Valéry Hédouin

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum.

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C18 reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50-1,000 ng/ml for fluvoxamine, 15-1,000 ng/ml for fluoxetine, 25-500 ng/ml for norfluoxetine, 50-500 ng/ml for sertraline, 20-500 ng/ml for paroxetine, 25-550 ng/ml for citalopram, 25-750 ng/ml for desmethylcitalopram, 25-800 ng/ml for didesmethylcitalopram, 25-650 ng/ml for milnacipran, and 25-500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.

Entomofauna of buried bodies in northern France

Autopsies of exhumed cadavers can reveal important evidence for clarification of medical insurance and social issues. This study concerns insects sampled on 22 exhumed cadavers in the Lille area. For each corpse, the species and the stages of development were noted, as well as the time elapsed after burial, the location of the cemetery, the stage of decay and possible preservation treatment. A total of eight Diptera and two Coleoptera species were sampled on the corpses. The relationships between entomofauna and conditions of burial are discussed. Three species were regularly found because of their preference for underground environments or closed environments: Conicera tibialis, typically associated with buried bodies, Leptocera caenosa which is known to be associated with human faeces, water closets, caves and cracked soil pipes, and Ophyra capensis, sometimes found on human bodies kept indoors for several months, where blowflies have not had access. Triphleba hyalinata, which is associated with human bodies in wooden coffins, was found only twice.

EFFECTS OF MORPHINE IN DECOMPOSING BODIES ON THE DEVELOPMENT OF LUCILIA SERICATA (DIPTERA : CALLIPHORIDAE)

This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Students T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.

Morphine extraction in necrophagous insects remains for determining ante-mortem opiate intoxication

To study the potential use of insects remains as toxicological indicators, we measured morphine in desiccated entomological materials using a radioimmunoassay method (Coat-A-Count Serum Morphine, Dade Behring, France) following enzymatic hydrolysis.First, enzymatic cuticle extraction of morphine (pronase digestion preceded by a 2h incubation with a dithiothreitol solution) was performed on various substrates (Calliphoridae puparial cases and desiccated adults, and desiccated pupae of Dermestidae) which were then tested to identify possible interferences with the radioimmunoassay procedure. This same procedure was performed on puparial cases and desiccated adults of Lucilia sericata (Diptera, Calliphoridae) previously reared on minced meat containing various morphine concentrations. Morphine was detected only in cuticle powder from insects reared on meats containing 100 and 1000mg/kg. Higher concentrations were measured in puparial cases. Rearings on psoas from eight heroin overdose victims confirmed previous experimental results. Remains of necrophagous insects, particularly puparial cases, are often preserved for a long time, and consequently can serve as late alternative specimens for toxicological analysis when suitable tissues are not available.

Flies eggs: a new method for the estimation of short-term post-mortem interval?

Three modeling methods were used to estimate the time for laying of Lucilia sericata eggs (Diptera, Calliphoridae) after measurements of hatching times at several constant temperatures. These models were tested first under controlled conditions with 19 profiles of fluctuating temperatures, and on the other hand under field conditions on mice. All three models were allowed to determine the time of laying within a period of about 2h, and consequently this determines the time of death, as this species lays immediately after death under favorable conditions. Rearing of eggs sampled on a corpse may therefore contribute to the determination of a short post-mortem interval (PMI).

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology.

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.

Determination of Drug Levels in Larvae of Lucilia sericata (Diptera: Calliphoridae) Reared on Rabbit Carcasses Containing Morphine

This study concerns the determination of morphine concentrations in fly larvae reared on rabbits administered different concentrations of morphine and a correlation between concentrations of the drug in larvae and tissues. Three rabbits (R1, R2 and R3) were given dosages of 12.5, 25.0 and 50.0 mg/h of morphine over a 3 h period via continuous ear artery perfusion. These dosages and time of perfusion were calculated to create tissue concentrations of morphine similar to those encountered in human death due to overdose. Morphine blood level plateau was attained after 1 h of perfusion. A fourth rabbit was used as a control. To evaluate drug concentrations, tissues were sampled using a coelioscopic technique. Approximately 400 eggs of Lucilia sericata, all of the same age category, were placed in eyes, nostrils and mouth of each rabbit carcass. Larvae and puparia were regularly collected from each rabbit for toxicological analysis. The concentrations of the drug in the tissues sampled were determined to be similar to those normally encountered in human overdoses and were correlated with the dosage of morphine that had been administered. Morphine was detected in all larvae and pupae fed on tissues from carcasses administered morphine, except for puparia from the colony fed on the R1 animal which received 12.5 mg/h dosage of morphine. All samples from the control rabbit were negative for morphine. Concentrations of morphine in larvae reared on rabbit carcasses containing morphine were 30 to 100 times lower than the concentrations found in the tissues. A correlation between the tissue concentrations and larval concentrations was found in only 3rd instar larvae (80 to 140 h following hatching). No correlations were found between administered dosages, tissue concentrations and younger larvae, prepuparial larvae or puparia.

