Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

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.

A unique case of death by MDPHP with no other co-ingestion: a forensic toxicology case.

Synthetic cathinones are a class of psychoactive drugs that have become, in recent years, of quite common observation in medical and toxicological forensic field. MDPHP (3,4-methylenedioxy-α-pyrrolidinohexanophenone) belongs to this class of substances but lethal acute intoxication caused by this specific substance without other co-ingestions has never been described in literature. We are presenting the unique case of a fatal acute intoxication caused by MDPHP in a 48-year-old man carried to the A&E department of a big Milanese hospital where he suddenly deceased after brief time. Clinical records, autopsy examination, histological findings, and toxicological analysis, assessed via a Q-Exactive Orbitrap with a HPLC system and LC/MS-MS system, are discussed. This case report may represent the first case of this type of intoxication and might help in improving recognition and treatment of these particular cases.

Detecting drugs in dry bone: a pilot study of skeletal remains with a post-mortem interval over 23 years.

In decomposed or skeletonized bodies, conventional matrices used in forensic toxicology may no longer be available for analysis. The aim of this paper was to test the survival and detection of toxicological substances in dry bone samples with over 23 years of post-mortem interval. In this perspective, bone samples from the cranium, ribs, and vertebrae of seven skeletons from the CAL Milano Cemetery Skeletal Collection, buried for over 23 years, fully decomposed and altered by taphonomic factors were selected based on their ante-mortem data, which included verified or suspected drug addictions or overdose. Qualitative and quantitative analyses were performed with Dionex™ ASE™ 350 Accelerated Solvent Extractor and Q-Exactive Orbitrap-mass spectrometry with a HPLC system. Positive results were obtained in six of the seven cases, and different psychoactive drugs (and in some cases their active metabolites) were detected, including analgesic (two opioids: methadone and buprenorphine) and anxiolytic drugs (benzodiazepines, in particular delorazepam, diazepam, nordiazepam, and lorazepam), a cannabinoid metabolite (THCCOOH) as well as metabolites of stimulants (benzoylecgonine and MDA). Consequently, this research shows that toxicological substances may be found in bone tissue after over 23 years of post-mortem interval.

Towards an integrative approach to the biological profile.

One of the most important tasks in forensic anthropology is the construction of the biological profile, classically defined as a set of four basic biological descriptors: biological sex, age-at-death, ancestry, and stature. Yet, our empirical and technological abilities in reconstructing the life experiences and health from skeletal remains far exceed these four parameters and forensic anthropology could benefit from further descriptors in the search for an identity. In this paper, we propose the inclusion of two other investigations to forensic anthropology practice to implement the already known biological profile: the interpretation of bone disease and lesions, and forensic toxicology on unconventional biological matrices. These analyses can provide information regarding health, habits, and disease burden, and by implementing them in our practice of forensic anthropology, they have the potential to improve the biological profile. We also propose a new term that can include not only the classical biological profile but also further descriptors, namely, the "biocultural profile".

Work-related and non-work-related fatal road accidents: Assessment of psychoactive substance use in commuting.

Commuting road traffic collisions (RTCs) are one of the main causes of occupational death worldwide, including Italy. Among the prominent yet relatively understudied personal risk factors in the subpopulation of workers, there is the use of psychoactive substances. Since the phenomenon of driving under alcohol and drugs effects in the commuter sub-population has so far been poorly studied, we carried out a study by comparing results obtained from the analysis of commuters and non-commuters. The percentage of commuting RTCs victims was 10.4% out of all the RTCs. Commuter victims were mostly men, 51-60 years-old, motorcyclists (32.1%) or car drivers (28.6%), employees, deceased during Fridays and Saturdays, in the afternoon (35.7%) and in the evening (32.1%). It was possible to perform toxicological analyses in 60.7% of commuter cases: approximately 40% tested positive, always and only for ethanol (median Blood Alcohol Concentration, BAC, of about 1.03 g/L). Values above the legal limit were observed in 23.5% of the toxicological samples analyzed from commuter accidents. Our findings contribute to bridging the gap in knowledge in the area of RTCs and commuting and emphasize the importance of carrying out toxicological investigations, with possible repercussions on both insurance issues and public health strategies, which are relevant for analyzing the phenomenon structurally.

