The German Stain Commission: recommendations for the interpretation of mixed stains.

In the course of forensic DNA analysis, the interpretation of DNA profiles of mixed stains, i.e. cell material from more than a single donor, has become increasingly more important. The German Stain Commission, a joint commission of Institutes of Forensic Science and Legal Medicine, has therefore developed guidelines aiming to harmonize the evaluation of mixed stains in German criminal cases.

History of forensic serology and molecular genetics in the sphere of activity of the German Society for Forensic Medicine.

In the field of forensic serology, essential developmental impulses have come from the sphere of activity of the German Society for Forensic Medicine. Among these are the orientating enzyme-reactive and specific tests for blood using crystallization tests, the determination of the species-specificity of the donor of the stain and the beginning of the individualization of a stain to its donor. Pioneering work has also been done in the development of blood group serology of the conventional markers. DNA analysis originated in the Anglo-Saxon region. The German Society for Forensic Medicine also contributed to its further progress and essentially influenced it e.g. by the discovery of microsatellite markers, including STRs, by the validation of numerous test methods, by optimization of these methods and by preliminary work for the use of mass spectrometry in DNA analysis.

The use of clinical CCT images in the forensic examination of closed head injuries.

The forensic evaluation of clinical cranial computed tomographies (CCT) frequently is the only reliable source of morphological evidence in head injuries when the injured individual survives or when death is delayed and autopsy findings are characterized by secondary changes. We have reviewed 21 cases where clinical CCT examinations were used to establish a medico-legal diagnosis. In 18 cases falls (n = 13) could be distinguished from blows (n = 5) due to the presence and/or absence of coup and contrecoup lesions and linear or depressed skull fractures. In two cases the striking object could be identified by digital superimposition. The minimum number of blows could be determined in 1 case. Only in 3 remaining cases the results were inconclusive. In our experience, CCT scans provide an important source of information for the forensic expert. To have unbiased access to these information, it is useful to evaluate the CT scans personally which requires a basic knowledge of traumatic changes found on radiographs.

A method for simultaneous RNA and DNA isolation from dried blood and semen stains.

The origin of forensic stains can be determined by detection of cell- and tissue-specific messenger-RNA (mRNA) using reverse transcription-polymerase chain reaction (RT-PCR) as demonstrated recently by the authors. However, in cases with limited sample amount DNA analysis has priority in routine forensic laboratory work. The objective of this study was to develop an easy-to-perform method for the simultaneous isolation of RNA and DNA from the same sample. Due to the common nucleic acid structure RNA and DNA isolation procedures are very similar. The investigation of blood and semen stains has shown that DNA suitable for STR analysis can be recovered by ethanol precipitation after acid phenol/chloroform extraction and removal of RNA. Although the sensitivity of this method is lower than direct DNA isolation with chelex extraction, it is sufficient for most cases as experiments with highly diluted stains have demonstrated. This time- and cost-saving method therefore may be useful when RNA and DNA analysis have to be performed from the same sample.

Protamine mRNA as molecular marker for spermatozoa in semen stains.

Cytological detection of spermatozoa with subsequent DNA analysis is the most important biological evidence in sexual crimes when suitable samples are available. Immunological and enzymatic detection of semen-specific proteins may be helpful but cannot replace specific identification of spermatozoa. We have recently shown that detection of cell-specific gene expression can be used to identify menstrual blood. In this paper we demonstrate that the basic nucleoproteins protamine 1 and 2 are suitable markers for spermatozoa because they are exclusively expressed in the haploid genome and that protamine mRNA can be detected in semen stains by the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). With semi-nested PCR, 10-100 spermatozoa are theoretically sufficient to provide positive amplification results, with hot-start PCR at least 100-1,000 cells are required corresponding to an average semen volume of 0.01-0.1 microl. This new method thus allows specific identification of spermatozoa with molecular biology tools and may broaden the spectrum of investigations in the forensic laboratory.

Sudden death due to pituitary apoplexy.

A case of sudden, unexpected death due to hemorrhage into a large, previously undiagnosed pituitary adenoma (pituitary apoplexy) in a 41-year-old man is presented. Pituitary adenomas are frequent intracranial tumors with usually benign prognosis. Acute hemorrhage into the tumor is a severe and potentially fatal complication, if not diagnosed and treated early, and occurs in up to 10% of cases. Sudden tumor enlargement due to the hemorrhage with compression of vessels and neural tissue at the skull base may result in coma and death without preceding symptoms leading to forensic investigations as demonstrated by this case report.

