Forensic Sciences

Background/Objectives: Personal identification is a cornerstone of Forensic Medicine. Historical records indicated that 27 Italian soldiers were executed by Tito’s army in April 1945 and buried in the mass grave of Ossero (Cres Island, Croatia). Methods: The remains, exhumed in 2019 by Croatian authorities and transferred to the Sacrario d’Oltremare (Bari, Italy), underwent radiographic and anthropological analyses. Genetic analysis was later performed on 147 bone samples, leading to the identification of 10 soldiers. Results: Anthropological analyses revealed commingled remains of at least 34 individuals (15–45 years; 161–181 cm), including eight skeletal elements of female sex. Forensic examination confirmed cranial and post-cranial gunshot wounds. Conclusions: Extensive commingling and fragmentation of the remains prevented full reconstruction of individual skeletons, yet comparison with ante-mortem data supported their identification as the aforementioned soldiers. Unexpected findings, including historically undocumented females, were confirmed by DNA analysis. These findings underscore the value of a multidisciplinary approach to optimize recovery and subsequent forensic and genetic investigations.

Background/Objectives: DNA profiling in forensic investigation typically compares genetic profiles, usually derived from the analysis of STR markers. However, this method has limitations when there is no biological reference sample or match in the DNA database. The aim of the current study is thus to replicate, in an Italian cohort, epigenetic markers previously identified in the literature for distinguishing tobacco smokers from non-smokers or estimating chronological age, so as to help narrow down the pool of suspects. Methods: DNA methylation at four CpG dinucleotides located around the cg05575921 site of the AHRR gene, widely associated with tobacco consumption, was measured. Additionally, five CpG dinucleotides in the ELOVL2, FHL2, KLF14, TRIM59, and C1orf132 genes were examined for chronological age estimation in buccal swab samples of 102 volunteers through pyrosequencing. Results: A multiple linear regression model for estimating chronological age shows that ELOVL2-C7, C1orf132-C1, and TRIM59-C7 have a significant effect on age. In this model, the prediction error increases with age. Two logistic regression models were used for determining smoker/non-smoker status, proving that two CpG sites significantly influence the odds of being classified as a smoker. When ex-smokers are included in the non-smoking group, the model correctly classifies the two conditions in about 80% of cases. Conclusions: The results demonstrate that the models generated from pyrosequencing data are useful for identifying tobacco smokers and estimating an individual’s chronological age, particularly for younger subjects. Further studies are needed to develop models with higher predictive accuracy and to integrate these tools into regular forensic practice.

Background/Objectives: Rapid DNA systems accelerate STR profiling but often require the consumption of the entire swab, limiting confirmation testing or downstream analyses. We previously validated a simple subsampling protocol for blood swabs on the RapidHIT^TM ID, using a rigid subungual mini-swab (Copan Italia S.p.A). A new version of this instrument has recently been released, featuring redesigned software and consumables. The RapidINTEL^TM Plus sample cartridge now enables two distinct lysis/extraction protocols, expanding analytical possibilities for rich biological traces. We evaluated subsampling performance using the subungual mini-swab and microFLOQ^® swabs (Copan Italia S.p.A), and assessed feasibility for both blood and buccal reference swabs. Methods: Whole blood from four donors was deposited onto regular Copan swabs (10 µL) or microFLOQ^® swabs (1 µL). A comparison was performed between the direct analysis of blood swabs using a RapidHIT^TM ID V1 (RapidINTEL^TM cartridge) and a RapidHIT^TM ID V2 (RapidINTEL^TM Plus cartridge, GENERAL protocol). Subsequently, both the GENERAL and SPECIALIZED protocols were tested after subsampling from primary blood or buccal swabs dried for 24 h using either a subungual mini-swab or a microFLOQ^®. Results: Blood-swab subsampling on the V2 produced usable STR profiles with both the subungual mini-swab and the microFLOQ^®. The subungual mini-swab was compatible with both the GENERAL and SPECIALIZED protocols. For blood applications, microFLOQ^® fiber treatment showed no inhibitory effects. Reference buccal swabs were successfully analyzed with the RapidINTEL^TM Plus cartridge, either directly (regular swab) or via subungual subsampling under both protocols. In contrast, in this feasibility dataset (single analysis per donor per condition), subsampling a reference swab with microFLOQ^® did not yield suitable profiles for RapidINTEL^TM Plus analysis under the tested conditions. Conclusions: This feasibility study indicates that the subsampling strategy can be applied on the RapidHIT^TM ID V2, particularly using subungual mini-swabs, to retain the primary swab for potential downstream testing while maintaining usable STR profile quality for blood and buccal reference workflows under the tested conditions.