Toxicological analysis in victims of sexual and domestic violence: A retrospective study of a 3-year period (2018-2020).

This manuscript presents an epidemiological investigation carried out on abuse victims who accessed the Sexual and Domestic Violence Service (SVS&D) of IRCCS Ca' Granda in Milan, Italy. The focal point of this research was the detection of alcohol, prescription medications, and illicit substances in victims who solicited help from the SVS&D center between 2018 and 2020. Over this three-year span, biological samples of blood and urine were procured from 207 victims, out of a patient pool of 2470. All collected samples were analyzed via High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography - Mass Spectrometry (GC-MS). Toxicological examination results demonstrated that 43% of the cases tested positive for substances in 2018, 39% in 2019 and 60% of the cases in 2020. Overall, 45% of the victims tested resulted positive to some substance over a 3-year period, equivalent to 3.6% of the overall cases (2470 victims). Substances of toxicological interest were detected in 104 samples (out of 377, corresponding to 27.6%) belonging to 94 patients. The most detected classes of drugs were stimulants, antidepressants, benzodiazepines and antipsychotics. Moreover, BAC (Blood Alcohol Concentration) indicated positivity in 25 cases (out of 184 cases analyzed - 14% of positive cases). Based on this study's findings, we recommend broadening the range of substances evaluated in drug-facilitated sexual assaults and establishing standardized protocols for both national and international implementation. Implementing procedures would significantly enhance forensic support provided to victims of abuse seeking healthcare services post-incident.

Papaver somniferum in seventeenth century (Italy): archaeotoxicological study on brain and bone samples in patients from a hospital in Milan.

In this paper, we present the results of toxicological analyses of preserved brain tissue and bone samples from the remains of the seventeenth century patients of the Ospedale Maggiore, the main hospital in Milan and one of the most innovative hospitals in Europe from the Renaissance period. Beneath it, the crypt functioned as the burial place for the deceased of the hospital. In this multidisciplinary study of the remains, toxicological analyses in particular were performed with HPLC-MS/MS on different biological samples from nine individuals. Anthropological, paleopathological, histological, radiological examinations and radiocarbon dating were also carried out. As a result, archeotoxicological analyses revealed the presence of codeine, morphine, noscapine and papaverine, derived from Papaver somniferum, a plant present in the hospital pharmacopeia used as a narcotic, analgesic, astringent, coagulant, and antitussive agent. Such analyses have shed light on the pharmacological therapies administered to the patients near the time of death and have implemented our knowledge of medical treatment and drug administration in the 1600's.

Postmortem forensic toxicology cases: A retrospective review from Milan, Italy.

We are presenting a study on 136 cases performed in a 2-year period (2018-2019) at the Bureau of Legal Medicine of the University of Milan for which toxicological analyses were requested and we are making a detailed interpretation of clinical records and discussing toxicological results from each case included in the study. Total number of autopsies was 1323 and in 10.3% of the cases, toxicological analyses were requested to obtain further information. Analyses were assessed with High-Performance Liquid Chromatography-Mass Spectrometry system and Gas Chromatography-Mass Spectrometry analyses. Additionally, Blood Alcohol Concentration and detection of volatile substances were obtained with Head Space-Gas Chromatography-Mass Spectrometry system. From these analyses, 101 cases out of 136 provided positive results (74.3%). Main substances detected were cocaine, diazepam, morphine, and ethanol. The most representative profiles of individuals that emerged from this study were: a Caucasian male, age 41-50, that died for cocaine acute intoxication or was killed; a Caucasian male or female with a range-of-age of 31-50 deceased for simple suicide caused by acute intoxication or by complex suicide caused by acute intoxication and suffocation; and finally, a Caucasian male with a range-of-age 21-40 that died in a car accident without any toxicological evidence. From the results, acute intoxication at the time of death was confirmed in 54 cases and in 57 cases the toxicological analyses helped in the determination of the cause and manner of death. From this study, the importance of toxicological data among forensic sciences is confirmed.

