Knife wound or nosebleed-where does the blood at the crime scene come from?

Secretion analysis is a useful tool in forensic genetics, since it establishes the (cellular) origin of the DNA prior in addition to the identification of the DNA donor. This information can be crucial for the construction of the crime sequence or verification of statements of people involved in the crime. For some secretions, rapid/pretests already exist (blood, semen, urine, and saliva) or can be determined via published methylation analyses or expression analyses (blood, saliva vaginal secretions, menstrual blood, and semen). To discriminate nasal secretion/blood from other secretions (like oral mucosa/saliva, blood, vaginal secretion, menstrual blood, and seminal fluid), assays based on specific methylation patterns at several CpGs were set up in this study. Out of an initial 54 different CpG markers tested, two markers showed a specific methylation value for nasal samples: N21 and N27 with a methylation mean value of 64.4% ± 17.6% and 33.2% ± 8.7%, respectively. Although identification or discrimination was not possible for all nasal samples (due to partial overlap in methylation values to other secretions), 63% and 26% of the nasal samples could be unambiguously identified and distinguished from the other secretions using the CpG marker N21 and N27, respectively. In combination with a blood pretest/rapid test, a third marker (N10) was able to detect nasal cells in 53% of samples. Moreover, the employment of this pretest increases the proportion of identifiable or discriminable nasal secretion samples using marker N27 to 68%. In summary, our CpG assays proved to be promising tools in forensic analysis for the detection of nasal cells in samples from a crime scene.

About the influence of environmental factors on the persistence of DNA - a long-term study.

DNA persistence and DNA transfer are important features in the assessment of a crime scene. The question how long DNA may persist at a certain location is similarly important as the one how the DNA has been transferred to this location. Depending on the source of the DNA as well as the conditions at the crime scene, the answer to this question is quite difficult. In this study, persistence of DNA from epithelial abrasions, blood cells, and saliva cells in indoor and outdoor scenarios has been investigated with regard to exposure time and exposure conditions including sunlight, temperature, and humidity in summer and winter scenarios. Overall, we generated 338 epithelial samples, 572 blood samples, and 572 saliva samples. A complete profile of the cell/DNA donor after exposure could be obtained in 47%, 65%, and 58% of epithelial abrasions, blood samples, and saliva samples, respectively. Regarding blood samples, there were no differences between supporting materials cloth and plastic; however, the percentage of complete profiles was higher for saliva samples on plastic and for epithelial samples on cloth. In indoor scenarios, complete profiles could be recovered from nearly all blood and saliva samples up to 9 months, whereas the amount of epithelial complete profiles already started to decline after 3 months. In outdoor scenarios, we observed a tipping point at an exposure time of 3 months. Blood and saliva samples collected after this period displayed complete profiles in less than 25% of samples. After 12 months, no outdoor sample showed a complete profile. The results of this study facilitate decisions on the relevance of recovered DNA from crime scenes.

Vibration as a pitfall in pyrosequencing analyses.

Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful validation and establishment of several pyrosequencing assays using a PyroMark® Q48 Autoprep instrument (Qiagen, Hilden, Germany), we decided to expand the method further purchasing a second instrument. But after initializing this second instrument side by side with the first, the majority of analyses failed (97 samples of 133 samples (73%)). The number of error messages increased rapidly and the average RFU values decreased. After purchasing two anti-vibration weighing tables for the PyroMark® instruments and repeating the analyses under the same conditions and with identical samples the results improved considerably, 115 samples of 130 samples (88%) showed successful and reproducible results. These findings demonstrate the impact of vibrations and percussions on PyroMark® Q48 Autoprep performance and the reliability of methylation analyses.

[Student training in forensic medicine in Germany: testing and evaluation]. / Studentische Ausbildung im Fach Rechtsmedizin in Deutschland: Prüfungen und Evaluation.

The implementation of the Medical Licensure Act in 2002 led to remarkable changes in teaching, testing and evaluation in undergraduate medical education. Using an online questionnaire the current situation among German institutes for forensic medicine was evaluated. The return rate of the questionnaires was 80%. The results point at a preponderance of testing of factual knowledge. A change to testing of practical skills appears necessary to match the learning objectives of practical teaching. The evaluation results represent a high level of student contentment with teaching in forensic medicine. Clinical electives can be offered by more of 90% of the institutes. Teaching time in forensic medicine is thought to be inadequate by a relevant number of institutes.

