An mRNA Profiling Study of Vaginal Swabs from Pre- and Postmenopausal Women.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6-20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay's success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

Taphonomic study on drowned victims in a non-sequestered aquatic environment in the Mediterranean Sea.

Human decomposition in sea water poses several challenges to forensic practitioners tasked with the analysis of drowned bodies. Postmortem changes in the marine environment have not been extensively investigated and the current literature is mainly based on bodies retrieved from shallow waters or on limited samples. On 18 April 2015, a fishing boat carrying allegedly 1,000 migrants sank in the Mediterranean Sea. In a fifteen-month time span, humanitarian missions were carried out to recover the bodies from the sea. The present study investigates postmortem changes on the drowned victims in a non-sequestered environment in the Mediterranean Sea. A retrospective study was performed by two observers on the autopsy photographic records in the series of bodies recovered from the open sea. For 184 bodies, the postmortem changes were evaluated according to facial, body, limb, and total aquatic decomposition score (FADS, BADS, LADS, TADS, respectively). Furthermore, a modification to the current scoring system that divides upper and lower limbs was suggested. The interobserver agreement was assessed using Krippendorff's alpha coefficient. Possible relations between the decomposition scores and PMSI were investigated with Pearson correlation analysis. According to the sequence of the recovery missions, increasing FADS, BADS, LADS, and TADS were observed. The modified scoring system showed a strong agreement between observers, allowing a more accurate description of the actual extent of decomposition. The scores showed a significant relation with the PMSI (p < 0.01). Prolonged submersion in the open marine environment was confirmed to show increasing decomposition rates, from moderate decay to full disarticulation. This study provides a descriptive unicum of the postmortem changes in the open sea, which may contribute to strengthen the discipline and aid description of bodies recovered in similar circumstances, especially if a body needs to be associated to a disaster or period with respect to another, thus facilitating families or authorities in the search for specific victims.

Forensic DNA Typing From Femurs and Bones of the Foot: A Study of 3 Cases.

ABSTRACT: Evidence has been accumulating in the sense that femur may not always be the best option for DNA typing of skeletal remains. Recent studies have shown that bones of the hands and feet appear to be a superior source of preserved DNA. The current study reanalyzed DNA quantitation, degradation, and short tandem repeat typing in femurs, lateral cuneiforms, and distal foot phalanges. Data from 3 human identification cases involving corpses in an advanced decomposition state were collected. We found that in the studied cases, the femur provided equal or inferior results, recovering 84.9% of true alleles. Lateral cuneiforms (99.2%) and distal foot phalanges (96.8%) yielded higher percentages. In addition, more drop-ins and drop-outs were detected in femurs than cuneiforms and phalanges. This study adds to current findings that advocate for further investigation into bone selection for use in forensic practice. The impacts of our findings are limited by the small number of individuals studied and may not apply to old and degraded bones.

Large fragment demineralization: an alternative pretreatment for forensic DNA typing of bones.

In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories.

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

Complete decapitation in suicidal hanging - a case report and a review of the literature.

Complete decapitation as a result of a suicidal hanging is rarely observed in forensic practice. The authors report a case of a 38-year-old man hanging from a bridge with a 4.2 m nylon rope. The man, weighing 70 kg, was wearing a 6.5 kg rucksack. The headless body was found lying in the river below the bridge and his head was found the next day in the river 6 m away from the bridge. Autopsy findings recorded a regular and concentric skin abrasion pattern and cervical spine transection between C2 and C3. Radiologic study depicted C2-C3 neck transection and the fracture of the left lamina and the spinous process of the axis. The force applied to the neck and the kinetic energy were calculated.

Streptococcus Pneumoniae Detection Long Time After Death in a Fatal Case of Waterhouse-Friderichsen Syndrome.

We report a fatal case of Waterhouse-Friderichsen syndrome in a 64-year-old man. The diagnosis, suspected during the autopsy (performed 63 hours after death), was confirmed through the successful detection of Streptococcus pneumoniae DNA and antigens in samples (blood and liver) collected during the autopsy. These results conformed with blood cultures performed antemortem, which became available only the day after the autopsy. The case underlines the need to collect biological material (liver and blood samples) during autopsy for microbiological investigations, although the collection is performed a long time after the death, suggesting that a liver sample works for DNA and liver and blood work for Streptococcus pneumoniae antigen detection.

PowerPlex® Fusion 6C System: evaluation study for analysis of casework and database samples.

