Body fluid and tissue analysis using filter paper sampling support prior to LC-MS/MS: application to fatal overdose with colchicine

Because of the various matrices available for forensic investigations, the development of versatile analytical approaches allowing the simultaneous determination of drugs is challenging. The aim of this work was to assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform allowing the rapid quantification of colchicine in body fluids and tissues collected in the context of a fatal overdose. For this purpose, filter paper was used as a sampling support and was associated with an automated 96-well plate extraction performed by the LC autosampler itself. The developed method features a 7-min total run time including automated filter paper extraction (2 min) and chromatographic separation (5 min). The sample preparation was reduced to a minimum regardless of the matrix analyzed. This platform was fully validated for dried blood spots (DBS) in the toxic concentration range of colchicine. The DBS calibration curve was applied successfully to quantification in all other matrices (body fluids and tissues) except for bile, where an excessive matrix effect was found. The distribution of colchicine for a fatal overdose case was reported as follows: peripheral blood, 29 ng/ml; urine, 94 ng/ml; vitreous humour and cerebrospinal fluid, < 5 ng/ml; pericardial fluid, 14 ng/ml; brain, < 5 pg/mg; heart, 121 pg/mg; kidney, 245 pg/mg; and liver, 143 pg/mg. Although filter paper is usually employed for DBS, we report here the extension of this alternative sampling support to the analysis of other body fluids and tissues. The developed platform represents a rapid and versatile approach for drug determination in multiple forensic media.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Simultaneous detection of aneuploidies for chromosomes 4, 6, 10 and 17 by automated four colour I-FISH in high hyperdiploid acute lymphoblastic leukemia : diagnostic assessment, clonal heterogeneity and chromosomal instability in adults

SummarySimultaneous detection of aneuploidies for chromosomes 4, 6,10 and 17 by automated four color l-FISH in high hyperdiploid acute lymphoblastic leukemia: diagnostic assessment, clonal heterogeneity and chromosomal instability in adultsAnna Talamo BlandinService de Génétique Médicale, Unité de Cytogénétique du Cancer, CHUVAcute lymphoblastic leukemia (ALL) is a malignant hemopathy characterized by the accumulation of the immature lymphoid cells in the bone marrow and, most often, in the peripheral blood. ALL is a heterogeneous disease with distinct biological and prognostic entities. At diagnosis, cytogenetic and molecular findings constitute important and independent prognostic factors. High hyperdiploidy with 51-67 chromosomes (HeH), one of the largest cytogenetic subsets of ALL, in childhood particularly, is generally associated with a relatively favorable outcome. Chromosome gain is nonrandom, extracopies of some chromosome occurring more frequently than those of others. Concurrent presence of trisomy for chromosomes 4, 10 and 17 confers an especially good prognosis. The first aim of our work was to develop an automated four color interphase fluorescence in situ hybridization (l-FISH) methodology and to assess its ability to detect concurrent aneuploidies 4, 6, 10 and 17 in 10 ALL patients. Various combinations of aneuploidies were identified. All clones detected by conventional cytogenetics were also observed by l-FISH. However, in all patients, l-FISH revealed numerous additional abnormal clones, leading to a high level of clonal heterogeneity. Our second aim has been to investigate the nature and origin of this clonal heterogeneity and to test for the presence of chromosome instability (CIN) in HeH ALL at initial presentation. Ten HeH ALL and 10 non-HeH ALL patients were analysed by four colour l-FISH and numerical CIN values were determined for all four chromosomes together and for each chromosome and patient group, an original approach in ALL. CIN values in HeH ALL proved to be much higher than#iose in non-HeH ALL, suggesting that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by l-FISH. Our third aim has been to study the evolution of these cytogenetic features during the course of the disease in 10 HeH ALL patients. Clonal heterogeneity was also observed again during disease progression, particularly at relapse. Clones detected at initial presentation generally reappeared in relapse, in most cases with newly generated ones. A significant correlation between the number of abnormal clones and CIN suggested that the higher the instability, the larger the number of abnormal clones. Whereas clonal heterogeneity and its evolution most probably result from underlying chromosome instability, operating processes remain conjectural.RésuméLa leucémie lymphoblastique aiguë (LLA) est une hémopathie maligne qui résulte de l'accumulationde cellules lymphoïdes immatures dans la moelle osseuse, et, le plus souvent, dans le sangpériphérique également. La LLA est une affection hétérogène au sein de laquelle se distinguentplusieurs entités biologiques et pronostiques. Les données cytogénétiques et moléculaires font partieintégrante du diagnostic et jouent un rôle essentiel dans l'évaluation du pronostic. L'hyperdiploïdieélevée à 51-­67 chromosomes (HeH), relativement fréquente, en particulier chez l'enfant, s'associe àun pronostic favorable. Le gain de chromosomes ne relève pas du hasard, certains chromosomesétant plus fréquemment impliqués que d'autres. La présence simultanée des trisomies 4, 6, et 17s'associe à un pronostic particulièrement bon. Le premier but du travail a été de développer uneméthode d'analyse automatique par hybridation in situ fluorescente interphasique (I-­FISH) à 4couleurs et de tester sa capacité à identifier la présence simultanée d'aneuploïdies 4, 6, 10 et 17 dans10 cas de LLA. Différentes combinaisons d'aneuploïdies ont été identifiées. Tous les clones détectéspar cytogénétique conventionnelle l'ont été par I-­FISH. Or, chez tous les patients, l'I-­FISH a révélé denombreux clones anormaux additionnels générant un degré élevé d'hétérogénéité clonale. Notredeuxième but a été d'investiguer la nature et l'origine de cette hétérogénéité et de tester la présenced'instabilité chromosomique (CIN) chez les patients avec une LLA HeH en presentation initiale. DixLLA HeH et 10 LLA non-­HeH ont été analysées par I-­FISH et les valeurs de CIN numérique ont étédéterminées pour les 4 chromosomes ensemble et pour chaque chromosome et groupe de patients,approche originale dans la LLA. Ces valeurs étant beaucoup plus élevées dans la LLA HeH que dansla LLA non-­HeH, elles favorisent l'hypothèse selon laquelle la CIN serait à l'origine de l'hétérogénéitéclonale révélée par I-­FISH. Le troisième but de notre travail a été d'étudier l'évolution de cescaractéristiques cytogénétiques au cours de la maladie dans 10 cas de LLA HeH. L'hétérogénéitéclonale a été retrouvée lors de la progression de la maladie, en particulier en rechute, où les clonesanormaux détectés en présentation initiale réapparaissent, généralement accompagnés de clonesnouveaux. La corrélation existant entre nombre de clones anormaux et valeurs de CIN suggère queplus l'instabilité est élevée, plus le nombre de clones anormaux est grand. Bien que l'hétérogénéitéclonale et son évolution résultent très probablement de l'instabilité chromosomique, les processus àl'oeuvre ne sont pas entièrement élucidés.