Histological paraffin-embedded block: a good alternative specimen to detect the use of opiates at least 20 years ago.

PURPOSE: Since the solely certain remnants of a performed autopsy are formalin-fixed paraffin-embedded (FFPE) samples, stored in the archives of every institute of legal medicine, we managed to extract molecules of toxicological interest from these specimens. METHODS: We assessed the analysis of ten fresh liver samples collected from heroin-related deaths and then histologically processed the same samples. The embedded blocks were then extracted by means of a new extracting method and the eluates were measured. We also selected five toxicological cases of heroin-related fatalities that were examined 20 years ago, collected the toxicological result documents of the analysis that were carried out at the time and then processed the corresponding FFPE liver samples that were stored in the archives. RESULTS: We managed to isolate heroine-related metabolites from 20-year-old paraffin-embedded blocks and calculated ratios to evaluate the performance of our new extraction. CONCLUSIONS: According to our study, it is feasible to carry out a toxicological examination on old histological samples and, therefore, this matrix can be considered as a new alternative specimen for chemical-analytical evaluations of past cases or when fresh samples are not available anymore. The new extractive method was evaluated as efficient in treating these complex, paraffin-embedded samples. It was surprising that the target compounds could be quantitated from FFPE bocks created as long as 20 years ago.

How Do Drugs Affect the Skeleton? Implications for Forensic Anthropology.

Forensic anthropologists rely on a number of parameters when analyzing human skeletal remains to assist in the identification of the deceased, predominantly age-at-death, sex, stature, ancestry or population affinity, and any unique identifying features. During the examination of human remains, it is important to be aware that the skeletal features considered when applying anthropological methods may be influenced and modified by a number of factors, and particular to this article, prescription drugs (including medical and non-medical use) and other commonly used drugs. In view of this, this paper aims to review the medical, clinical and pharmacological literature to enable an assessment of those drug groups that as side effects have the potential to have an adverse effect on the skeleton, and explore whether or not they can influence the estimation of age-at-death, sex and other indicators of the biological profile. Moreover, it may be that the observation of certain alterations or inconsistencies in the skeleton may relate to the use of drugs or medication, and this in turn may help narrow down the list of missing persons to which a set of human remains could belong. The information gathered from the clinical and medical literature has been extracted with a forensic anthropological perspective and provides an awareness on how several drugs, such as opioids, cocaine, corticosteroids, non-steroidal anti-inflammatory drugs, alcohol, tobacco and others have notable effects on bone. Through different mechanisms, drugs can alter bone mineral density, causing osteopenia, osteoporosis, increase the risk of fractures, osteonecrosis, and oral changes. Not much has been written on the influence of drugs on the skeleton from the forensic anthropological practitioner perspective; and this review, in spite of its limitations and the requirement of further research, aims to investigate the current knowledge of the possible effects of both prescription and recreational drugs on bones, contributing to providing a better awareness in forensic anthropological practice and assisting in the identification process of the deceased.

Drugs in bone: Detectability of substances of toxicological interest in different states of preservation.

In forensic contexts of advanced decomposition, when conventional matrices are no longer available for toxicological analyses, finding alternative matrices is necessary. The skeleton, which is fundamental for anthropologists and geneticists, could be useful also for toxicological purposes. The present study aims to examine what kind of information toxicological analysis performed on bones (the cranium and the ribs) in different states of preservation could provide to the forensic practitioner. Thirty cadavers with known pharmacological history, subjected to forensic autopsy at the Institute of Legal Medicine of Milan, were selected. Rib and cranium samples were collected from each body and separated into two parts in order to create two different states of preservation: One was cleaned from soft tissues and analyzed as a well-preserved bone sample; the other was submitted to a long maceration process, simulating complete skeletonization. All specimens were then processed with accelerated solvent extraction and the eluates analyzed using Q-Exactive™ Orbitrap™ Mass Spectrometer. The analysis of blood and skeletal matrices showed positive results for the tested substances in 63% of cases, mainly benzodiazepines, antidepressants, and drug abuse. Significant Pearson correlations were observed between non-macerated vs. macerated bone samples: r = 0.79 for rib samples, r = 0.61 for cranium samples, and r = 0.69 for all bone samples. As a consequence, the positive results confirm the potential of the bone tissue as an alternative matrix in forensic toxicology, even in cases of extremely decomposed bodies. This study also highlighted important elements for reconstructing the biological profile in cases of forensic anthropological concern.

