Characterization of subdural collections in initial neuroimaging of abusive head trauma: Implications for forensic age diagnostics and clinical decision-making.

PURPOSE: Subdural collections (SDCs) represent a key finding in abusive head trauma (AHT), a serious form of child physical abuse. Common SDC entities in this context are subdural hematoma (SDH), subdural hygroma (SDHy), subdural hematohygroma (SDHHy), and chronic subdural hematoma (cSDH). The present study examines the prevalence of the different SDC entities and investigates the influence of the SDC diagnosis on the forensic age estimation of the injury. METHODS: In this retrospective multi-center study from three German university hospitals of a 10-year period, the initial neuroimaging material (CT or MRI) of 56 children (36 males, 20 females; age median 3.9 months) with medico-legally well-documented AHT was analyzed. SDCs were characterized by determining presence, location, extension, and visual appearance, by assigning to one of the five entities, and by categorizing with three different classification systems, one of which represents a novel system based on focality and Mixed Appearance Pattern and especially developed for children with AHT. The data were correlated with demographic and clinical data. By means of court files, AHT cases were also sub-divided into confession (n = 14) and non-confession cases (n = 42) and then compared. RESULTS: Most cases showed a multifocal presence of SDCs (96.4%) and the presence of a Mixed Appearance Pattern (82.1%). The most common SDC entity was the heterogeneous variant of the SDHHy (66.1%). The cSDH occurred infrequently only (3.6%). Our novel classification system illustrates that unifocal SDCs rarely occur in AHT, and that more complex SDC patterns are common. In nearly all cases (94.6%), additional signs of recently caused brain injury were present beside the SDCs. Comparison between confession and non-confession groups did not reveal any significant differences, indicating that the diagnostic criteria of AHT are robust. CONCLUSIONS: Although precise dating of SDCs based on initial neuroimaging alone remains unrealistic, the exact diagnosis of the SDC entity provide an important basis for differentiation between acute trauma and chronic post-traumatic state. Therefore, especially the confirmation or exclusion of subdural neomembranes, that define the cSDH, should be considered indispensable.

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.

Fractures and skin lesions in pediatric abusive head trauma: a forensic multi-center study.

Abusive head trauma (AHT) and its most common variant, the shaken baby syndrome (SBS), are predominantly characterized by central nervous system-associated lesions. Relatively little data are available on the value of skeletal and skin injuries for the diagnosis of SBS or AHT. Thus, the present study retrospectively investigated 72 cases of living children diagnosed with the explicit diagnosis of SBS during medico-legal examinations at three German university institutes of legal medicine. The risk of circular reasoning was reduced by the presence of 15 cases with confession by perpetrators. Accordingly, the comparison with the 57 non-confession cases yielded no significant differences. Skeletal survey by conventional projection radiography, often incomplete, was found to be performed in 78% of the cases only. Fractures were found in 32% of the cases. The skull (43%) and ribs (48%) were affected most frequently; only 8% of the cases showed classic metaphyseal lesions. In 48% of the cases, healing fractures were present. Skin lesions (hematomas and abrasions) were found in 53% of the cases with the face (76%), scalp (26%), and trunk (50%) being the major sites. In 48% of the cases, healing skin lesions were observed. Nearly 80% of the cases with fractures also showed skin lesions. The data prove that SBS is frequently accompanied by other forms of physical abuse. Therefore, skeletal survey is indispensable and should always be done completely and according to existing imaging guidelines if child abuse is suspected.

Abusive head trauma in court: a multi-center study on criminal proceedings in Germany.

The shaken baby syndrome (SBS) is a common variant of abusive head trauma (AHT) in infants and toddlers. Data on the legal outcome of such cases are still sparse. By means of a retrospective multi-center analysis, 72 cases of living children diagnosed with SBS/AHT from three German university institutes of legal medicine were identified. Forty-six of these cases with 68 accused individuals were available and could be evaluated with regard to basic data on the course of the criminal proceedings as well as the profile of the defendants (sub-divided into suspects, convicts, and confessed perpetrators). Criminal proceedings predominantly commenced with a complaint by the treating hospital (62%) and were found to be closed (without judgment) in 50% of the cases, mostly due to a "lack of sufficient suspicion." Of the 23 cases with judgment, the court decided on acquittal in 4 cases (17%). Imprisonment was the most frequent sentence (16 out of 19 cases with conviction, 84%), whereby the sentence has been suspended on probation in 63% of the cases. Suspects and perpetrators were mostly male and derived from the close family environment of the injured children. All confessed perpetrators stated an "excessive demand" as the reason for the violent shaking of the child. The results of the present study are in line with data from other studies with other legal systems. As many criminal proceedings were closed and the 4 acquittals occurred because the perpetration could not be ascribed to a specific perpetrator, improving the forensic methods for such an unequivocal assignment would be desirable.

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.

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.

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.

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.

DNA transfer-a never ending story. A study on scenarios involving a second person as carrier.

The transfer of DNA directly from one item to another has been shown in many studies with elaborate discussions on the nature of the DNA donor as well as material and surface of the items or surrounding features. Every DNA transfer scenario one can imagine seems to be possible. This evokes more and more intricate scenarios proposed by lawyers or attorneys searching for an explanation of the DNA of a certain person on a distinct item with impact on a crime. At court, the forensic genetic scientist has to comment on the probability of these scenarios thus calling for extensive studies on such settings. Here, the possibility of an involvement of a second person as a carrier of the donor's DNA in a variety of different scenarios including three pairs of people and two kinds of items (textiles and plastic bags) was investigated. All transfer settings were executed with and without gloves on the carrier's hands. DNA left on the items was isolated and analyzed using the Powerplex® ESX17 kit. In 21 out of 180 samples, all alleles of the donor DNA could be obtained on the second item (12%), on eight samples, the donor's DNA was dominant compared to all other alleles (38% of samples with complete donor profile). Additionally, 51 samples displayed at least more than half of the donor's alleles (28%). The complete DNA profile of the carrier was found in 47 out of 180 samples (42 partial profiles). In summary, it could be shown that a transfer of donor DNA from epithelial cells through a carrier to a second item is possible, even if the carrier does not wear gloves.