Dimensional accuracy of structured light scans and 3D prints of various human skeletal elements.

Three-dimensional (3D) structured light scanning is a beneficial documentation technique in forensic anthropology because such models facilitate continued analysis and data sharing; they can also be 3D printed for demonstrative purposes in legal proceedings and training, without risk of damage to the original skeletal material. As its application in forensic anthropology is relatively novel, the aim of the present study is to statistically evaluate the dimensional accuracy of 3D structured light scans and 3D prints for ten bone types, including the cranium, mandible, 2nd cervical vertebra (C2), clavicle, scapula, capitate, 2nd metacarpal, os coxae, femoral head, and patella. Standard linear measurements are acquired in each physical bone, 3D virtual model, and 3D print of the same bone specimen. Variances between measurements of physical, virtual, and printed bones are quantified using the technical error of measurement (TEM), relative TEM (rTEM), and coefficient of reliability (R). Measurements acquired in the virtual models and prints were found to be within ±2 mm average of the same measurements in the physical bones, with a tendency to underestimate true value. rTEM and R values for the virtual clavicle, capitate, scapula and C2, and rTEM for the printed clavicle and capitate, were comparatively less reliable than for other bone types; although all bones were reproduced to within acceptable anthropological error standards (rTEM≤5 %; R≥0.95). This study reaffirms the use of 3D structured light scanning and 3D printing to complement traditional skeletal documentation in forensic anthropology.

Sex Determination via the Second Cervical Vertebra and Odontoid Process: A Case Report and a Review of the Literature.

Determining an individual's sex is crucial in several fields, such as forensic anthropology, archaeology, and medicine. Accurate sex estimation, alongside the estimation of age at death, stature, and ancestry, is of paramount importance for creating a biological profile. This profile helps narrow the potential pool of missing persons and aids identification. Our research focuses on the second cervical vertebra and odontoid process, which is particularly valuable due to their high sexual dimorphism. This brief research is structured as follows: we provide an overview of morphometric analysis of the second cervical vertebra for accurate sex estimation in forensic anthropology. We then delve into a case report to explore sexual dimorphism of the C2 vertebrae. Moreover, we discuss some of these studies that showed a significant correlation between the dimensions of the second cervical vertebrae and height, suggesting that the C2 can be used as a reliable indicator for stature estimation. The high accuracy rate of sex estimation using the second cervical vertebrae suggests that this method is a valuable tool for forensic anthropologists. Its practical application can significantly contribute to identifying and profiling individuals in a forensic context, thereby aiding in the identification process.

Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

No Bones About It: Sex Is Binary.

Anthropologists have led the way in formulating techniques that reveal skeletal differences between males and females. Understanding of physical differences in the pelvis related to childbirth, hormonal impacts on bones, and extensive comparative studies have provided anthropologists with an array of traits and measurements that help them estimate sex using just bones. Forensic anthropologists and bioarcheologists are improving their ability to differentiate males and females by increasing research on a variety of postcranial bones and through the use of molecular data, especially new methods called proteomics, to identify sex in prepubescent juveniles. As remains from more cultures and time periods are studied, sex identification will continue to improve, because skeletal sex differences are in large part biologically determined. Yet, anthropologists have also been at the forefront of arguing that sex lies on a spectrum. Anthropologists who view sex as on a spectrum may deter sex identification progress; from their perspective, an individual of an undetermined sex may just be a nonbinary individual. Anthropologists who consider sex is on a spectrum are coming to this conclusion in part because they are looking for anatomical ideals, mistaking pathology for variation, and confusing independent variables with dependent variables. Nonetheless, anthropologists need to continue to improve sex identification techniques to reconstruct the past accurately, which may reveal less strict sex roles than previously presumed and help with the identification of crime victims. Forensic anthropologists should also increase their efforts to identify whether individuals have undergone medical procedures intended to change one's gender due to the current rise in transitioning individuals.

Y-STR analysis of highly degraded DNA from skeletal remains over 70 years old.

The goal of the following study is to clarify whether the skeletal remains over 70 years old from missing persons and their alleged relatives shared identical Y-STR loci. Nowadays, advances in ancient DNA extraction techniques and approaches of using multiple different Y-STRs have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Given the ages and conditions of the skeletal remains, ancient DNA extraction methods can be used to maximize the probability of DNA recovery. Considering that information about distant relatives is more relevant for long-term missing persons and alleged family members are male, Y-STR loci analysis is considered the most appropriate and informative approach for determining paternal lineage relationship. In this study, Y-STR genotypes obtained from these alleged relatives were identical to each other and to the alleles of missing persons' consensus profiles at more than 22 loci examined, whilst not being found in Y-STR population database from Y-Chromosome STR Haplotype Reference Database. Therefore, Missing Person No.7 and Missing Person No.18 have a patrilineal relationship with reference samples from Family1 and Family2, respectively. In addition, the fact that Y-STR haplotypes obtained from skeletal remains of missing persons and reference samples are not found in the Han Chinese people from East Asian demonstrates its rarity and further supports a paternal lineage relationship amongst them.