Detection of epithelial cells in dried blood stains by reverse transcriptase-polymerase chain reaction.

The identification of menstrual blood stains can be improved by detection of messenger ribonucleic acid (mRNA) specific for epithelial (endometrial) cells. RNA molecules, however, are believed to be unstable and require careful sample processing. In this study, we have investigated the extraction of RNA suitable for reverse transcriptase-polymerase chain reaction (RT-PCR) from dried blood stains stored for up to six months. With a modified RNA isolation protocol, it was possible to obtain RNA from dried blood stains with at least 5 x 10(2) leukocytes. In an additional experiment, we evaluated the RNA isolation from mixed stains composed of leukocytes and T47D cells, a breast cancer-derived cell line with epithelial origin. Detection of 10(2) T47D cells in a total number of 10(5) leukocytes was possible by amplification of cytokeratin 19 mRNA and progesterone receptor-mRNA specific for hormonally regulated epithelial cells. In both experiments amplification results were not dependent on storage time with similar data from one day to six months. Furthermore, it was possible to identify dried menstrual blood samples by showing the presence of mRNA specific for epithelial cells. These results demonstrate for the first time, that RNA suitable for RT-PCR, can be isolated from forensic specimens stored up to at least six months, and that a small number of epithelial (endometrial) cells can be identified in dried blood specimens. Using this method, it will be possible to identify the origin of small and partially degraded blood samples which can be especially useful in forensic evaluation of cases with sexual offense.

Fulminant liver failure in a young child following repeated acetaminophen overdosing.

Acetaminophen (paracetamol), a widely used analgetic drug, is well tolerated at therapeutic doses, but may cause severe hepatotoxicity when ingested in large overdose. Self-poisoning is still very popular in adults and accidental ingestion of one single overdose occurs occasionally in children. In contrast, lethal intoxication in children after repeated administration of therapeutic doses is a very rare event. This case report describes an iatrogenic acetaminophen overdosing in a 5-year-old child receiving 8.5 g acetaminophen in 48 h. Fulminant liver failure developed within 60 h. Autopsy findings included panlobular liver cell necrosis. Acetaminophen serum levels were rather low compared to cases with ingestion of one single overdose. Postmortem diagnosis of chronic acetaminophen intoxication as cause of death should include the clinical history as well as, if available, the calculated drug serum half-life.

Population data for the STR systems HumTH01, HumVWA and FES/FPS in a population sample from lower Franconia.

Population data studies for the three tetranucleotide STR systems HumTH01, HumVWA and FES/ FPS were carried out on a Caucasian population sample from Lower Franconia (Germany). The observed heterozygosities were 0.83, 0.80 and 0.73, respectively, and the discrimination power of the triplex was 0.9995. All loci were in accordance with Hardy-Weinberg equilibrium tested using the chi 2-analysis.

Extraction of high quality DNA from bloodstains using diatoms.

A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.

[Bolus death and drowning--expression of central nervous system dysregulation?]. / Bolus- und Ertrinkungstod--Ausdruck einer zentralnervösen Regulationsstörung?

According to the author's own investigations and to findings reported in the literature, almost all bolus deaths and highly numerous accidental deaths by drowning occur to adults and adolescents under the impact of alcohol. An attempt was made to elucidate the mechanism by which alcohol actually play its role in bolus or drowning death. These two causes of death have in common complete or partial obstruction of respiratory tract. Yet, safety against that life-threatening condition were devised by evolution very early in phylogenesis (respiratory block, cough reflex, circulatory centralisation). They are reflex-triggered for immediate effect and take that effect through the vegetative nervous system. These findings have been secured by animal experiments. However, on account of considerable interference of alcohol with vegetative activity, via action on transmitter metabolism (transmitter synthesis, storage, release, rebonding and bonding by means of products of alcohol metabolism), symptoms of extreme vagotomia may develop, such as bradycardia with circulatory centralisation and apnoea of alcohol-related irreversibility, finally resulting in cardiac arrest. The "reflex-related" process of bolus death has often been described, with reference being regulatory made to individuals staying in water and drowning without any self-rescue attempt.

The genetic haptoglobin polymorphism: relevance of paternity assessment.

A practical method for haptoglobin subtyping is described utilizing fast sample preparation by means of batch adsorption to DEAE-cellulose and subsequent isoelectric focusing of reductively cleaved samples. The expanded haptoglobin polymorphism leads to an increase of the theoretical paternity exclusion rate to approximately 33%. Hence, the system appears to be highly attractive for paternity assessment.