AIM: To report on the successful analysis of amplicons obtained with PowerPlex® Fusion 6C System, a highly robust 27-plex genotyping kit developed for human identification laboratories, on the Applied Biosystems® 3500 Genetic Analyzer. METHOD: We performed characterization and evaluation studies following the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines, examining several critical areas of kit performance. We report the results of sensitivity, robustness, heterozygous peak height ratio, precision, concordance, caseworks, and mixture interpretations. We tested sensitivity, using serial dilutions of control DNA. RESULTS: The minimum amount of input DNA resulting in a full profile was 125 pg. Inhibition, inducted by urea, showsed a progressively fragmentation of DNA and a full profile was obtained until 1M of inhibitor factor. To test the profile quality, casework samples were extracted with different extraction methods: Chelex®100, QIAmp DNA Micro Kit and Phenol-Chloroform extraction. The results demonstrated that extraction chemistries do not have affect on amplification performance. Concordance check was performed by typing some casework samples and comparing the typing results with those obtained with other available kits. Thus, concordance was expected and supported by the data. CONCLUSION: Reliable DNA typing results can be obtained using this new kit, demonstrating its effectiveness and utility in forensic analysis.

Hanging Deaths: A Retrospective Study Regarding 260 Cases.

Hanging is a form of asphyxia caused by external pressure on the upper airways deriving from a band tightened by the gravitational weight of the body. During a 33-year period from 1983 to 2015, there were 260 hanging deaths examined at the Brescia Institute of Forensic Medicine (Northern Italy), which represented approximately 40% of all the asphyxial deaths recorded during this period. Almost all of the cases were represented by suicidal events, 1 case was a homicide event and there also was a case of sexual asphyxia. Eighty-two percent of the victims were men; 18% were women. There was an increasing tendency in the ages between 20 and 60 years, and the highest number of cases was observed in the 41- to 50-year age group (23% of the cases). The most common place chosen by the victim was the home setting followed by isolated outdoor places. The most common ligature material was a rope. The pathological findings were evaluated in all cases.

Standard of care and guidelines in prevention and diagnosis of venous thromboembolism: medico-legal implications.

Concerning recent Italian laws and jurisprudential statements, guidelines application involves several difficulties in clinical practice, regarding prevention, diagnosis and therapy of venous thromboembolism. International scientific community systematically developed statements about this disease in order to optimize the available resources in prophylaxis, diagnosis and therapy. Incongruous prevention, missed or delayed diagnosis and/or inadequate treatment of this disease can frequently give rise to medico-legal litigation.

Hydranencephaly in a case of suspected infanticide.

An abandoned fetus with suspicious skin injuries was found dead, lying on the grass of a garden, near a private house. Suspecting infanticide, the prosecutor ordered a medico-legal autopsy. The cause of death was identified as a congenital malformation of the central nervous system such as hydranencephaly, and infanticide was excluded.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Homicide by blunt trauma in Brescia county (northern Italy) between 1982 and 2012.

Blunt force injuries are produced by a variety of objects, including hands and feet (which is human strength) and many different weapons. Some of these objects produce distinctive patterns of injury of potential evidentiary value. This article presents a series of 53 homicides by blunt trauma along the period from 1982 to 2012, representing 16% of all homicides treated by the University Institute of Forensic Medicine in Brescia (northern Italy). Fifty-seven percent (30 cases) of the victims were male. The mean age of the victims was 47.9 years. The weapon most frequently involved was human strength. Not surprisingly, head trauma was the most common cause of death (66%). Forty-seven percent (25 cases) of the victims survived their assaults for varying periods. Homicides due to blunt trauma are still a relevant challenge for the forensic pathologist, who must obtain a complete and accurate history of the crime (including details regarding the crime scene), interpret patterns of injury and other findings at autopsy, and correlate all of the findings to make an accurate ruling of the cause and manner of death.

Assessing the usefulness of Raman spectroscopy and lipid analysis of decomposed human bones in forensic genetics and molecular taphonomy.

Bones are among the structures most likely to be recovered after death. However, the low quantity of preserved DNA and complex processing from sample to DNA profile make forensic DNA analysis of bones a challenging task. Raman spectroscopy and gas chromatography-mass spectrometry (GC/MS), have the potential to be useful as screening tools for DNA analysis and in decomposition studies. The objective of this research was to assess the usefulness of such molecular investigations. Femur samples collected from 50 decomposing human bodies were subjected to Raman spectroscopy and GC/MS. Assessment of nuclear DNA quantity and short tandem repeat (STR) genotyping efficiency were also performed. Raman parameters (crystallinity, carbonate-to-phosphate ratio, mineral-to-matrix ratio) and detected lipids were recorded. Background fluorescence proved problematic for Raman analysis of forensic bones. Regardless, it was not associated with less preserved DNA or less detected STR alleles. Fatty acids, hydrocarbons, and five types of fatty acid methyl esters (FAMEs) were detected. The main phosphate peak position in Raman spectra was significantly correlated with preserved DNA (p = 0.03713), while significantly more STR alleles were detected in bones containing methyl hexadecenoate (p = 0.04236). Detection of FAMEs in the bone matrix suggests a reaction between methanol produced by bacteria and free fatty acids, which are not associated with the level of preservation of endogenous DNA. The techniques assessed have shown to be useful in molecular taphonomy studies and forensic genetics.