Handheld hyperspectral imaging as a tool for the post-mortem interval estimation of human skeletal remains.

In forensic medicine, estimating human skeletal remains' post-mortem interval (PMI) can be challenging. Following death, bones undergo a series of chemical and physical transformations due to their interactions with the surrounding environment. Post-mortem changes have been assessed using various methods, but estimating the PMI of skeletal remains could still be improved. We propose a new methodology with handheld hyperspectral imaging (HSI) system based on the first results from 104 human skeletal remains with PMIs ranging between 1 day and 2000 years. To differentiate between forensic and archaeological bone material, the Convolutional Neural Network analyzed 65.000 distinct diagnostic spectra: the classification accuracy was 0.58, 0.62, 0.73, 0.81, and 0.98 for PMIs of 0 week-2 weeks, 2 weeks-6 months, 6 months-1 year, 1 year-10 years, and >100 years, respectively. In conclusion, HSI can be used in forensic medicine to distinguish bone materials >100 years old from those <10 years old with an accuracy of 98%. The model has adequate predictive performance, and handheld HSI could serve as a novel approach to objectively and accurately determine the PMI of human skeletal remains.

Transcending time: the forensic anthropological case study of three unidentified transgender women in Italy in the early 1990s.

In forensic anthropology, the estimation of skeletal sex results in a dichotomous outcome: male or female. This poses challenges when dealing with transgender individuals. Italy has the highest rate per capita of transgender murders, making their identification a compelling issue. This study presents three cases of unidentified skeletal remains of transgender women (MtF) that underwent autopsies in the 1990s at the Institute of Legal Medicine of Milan. The transgender status of the individuals was determined from the autopsy records which indicated the co-existence of breast implants and male genitalia. The biological profile was created by estimating sex, age, ancestry, and stature, and the bones were investigated looking for evidence of Facial Feminization Surgery (FFS). Anthropological estimations revealed that the skeletons belong to male, adults, European individuals, between 164 and 184 cm. Fragmented gelatinous masses were found in association with two skeletons, which were interpreted as remnants of breast implants. In two cases, signs of remodeling of the cortical surface were observed on the zygomatic bones, although the bone marks observed here were not specific enough to link them to FFS. Despite some limitations, this study highlights the need for greater awareness among practitioners about this limitedly addressed issue, advocating for a more inclusive forensic anthropology that strives to improve methods and interpretation of evidence for the identification of transgender individuals.

Use of strontium isotope ratios in potential geolocation of Ajnala skeletal remains: a forensic archeological study.

Stable isotope methods for provenance of unidentified human remains are relatively a newer field of enquiry in forensic archeology. It is of great interest for forensic experts these days. The application of strontium isotope analyses for estimating geolocation of archeological remains is of great interest in bioarcheology and modern forensics. The strontium (Sr) isotope composition of human bones and teeth has been widely used to reconstruct an individual's geo-affiliation, residential mobility, and migration history. Thousands of unknown human remains, reportedly belonging to 282 Indian soldiers of 26th Native Bengal regiment and killed in 1857, were exhumed non-scientifically from an abandoned well situated underneath a religious structure at Ajnala (Amritsar, India). Whether these remains belonged to the individuals, local or non-local to the site, was the important forensic archeological question to be answered by doing their thorough forensic anthropological examinations. In the present study, 27 mandibular teeth (18 s molars, 6 first molars, and 3 premolars) collected from the Ajnala skeletal assemblage were processed for strontium isotope analysis, and the measured ratios were compared with published isotope baseline data to estimate the locality status of these remains. The Sr isotopic values were concentrated in the range of 0.7175 to 0.7270. The comparative analysis of isotopic ratios revealed that most individuals buried in the Ajnala well have 87Sr/86Sr values close to the river as well as groundwater of the Gangetic plain (less radiogenic 87Sr/86Sr ~ 0.716); most likely originated near Varanasi (Uttar Pradesh, India) region, whereas the individuals with higher 87Sr/86Sr ratios (~ 0.7200) probably resided in the West Bengal and Bihar areas where the river as well as groundwater of the Gangetic plain is relatively more radiogenic. Thus, the strontium isotope results reveal that the Ajnala individuals did not grow up or live in the Amritsar region during their childhood, and this observation complemented the previous forensic anthropological and molecular findings. There is very little Indian data on the bioavailable strontium, so the inferences from the present study estimating Sr isotope abundances are expected to provide baseline data for future forensic provenance studies that will contribute to the global efforts of mapping Sr isotope variations by the isotope community.

