The human intervertebral disc as a source of DNA for molecular identification.

Genetic analyses such as STR-typing are routinely used for identification purposes in forensic casework. Although genotyping techniques only require a minimum amount of DNA to provide a genetic profile, DNA quality differs not only between but also within tissues during ongoing decomposition. Initiated by a recent case where, due to the constitution of the body, preferred tissue was not available or only resulted in a partial and not usable DNA profile, the analysis of intervertebral discs as a source of DNA was considered. As the analysis of this tissue resulted in a high quality DNA profile a further study was performed in which thirty intervertebral discs dissected from bodies in different stages of decay were analyzed. All samples yielded good quality DNA in quantities suitable for STR-based amplification with no or only low degradation indices, resulting in complete genetic profiles. These results demonstrate the robustness of human intervertebral disc tissue as a source of DNA for molecular identification purposes.

Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Quantitative low-volume assay for simultaneous determination of fentanyl, norfentanyl, and minor metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A rapid and sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for simultaneous quantification of fentanyl (F), norfentanyl (NF), despropionylfentanyl (DPF), and hydroxynorfentanyl (OHNF) in human plasma and urine specimens has been developed and validated according to international guidelines. Analytes were extracted from 250-µL plasma or urine by liquid-liquid extraction. OHNF in urine affords a second extraction step and analysis with a different column. Calibration curves in plasma were linear from 0.05-10 ng/mL for F, 0.07-0.5 ng/mL for NF, 0.02-1.0 ng/ml for DPF, and 0.67-3.0 ng/mL for OHNF; in urine, from 0.09-10.0, 0.17-50, 0.08-1.0, and 1.0-5.0 ng/mL for F, NF, DPF, and OHNF, respectively. Analytical bias and intra- and inter-assay imprecision were within ± 15 % of target, except for OHNF in plasma and DPF in urine at the respective lower quality control level. All analytes were stable in processed samples when stored for 24 h at room temperature. Recoveries and process efficiencies were above 82.9 and 75.1 % for all analytes in plasma and urine. The low level of DPF in plasma indicated with a matrix effect of 71.3 % moderate ion suppression, all other analytes in plasma and urine showed no matrix effects. The lower limit of quantification (LOQ) in plasma was 0.05, 0.07, 0.02 and 0.67 ng/mL for F, NF, DPF, and OHNF, respectively. In urine, the LOQ of F, NF, DPF, and OHNF were 0.09, 0.17, 0.08, and 1.28 ng/mL, respectively. This assay has been applied to human specimens collected during a clinical drug-drug interaction study.