Post-mortem genetic investigation of cardiac disease-associated genes in sudden infant death syndrome (SIDS) cases.

The sudden infant death syndrome (SIDS) is one of the leading causes of postneonatal infant death. It has been shown that there exists a complex relationship between SIDS and inherited cardiac disease. Next-generation sequencing and surveillance of cardiac channelopathy and cardiomyopathy genes represent an important tool for investigating the cause of death in SIDS cases. In the present study, targeted sequencing of 80 genes associated with genetic heart diseases in a cohort of 31 SIDS cases was performed. To determine the spectrum and prevalence of genetic heart disease associated mutations as a potential monogenic basis for SIDS, a stringent variant classification was applied and the percentage of rare (minor allele frequency ≤ 0.2%) and ultra-rare variants (minor allele frequency ≤ 0.005%) in these genes was assessed. With a minor allele frequency of ≤ 0.005%, about 20% of the SIDS cases exhibited a variant of uncertain significance (VUS), but in only 6% of these cases, gene variants proved to be "potentially informative." The present study shows the importance of careful variant interpretation. Applying stringent criteria misinterpretations are avoided, as the results of genetic analyses may have an important impact of the family members involved.

Inter-laboratory adaption of age estimation models by DNA methylation analysis-problems and solutions.

In recent years, a lot of age prediction models based on different CpG motives in different cell types were published determining the biological age of a person by DNA methylation. For a general employment of this technique, maybe even as a routine method, the cross-laboratory application of such models has to be examined. Therefore, we tested two different published age prediction models for blood and mouth swab samples with regard to prediction accuracy (Bekaert et al Epigenetics 10:922-930, 2015a; Bekaert et al Forensic Sci Int Genet Suppl Ser 5:e144-e145, 2015b). Both models are based on CpG sites of four genes (ASPA, EDARADD, PDE4-C, and ELOVL2), but with a different combination of CpGs for the two tissue types. A mean absolute difference (MAD) between chronological and predicted age of 9.84 and 8.32 years for blood and buccal swab models could be demonstrated, respectively, which is significantly worse than the published data, probably due to higher DNA methylation variances in some CpGs. By retraining both prediction models, the prediction accuracy could be improved to a MAD of 5.55 and 4.65 years for the renewed blood and buccal swab model, respectively. This study demonstrates the usefulness of effective DNA standards to normalize DNA methylation data for better comparison of study results.

Cleaning a crime scene 2.0-what to do with the bloody knife after the crime?

The persistence of DNA on washed items as well as the DNA transfer has become a major subject of research in recent years, especially after the detectability of minor DNA traces was heavily increased by sensitive analysis methods. Nowadays, the attribution of a DNA trace to an individual is only rarely questioned, whereas the way of application of this DNA to an item is subject to much discussion and speculation. Additionally, the removal of DNA by cleaning or its possible persistence on an item despite a cleaning process are often important problems in court. The aim of this study was to investigate whether DNA traces (blood, saliva, epithelial cells) on different objects (knives, plates, glasses, and plastic lids) can persist on the surface despite cleaning by different methods like hand-washing or the use of a dishwasher. In total, 120 samples were collected from artificially constructed blood, saliva, and epithelial cell stains on objects with smooth surfaces after washing and analyzed by STR amplification. Samples taken after rinsing or hand-washing resulted mainly in complete DNA profiles (62.5% of samples), while cleaning in the dishwasher rendered almost everything completely DNA-free. Since in the hand-washing experiments a secondary transfer of DNA through the water could not be ruled out, additional transfer experiments were conducted with blood and saliva samples on plates. Here, a carryover of DNA traces could be demonstrated up to the fifth washed item.

[External post-mortem examination according to S1 guideline 2017 of the German Society of Legal Medicine]. / Durchführung der ärztlichen Leichenschau gemäß S1-Leitlinie 2017 der Deutschen Gesellschaft für Rechtsmedizin.

