Analysis of eight STR loci in two Hungarian populations

A collection of eight STR loci (D3S1358, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) was used to generate allele frequency databases for two Hungarian population samples, Caucasians from the Budapest area and Romanies from Baranya county. During the analysis two intermediate sized alleles and a sequence variant allele were observed at the D7S820 locus. All three types of allelic variants were found to have modification (deletion, insertion, transversion) in the same block of a (T)(9) stretch located within the 3' flanking region of each allele, which may indicate a possible higher mutation rate of this (T)(9) block. For the loci D3S1358 and D7S820 the Romany population database showed departures from Hardy-Weinberg equilibrium. The forensic efficiency values for the Romany population were slightly different from those found in the Hungarian Caucasian population. Comparing the allele frequency values by G-statistic, calculating the F(st) indices and with the pairwise comparisons of inter-population variance, the two Hungarian populations could be distinguished using data of the eight STR loci.     

Human autosomal short tandem repeat types in Jat Sikhs from North India

This study provides Jat Sikhs population data in North India for nine short tandem repeat (STR) loci.     

Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.     

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.     

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.     

Application of mtDNA sequence analysis in forensic casework for the identification of human remains

In four forensic cases of unidentified skeletal remains investigated in the last year, we were able to attach three to missing persons. In one case we could show that the discovered bone sample did not fit to a missing child. The method for mitochondrial DNA analysis for the routine identification of skeletal remains was established in our institute by typing bone samples of defined age obtained from Frankfurt's cemetery. Reproducible results were obtained for bones up to 75 years old. For analysis the bone samples were pulverised to fine powder, decalcified and DNA was extracted. From the DNA we amplified a 404-bp fragment from HV-1 and a 379-bp fragment from HV-2 of the mtDNA control region. After sequencing of the PCR products, the results were compared to the Anderson reference sequence and to putative maternal relatives.