Factor B (BF) subtyping by isoelectric focusing: methods, nomenclatures, genetics and forensic application.

Usually factor B (BF) typing is performed by means of the traditional agarose gel electrophoresis. Using isoelectric focusing, the system can be extended by two common subtypes of BF F. The existence of BF F subtypes has in the meantime been confirmed by various authors and in different populations. Their inheritance has been proven by family- and mother/child analyses and molecular-genetic studies (correlation with restriction fragment length polymorphism). Different typing methods as well as different nomenclatures seem to indicate that the subtypes FA and FB (according to Geserick et al.) are identical with the Fb and Fa subtypes (according to Teng and Tan). At present, some confusion still exists for the less frequent variants and subtypes which possibly could be identified by direct comparison of the patterns. The BF system is a valuable marker in paternity testing. Its chance for exclusion of paternity in Caucasian populations has been calculated to be about 14% for agarose gel electrophoresis and increases to about 16% for BF F subtyping by isoelectric focusing. Preliminary results indicate that BF may also be used for typing of bloodstains (up to 2 weeks old).

Haptoglobin subtypes in Berlin, GDR. A simple procedure for haptoglobin purification and subtyping.

Sera were obtained from 1,275 blood donors in Berlin, probands involved in paternity tests, and from 119 families with 235 children; the sera were subtyped by isoelectric focusing, following preparation and reductive molecular cleavage of haptoglobin. In this paper, an uninvolved preparation technique is described for routine testing. Allelic frequencies are: Hp *1F = 0.1471; *1S = 0.2502; *2FF = 0.0020; *2FS = 0.5753; *2SS = 0.0251. Only one deviation from autosomal codominant inheritance was recorded in the family examinations, with illegitimacy considered possible. In the region of Berlin, the changes of ruling out uninvolved individuals in paternity suits have gone up from 18% (conventional technique recording two frequent alleles) to 33% (subtyping).

[Demonstration of a "new" BF-variant (BF S Berlin) using isoelectric focusing]. / Nachweis einer "neuen" BF-Variante (BF S Berlin) mittels Isoelektrofokussierung.

Using the islelectrofucising in polyacrylamide gel for BF typing in the population from Berlin (GDR) a "new" variant was found. This variant is situated cathodically of the BF S line, but it is not detectable by means of agarose gel electrophoresis. Therefore the common BF nomenclature is not applicable. The designation "S Berlin" is proposed. Typing the family of the proposita the same phenotype (S Svar .) was found in the serum of her daughter. This result confirms a codominant inheritance of the new allele. The observed allele frequency is 0.0017.

Isoelectrofocusing in the study of the Bf system: existence of two common subtypes of the BfF allele.

The inherited polymorphism of the properdin factor B (Bf system) was studied by isoelectrofocusing in polyacrylamide gel (PAGIF, pH range 5.0-8.0), followed by immunofixation. Using the described technique, two different subtypes of F bands are visible (designated as F' and F"). Studies of 13 families and 48 mother-child pairs are not in contradiction to the assumption of a codominant inheritance by two alleles (BfF' and BfF") at a single locus.

[Gc subtypes: demonstration using isoelectric focusing. Study of population and formal genetics]. / Die Gc-Subtypen: Darstellung mittels isoelektrischer Fokussierung. Studie zur Populations- und Formalgenetik.

Gc-subtypes were determined by isoelectric focusing and immunfixation on samples from 492 unrelated blood donors from Berlin. The frequency of the three genes was found to be GcIF = 0.1270, GcIS = 0.6006, Gc2 = 0.2724. Analysis of 78 parents with 190 children did not show deviations from the expected mode of inheritance. Investigation of the adults from paternity cases and of their children on the other hand obtained similar results. No rare alleles were observed.

[Identification of subtypes of the Bf system: distribution in a random sample from the population of Berlin (GDR)]. / Zum Nachweis von Subtypen des Bf-Systems: Verteilung in einer Stichprobe der Berliner Bevölkerung (DDR).

Using isoelectrofocusing in polyacrylamide gel (pH range 5.0-8.0) for the classification of Bf types two different subtypes of F bands are visible. The extended polymorphism is composed of six common phenotypes (Bf F', F'F", F", F'S, F"S, and S). The distribution of Bf subtypes was studied in the population (n = 584) from Berlin (GDR). The allele frequencies observed were: BfF' = 0.0916, BfF" = 0.0753, BfS = 0.8202, BfF1 = 0.0060, BfS0.7 = 0.0043, BfS0.3 = 0.0009, BfSvar(new) = 0.0017.