The appearance of breast cancer metastases on dry bone: Implications for forensic anthropology.

Breast carcinoma is a major cause of morbidity and mortality in women. The study of bone pathologies presents considerable potential in anthropology, paleopathology, forensic science and medicine. In this paper, we present and discuss metastatic lesions found in the skeletons of known individuals from the CAL Milano Cemetery Skeletal Collection, clinically diagnosed with breast cancer during life. Fourteen skeletons from a contemporary and identified collection were macroscopically studied and metastases were identified by comparison with clinical literature. As a result, bone metastases were observed in 43% of the study sample. They were located most commonly on the ribs (28.1%), pelvic girdle (19.8%), vertebrae (15.6%), skull (15.6%), scapulae (10.2%) as well as proximal segment of the femora (8.4%) and humeri (2.4%) respectively, favoring sites of high vascularization. The majority of the lesions were osteolytic, although osteoblastic and mixed metastases did occur. Osteolytic metastases appear as coalescent porosity or round to oval perforating lesions on bones with denticulated margins and pitted surrounding bone, whereas osteoblastic metastases thickened the existing trabecula (spongiosclerosis). Mixed metastases were perforating lytic lesions exposing the osteoblastic activity in the underlying trabecular bone. These results, consistent with the data from the literature, strengthen the diagnostic criteria for metastases and illustrate the aspect of bone metastases in breast carcinoma.

The overlooked primary: bladder cancer metastases on dry bone. A study of the 20th century CAL Milano Cemetery Skeletal Collection.

OBJECTIVE: The aim of this article is to provide additional documentation of bone metastases to help anthropologists recognize the condition and potentially suggest the diagnosis of bladder carcinoma in differential diagnosis. MATERIALS AND METHODS: Thirteen individuals clinically diagnosed with bladder carcinoma from the 20th century Milano Cemetery Skeletal Collection were macroscopically studied to document bone metastases in bladder cancer. RESULTS: Bone metastases were found through macroscopic observation in three individuals or 23% of the study sample. Metastases were mostly of a mixed nature (45%), although both osteoblastic (13%) and osteolytic (9%) also occurred. In particular, mixed and osteoblastic metastases exhibited different distribution patterns, even when affecting the same bones. The vertebrae (24.7%), skull (12.9%), ribs (11.7%), proximal humeri (7.8%), pelvis (5.2%), proximal femora (2.6%), sacrum (1.3%) and sternum (1.3%) were the most commonly affected. Osteolytic lesions included coalescing superficial pits or lesions perforating the bone cortex. Proliferative lesions manifested as spongiosclerosis or periosteal new bone. Mixed metastases were osteolytic lesions exposing a thickened trabecular bone or coalescent porosity with reactive new bone. CONCLUSIONS: Bladder carcinoma metastases were mostly mixed, exhibiting periosteal reactions, perforations of bone cortex, spongiosclerosis and coalescing porosity. SIGNIFICANCE: Bladder carcinoma is rarely considered in the differential diagnosis of the primary organ. This study reports the macroscopic aspect of bone metastases in bladder carcinoma and may help anthropologists diagnose the condition in skeletons. LIMITATIONS: Absence of evidence is not evidence of absence; some lesions may have been hidden from macroscopic observation and therefore missed. FURTHER RESEARCH: Radiographic analysis and comparison with other neoplasms should provide additional details for the diagnosis of bladder cancer bone metastases.