Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial

A qualitative and quantitative analytical method was developed and validated for the determination of 49 licit and illicit drugs in oral fluid. Small oral fluid samples, volume 1mL, were collected from volunteers using a modified Omni-Sal device and the analytes were extracted from an oral fluid/buffer mixture using a single Bond Elut Certify solid phase extraction cartridge. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) and gas chromatography-repetitive full scan mass spectrometry (GC-MS) were used in parallel to analyze the extracts for the targeted drugs. Extracts were analyzed by GC-MS in their underivatized form and as their pentafluoropropionyl derivatives. Deuterated internal standards were used for quantification of drugs of abuse by LC-MS-MS to minimize matrix effects. Methadone-d(9) and tumoxetine were used as the internal standards for quantification of non-derivatized and derivatized analytes respectively by GC-MS. Linearity was demonstrated over the range 5-200 ng/mL and limits of detection were less than 4 ng/mL for each drug analyzed. The method demonstrated acceptable recoveries for most of the analytes and good intra- and inter-day precision. Acquisition of data by repetitive full scan GC-MS allows the addition of further analytes to the target menu.     

Study of cathepsin A, B and D activities in the skin wound edges. Its application to the differential diagnosis between vital and postmortem wounds

The aims of the authors in this paper has been to check the diagnostic ability of the Cathepsin A, B and D concentrations in the skin wound edges to the differential diagnosis between vital and postmortem wounds. We have studied 56 domestic pigs grouped in seven experimental series consisting of 8 animals in each, according to the time (0, 5, 15, 30 min and 1, 3 and 6 h) after the injury. The enzymatic activities were investigated following the methods by Bowen and Davison Biochem. J., 131 (1973) 417-419, Suhar and Marks J. Biochem., 101 (1979) 23-30 and by Anson (modified by Yamamoto, Eur. J. Biochem., 95 (1979) 459-467) for Cathepsin A, B and D, respectively. For the differential diagnosis between vital and postmortem wounds, our results showed that the most useful markers studied are the Cathepsin A and D activities, Cathepsin D that of the first one.     

Intentional mixed buccal cell reference sample in a paternity case

We report a case where an alleged father (AF) attempted to substitute someone else's saliva sample for his reference sample in a paternity analysis. Buccal cells were collected from the AF and the child, and DNA analysis was performed using an autosomal STR loci (Identifiler). The profile from the AF showed extra peaks in some loci, as well as a much higher "X" allele peak relative to the "Y" allele peak at the amelogenin locus. After conducting reanalysis by another technician with another set of positive and negative controls, it was concluded that the only source of the mixed profile was by intentional introduction by the AF, at the time of sampling, of some foreign human biological material, most likely saliva from a woman. Owing to the inconclusive results, when the AF was called back to the lab and the peculiar results were explained to him, he admitted that he had introduced into his mouth saliva from another person in an attempt to be excluded as the father of the child. Although tampering with DNA reference samples is not common, some individuals may attempt to contaminate or otherwise adulterate specimens before DNA tests. Personnel responsible for sampling should be aware of this possibility and should try to establish procedures to avoid the problem.     

Cathepsin D: a new marker of the vitality of the wound. Comparative study with histamine and serotonin

We have studied the variation of specific activity of the most important lysosomal enzyme: Cathepsin D. This study has been developed in pig skin incision wounds for its application to the differential diagnosis between vital and postmortem wounds. At the same time, we have determined histamine and serotonine levels to make a comparative study with Cathepsin results. Our results show the utility of this new marker to determine the vitality of wounds, even when the time elapsed until death has been very short. Nevertheless, there is no correlation between Cathepsin D results and those of histamine or serotonin, a fact that induces us to think that this new marker is not useful to determine wound age.     

Postmortem stability of lung surfactant phospholipids.

The postmortem stability of the main phospholipids of lung surfactant-phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl serine (PS) and sphingomyelin (S) in three different deaths; one caused by fresh-water drowning, one by salt-water drowning, and one from a sodium-pentobarbital overdose has been studied. The drug overdose was considered the control because there was no surfactant involvement. The results show the stability of these kinds of lipids in the first 24 h, with a progressive decrease from 48 h on until 96 h, with a significant correlation to the time of P less than 0.01 in most cases.