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.

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.

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.

Optimization and validation of a new approach based on CE-HRMS for the screening analysis of novel psychoactive substances (cathinones, phenethylamines, and tryptamines) in urine.

The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75 µm with a total length of 100 cm, by applying a constant voltage of 15 kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.

The prognostic value of muscle StO2 in septic patients.

OBJECTIVE: To quantify sepsis-induced alterations in changes in muscle tissue oxygenation (StO(2)) after an ischemic challenge using near-infrared spectroscopy (NIRS), and to test the hypothesis that these alterations are related to outcome. DESIGN: Prospective study. SETTING: Thirty-one-bed, university hospital Department of Intensive Care. PATIENTS: Seventy-two patients with severe sepsis or septic shock, 18 hemodynamically stable, acutely ill patients without infection, and 18 healthy volunteers. INTERVENTIONS: Three-minute occlusion of the brachial artery using a cuff inflated 50[Symbol: see text]mmHg above systolic arterial pressure. MEASUREMENTS AND MAIN RESULTS: Thenar eminence StO(2) was measured continuously by NIRS before (StO(2)baseline), during, and after the 3-min occlusion. Changes in StO(2) were assessed by the slope of increase in StO(2) during the first 14 s following the ischemic period and by the difference between the maximum StO(2) and StO(2)baseline (Delta). The slope was lower in septic patients than in controls and volunteers [2.3 (1.3-3.6), 4.8 (3.5-6.0), and 4.7 (3.2-6.3) %/s, p < 0.001]. Delta was also significantly lower in septic patients than in the other groups. Slopes were lower in septic patients with than without shock [2.0 (1.2-2.9) vs 3.2 (1.8-4.5) %/s, p < 0.05]. In 52 septic patients, in whom the slope was obtained every 24 h for 48 h, slopes were higher in survivors than in non-survivors and tended to increase in survivors but not in non-survivors. CONCLUSIONS: Altered recovery in StO(2) after an ischemic challenge is frequent in septic patients and more pronounced in the presence of shock. The presence and persistence of these alterations in the first 24[Symbol: see text]h of sepsis are associated with worse outcome.