Identification of menstrual blood by real time RT-PCR: technical improvements and the practical value of negative test results.

The identification of body fluids can be of crucial importance in forensic casework. Recently we have shown that the detection of cell- and tissue-specific mRNAs is a suitable technique to identify menstrual blood and semen. In this paper we present an improved method using real time-PCR and the menstrual blood marker MMP-7 which increases sensitivity and specificity. We furthermore demonstrate case examples described showing that the correct identification of samples as non-menstrual is likely to be in the focus of criminal investigations. Although negative test results may have a number of other reasons, the new technique provides quantitative results thus allowing a cut-off value for the control marker GAPDH to be defined, below which negative results for the menstrual blood marker MMP-7 allow the conclusion that the sample was indeed not menstrual blood. Using these data, it is possible to apply the molecular identification of menstrual blood to forensic casework considering both the limitations and the capabilities of the method.

Digital imaging of the dissection and sexual abuse of a corpse - an exceptional case of necrophilia.

Regular necrophilia refers to the sexually motivated abuse of corpses and is not considered as severe crime in many western countries. However, the risk of "switching" to necrophilic homicides, i.e., committing a homicide to obtain a dead body, has to be assessed by forensic experts. We present a case of semi-professional dissection, preservation and sexual abuse of the body and body parts of a 14-year-old girl. Every step was documented by the offender on thousands of digital images thus allowing an exact reconstruction of necrophilic acts and fantasies. Three months after the disappearance of the body the remains could be recovered and linked to the deceased by pathological examination and DNA analysis. The offender had excessively used the internet for downloading files with sadistic and necrophilic contents including autopsy instructions. The psychiatric examination of the socially integrated and married patient revealed a severe personality disorder. Two other, previously unsolved cases could be attributed to him showing a clear progression of fantasies and acts.

[Suicidal buflomedil intoxication]. / Suizidale Intoxikation mit Buflomedil.

A suicidal intoxication of a young woman following an overdose of buflomedil is reported. She died in a hospital 17 hours after ingestion. In various body fluids the following buflomedil concentrations were determined: heart blood 24.5 microg/ml, liquor 21.3 microg/ml, bile 39.1 mg/ml and urine 138.6 mg/ml. Additionally the results of autopsy and histology are presented. Anemia of the internal organs was conspicuous; this finding is attributed to the vasodilating effect of buflomedil on the peripheral vessels.

STR analysis of artificially degraded DNA-results of a collaborative European exercise.

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.

Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?

In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. We have developed a semi-quantitative duplex reverse transcription-polymerase chain reaction (RT-PCR) assay which, in combination with competitive RT-PCR using an external standard, allows quantification of RNA degradation levels. Using this method, we have investigated 106 bloodstains stored up to 15 years. The distribution of the peak area quotients of standard and target messenger-RNA (mRNA) as measured by laser-induced fluorescence capillary electrophoresis was closely correlated with the age of the samples. Further statistical analysis showed that bloodstains with age differences of 5 years and more exhibit statistical significant variances in peak area quotients of both housekeeping genes included in this study, beta-actin and cyclophilin. This can be of value when evidence from old cases is re-investigated. Our data show, that, although RNA continues to be degraded in dried bloodstains, mRNA suitable for RT-PCR can be isolated from samples stored for at least 15 years.

Quantification of mRNA degradation as possible indicator of postmortem interval--a pilot study.

Postmortem RNA degradation is a complex process which has not been studied systematically up to now. RNA, in contrast to DNA, is degraded in vivo to control translation and is believed to be less stable than DNA in vitro and postmortem due to ubiquitously present ribonucleases. Quantification of the fragmentation level of human mRNA might be a tool for determination for the time since death. We have developed a method for quantitating mRNA degradation by multiplex-RT-PCR coupled to laser-induced fluorescence capillary electrophoresis. The study included whole blood samples from living individuals and postmortem blood and brain samples which were stored refrigerated for up to 5 days. It could be shown that RNA degradation is significantly correlated with the storage interval of blood samples taken from living individuals and with the postmortem interval (PMI) in autopsy cases. This technique might become a parameter for the assessment of RNA quality in postmortem samples used for diagnosis and research and for the determination of the PMI. However, rather large confidence intervals have to be considered so that the use of this method in forensic casework requires further research.

Evaluation of mRNA markers for the identification of menstrual blood.

The detection of epithelial cells in dried bloodstains by reverse transcriptase-polymerase chain reaction is based on cell- and tissue-specific gene expression. In this paper mRNA markers suitable for the identification of menstrual blood were evaluated. RNA isolated from autopsy tissue samples including endometrium, vaginal mucosa, and blood were screened for tissue-specific expression patterns using RT-PCR with primers for hormone receptors, intermediate filaments, matrix metalloproteinases, heat shock proteins, cytokines, and growth factors. Matrix metalloproteinase (MMP) mRNA could be detected in endometrium but not in blood and other epithelia. This was confirmed in further studies with artificial menstrual bloodstains, indicating that the detection of MMP expression in bloodstains may serve as a forensic marker for menstrual blood.

Intracranial stab injuries: case report and case study.

Non-missile penetrating brain injuries are rare events in western countries. We report a case with lethal stab injury of the brain and identification of the weapon used in the assault by digital superimposition on CT scans taken at admission of the victim to a hospital. Furthermore, all cases with knife stab wounds of the skull between 1971 and 2000 were analyzed and compared with literature reports. Results of this study show that there is no region preference despite of differences in bone thickness, that stab wounds of the brain are almost invariably associated with multiple stab wounds to the trunk and that the wound tract may correspond to the dimensions of the blade allowing the identification of the weapon by digital image analysis.

Paternity testing after pregnancy termination using laser microdissection of chorionic villi.

We report an unusual case of paternity testing from residues of chorionic villi 5 weeks after pregnancy termination. The autopsy of a 32-year-old female homicide victim revealed the presence of intact chorionic villi at the former placenta implantation site. Fetal cells were selectively isolated by laser-induced microdissection of the remaining villi to avoid contamination with maternal DNA. Simultaneous amplification of 12 STR loci in 2 PCR reactions resulted in a combined probability of paternity of 99.94%. This case demonstrates that laser-assisted microdissection and multiplex STR typing provide tools for paternity testing performed on endometrial mucosa long after the product of conception was removed by therapeutic abortion.