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish.

Insights into low fish mercury bioaccumulation in a mercury-contaminated reservoir, Guizhou, China.

We examined Hg biogeochemistry in Baihua Reservoir, a system affected by industrial wastewater containing mercury (Hg). As expected, we found high levels of total Hg (THg, 664-7421 ng g(-1)) and monomethylmercury (MMHg, 3-21 ng g(-1)) in the surface sediments (0-10 cm). In the water column, both THg and MMHg showed strong vertical variations with higher concentrations in the anoxic layer (>4m) than in the oxic layer (0-4 m), which was most pronounced for the dissolved MMHg (p < 0.001). However, mercury levels in biota samples (mostly cyprinid fish) were one order of magnitude lower than common regulatory values (i.e. 0.3-0.5 mg kg(-1)) for human consumption. We identified three main reasons to explain the low fish Hg bioaccumulation: disconnection of the aquatic food web from the high MMHg zone, simple food web structures, and biodilution effect at the base of the food chain in this eutrophic reservoir.

Fish populations surviving estrogen pollution.

Among the most common pollutants that enter the environment after passing municipal wastewater treatment are estrogens, especially the synthetic 17α-ethinylestradiol that is used in oral contraceptives. Estrogens are potent endocrine disruptors at concentrations frequently observed in surface waters. However, new genetic analyses suggest that some fish populations can be self-sustaining even in heavily polluted waters. We now need to understand the basis of this tolerance.

Which is the optimal sampling strategy for habitat suitability modelling

Designing an efficient sampling strategy is of crucial importance for habitat suitability modelling. This paper compares four such strategies, namely, 'random', 'regular', 'proportional-stratified' and 'equal -stratified'- to investigate (1) how they affect prediction accuracy and (2) how sensitive they are to sample size. In order to compare them, a virtual species approach (Ecol. Model. 145 (2001) 111) in a real landscape, based on reliable data, was chosen. The distribution of the virtual species was sampled 300 times using each of the four strategies in four sample sizes. The sampled data were then fed into a GLM to make two types of prediction: (1) habitat suitability and (2) presence/ absence. Comparing the predictions to the known distribution of the virtual species allows model accuracy to be assessed. Habitat suitability predictions were assessed by Pearson's correlation coefficient and presence/absence predictions by Cohen's K agreement coefficient. The results show the 'regular' and 'equal-stratified' sampling strategies to be the most accurate and most robust. We propose the following characteristics to improve sample design: (1) increase sample size, (2) prefer systematic to random sampling and (3) include environmental information in the design'

Fish oil for prevention of sudden death in hemodialysis patients?

