Internal validation study to assess the SeqStudio™ for human identification's performance.

The SeqStudio™ for human identification (HID) is a new benchtop capillary electrophoresis (CE) platform recently developed by Applied Biosystems for genotyping and sequencing short tandem repeat (STR) fragments. Compared to the previous series of CE systems developed by this maker, it is more compact and easier to use. Moreover, by allowing the detection of 4 to 8 fluorescent dyes, it seems to be fully compatible with the different kits of autosomal and gonosomal STR markers usually used in forensic genetics, which are available in trade and supplied by various manufacturers. However, being a new CE model, before its routine use in forensic genetics applications, it should undergo appropriate analytical validation studies in its own laboratories to understand its potential and limitations. A series of experiments on DNA samples coming from cell line controls, using the GlobalFiler™ IQC Amplification Kit, were carried out to meet this purpose. The SeqStudio™ Genetic Analyzer for HID's findings on genotyping reproducibility (precision and accuracy of sizing), sensitivity, signal variability between dyes (intra- and inter-color channel balance), and stutter ratios are reported. These findings confirm the validity of this new CE system and its capability to generate reliable results.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system.

BACKGROUND: Massively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses. METHODS AND RESULTS: DNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened. CONCLUSIONS: Although the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.

A hard way to die: when multiple lethal gunshots mean suicide.

Multiple suicidal gunshot wounds are rare and often present a challenging issue for forensic pathologists in determining the manner of death.We describe three cases of suicidal multiple gunshot wounds in which crime scene investigation, cadaveric examination, and autopsy were provided. In all cases, integration of circumstantial data and crime scene investigation suggested a suicidal event.Firstly, we describe the self-infliction of two gunshots to the chest and head in a 90-year-old man by a revolver with wounds to the left lung and lethal wound to both frontal and temporal lobes. The second case concerns a 97-year-old man found dead with two gunshot wounds to the chest by a semi-automatic weapon and bullets penetrated to the left lung and heart. The last case, exceptional in literature, is a 41-year-old man with three self-inflicted gunshots, all-penetrating the left lung and the heart, using a revolver. This paper illustrates that immediate incapacitation can lack even in case of multiple fatal gunshot wounds on instantaneously lethal targets. Therefore, a complete investigation is required for a correct interpretation of the manner of death.An extensive review of literature is also provided.

Spinal cord injury as an indicator of abuse in forensic assessment of abusive head trauma (AHT).

Abusive head trauma (AHT) in children is notoriously one of the most challenging diagnoses for the forensic pathologist. The pathological "triad", a combination of intracranial subdural haematoma, cerebral oedema with hypoxic-ischaemic changes and retinal haemorrhages, is frequently argued to be insufficient to support a corroborated verdict of abuse. Data from all available English-language scientific literature involving radiological and neuropathological spinal cord examination is reviewed here in order to assess the contribution of spinal cord changes in differentiating abusive from accidental head trauma. In agreement with the statistically proven association between spinal subdural haemorrhage (SDH) and abuse (Choudhary et al. in Radiology 262:216-223, 2012), spinal blood collection proved to be the most indicative finding related to abusive aetiology. The incidence of spinal blood collection is as much as 44-48% when all the spinal cord levels are analysed as opposed to just 0-18% when the assessment is performed at cervical level only, in agreement with the evidence of the most frequent spinal SDH location at thoracolumbar rather than cervical level. In this review, the source of spinal cord blood collection and how the age of the child relates to the position of spinal cord lesions is also discussed. We concluded that the ante mortem MRI examination and post mortem examination of whole-length spinal cord is of fundamental interest for the assessment of abuse in the forensic setting.

Are rapidly mutating Y-short tandem repeats useful to resolve a lineage? Expanding mutability data on distant male relationships.