In Germany, the system of external post-mortem examinations is regulated by state law. In order to standardize the performance of external post-mortem examinations as far as possible throughout Germany, the German Society of Legal Medicine developed the S1 guideline "Rules for the performance of external post-mortem examination." The current version of this guideline was published on the Association of the Scientific Medical Societies in Germany (AWMF) homepage in November 2017. The guideline explains in detail the reliable determination of death, the diagnosis of the cause of death, the classification of the manner of death, the estimation of the time of death, and the reporting obligations of the post-mortem examination physician. Detailed information is provided on the examination of the corpse. A careful performance of the external post-mortem examination avoids false death determinations, lies in the presumed will of the deceased, and serves to protect the interests of the bereaved. In addition, it is of importance to society as a whole in that communicable diseases and legally relevant deaths are identified. Finally, the validity of cause-of-death statistics is improved, which can have far-reaching consequences for health-related policy decisions. In this article the main rules of the guideline are described.

Unintentional effects of cleaning a crime scene-when the sponge becomes an accomplice in DNA transfer.

DNA transfer in aqueous solutions as well as the persistence of DNA on washed items has become a major subject of research in recent years and is often a significant problem in court. Despite these approaches, the question about the "mobility" of DNA especially in capital offenses cannot be answered in every case, since a variety of scenarios for DNA transfer are possible. The aim of this study was to investigate whether DNA traces could be distributed by cleaning an object. For this purpose, a large table surface and fabric piece were artificially provided with skin contact traces and body fluids (saliva and blood) in two series of experiments and then wiped off with water or with soap water (218 samples in total). These experiments resulted in a clear "carry over" of DNA traces especially for body fluid samples (100% of blood samples and 75% of saliva samples led to a complete profile). The results could be confirmed in a second experimental set-up with 384 samples using different cleaning agents and more intense cleaning actions. Even small amounts of 5-10 µl body fluid led to complete profiles in around 45% of the samples, while 20 µl led to nearly 65% complete profiles. A strong impact of the amount of traces and the chosen surface could be demonstrated, while the active component of the cleaning agent seemed to be of less influence with the explicit exception of chloric agents which rendered almost everything completely DNA-free. In summary, a distribution of DNA traces by wiping or scrubbing an object could be clearly proven.

Persistence of DNA on clothes after exposure to water for different time periods-a study on bathtub, pond, and river.

DNA traces on clothes of drowned bodies can provide important evidence for police investigations, especially in cases of suspected suicides or homicides. However, it is generally assumed that the water "erodes" a large part of the DNA depending especially on the exposure time. In forensic casework, DNA of suspects could be found frequently on clothes of drowned bodies after hours, sometimes days of exposure to water. This study was conducted to attempt a general statement about the conditions under which sufficient DNA remains can be expected for molecular genetic analysis. For this purpose, different scenarios were designed including DNA from three to five people, different types of waters (tap, pond, bathtub and river) for various time periods, with higher water pressure, different temperature, and soapy water (bathtub). Epithelial cells and blood cells were mounted on cotton cloths, and the DNA left after exposure was analyzed using the Powerplex® ESX17fast kit. In the indoor experiments, complete profiles could be seen even after 10 min rinsing of clothes under the tap and after 1 week in the bathtub. Outdoors, the results differed considerably between summer and winter as well as between pond and river. The longest exposure time still resulting in a complete profile was 2 weeks for a sample with skin cells in the pond during winter. In summer, the time period for erasing the bulk of DNA was 4 hours regarding epithelial samples and more than 1 day for blood samples in pond and river environments. All in all, the results demonstrate that DNA could still be recovered from clothes exposed to water for more than 1 week.

The prone sleeping position and SIDS. Historical aspects and possible pathomechanisms.

The incidence of SIDS decreased during the previous 25 years significantly. This is mainly due to epidemiological research identifying important risk factors such as prone sleeping position and subsequent campaigns to reduce this risk factor.Originally, the prone sleeping position for babies had been strongly recommended in the sixties and seventies despite previous publications pointed to the associated risk. Worldwide, many infants died of SIDS whose deaths could have been avoided. Today, the recommendation that infants should sleep in supine position has been scientifically verified. In supine sleeping position, pathophysiological mechanisms can be avoided which may lead to hypoxia and death in prone position. Such mechanisms could be occlusion of airways (in particularly associated with face-down position), elevated diaphragm, positional cerebral hypoxia caused by constriction of arteries, rebreathing CO2, and overheating.Irrespective of the specific pathomechanism leading to death in individual cases, it has been established that the prone position is the most important risk factor for SIDS and therefore should be incorporated in the definition of the term SIDS.

Impact of several wearers on the persistence of DNA on clothes-a study with experimental scenarios.