Performance comparison of four qPCR and three autosomal STR commercial kits from degraded skeletal remains.

This research evaluates the current DNA quantification (Quantifiler™ Trio, PowerQuant®, Investigator® Quantiplex® Pro and InnoQuant® HY Fast) and autosomal STRs amplification kits (GlobalFiler™, PowerPlex® Fusion 6 C, Investigator® 24Plex QS) using 62 degraded skeletal remains from armed conflicts (petrous bone, femur, tibia, and tooth) with several parameters (autosomal small, large, and male target, degradation index, probability of degradation, number of alleles above analytical threshold, number of alleles above stochastic threshold, RFU, peak height ratio, number of reportable loci). The best qPCR/autosomal STRs amplification tandem was determined by comparing quantification results by a DNA quantity estimation based on sample average RFU. InnoQuant® HY Fast was the most sensitive kit, and no significative differences were observed among amplification kits; however, Investigator® 24 Plex QS was found to be the most sensitive in our samples. That is why InnoQuant™ and Investigator® 24Plex QS were determined to be the best tandem.

Retrospective analysis of factors affecting rate of skeletonization within a tropical climate.

Estimating the post mortem interval (PMI) in skeletonized cases is an extremely challenging exercise. Sri Lanka lacks adequate taphonomic research which is a serious limitation when assessing PMI in forensic death investigations. Methods that have been proposed to estimate PMI using the total body score (TBS) and accumulated degree days (ADD) are mainly based on data from continental and temperate climates and have shown less reliability in tropical climates. With the intention of developing a region-specific, evidence-based guide which would be applicable to tropical climates like Sri Lanka, we selected thirteen skeletonized remains with known PMIs from forensic case records and analysed their taphonomy in relation to selected weather data. We also compared the ADD values within our dataset with reference ranges calculated using published formula. All except one were found from outdoor locations. The TBS ranged from 24 to 32 and had a weak positive correlation with the PMI. The earliest appearance of skeletonization was 15 days in a body found indoors. The highest rate of skeletonization was seen in a body with a TBS of 32 and a PMI of 23 days. The average daily temperature and relative humidity were similar across all the cases however, the amount of rainfall varied. Bodies exposed to monsoon rains (n = 6) had a lower mean rate of skeletonization compared to those that were not exposed (n = 4) suggesting lower rates of decomposition during periods of heavy rainfall. No correlation was found between ADD and TBS. In 9 (69.2%) cases, the actual ADD was much lower than reference ADD ranges for TBS values, indicating poor applicability of TBS and ADD based formulae in estimating PMI within the Sri Lankan climate. Our study shows a strong need for taphonomic and entomological research in tropical climates to further explore the impact of monsoons on biotic and abiotic factors affecting skeletonization.

Comparison of Morphological Sex Assessment and Genetic Sex Determination on Adult and Sub-Adult 17th-19th Century Skeletal Remains.

The first step in the analysis of human skeletal remains is the establishment of the biological profile of an individual. This includes sex assessment, which depends highly on the age of the individual and on the completeness and preservation state of the remains. Macroscopic methods only provide the assessment of sex, while for sex determination, molecular methods need to be included. However, poor preservation of the remains can make molecular methods impossible and only assessment can be performed. Presented research compares DNA-determined and morphologically assessed sex of adult and non-adult individuals buried in a modern-age cemetery (17th to late 19th century) in Ljubljana, Slovenia. The aim of the study was to assess the accuracy of commonly used macroscopic methods for sex assessment on a Slovenian post-medieval population. Results demonstrate that for adults, macroscopic methods employed are highly reliable and pelvic morphology, even the sciatic notch alone, is more reliable than skull. In non-adults, macroscopic methods are not as reliable as in adults, which agrees with previous research. This study shows how morphological and molecular methods can go hand in hand when building a biological profile of an individual. On their own, each methodology presented some individuals with undetermined sex, while together, sex of all the individuals was provided. Results confirm suitability of sex assessment based on skull and especially pelvic morphology in Slovenian post-medieval adults, while in the non-adult population molecular methods are advised.