BACKGROUND: Y-chromosomal short tandem repeats (Y-STRs) are essential to relate male lineages in forensic and evolutionary studies. Although large panels of Y-STR markers are now available, none possess sufficient discrimination power to distinguish close male relatives. This limit may be overcome by the use of rapidly mutating Y-STRs (RM Y-STRs), characterized by mutation rates higher than common Y-STRs. Recently, multicenter studies evaluated the ability of RM Y-STRs to differentiate father-son pairs; however, more extensive data on distantly related males are needed. STUDY DESIGN AND METHOD: A total of 157 male relative pairs separated by two to seven meiotic events, originating from Italy, were analyzed by 13 RM Y-STRs and 23 Y-STRs. RESULTS: Overall, 154 mutational events were observed at RM Y-STR loci and the estimated mutation rate was of 2.59 × 10(-2) (95% confidence interval, 2.16 × 10(-2) -2.97 × 10(-2) ). A total of 105 male relative pairs showed at least one mutation in at least one locus and differentiation rates increased from 52.8% to 88.9% from the second to the fourth generation, while 23 Y-STRs provided much lower values, spanning from 10.1% to 29.6%. CONCLUSIONS: These findings confirmed the higher capability of RM Y-STRs than conventional Y-STRs to resolve male lineages, thus suggesting a possible future use for forensic male individual identification.

Kinship analysis: assessment of related vs unrelated based on defined pedigrees.

The study aimed at evaluating whether the adoption of enlarged batteries of STR markers in kinship analysis may provide LR values suitable for discrimination of relatives from non-relatives, in comparison to conventionally used STR panels. The presence of LD among some loci and its effects on LR values were also assessed. Three hundred pairs of related and unrelated individuals, each separated from 1-3 generations and residing in North Italy were genotyped with the Investigator HDplex STR kit (Qiagen), AmpFlSTR Identifiler (Applied Biosystems), and PowerPlex Fusion System (Promega). Loci and alleles shared between each pair and within groups of relatives were compared. Also, combined LR values with and without loci in LD, sensitivity and specificity were calculated for each commercial kit and their combinations. Full siblings displayed the largest number of shared loci and alleles, with a proportion of LR ≥ 10 results significantly higher than other degrees of relatedness and, consequently, with the lowest percentage of inconclusive and false negative results. Only minor differences were detected in the combined LR distributions, after including or omitting loci in LD. However, these became only appreciable when analyzing more distant relative pairs.The implementation of additional STRs into the LR calculation allowed a complete and robust discrimination between relatives and non-relatives only for full siblings, by removing the typical uncertainty of the "grey zone", while this was not achieved among other degrees of relatedness. Furthermore, the presence of loci in LD seems to not significantly affect LR distributions within each generation.

Haplotype data and mutation rates for the 23 Y-STR loci of PowerPlex® Y 23 System in a Northeast Italian population sample.

The PowerPlex® Y 23 System (Promega) is a short tandem repeat (STR) multiplex that allows co-amplification of 23 gonosomal Y-STRs, combining 17 loci commonly included in commercially available kits (DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS438, DYS437, DYS635, DYS390, DYS439, DYS392, DYS393, DYS458, DYS385a/b, DYS456, and Y-GATA-H4) and six new loci (DYS481, DYS549, DYS533, DYS643, DYS576, and DYS570) with the last two being rapidly mutating Y-STRs (RM Y-STRs). In order to assess the possible gain in forensic efficiency provided by the six additional markers, a population sample of 410 unrelated healthy males originating from Northeast Italy (Veneto, Trentino Alto Adige, Lombardia, and Friuli Venezia Giulia regions) was typed. The data (335 of the 410 samples) are available in the Y chromosome haplotype reference database under accession number YA003327. Overall, 410 unique haplotypes were found corresponding to a global haplotype diversity (HD) of 0.999994 with a discriminatory capacity (DC) of 100%. Allelic microvariants, null alleles, and duplications were detected. Pairwise genetic distances (R(ST)) calculated among neighboring European reference populations revealed no significant differences. Furthermore, for studying Y-STR mutation rates, 90 father-son pairs, in which the fathers were already included in the full dataset, were tested. On a total of 2,070 meioses considered, eight single-step mutational events were observed, two of which within the same father-son pair and the average mutation rate was 3.38 × 10(-3) per locus per generation (95% confidence interval, 1.36 × 10(-3)-6.95 × 10(-3)).

