Delimitation of the time of death by immunohistochemical detection of thyroglobulin

To improve the possibilities to delimit the time of death after longer laytime it was examined if this is possible by immunohistochemical detection of thyroglobulin. The results show that in our examination material the colloid and the follicular cells of the thyroid glands of up to 5-day-old corpses produce a positive immunoreaction towards thyroglobulin in all cases whereas none of the corpses older than 13 days show such a reaction. This means that in case of a negative immunoreaction the time of death can be assumed to lie more than 6 days before the autopsy. The fact that a negative immunoreaction occurs consistently after 13 days leads to the conclusion that when thyroglobulin has been stained in a specimen, the death of the respective person must lie a maximum of 12 days earlier, whereby these time-limits may change in considerably different surrounding conditions.     

Immunohistochemical detection of methadone in the human brain

To develop a method of detecting methadone in the human brain by immunohistochemistry, brain tissue of frontal cortex, cerebellum, hippocampus, basal ganglions and brain stem from victims of a lethal methadone overdose was examined. The staining was performed with a monoclonal anti-methadone antibody from the mouse, originally developed for immunochemichal purposes (ELISA). With the help of the DAKO((R)) Catalyzed Signal Amplification (CSA) System, a specific positive immunoreaction was obtained in the neurons of the frontal cortex and hippocampus, as compared with specimen from deaths without exposition to methadone. Thus, along with metamphetamine, phenobarbital, morphine and insulin, immunohistochemical detection is also possible for methadone and the intake of this medicament can now be proven morphologically.     

Mass screening in low-income populations: the challenges of securing diagnostic and treatment services in a national cancer screening program

Funding for many mass screening programs for low-income and uninsured populations provides resources for screening tests, yet only rarely does it provide coverage for necessary follow-up diagnostic and treatment services. The National Breast and Cervical Cancer Early Detection Program (NBCCEDP), a federally funded initiative that provides cancer screening to low-income uninsured and underinsured women, covers some diagnostic follow-up tests and no treatment services. We conducted in-depth case studies of seven state programs participating in the NBCCEDP to investigate the strategies and approaches being used to secure diagnostic and treatment services. The results suggest that the program relies on a patchwork of resources--at state and local levels--to provide diagnostic and treatment services. This includes a number of components of local safety nets, all of which are unstable and have uncertain futures. Public health disease-screening initiatives need to reconsider the feasibility of continued reliance on case-by-case appeals to the local safety net for diagnostic follow-up and treatment services.     

Streifenlichttopometrie (SLT): a new method for the three-dimensional photorealistic forensic documentation in colour

By means of the new method of Streifenlichttopometrie (SLT) it is possible to record the complete body surface of casualties in a practically photorealistic fashion, i.e. three-dimensionally and in colour. In comparison with the classic method of Photogrammetry Streifenlichttopometrie (SLT) is remarkably faster (10,000 points/s instead of 1 point/s) and in addition the colour of every point measured upon the corpse's surface is instantly recorded. Taking into consideration the resolution required and the qualities of the camera system the body surface is recorded in 'patches', i.e., areas of a defined extension (in the present case 500 mmx500 mmx200 mm) which are marked with a body fixed reference frame to grant the exact matching of the data after the recording process. Length, perimeter, square and volume of the body segments and injuries can be determined. Furthermore the natural colour of the wounds can be used for an immediate classification according to the intensity of the impact forces. In addition the 3-D coordinates of the body surface including the wounds can be transferred into an animated computer simulation for the reconstruction of the traumatic events.     

Comparison of nonradioactive microtiter plate enzyme immunoassays for the sensitive detection of fentanyl

Fentanyl is a very strong opioid with analgesic properties that are approximately 80 times stronger than those of morphine and therefore is used in major surgery and treatment of pain in tumor patients. Cases of fentanyl abuse by intravenous injection, inhalation, oral or nasal application have been reported especially in the USA. Therapeutic levels of fentanyl are as low as 1 ng/ml of serum and therefore a screening test must have a detection limit below that concentration. Recently three non-radioactive enzyme immunoassays (EIAs) have become commercially available from COZART, STC and DIAGNOSTIX, all of them supplied by MAHSAN Diagnostika for evaluation with serum samples from forensic and clinical cases. A calibration curve is obtained with samples that contain 0, 0.1, 0.5, 1 and 5 ng fentanyl per ml of negative serum. The calibration curve of COZART is especially in the low range, steeper than those of STC and DIAGNOSTIX. The cut-off for all these EIAs, however, can be set at 0.5 ng/ml. After the administration of therapeutic doses, fentanyl concentrations were between 3 and more than 5 ng/ml as determined with the EIAs. The presence of the typical drugs of abuse, e.g. heroin, methadone, cocaine, cannabinoids and amphetamines including the derivatives of methylenedioxyamphetamine, don't generate false-positive results. No cross-reactivity was also observed at toxic levels of benzodiazepines and paracetamol and therapeutic levels of barbiturates, phenothiazines, antidepressants and analgesics. The EIAs tested so far appear to be suitable for the detection of fentanyl at therapeutic levels. False-positive results or cross-reactivity towards other compounds have not been observed.     

Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry

Benzodiazepines are one of the most widely prescribed drugs for the treatment of a wide spectrum of clinical disorders. They are used as anticonvulsants, anxiolytics, hypnotics or muscle relaxants with different duration of action. In this paper, a simple and sensitive method for the determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry (GC/MS) is described. The drugs spiked in whole blood were extracted with an Oasis HLB solid-phase extraction cartridge (Waters), which contains a copolymer designed to have a hydrophilic-lipophilic balance. GC/MS analysis was performed using a Shimadzu QP-5000 equipped with a BPX5 capillary column (15 mx0.32 mm I.D., film thickness 0.25 microm, SGE). Nineteen benzodiazepines and two thienodiazepines were well separated from each other on their SIM chromatograms and also on the TIC with the exception of oxazolam to cloxazolam separation. The blank extract from whole blood gave no peaks that interfered with all benzodiazepines and thienodiazepines on the chromatogram. The calibration curves for selected benzodiazepines with fludiazepam as an internal standard showed excellent linearity over the concentration range 5-500 ng/ml blood with a correlation coefficients of >0.995. The detection limits ranged from 0.2 to 20 ng/ml blood. The method is simple and sensitive for the determination of benzodiazepines in whole blood and seems to be useful in the practice of forensic science.     

Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS

A fast method using liquid-liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid beta-glucuronide (THC-COOH-glucuronide) and 11-Nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. This highly specific method, which combines chromatographic separation and MS/MS analysis, can be used for the confirmation of positive immunoassay results even without hydrolysis of the sample or derivatisation of extracts. Liquid-liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH(+), m/z 521) and THC-COOH (MH(+), m/z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/z 345 (for THC-COOH-glucuronide) and m/z 327 as well as m/z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with beta-glucuronidase/arylsulfatase (37 degrees C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D(3) was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.     

Numerical density of delta-opioid receptor expressing neurons in the frontal cortex of drug-related fatalities

In animal experiment and in cell culture, chronic morphine treatment has been followed by a reduction as well as an increase of the delta-opioid receptor (OR) number. The present postmortem morphometric study of morphine-related fatalities of drug addicts (n=12, 22-35 years old, with blood unconjugated morphine levels from 27.1 to 407 ng/ml, m.v. 176.9 ng/ml) versus a non-addicted control group (n=13, 10-44 years old) is intended to examine whether chronic opiate exposure also affects the numerical density of deltaOR expressing neurons in the human neocortex (area 10 according to Brodmann (Vergleichende Lokalisationslehre der Grosshirnrinde (1909) Johann Ambrosius Barth, Leipzig)). For the immunohistochemical procedure, vibratome sections (100 microm) were incubated with a monoclonal antibody against the deltaOR diluted 1:100, and immunoreactive sites were visualized using an immunoperoxidase protocol. The numerical densities of OR expressing and Nissl-stained neurons were assessed morphometrically (camera lucida drawings). In both collectives, the anti deltaOR immunoreactivity was predominantly localized in pyramidal neurons of layers (L) II/III and V as well as in round and ovoid neurons of L VI. In the drug-related fatalities, the density of neurons expressing deltaOR protein amounted for 2515+/-240/mm(3), in the control group for 2616+/-204/mm(3), thus displaying no statistically significant difference. These findings go along with the binding behavior of opioid ligands in postmortem brains of heroin addicts revealing similar receptor densities and affinities in the control subjects and addicts.     

Methamphetamine-related fatalities in forensic autopsy during 5 years in the southern half of Osaka city and surrounding areas

To outline the recent features of methamphetamine-related fatalities from the medico-legal point of view, a retrospective investigation of forensic autopsy cases involving methamphetamine during a 5-year period (1994-1998) in the southern half of Osaka city and surrounding areas (about 1.57 million population) was undertaken. Among 646 autopsy cases, methamphetamine was detected in 15 victims (nine males, six females; 16-71 years of age; most frequently in males in their thirties). Primary scenes of fatal events were concentrated in the middle of the city. About half of them were transferred from emergency medical centers (survival time, up to 30 h). The cause and manner of death were: methamphetamine poisoning (n=4), homicide (n=4), accidental falls and aspiration from drug abuse (n=4), fire death (n=1), myocardial infarction (n=1), and cerebral hemorrhage (n=1) under drug influence. Usually injection scars and fresh puncture sites were found. Blood methamphetamine concentrations were 2.29-17.05 micromol/dl in the fatal poisoning, 0. 44-3.80 micromol/dl in deaths from other extrinsic causes (trauma), and 1.35-2.17 micromol/dl in cardio- and cerebrovascular strokes. Common complications were cardiomyopathy, cerebral perivasculitis and liver cirrhosis/interstitial hepatitis. Fatal and nonfatal methamphetamine poisonings are separately dealt with by the administrative medical examiner's office and in emergency medical centers. Tightly cooperative approaches of clinical and medico-legal experts are required for the effective social and medical management of drug abuse.     

Extraction and amplification of authentic DNA from ancient human remains

A total of 36 ancient human remains from 12 individuals (three tooth/bone samples each) and one sample each of three individuals from the newest time was typed in a blind study using the amelogenin sex test. Prior to this molecular sex determination the sex of the individual was determined morphologically. The success rate of the amelogenin amplifications was >90%. For every individual an unambiguous molecular sex typing result was obtained. Furthermore, the morphological and molecular sex determinations were in accordance with each other, giving evidence for the authenticity and ancient origin of the polymerase chain reaction (PCR) amplifications.