Measure quantity of mitochondrial DNA in aged bones or calculate it from nuclear DNA quantitative PCR results?

Mitochondrial DNA (mtDNA) is of great value in forensics to procure information about a person when a next of kin, personal belongings, or other sources of nuclear DNA (nDNA) are unavailable, or nDNA is lacking in quality and quantity. The quality and reliability of the results depend greatly on ensuring optimal conditions for the given method, for instance, the optimal input of the copy number (CN) in next-generation sequencing (NGS) methods. The unavailability of commercial quantitative PCR (qPCR) methods to determine mtDNA CN creates the necessity to rely on recommendations to infer mtDNA CN from nDNA yield. Because nDNA yield varies between individuals, tissues, parts of the same tissue, and because mtDNA CN varies between tissues, such assumptions must be examined for a specific context, rather than be generalized. This study compares mtDNA CN calculated from nDNA yield and qPCR measured mtDNA CN. Seventy-five femurs from the Second World War victims were used as samples; they were cut below the greater trochanter, surface contaminants were removed by mechanical and chemical cleaning, samples were fully demineralized, and DNA was isolated. PowerQuant® Kit (Promega) was used to analyze DNA yield. An in-house method was used to determine mtDNA CN. Comparison of mtDNA CN from nDNA derived calculations and measured mtDNA CN highlighted vast differences. The results emphasize the need to perform qPCR to assess mtDNA CN before NGS analyses of aged bones' mitogenomes rather than estimating mtDNA CN from nDNA yield to ensure the quality and reliability of the results of NGS analysis.

A comparison between petrous bone and tooth, femur and tibia DNA analysis from degraded skeletal remains.

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15-30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.

A Multisample Approach in Forensic Phenotyping of Chronological Old Skeletal Remains Using Massive Parallel Sequencing (MPS) Technology.

It is very important to generate phenotypic results that are reliable when processing chronological old skeletal remains for cases involving the identification of missing persons. To improve the success of pigmentation prediction in Second World War victims, three bones from each of the eight skeletons analyzed were included in the study, which makes it possible to generate a consensus profile. The PowerQuant System was used for quantification, the ESI 17 Fast System was used for STR typing, and a customized version of the HIrisPlex panel was used for PCR-MPS. The HID Ion Chef Instrument was used for library preparation and templating. Sequencing was performed with the Ion GeneStudio S5 System. Identical full profiles and identical hair and eye color predictions were achieved from three bones analyzed per skeleton. Blue eye color was predicted in five skeletons and brown in three skeletons. Blond hair color was predicted in one skeleton, blond to dark blond in three skeletons, brown to dark brown in two skeletons, and dark brown to black in two skeletons. The reproducibility and reliability of the results proved the multisample analysis method to be beneficial for phenotyping chronological old skeletons because differences in DNA yields in different bone types provide a greater possibility of obtaining a better-quality consensus profile.

Eye and hair color prediction of an early medieval adult and subadult skeleton using massive parallel sequencing technology.

Phenotypic trait prediction in ancient DNA analysis can provide information about the external appearance of individuals from past human populations. Some studies predicting eye and hair color in ancient adult skeletons have been published, but not for ancient subadult skeletons, which are more prone to decay. In this study, eye and hair color were predicted for an early medieval adult skeleton and a subadult skeleton that was anthropologically characterized as a middle-aged man and a subadult of unknown sex about 6 years old. When processing the petrous bones, precautions were taken to prevent contamination with modern DNA. The MillMix tissue homogenizer was used for grinding, 0.5 g of bone powder was decalcified, and DNA was purified in Biorobot EZ1. The PowerQuant System was used for quantification and a customized version of the HIrisPlex panel for massive parallel sequencing (MPS) analysis. Library preparation and templating were performed on the HID Ion Chef Instrument and sequencing on the Ion GeneStudio S5 System. Up to 21 ng DNA/g of powder was obtained from ancient petrous bones. Clean negative controls and no matches with elimination database profiles confirmed no contamination issue. Brown eyes and dark brown or black hair were predicted for the adult skeleton and blue eyes and brown or dark brown hair for the subadult skeleton. The MPS analysis results obtained proved that it is possible to predict hair and eye color not only for an adult from the Early Middle Ages, but also for a subadult skeleton dating to this period.

Colonization of diatoms in and on porcine bone substrate and considerations of diatom ecology for forensic science.