Investigator HDplex markers: allele frequencies and mutational events in a North Italian population.

Autosomal short tandem repeats (STRs) analysis represents the method of election in forensic genetics and up to now, 23 STRs are available for these purposes. However, in particular circumstances such as human identification or complex kinship cases, examination of additional STRs may be required in order to obtain reliable conclusions. For this purpose, a new multiplex STR system, namely Investigator® HDplex kit (QIAGEN) that coamplifies a set of 12 autosomal loci, 9 of which, represents novel supplementary STRs, was recently developed. A population sample of 359 unrelated healthy subjects residing in North Italy was typed to determine allele frequencies, forensic parameters and genetic distances among European populations. Furthermore, to evaluate the suitability of the HDplex kit as an auxiliary tool for paternity testing, mutation rates were estimated on 84 confirmed family trios. The 12 loci resulted highly informative with a combined power of discrimination of 0.999998 and no departures from Hardy-Weinberg equilibrium were observed with the sole exception of locus D4S2366. From the comparison of our population sample and European reference populations, a single significant difference was revealed with the Poland population at D4S2366 locus. With regard to the mutation rate study, on a total of 2,016 meioses considered, six single-step mutational events were observed and the average mutation rate calculated was of 2.94 × 10(-3) per locus per generation (95% confidence interval, 1.08 × 10(-3)-6.39 × 10(-3)).

Deletion and duplication at DYS448 and DYS626 loci: unexpected patterns within the AZFc region of the Y-chromosome.

Increasing the knowledge of multiple and microstructural events within the Y-chromosome may prove useful to better characterize abnormal short tandem repeats patterns complicating DNA profile interpretation. On the long arm of the human Y-chromosome, such structural rearrangements were observed in azoospermia factor regions (AZFa, AZFb, AZFc) that play an important role in male fertility and also host Y-STRs commonly used in forensic genetics. Here, we describe two cases, involving two males formerly included in an Italian population study, where DYS448 and DYS626 loci, located within the AZFc region, simultaneously displayed a double deletion in one case and a double duplication in the other. With the aim of better defining the size of both events, low and high-resolution mapping by means of 16 sequence-tagged sites was performed, and unexpected discontinued patterns within the palindromic segments b1/b3 of the AZFc were identified. Extending the analysis to their respective male relatives revealed unaltered transmission of the patterns along the two pedigrees. Reviewing literature data describing DYS448-DYS626 deletion and duplication suggested no close correlation between the occurrence of multiple/microstructural events and geographical origin.

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.

Evaluation of genetic parameters of 22 autosomal STR loci (PowerPlex® Fusion System) in a population sample from Northern Italy.

The PowerPlex® Fusion System (Promega, Madison, WI) is a short tandem repeat (STR) multiplex that allows co-amplification of 22 autosomal STRs, including the CODIS core and the European Standard Set loci, plus amelogenin for gender determination and DYS391 male specific marker included in order to avoid errors in gender assignment when null Y-alleles or deletions of the Y-chromosome short arm involve the amelogenin locus. Allele frequencies and forensic efficiency parameters were estimated in a population sample of 303 unrelated healthy individuals living in Northern Italy. No significant deviations from Hardy-Weinberg expectations were observed after applying Bonferroni's correction for multiple testing. The combined power of discrimination was 0.999999999999 and the combined power of exclusion was 0.9999956. A rare 28 allele at locus D12S391 was observed, while one tri-allelic pattern at Penta E locus was detected. Population differentiation test revealed significant genetic diversity between our population sample and other European populations considered. The results showed that the PowerPlex® Fusion System is one of the most informative kit available in forensic genetics and may prove useful in both human identification and kinship analysis.

The ménage à trois of healthcare: the actors in after-AI era under patient consent.

