Discrepancy between CYP2D6 phenotype and genotype derived from post-mortem dextromethorphan blood level

OBJECTIVE: To describe the death of a toddler after a therapeutic dose of dextromethorphan and its investigation. STUDY DESIGN: Case report, cytochrome P450 phenotype and genotype determination in the victim and post-mortem drug redistribution study performed in rats. RESULTS: A 20-month Asian male who received 3 mg of dextromethorphan once at 09:00 h and again at 22:00 h was found dead at 04:35 h. Post-mortem examination showed signs of early bronchopneumonia (bacterial cultures were negative); dextromethorphan and dextrorphan blood concentrations taken from the heart cavity were 500 ng/ml (1. 84 micromol/l) and 200 ng/ml (0.78 micromol/l), respectively. Despite the dextromethorphan level being almost 100-fold higher than expected after therapeutic doses, intentional or unintentional overdose was extremely unlikely; other potential causes were investigated. Post-mortem drug redistribution study performed in rats showed that dextromethorphan does not undergo extensive redistribution after death (6+/-5-fold increase) and could not explain the observed dextromethorphan level. The dextromethorphan/dextrorphan concentration ratio of 2.5 found in this toddler was compatible with a slow CYP2D6 metabolizer phenotype. However, the toddler exhibited a fast metabolizer genotype. Potential reasons for this discrepancy are discussed. CONCLUSION: CYP450 phenotypes derived from post-mortem blood levels should be interpreted with caution and preferably confirmed by a genotype analysis.     

Pulmonary edema in fatal heroin overdose: immunohistological investigations with IgE, collagen IV and laminin - no increase of defects of alveolar-capillary membranes

Pulmonary edema complicating heroin overdosage is a well recognized entity and regarded as the major mechanism contributing to death in heroin addicts. It's pathogenesis is unknown, several mechanisms are discussed: hypoxia-induced increase of pulmonary capillary permeability, depressed myocardial contractility, centrally induced respiratory depression, primary toxic effects on the alveolar capillaries and acute anaphylactic shock. The present study included opiate-related deaths (n=23) and a control group of sudden cardiovascular deaths (n=12) to verify the hypothesis, that defects of the alveolar capillary membranes and/or an acute anaphylactic reaction leads to pulmonary congestion, edema and hemorrhages. Lung specimens were obtained from these 35 autopsies of persons autopsied in the Institute of Forensic Medicine, University of Bonn, in 1997 and 1998. All specimens were examined with hematoxylin-eosin, prussian blue and investigated with immunohistological methods using primary antibodies against collagen IV, laminin and IgE. Defects of the basal laminae of the alveoli were found, demonstrated by laminin and collagen IV, and the number of IgE-positive cells was counted in both groups. There was an increased but not significant number of IgE-positive cells in the heroin-group and defects of the epithelial and endothelial basal laminae were found in both groups without significant differences.     

Alternative primers for DYS391 typing: advantages of their application to forensic genetics

The amplification of the STR DYS391, using the primers described in the Genome Data Base (GDB: G00-365-251), shows not only an additional band to the Y-specific one in males with a size range of 26 bp less than those of DYS391 locus alleles, but also a polymorphic pattern in females in the same size range as the additional band observed in males. The DYS391 pattern in families reflects a Y-specific linked locus and also a polymorphic X locus with an X-linked pattern of inheritance. A first screening in the X homologous locus allowed the identification of five different alleles. Allele frequencies were explored in different population groups for both the Y locus and the homologous locus in the X chromosome showing a similar allele distribution pattern in the X and Y homologous loci. An alternative reverse primer was designed to amplify the Y-chromosome specific STR in order to improve the specificity and applicability of this system to forensic genetics. Comparative results of the amplification with the new and the previously described primers proved that with this new primer there is a substantial increase in the specificity of the amplification. Moreover, a smaller fragment is amplified with a size out of the range of the alleles of the other Y-STRs usually used in forensic applications, therefore simplifying its inclusion in multiplex systems.     

The use of supercritical fluid extraction for the determination of amphetamines in hair

A laboratory study interested in the analysis of human hair for drugs-of-abuse was conducted to determine if drugs could be detected and quantified from hair. Supercritical fluid extraction (SFE) techniques followed by GC-MS analysis were applied to extract amphetamines from hair. The group of amphetamines included methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and internal standard mephentermine (MP). To validate information on amphetamine use in hair, powdered hair samples free from drugs were collected and soaked in a known amphetamine standard solution. Authentic fortified case hair samples taken from known drug users known to have consumed amphetamines were also analyzed for amphetamine. Results from this study show that amphetamine use can be detected in spiked and authentic fortified human hair using SFE techniques for qualitative and quantitative reproducible results.     

Computer-assisted facial image identification system using a 3-D physiognomic range finder

This system consists of a 3-D physiognomic range finder and a computer-assisted facial image superimposition unit. The 3-D range finder is composed of a detector for measuring facial surface and its control computer. The detector has two sinusoidal grating projection devices and two CCD cameras. The computer-assisted facial image superimposition unit consists of a host computer including a proprietary software, a flat surface color display and a color image scanner for inputting 2-D facial images of a criminal. The 3-D facial shape and texture of a suspect is obtained by using the range finder. To make the comparison between the 3-D facial image and the 2-D facial image, the 3-D facial image is first reproduced on a display of the host computer from a MO disk and then the 2-D facial image is taken with the color image scanner and reproduced on the display. The 3-D facial image is exactly adjusted to match the orientation and size of the 2-D facial image under the fine framework mode, and then the fine framework mode of 3-D facial image is converted to the fine texture image. The shape and positional relationships of facial components between the 3-D and 2-D facial images are examined by the fade-out or wipe image mode. The distance between the selected two points and angle among the selected three points on the 3-D and 2-D facial images are automatically measured for the assessment of anthropometrical data between both images. For evaluating the fit between the anthropometrical points on the 3-D and 2-D facial images, the reciprocal point-to-point difference between both images is compared.     

Post-mortem analysis of apoptotic changes associated with human skin bruises

The estimation of the age of skin bruises is of importance in forensic medicine, especially in child abuse cases. Time-dependent changes in bruise colour and/or associated histological features have been used with a limited degree of success. An increased rate of apoptosis in the injured tissue has been considered as a novel time-dependent marker of cell death, by injury inflicted in a rat model. The object of the present study was to apply the TUNEL method of DNA end labelling to identify and enumerate apoptotic cells in bruised and normal skin in order to study the relationship of apoptotic cell density with the age of the bruise. A commercially available DNA end labelling kit, TUNEL method, was standardised, validated and used for this purpose. Twenty unselected post-mortem cases with bruises due to a variety of causes were studied. The apoptotic cells stained with TUNEL reaction were counted in 10 high power fields in the epidermis, as well as in the dermis of formaldehyde-fixed paraffin-embedded skin specimens. The mean positive cell densities (+/- 1 S.E.) were compared with respect to the age of the bruise. In the epidermis, the mean apoptotic cell count was statistically significantly greater in the bruised skin compared to normal skin in 2- to 6-day-old bruises; whilst in the dermis the same was true in 3- to 8-day-old bruises. The overall findings suggest that there is a quiescent period prior to the increase in the apoptotic cell activity that is seen following skin bruising. This is so provided the post-mortem skin samples were collected within a lapse of 6 days or less between the time of death and formalin fixation and paraffin embedding to avoid the bias made by the difference of length of post-mortem interval.     

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.