Friedman et al. report that hemodialysis patients with the highest levels of n-3 fatty acids had impressively low odds of sudden cardiac death. The study is limited by a small sample size, and the analysis relies on only a single baseline measurement of blood levels. Recent randomized evidence indeed fails to support that n-3 fatty acids may prevent sudden death in nonrenal patients. More evidence is needed to advocate fish oil in this setting.

Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy men.

High-fructose diet stimulates hepatic de novo lipogenesis (DNL) and causes hypertriglyceridemia and insulin resistance in rodents. Fructose-induced insulin resistance may be secondary to alterations of lipid metabolism. In contrast, fish oil supplementation decreases triglycerides and may improve insulin resistance. Therefore, we studied the effect of high-fructose diet and fish oil on DNL and VLDL triglycerides and their impact on insulin resistance. Seven normal men were studied on four occasions: after fish oil (7.2 g/day) for 28 days; a 6-day high-fructose diet (corresponding to an extra 25% of total calories); fish oil plus high-fructose diet; and control conditions. Following each condition, fasting fractional DNL and endogenous glucose production (EGP) were evaluated using [1-13C]sodium acetate and 6,6-2H2 glucose and a two-step hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity. High-fructose diet significantly increased fasting glycemia (7 +/- 2%), triglycerides (79 +/- 22%), fractional DNL (sixfold), and EGP (14 +/- 3%, all P < 0.05). It also impaired insulin-induced suppression of adipose tissue lipolysis and EGP (P < 0.05) but had no effect on whole- body insulin-mediated glucose disposal. Fish oil significantly decreased triglycerides (37%, P < 0.05) after high-fructose diet compared with high-fructose diet without fish oil and tended to reduce DNL but had no other significant effect. In conclusion, high-fructose diet induced dyslipidemia and hepatic and adipose tissue insulin resistance. Fish oil reversed dyslipidemia but not insulin resistance.

The weaker points of fish acute toxicity tests and how tests on embryos can solve some issues.

Fish acute toxicity tests play an important role in environmental risk assessment and hazard classification because they allow for first estimates of the relative toxicity of various chemicals in various species. However, such tests need to be carefully interpreted. Here we shortly summarize the main issues which are linked to the genetics and the condition of the test animals, the standardized test situations, the uncertainty about whether a given test species can be seen as representative to a given fish fauna, the often missing knowledge about possible interaction effects, especially with micropathogens, and statistical problems like small sample sizes and, in some cases, pseudoreplication. We suggest that multi-factorial embryo tests on ecologically relevant species solve many of these issues, and we shortly explain how such tests could be done to avoid the weaker points of fish acute toxicity tests.

Effects of fish oil on the neuro-endocrine responses to an endotoxin challenge in healthy volunteers