The detection of DNA of a certain person on the inside of a piece of clothing involved in a crime scene is usually seen as confirmation that this person is the owner or bearer and therefore participated in this crime. However, besides the possibilities of secondary or even tertiary transfer of DNA, the accused often argues that he lent the garment to another person who by chance did not leave any DNA while committing the crime. Then, forensic genetic scientists have to answer the question how long DNA persists on an item used in daily routine and how long a piece of clothing must be worn to definitively leave detectable DNA behind. In an attempt to answer these questions, several scenarios with two or three individuals wearing the same sweatband for different time periods were set up. DNA left on the sweatbands was isolated, quantified, and then analyzed using the Powerplex® ESX17fast kit. The majority of samples displayed all alleles of both/all three wearers on the outside (67%) as well as on the inside (80%) of the sweatbands. In contrast, a single profile of the first wearer could only be found once among all 204 samples, a single profile of the second wearer in 7% of samples. Wearing the sweatband for only 10 min was enough to result in a complete profile of the second wearer in 79% of samples. So, it is highly unlikely to wear/use a piece of clothing for even a short period of time without leaving own DNA behind.

Methodology for the identification of vulnerable asylum seekers.

Asylum seekers often experience situations of vulnerability, being frequently exposed to a heightened risk of harm, and thus require special care, support and protection. The categories of "vulnerable persons", identified by International Legislation, and an individual's classification as a "vulnerable asylum seeker", have important implications in the reception procedures, in the decision-making phase and in the definition of therapeutic needs and rehabilitation. The Istanbul Protocol, the first international guideline approved by the United Nations and applied in different contexts, is not applicable for the assessment of the totality of the conditions (medical and otherwise), and therefore, the identification and assessment of conditions of vulnerability is largely delegated to questionnaires administered by non-medical personnel. The proposed methodology, based on the modificatory reworking of the Guidelines of the International Academy of Legal Medicine concerning the "medicolegal ascertainment of personal injury and damage on the living person", takes into consideration all the medical issues relevant for the decision concerning the applicant, both in the reception procedures and in the outcome of the asylum application.

Comparison of imaging planes during CT-based evaluation of clavicular ossification: a multi-center study.

Determining the ossification stage of the medial clavicular epiphysis by computed tomography represents the currently recommended methodology for the question of whether a living individual has completed the 18th or 21st year of life. In the present study, thin-slice CT scans of 1078 sternoclavicular joints were reconstructed in axial and coronal image series and evaluated according to the two classification systems established for age diagnostics using the clavicle. Both image series (axial and coronal) were analyzed separately. When comparing the results of axial and coronal view, a different ossification stage was found in 35.6% of the clavicles. The results suggest an influence of the imaging plane on the process of stage determination. In order to further approximate the three-dimensional and asymmetrical structure of the epiphyseal ossification center, the usage of at least two different reformation types may be recommended. In practice, only those reference studies should be applied which exactly employed the same number and orientations of the reformation types that are going to be used in the respective routine case.

Back to the Future - Part 1. The medico-legal autopsy from ancient civilization to the post-genomic era.

Part 1 of the review "Back to the Future" examines the historical evolution of the medico-legal autopsy and microscopy techniques, from Ancient Civilization to the Post-Genomic Era. In the section focusing on "The Past", the study of historical sources concerning the origins and development of the medico-legal autopsy, from the Bronze Age until the Middle Ages, shows how, as early as 2000 BC, the performance of autopsies for medico-legal purposes was a known and widespread practice in some ancient civilizations in Egypt, the Far East and later in Europe. In the section focusing on "The Present", the improvement of autopsy techniques by Friedrich Albert Zenker and Rudolf Virchow and the contemporary development of optical microscopy techniques for forensic purposes during the 19th and 20th centuries are reported, emphasizing, the regulation of medico-legal autopsies in diverse nations around the world and the publication of international guidelines or best practices elaborated by International Scientific Societies. Finally, in "The Future" section, innovative robotized and advanced microscopy systems and techniques, including their possible use in the bio-medicolegal field, are reported, which should lead to the improvement and standardization of the autopsy methodology, thereby achieving a more precise identification of natural and traumatic pathologies.

Back to the Future - Part 2. Post-mortem assessment and evolutionary role of the bio-medicolegal sciences.