Introduction: Artificial intelligence has become an increasingly powerful technological instrument in recent years, revolutionizing many sectors, including public health. Its use in this field will inevitably change clinical practice, the patient-caregiver relationship and the concept of the diagnosis and treatment pathway, affecting the balance between the patient's right to self-determination and health, and thus leading to an evolution of the concept of informed consent. The aim was to characterize the guidelines for the use of artificial intelligence, its areas of application and the relevant legislation, to propose guiding principles for the design of optimal informed consent for its use. Materials and methods: A classic review by keywords on the main search engines was conducted. An analysis of the guidelines and regulations issued by scientific authorities and legal bodies on the use of artificial intelligence in public health was carried out. Results: The current areas of application of this technology were highlighted, divided into sectors, its impact on them, as well as a summary of current guidelines and legislation. Discussion: The ethical implications of artificial intelligence in the health care system were assessed, particularly regarding the therapeutic alliance between doctor and patient, and the balance between the right to self-determination and health. Finally, given the evolution of informed consent in relation to the use of this new technology, seven guiding principles were proposed to guarantee the right to the most informed consent or dissent.

Medical error disclosure in the Italian healthcare context: A delicate balance between ethical obligations and the principle of non-self-incrimination.

Surprisingly, Italian legal doctrine and jurisprudence never systematically address the medical error disclosure issue. The topic of medical error communication represents a non-negligible profile of interest, relating to the possible detrimental effects on doctors who accuse themselves of conduct that does not comply with the lex artis. The effects of error disclosure on the effectiveness of insurance guarantees in civil and administrative liability are particularly relevant, as are the implications for ethical liability. Although the burden of reporting an error falls within the wide range of informative duties doctors hold, it still seems far from having found a factual statement in clinical practice, especially in Italy. This applies whether the error has a marginal or significant impact on the patient's health. The reasons lay in a very contradictory legal framework. Doctors tend with increasing ease not to comply with their information obligation - especially in cases where fulfilling this duty means admitting a personal, professional error - to preserve the integrity of their professional images. This article aims to offer a brief overview of that topic in the context of Italian healthcare.

Unusual Lichtenberg figures in a lightning strike's victim: Case report and literature review.

Lichtenberg figures are pathognomonic markings commonly described as transient pink-red fractal pattern marks on the skin of people struck by a lightning. Their nature and the underlying physiopathology are still largely unknown. Here we describe a Lichtenberg figure in a 45-year-old woman who died due to a lightning strike during a thunderstorm. On autopsy, five days after the fatal event, she presented atypical green Lichtenberg figures on the skin of her abdomen. This appearance has not previously been described in the scientific literature, so a review of literature was done to support our observation.

Preliminary Study on the Possibility to Detect Virus Nucleic Acids in Post-Mortem Blood Samples.

BACKGROUND: In many forensic cases, the medical records of the deceased are not available at the time of the autopsy; therefore, no information about the deceased's state of health, including any infectious diseases contracted during life, is accessible. The detection of some of the principal viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1), could contribute to determining causes of death and interesting applications could be found in medico-legal practice, such as occupational risk assessment. To date, accurate and sensitive serological and molecular assays capable of detecting these viruses have been validated on biological samples taken from living beings, while their efficiency on forensic post-mortem biological samples has yet to be thoroughly assessed. To further this aim, this study evaluated whether the nucleic acid amplification techniques (NAATs) for the detection of viral genomes that are applied in clinical settings can be used, with the same success rate, for these latter samples. METHODS: Manual viral nucleic acid extraction processes and fully-automated amplification-based detection techniques developed in-house were evaluated on blood samples taken during the routine autopsies of 21 cadavers performed 2 to 9 days after death. Information on HBV, HCV, and HIV-1 seropositive status was previously known for only four of these cadavers. RESULTS: Using automated quantitative real-time PCR (qPCR) and qualitative PCR (end-point) analyses, it was possible to confirm the presence of viral genomes in the four post-mortem whole blood samples with previously reported specific serological positivity. In addition, the genomes of HCV and/or HIV-1 genomes were detected in three other blood samples with unknown serological status at the time of autopsy. CONCLUSIONS: Therefore, our findings suggest that molecular assays may detect the presence of viral genomes in forensic post-mortem blood samples up to five days after death. This provides an additional means of investigation that can contribute to the determination of the deceased's cause of death.