Bitemarks and 3D scanner: An objective comparison for bitemarks. A pilot study.

The marks left by the bite of a subject (bitemark) represent an unequivocal character, being useful in identifying the possible perpetrator of a crime. To date, the analysis of bitemarks is mainly based on the direct vision of photographic finds and on the visual comparison with the teeth of the hypothetically responsible person. However, the bitemark also retains three-dimensional characteristics detectable with a 3D scanner. In this pilot study, an innovative method of bitemark analysis, utilizing a three-dimensional scanner and some software, will be introduced, enabling a quantitative comparison of bitemarks and their corresponding human dentitions. For this purpose, 10 complete plaster models (human dentitions) of 10 adult subjects were used to make 20 experimental bitemarks on dentistry wax. All materials were individually scanned with the iTero® 3D scanner and reworked with MeshMixer software. A visual analysis of the characteristics and a computerized analysis with the CloudCompare software were also performed. Study showed a different trend of the distribution curves of the points which were obtained comparing the human dentition and coincident and non-coincident bitemarks. Current results support that the intraoral 3D scanner allows the fast record and the preservation of the three-dimensional characteristics of the bitemarks, and it allows computerized analyses to be carried out.

Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

Y-chromosome polymorphisms and ethnic group - a combined STR and SNP approach in a population sample from northern Italy.

AIM: To find an association between Y chromosome polymorphisms and some ethnic groups. METHODS: Short tandem repeats (STR) and single-nucleotide polymorphisms (SNP) on the Y chromosome were typed in 311 unrelated men from four different ethnic groups - Italians from northern Italy, Albanians, Africans from the Maghreb region, and Indo-Pakistanis, using the AmpFlSTR® Yfiler PCR Amplification Kit and the SNaPshot Multiplex Kit. RESULTS: STRs analysis found 299 different haplotypes and SNPs analysis 11 different haplogroups. Haplotypes and haplogroups were analyzed and compared between different ethnic groups. Significant differences were found among all the population groups, except between Italians and Indo-Pakistanis and between Albanians and Indo-Pakistanis. CONCLUSIONS: Typing both STRs and SNPs on the Y chromosome could become useful in determining ethnic origin of a potential suspect.

Medical malpractice and the professional legal responsibility of the sports physician.

As with other medical specialties, litigation in sports medicine appears to be on the increase. In most countries, the applicable legal standard is "good medical practice" as identified with reference to the physician's own field of specialisation: what is commonly done by physicians in the same specialty generally serves as the standard by which a physician's conduct is measured. To enhance the quality of sports medicine practice, medical societies have been issuing guidelines for use by sports physicians, and a number of courts have recognised guidelines as evidence of good medical practice. One potential field of malpractice in sports medicine relates to privacy issues: an athlete should be asked to fill in a consent form if the medical information needs to be shared with other parties. Another relevant field is doping: for any act of drug prescription to be legally sound, sports physicians have to be aware of the requirements of the World Anti-Doping Agency Code and its international standards. Ultimately, the best way for sports physicians to avoid sources of liability is for them to keep up to date with the latest research and to act in a careful and diligent manner.

Forensic remarks regarding 35 cases of complex suicides and 4 cases of complicated suicides investigated at the Institute of Legal Medicine of Brescia during the period 1983-2022.

The role of the forensic pathologist is central to both identifying the cause and determining the manner of death. Distinguishing a suicide from a homicide or accidental event is essential to define whether third parties are involved in death. Suicides are most frequently performed using a single method; therefore, they can be defined as simple. The term "complex suicide" refers to a form of suicide in which two or more methods are applied by the victim, simultaneously or in chronological succession, to achieve the death. The different methods may have been planned in advance to prevent failure of the first method or may occur because the first method was not effective or was too painful, so the victim quickly seeks another way to complete the suicide. "Complicated suicides", on the other hand, are characterised by an unintentional secondary trauma following the suicidal act. This study analyses 35 complex suicides and 4 complicated suicides investigated at the Institute of Forensic Medicine in Brescia (Italy) during the period 1983-2022. Some data about the cases are discussed. This study aims to demonstrate how multiple lesions on the victim's body are not in themselves indicative of the intervention of third parties in their production, but complex and complicated suicides must always be considered.