The presence of diatom algae in bone marrow has been used as forensic evidence of drowning for several decades; however, these studies are based on known or suspected recent drowning events. This study addresses the potential for diatoms to enter the bone marrow of skeletal remains, that is, de-fleshed long bones post-mortem. In laboratory and field experiments, bones were either inflicted with two access points by a cut and acid pitting or left intact. The bones were submerged in water for at least 1 week and up to 3 months. Samples of the bone surface and marrow were inspected for diatoms. The analysis considered the time required for diatoms to enter marrow and whether genus characteristics like size or mobility affect entry. The presence of an access point influenced diatom entry in that bones without an introduced access point had zero to one diatom present in the marrow, whereas a bone with an access point had >150 diatoms present in the marrow. The results of both laboratory and field phases suggest that diatoms will reliably colonize bone in as quickly as 1 week, establishing and maintaining communities for at least 3 months. However, the bone surface assemblages differ from the source community. Bone marrow displayed even more restrictive access to diatom colonization, resulting in communities dominated by small raphid diatoms. Based on these findings, we suggest some caveats on the use of diatoms as trace evidence in forensic science with recommendations for future avenues of research.

Estimation of Late Postmortem Interval: Where Do We Stand? A Literature Review.

Estimating time since death can be challenging for forensic experts, and is one of the most challenging activities concerning the forensic world. Various methods have been assessed to calculate the postmortem interval on dead bodies in different stages of decomposition and are currently widely used. Nowadays, the only well-recognized dating technique is carbon-14 radioisotope measurement, whereas other methods have been tested throughout the years involving different disciplines with different and sometimes not univocal results. Today, there is no precise and secure method to precisely determine time since death, and late postmortem interval estimation remains one of the most debated topics in forensic pathology. Many proposed methods have shown promising results, and it is desirable that with further studies some of them might become acknowledged techniques to resolve such a difficult and important challenge. The present review aims at presenting studies about the different techniques that have been tested in order to find a valuable method for estimating time since death for skeletal remains. By providing a comprehensive overview, the purpose of this work is to offer readers new perspectives on postmortem interval estimation and to improve current practice in the management of skeletal remains and decomposed bodies.

Comparing Genetic and Physical Anthropological Analyses for the Biological Profile of Unidentified and Identified Bodies in Milan.

When studying unknown human remains, the estimation of skeletal sex and ancestry is paramount to create the victim's biological profile and attempt identification. In this paper, a multidisciplinary approach to infer the sex and biogeographical ancestry of different skeletons, using physical methods and routine forensic markers, is explored. Forensic investigators, thus, encounter two main issues: (1) the use of markers such as STRs that are not the best choice in terms of inferring biogeographical ancestry but are routine forensic markers to identify a person, and (2) the concordance of the physical and molecular results. In addition, a comparison of physical/molecular and then antemortem data (of a subset of individuals that are identified during our research) was evaluated. Antemortem data was particularly beneficial to evaluate the accuracy rates of the biological profiles produced by anthropologists and classification rates obtained by molecular experts using autosomal genetic profiles and multivariate statistical approaches. Our results highlight that physical and molecular analyses are in perfect agreement for sex estimation, but some discrepancies in ancestry estimation were observed in 5 out of 24 cases.

A comparison of five DNA extraction methods from degraded human skeletal remains.

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

Water-related atmospheric agents and solubility: two parameters of validation in toxicological screening on clothing worn by skeletal remains.

In forensic toxicology, when conventional matrices are no longer available, alternative matrices can be used to assess toxicological investigations. Clothes worn by skeletal remains may be a good unconventional matrix for toxicological analyses considering that they have absorbed decomposition fluids and blood from a body. We hypothesized a scenario in which a skeleton, wearing clothes, was discovered in an open environment. From this starting point, an experimental study was developed on different textiles (cotton, wool, and polyester) to evaluate whether water-related atmospheric agents and molecule solubility can largely influence the detection of molecules of toxicological interest on this specific matrix, together with the characteristics of different garments chosen. The experimental study was performed on blood spots, previously spiked with 6-monoacetylmorphine and morphine, accurately placed on different textiles and washed with different quantities of deionized water adjusted at pH 5.6 with formic acid to simulate different rainfall conditions. Toxicological analyses were performed via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). From the experimental study morphine could not be detected on 100% cotton and 100% wool fabric after the passing of 500 mL of deionized water and in 100% synthetic polyester textile after washing with 250 mL of deionized water. In conclusion, when toxicological analyses are carried out on unconventional matrices as textiles worn by corpses exposed to different environmental conditions, it is of great importance, in using such substrates as evidence for the presence of molecules of toxicological interest, to evaluate chemical-physical characteristics of each analyte under investigation in order to correctly interpret the toxicological data obtained.