Analysis of cannabinoids and metabolites in dried urine spots (Dus)

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxico-logical analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabi-noids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites

Assessment of the Precision ID Identity Panel kit on challenging forensic samples

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the \u201csame donor\u201d or from a \u201cfirst degree relative\u201d was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine\u2122 (PGM\u2122) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50% and 35% of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23% when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37% of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 x 10-13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping

Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language

Clinal patterns of autosomal genetic diversity within Europe have been interpreted in previous studies in terms of a Neolithic demic diffusion model for the spread of agriculture; in contrast, studies using mtDNA have traced many founding lineages to the Paleolithic and have not shown strongly clinal variation. We have used 11 human Y-chromosomal biallelic polymorphisms, defining 10 haplogroups, to analyze a sample of 3,616 Y chromosomes belonging to 47 European and circum-European populations. Patterns of geographic differentiation are highly nonrandom, and, when they are assessed using spatial autocorrelation analysis, they show significant dines for five of six haplogroups analyzed. Clines for two haplogroups, representing 45% of the chromosomes, are continentwide and consistent with the demic diffusion hypothesis. Clines for three other haplogroups each have different foci and are more regionally restricted and are likely to reflect distinct population movements, including one from north of the Black Sea. principal-components analysis suggests that populations are related primarily on the basis of geography, rather than on the basis of linguistic affinity. This is confirmed in Mantel tests, which show a strong and highly significant partial correlation between genetics and geography but a low nonsignificant partial correlation between genetics and language. Genetic-barrier analysis also indicates the primacy of geography in the shaping of patterns of variation. These patterns retain a strong signal of expansion from the Near East but also suggest that the demographic history of Europe has been complex and influenced by other major population movements, as well as by linguistic and geographic heterogeneities and the effects of drift

Quantification of human DNA by Real Time PCR in forensic casework.

We extensively employed a real-time quantitative PCR system, together with a commercially available kit (Quantifilerk Human DNA Quantification Kit, Applied Biosystems), for the quantification of human DNA in a large variety of samples. The results we obtained were reliable, with a low deviation standard for the same sample; however, when the inhibitors in a sample were in high concentration and/or DNA degradation was present (like in postmortem matrices), we observed variable results of quantification in the same extract. Here, we report our experience using this real-time based method for quantification and typing of samples recovered in forensic casework

Quantification of human DNA by Real Time PCR in forensic casework

We extensively employed a real-time quantitative PCR system, together with a commercially available kit (Quantifilerk Human DNA Quantification Kit, Applied Biosystems), for the quantification of human DNA in a large variety of samples. The results we obtained were reliable, with a low deviation standard for the same sample; however, when the inhibitors in a sample were in high concentration and/or DNA degradation was present (like in postmortem matrices), we observed variable results of quantification in the same extract. Here, we report our experience using this real-time based method for quantification and typing of samples recovered in forensic casework

Y-chromosomal STR haplotypes in an Albanian population sample

Eight Y-chromosomal short tandem repeats (STRs), DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385, were typed in a population sample (n=101) of first-generation Albanian immigrants living in Italy

Paternal origin of the X chromosome in a 63,X Italian Trotter mare

In this work, we report information about a new case of a 63,X chromosome constitution in a mare and we established, for the first time in this specie, the parental origin of the only X chromosome present.The cytogenetic analysis of 100 metaphases, using both Giemsa staining, CBG banding and FISH technique, shown a 2n=63,X condition with no sign of mosaicism, while paternal origin of the only X chromosome present was ascertained analysing two high informative microsatellites: LEX024 and LEX003