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Violence against women is a violation of human rights, crossing all cultures, classes, levels of education, earnings, ethnic and age groups. We conducted a retrospective study to review forensic records of sexual assault examinations carried out in different Italian health facilities and to correlate these findings with the results of the forensic DNA analyses. The goal was to determine which factors could have affected the obtained results, to identify the fundamental aspects to search for while examining a sexual assault victim in order to gather useful evidence to identify the offender and reconstruct the dynamics of the fact. We analysed 102 cases that occurred between 2006 and 2017, coming from ten participating laboratories. Despite a relatively limited number of cases, this study shows that the ability to ascertain the presence of male biological material in the samples collected is not a problem for forensic laboratories and seems to be influenced by other factors, such as how much time elapsed between the event and the sampling, the availability of the aggressor's biological material on the victim and the identification of biological fluids/stains. Therefore, the need for health structures to adopt specific protocols has been highlighted. It is necessary for health structures to define specific pathways and adopt homogeneous procedures or operational protocols, and it is essential to provide adequate training for health personnel. The results of the study could be useful in drafting and revising protocols/guidelines implemented in Italian hospital. Issues related to the limited number of analyses requested by Italian Authorities are also discussed., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Storage conditions influence the integrity of the recoverable DNA from forensic evidence in terms of yield and quality. FTA cards are widely used in the forensic practice as their chemically treated matrix provides protection from the moment of collection to the point of analysis with current STR typing technology. In this study, we assess the recoverability and the integrity of DNA from 11-year-old saliva on FTA cards using a forensic quantitative real-time polymerase chain reaction (qPCR) commercial assay. The quality after long-term storage was investigated in order to evaluate if the FTA device could assure enough stability over time, applying some internally validated quality criteria of the STR profile. Furthermore, we used a 3D interpolation model to combine the quantitative and qualitative data from qPCR to calculate the minimum optimal DNA input (MODI) to add to the downstream PCR reaction based on the quantitative and qualitative data of a sample. According to our results, when saliva sample is properly transferred onto FTA cards and then correctly stored according to the manufacturer's instructions, it is possible to recover sufficient amounts of DNA for human identification even after more than a decade of storage at ambient temperature. Degradation affected the quality of results especially when the Degradation Index exceeds the value of 2.12, requiring modifications of the standard internal workflow to improve the genotyping quality. Above this value, the application of a "corrective factor" to the PCR normalization process was necessary in order to adjust the recommended manufacturer's PCR DNA input taking into account the degradation level. Our results demonstrated the importance to consider in predictive terms the parameters obtained with the real-time quantification assay, both in terms of quantity (DNA concentration) and of quality (DI, inhibition). Informatics predictive tools including qPCR data together with the variables of storage duration and conditions should be developed in order to optimize the DNA analysis process.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microsatellites or short tandem repeats (STRs) markers are important tools for mapping disease-causing genes by linkage, for performing investigations in forensic medicine, for population genetic studies and for studying genetic modifications in tumors. In forensic applications neoplastic tissues can be used as a source of genetic information for personal identification or paternity testing when no other specimen is available. Cancer tissues can show microsatellite instability (MSI) and loss of heterozygosity (LOH) also for the STRs used in the forensic field. In this study, we screened 56 sporadic gastrointestinal carcinomas in order to provide further data for the evaluation of the incidence of allelic alterations for 15 STR loci and the suitability of using cancerous tissues in forensic applications. Sixty-six percent of the cancerous tissues were found to possess allelic alterations of the microsatellites analyzed with a high incidence of MSI-L (microsatellite instability low) when compared to the corresponding normal tissue. The most frequently altered loci were D18S51, VWA, and FGA. From a forensic perspective, great care must be taken in evaluating the DNA typing results obtained from cancerous tissue samples.