Part 2 of the review "Back to the Future" is dedicated to the evolutionary role of the bio-medicolegal sciences, reporting the historical profiles, the state of the art, and prospects for future development of the main related techniques and methods of the ancillary disciplines that have risen to the role of "autonomous" sciences, namely, Genetics and Genomics, Toxicology, Radiology, and Imaging, involved in historic synergy in the "post-mortem assessment," together with the mother discipline Legal Medicine, by way of its primary fundament, universally denominated as Forensic Pathology. The evolution of the scientific research and the increased accuracy of the various disciplines will be oriented towards the elaboration of an "algorithm," able to weigh the value of "evidence" placed at the disposal of the "justice system" as real truth and proof.

Liver steatosis in pre-transplant liver biopsies can be quantified rapidly and accurately by nuclear magnetic resonance analysis.

Donor livers marginally acceptable or acceptable according to extended criteria are more frequently transplanted due to the growing discrepancy between demand and availability of donor organs. One type of marginally acceptable graft is a steatotic donor liver, because it is more sensitive to ischemia-reperfusion injury. Thus, quantitative assessment of steatosis is crucial prior to liver transplantation. Extent of steatosis of 49 pre-reperfusion liver biopsies from patients who received orthotopic liver transplantation was assessed by three techniques: semi-quantitative histological evaluation, computerized histomorphometry, and NMR-based estimation of fat content. The findings were correlated to clinical data and to histological examination of corresponding post-reperfusion biopsies for quantification of ischemia-reperfusion injury. We found that values obtained through all three assessment methods were positively correlated. None of the values obtained by the three applied methods correlated with clinical outcome or extent of ischemia-reperfusion injury. Quantitative evaluation of steatosis by NMR yields results comparable to histological and morphometrical assessment. This technique is rapid (<5 min), accurately quantifies fat in donor livers, and provides results that can be used when evaluation by a pathologist is not available.

The role of known variants of KCNQ1, KCNH2, KCNE1, SCN5A, and NOS1AP in water-related deaths.

Drowning is one of the most frequent causes of accidental deaths worldwide, and still it remains a diagnosis of exclusion. Moreover, sudden cardiac deaths (SCD) or, if no actual cardiac alterations can be found, sudden unexplained deaths (SUD) represent a major group within mortality statistics as well. This leads to the assumption that there might be a general underlying cause for at least some cases of drowning, SCD, or SUD, for example, genetic aberrations in arrhythmia-associated genes. In the present study, blood samples of 171 corpses found in water (drowning, death after almost drowning, and unclear deaths) were analyzed in 19 known variants of the genes KCNQ1, KCNH2, KCNE1, SCN5A, and NOS1AP by minisequencing. In three variants of NOS1AP, significant differences of allele and/or genotype frequencies could be demonstrated between victims of drowning and published controls as well as own controls. Moreover, similar differences were found comparing unexplained deaths in water and controls. Regarding the other genes, especially one single nucleotide polymorphism (SNP) of KCNQ1 could be associated with drowning. These results propose that performing a molecular autopsy analyzing known variants of arrhythmia-associated genes, in particular NOS1AP, may assist in establishing a cause of death for bodies found in water without clear drowning signs.

Does zero really mean nothing?-first experiences with the new PowerQuant(TM) system in comparison to established real-time quantification kits.

DNA quantification is an important step in the molecular genetic analysis of a forensic sample, hopefully providing reliable data on DNA content for a subsequent generation of reproducible STR profiles for identification. For several years, this quantification has usually been done by real-time PCR protocols and meanwhile a variety of assays are commercially available from different companies. The newest one is the PowerQuant(TM) assay by Promega Inc. which is advertised with the promise that a determined DNA concentration of 0 ng/µl in a forensic sample guarantees the impossibility to achieve true STR results, thus allowing to exclude such samples from STR analysis to save time and money. Thus, the goal of this study was to thoroughly verify the quantification step with regard to its suitability as a screening method. We have evaluated the precision and reliability of four different real-time PCR quantification assays by systematically testing DNA dilutions and forensic samples with various DNA contents. Subsequently, each sample was subjected to the Powerplex® ESX 17 fast kit to determine a reliable cutoff level for exclusion of definitely negative samples from STR analysis. An accurate quantification of different cell line DNA dilutions was not possible with any kit. However, at least the PowerQuant(TM) assay provided suitable data analyzing forensic samples, whereas in other systems up to 46 % of negative samples still displayed reliable STR analysis results. All in all, the PowerQuant(TM) assay represents a big step forward, but the evaluation of real-time PCR quantification results has still to be done with great care.