the semen samples from the case of azoospermia. This work provides a powerful and practical tool for species identification of semen stains in real forensic casework.