Résumé Introduction et hypothèse : Certains acides gras polyinsaturés de type n-3 PUFA, qui sont contenus dans l'huile de poisson, exercent des effets non-énergétiques (fluidité des membranes cellulaires, métabolisme énergétique et prostanoïdes, régulation génique de la réponse inflammatoire). Les mécanismes de la modulation de cette dernière sont encore mal connus. L'administration d'endotoxine (LPS) induit chez les volontaires sains une affection inflammatoire aiguë, comparable à un état grippal, associé à des modifications métaboliques et inflammatoires transitoires, similaires au sepsis. Ce modèle est utilisé de longue date pour l'investigation clinique expérimentale. Cette étude examine les effets d'une supplémentation orale d'huile de poisson sur la réponse inflammatoire (systémique et endocrinienne) de sujets sains soumis à une injection d'endotoxine. L'hypothèse était que la supplémentation d'huile de poisson réduirait les réponses physiologiques à l'endotoxine. Méthodes : Quinze volontaires masculins (âge 26.0±3.1 ans) ont participé à une étude randomisée, contrôlée. Les sujets sont désignés au hasard à recevoir ou non une supplémentation orale : 7.2 g d'huile de poisson par jour correspondant à un apport de 1.1 g/jour d'acides gras 20:5 (n-3, acide écosapentaénoïque) et 0.7 g/jour de 22:6 (n-3, acide docosahexaénoïque). Chaque sujet est investigué deux fois dans des conditions identiques : une fois il reçoit une injection de 2 ng par kg poids corporel de LPS intraveineuse, l'autre fois une injection de placebo. Les variables suivantes sont relevées avant l'intervention et durant les 360 min qui suivent l'injection :signes vitaux, dépense énergétique (EE) et utilisation nette des substrats (calorimétrie indirecte, cinétique du glucose (isotopes stables), taux plasmatique des triglycérides et FFA, du glucose, ainsi que des cytokines et hormones de stress (ACTH, cortisol, Adré, Nor-Adré). Analyses et statistiques :moyennes, déviations standards, analyse de variance (one way, test de Scheffé), différences significatives entre les groupes pour une valeur de p < 0.05. Résultats :L'injection de LPS provoque une augmentation de la température, de la fréquence cardiaque, de la dépense d'énergie et de l'oxydation nette des lipides. On observe une élévation des taux plasmatiques de TNF-a et IL-6, de la glycémie, ainsi qu'une élévation transitoire des concentrations plasmatiques des hormones de stress ACTH, cortisol, adrénaline et noradrénaline. L'huile de poisson atténue significativement la fièvre, la réponse neuro-endocrinienne (ACTH et cortisol) et sympathique (baisse de la noradrénaline plasmatique). Par contre, les taux des cytokines ne sont pas influencés par la supplémentation d'huile de poisson. Conclusion : La supplémentation d'huile de poisson atténue la réponse physiologique à l'endotoxine chez le sujet sain, en particulier la fièvre et la réponse endocrinienne, sans influencer la production des cytokines. Ces résultats soutiennent l'hypothèse que les effets bénéfiques de l'huile de poisson sont principalement caractérisés au niveau du système nerveux central, par des mécanismes non-inflammatoires qui restent encore à élucider.

The effects of dominance, regular inbreeding and sampling design on Q(ST), an estimator of population differentiation for quantitative traits.

To test whether quantitative traits are under directional or homogenizing selection, it is common practice to compare population differentiation estimates at molecular markers (F(ST)) and quantitative traits (Q(ST)). If the trait is neutral and its determinism is additive, then theory predicts that Q(ST) = F(ST), while Q(ST) > F(ST) is predicted under directional selection for different local optima, and Q(ST) < F(ST) is predicted under homogenizing selection. However, nonadditive effects can alter these predictions. Here, we investigate the influence of dominance on the relation between Q(ST) and F(ST) for neutral traits. Using analytical results and computer simulations, we show that dominance generally deflates Q(ST) relative to F(ST). Under inbreeding, the effect of dominance vanishes, and we show that for selfing species, a better estimate of Q(ST) is obtained from selfed families than from half-sib families. We also compare several sampling designs and find that it is always best to sample many populations (>20) with few families (five) rather than few populations with many families. Provided that estimates of Q(ST) are derived from individuals originating from many populations, we conclude that the pattern Q(ST) > F(ST), and hence the inference of directional selection for different local optima, is robust to the effect of nonadditive gene actions.

The sampling of ignitable liquids on suspects' hands

In arson cases, the collection and detection of traces of ignitable liquids on a suspect's hands can provide information to a forensic investigation. Police forces currently lack a simple, robust, efficient and reliable solution to perform this type of swabbing. In this article, we describe a study undertaken to develop a procedure for the collection of ignitable liquid residues on the hands of arson suspects. Sixteen different collection supports were considered and their applicability for the collection of gasoline traces present on hands and their subsequent analysis in a laboratory was evaluated. Background contamination, consisting of volatiles emanating from the collection supports, and collection efficiencies of the different sampling materials were assessed by passive headspace extraction with an activated charcoal strip (DFLEX device) followed by gas chromatography-mass spectrometry (GC-MS) analysis. After statistical treatment of the results, non-powdered latex gloves were retained as the most suitable method of sampling. On the basis of the obtained results, a prototype sampling kit was designed and tested. This kit is made of a three compartment multilayer bag enclosed in a sealed metal can and containing three pairs of non-powdered latex gloves: one to be worn by the sampler, one consisting of a blank sample and the last one to be worn by the person suspected to have been in contact with ignitable liquids. The design of the kit was developed to be efficient in preventing external and cross-contaminations.