Resizing reaction volumes for the ForenSeq™ DNA Signature Prep kit library preparation.

The introduction of next generation sequencing (NGS; also known as massively parallel sequencing) technology in the field of forensic genetics has been welcomed by the scientific community, above all because it complements the weaknesses of capillary electrophoresis (CE) in the analysis of genetic markers, such as single nucleotide polymorphism (SNP) typing. However, one of the main obstacles to its adoption does not seem to be the cost of the instrumentation, but rather the cost of the NGS library preparation kits. With the aim of reducing the cost of library preparation without compromising the quality of the results, we tried to scale down reaction volumes for the first two polymerase chain reactions in the amplification and enrichment phases of the targeted loci of library preparation using the ForenSeq™ DNA Signature Prep kit. We used 1 µL templated DNA input to a concentration of 1 ng/µL, instead of the 5 µL at 0.2 ng/µL recommended by the manufacturer. Our findings indicate that reduction of the library preparation volume using the ForenSeq™ DNA Signature Prep kit did not interfere with the quality and reproducibility of the DNA profiles obtained and can help lower the overall cost of NGS.

The Increasing Centrality of Robotic Technology in the Context of Nursing Care: Bioethical Implications Analyzed through a Scoping Review Approach.

At the dawn of the fourth industrial revolution, the healthcare industry is experiencing a momentous shift in the direction of increasingly pervasive technologization of care. If, up until the 2000s, imagining healthcare provided by robots was a purely futuristic fantasy, today, such a scenario is in fact a concrete reality, especially in some countries, such as Japan, where nursing care is largely delivered by assistive and social robots in both public and private healthcare settings, as well as in home care. This revolution in the context of care, already underway in many countries and destined to take place soon on a global scale, raises obvious ethical issues, related primarily to the progressive dehumanization of healthcare, a process which, moreover, has undergone an important acceleration following the outbreak of the COVID-19 pandemic, which has made it necessary to devise new systems to deliver healthcare services while minimizing interhuman contact. According to leading industry experts, nurses will be the primary users of healthcare robots in the short term. The aim of this study is to provide a general overview, through a scoping review approach, of the most relevant ethical issues that have emerged in the nursing care field in relation to the increasingly decisive role that service robots play in the provision of care. Specifically, through the adoption of the population-concept-context framework, we formulated this broad question: what are the most relevant ethical issues directly impacting clinical practice that arise in nursing care delivered by assistive and social robots? We conducted the review according to the five-step methodology outlined by Arksey and O'Malley. The first two steps, formulating the main research question and carrying out the literature search, were performed based on the population-context-concept (PCC) framework suggested by the Joanna Briggs Institute. Starting from an initial quota of 2,328 scientific papers, we performed an initial screening through a computer system by eliminating duplicated and non-English language articles. The next step consisted of selection based on a reading of the titles and abstracts, adopting four precise exclusion criteria: articles related to a nonnursing environment, articles dealing with bioethical aspects in a marginal way, articles related to technological devices other than robots, and articles that did not treat the dynamics of human-robot relationships in depth. Of the 2,328 titles and abstracts screened, we included 14. The results of the 14 papers revealed the existence of nonnegligible difficulties in the integration of robotic systems within nursing, leading to a lively search for new theoretical ethical frameworks, in which robots can find a place; concurrent with this exploration are the frantic attempts to identify the best ethical design system applicable to robots who work alongside nurses in hospital wards. In the final part of the paper, we also proposed considerations about the Italian nursing context and the legal implications of nursing care provided by robots in light of the Italian legislative panorama. Regarding future perspectives, this paper offers insights regarding robot engagement strategies within nursing.