Suicidal hanging resulting in complete decapitation--forensic, radiological, and anthropological studies: a case report.

Abstract:  The authors report a case of complete posthanging decapitation. The decapitated corpse lay against a pillar of a road bridge. The head had rolled 5 m from the trunk. The bridge was 7.2 m above the road level. The rope was 3.6 m long, its lower end was 3.6 m from the ground and its diameter was 10 mm. The noose used was a slip knot. Plain X‐rays of the skull and cervical spine were obtained. The skull X‐rays showed air in the meningeal spaces, in both lateral and third ventricles. The severance plane of the cervical spine was between the third and the fourth cervical vertebrae. No other cervical vertebral injuries were noted. At autopsy, the brain was macroscopically unremarkable except for air in the meningeal veins. The decapitation injuries of the head and the torso corresponded perfectly, without apparent loss of substance. The severance plane was confirmed. Dry bone study was carried out. Except for fractures of the extremities of the spinous processes of the second and third cervical vertebrae, no other bone injury of the spine was seen. The cervical vertebrae displayed numerous osteoarthritic lesions. The traditional hangman’s fracture was not found. To the best of our knowledge, this is the first report of complete posthanging decapitation with a severance plane between the third and fourth cervical vertebrae.

Fatal Accidental Intrathecal Injection of Vindesine

ABSTRACT: A 25‐year‐old woman being treated for non‐Hodgkins lymphoma was accidentally given vindesine intrathecally. The error was recognized immediately and a spinal cord washing was performed through syringing with isotonic saline. However, the patient died 6 weeks later with increasing paralysis, which was followed by neurologic failure. The deceased was autopsied and the central nervous system was removed for a microscopic examination. The results showed microscopic lesions extending from the lumbar to the thoracic portion of the spinal cord, which included pseudocystic transformation of the cells, degeneration of myelin, and microhemorrhages. The brain was edematous and, in the cerebellum, the vermis showed a loss of granule and Purkinje cells. The authors compare this report on vindesine toxicity with cases in the literature involving vincristine. The treating physician admitted responsibility and was sentenced to both a fine and imprisonment.

Repellent effect of some household products on fly attraction to cadavers

The most common task of a forensic entomologist is to determine an accurate minimum post-mortem interval (PMI) using necrophagous fly larvae found on carrion. More often, blowflies (Diptera: Calliphoridae) are the first insects to detect the cadaver and, if the circumstances are favourable, to leave eggs on the body. However, several studies reveal that products such as gas or paint found on the cadaver induce a delay in the colonisation of the body, leading to an under-estimate of the PMI. Six common household products (gas, mosquito citronella repellent, perfume, bleach, hydrochloric acid and soda) were added to dead rats (Rattus norvegicus) in a field (Lille Forensic Institute, France). The presence of necrophagous flies was checked at regular intervals during 1 month. This experiment was repeated at the same period for four consecutive years. Results clearly showed the repellent effect of three of the six tested substances: gas (petroleum spirit), perfume and mosquito citronella repellent, which resulted in a mean delay of several days in the appearance of the first Dipteran species. Experiments were then carried out in controlled conditions in order to confirm previous observations. An olfactometer was specially designed to observe the behaviour of female Calliphora vicina (Diptera: Calliphoridae) in response to mice (Mus musculus) cadaver odour stimuli combined with household products. Dead mouse odour was a strong attractive stimulus for most of the tested individuals. Furthermore, it was noticed that the presence of mosquito citronella repellent, perfume, hydrochloric acid and paradichlorobenzene produced a significant repellent effect on female flies. All these results together confirm the repellent effect of some household products on flies and the necessity for forensic entomologists to consider this hypothesis when estimating the PMI.