Showing papers in "International Journal of Legal Medicine in 2012"

Postmortem chemistry update part II

Abstract: Postmortem chemistry is becoming increasingly essential in the forensic pathology routine and considerable progress has been made over the past years. Biochemical analyses of vitreous humor, cerebrospinal fluid, blood and urine may provide significant information in determining the cause of death or in elucidating forensic cases. Postmortem chemistry may essentially contribute in the determination of the cause of death when the pathophysiological changes involved in the death process cannot be detected by morphological methods (e.g. diabetes mellitus, alcoholic ketoacidosis and electrolytic disorders). It can also provide significant information and useful support in other forensic situations, including anaphylaxis, hypothermia, sepsis and hormonal disturbances. In this article, we present a review of the literature that covers this vast topic and we report the results of our observations. We have focused our attention on glucose metabolism, renal function and electrolytic disorders.

Potential forensic application of DNA methylation profiling to body fluid identification.

Abstract: DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not currently determine the type and origin of the biological material. Recent advances in whole genome epigenetic analysis indicate that chromosome pieces called tDMRs (tissue-specific differentially methylated regions) show different DNA methylation profiles according to the type of cell or tissue. We examined the potential of tissue-specific differential DNA methylation for body fluid identification. Five tDMRs for the genes DACT1, USP49, HOXA4, PFN3, and PRMT2 were selected, and DNA methylation profiles for these tDMRs were produced by bisulfite sequencing using pooled DNA from blood, saliva, semen, menstrual blood, and vaginal fluid. The tDMRs for DACT1 and USP49 showed semen-specific hypomethylation, and the tDMRs for HOXA4, PFN3, and PRMT2 displayed varying degrees of methylation according to the type of body fluid. Preliminary tests using methylation-specific PCR for the DACT1 and USP49 tDMRs showed that these two markers could be used successfully to identify semen samples including sperm cells. Body fluid-specific differential DNA methylation may be a promising indicator for body fluid identification. Because DNA methylation profiling uses the same biological source of DNA for individual identification profiling, the determination of more body fluid-specific tDMRs and the development of convenient tDMR analysis methods will facilitate the broad implementation of body fluid identification in forensic casework.

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

Abstract: In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

Comparison of three methods of DNA extraction from human bones with different degrees of degradation

Abstract: There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol–chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol–chloroform protocols are more efficient for successful DNA extraction.

A validation study of the Qiagen Investigator DIPplex® kit; an INDEL-based assay for human identification.

Abstract: Marker sets that are based on small insertion/deletion (INDEL) alleles can serve as useful supplementary or stand-alone assays for human identification. A validation study has been performed on a human identification assay based on a panel of 30 INDELs and amelogenin using the Investigator DIPplex® kit (Qiagen). The assay was able to type DNA from a number of forensically relevant sample types and obtain full profiles with 62 pg of template DNA and partial profiles with as little as 16 pg of template DNA. The assay is reproducible, precise, and non-overlapping alleles from minor contributors were detectable in mixture analysis ranging from 6:1 to 19:1 mixtures. Population studies were performed on the 30 indels, and there were no significant departures from Hardy–Weinberg equilibrium or significant linkage disequilibrium between the markers (after correction for sampling). In all populations, the random match probability was 1.43 × 10−11 or less, and the power of exclusion was greater than .999999999. We also discovered several microvariant alleles in our population samples. The data support that the Investigator DIPplex® kit provides a powerful supplement or stand-alone capability for human identity testing.

The publication of population genetic data in the International Journal of Legal Medicine: guidelines

Abstract: Since the 10 or 11 century AD, Vineta has been a major trade city at the Baltic Sea. It is generally assumed that the population consists of Wends and Slavs. Here, we analyzed eleven X chromosomal STRs in 200 women and 300 men who could prove their ancestry in Vineta for at least twenty-five generations and compared the results with German, Polish, Finnish and Swedish populations. For almost 200 years Vineta was the most important harbour at the South of the Baltic Sea, and even after the Wendish Crusade in the 12 century AD, a part of the Christianisation efforts in this geographical region, it remained a major trade city. Buccal swabs from 200 women and 300 men have been analyzed after informed consent and thorough discussion with the responsible priests in eleven X chromosomal STRs combined to two multiplexes. Samples were obtained and analyzed after advice of the Medical Ethics Committee of the University of Vineta in accordance with the declaration of Helsinki. The anonymity of the individuals investigated was preserved corresponding to the rules of data protection of the Human Medical Faculty Vineta. DNA extraction, PCR, analysis on an ABI310 genetic analyzer and statistical evaluation have been done as described before [2]. The allele frequencies and the results of the forensic efficiency parameter calculations are given in Table S1 and Table S2. Combined forensic efficiency parameters were calculated for all STRs with the exception of DXS7424, for which linkage disequilibrium to DXS101 has previously been detected [3], as follows: combined power of discrimination for males 0.99999999, combined power of discrimination for females greater than 0.999999999 and combined mean exclusion chance for trios 0.999991. The frequency of DXS101–DXS7424 haplotypes is shown in Table S3. Linkage disequilibrium between DXS101 and DXS7424 has not been tested for our samples because the number of investigated individuals was too small. Two rare alleles were found in the samples from Vineta, the allele 61 in DXS8377 and the allele 7.3 in DXS9898. Both have been verified by sequencing as described before [2]. Genetic distances were estimated for each STR marker with the exception of DXS7424—due to the linkage disequilibrium to DXS101—to different European populations [4–8] and non-European populations [9, 10] as available (see Table S4 for results). In summary, there were only few differences between the peoples from Vineta and other Northern European populations, but major differences to the populations from Brazil and the Ivory Coast, as expected.

The application of traditional and geometric morphometric analyses for forensic quantification of sexual dimorphism: preliminary investigations in a Western Australian population

Abstract: A current limitation of forensic practice in Western Australia is a lack of contemporary population-specific standards for biological profiling; this directly relates to the unavailability of documented human skeletal collections. With rapidly advancing technology, however, it is now possible to acquire accurate skeletal measurements from 3D scans contained in medical databases. The purpose of the present study, therefore, is to explore the accuracy of using cranial form to predict sex in adult Australians. Both traditional and geometric morphometric methods are applied to data derived from 3D landmarks acquired in CT-reconstructed crania. The sample comprises multi-detector computed tomography scans of 200 adult individuals; following 3D volume rendering, 46 anatomical landmarks are acquired using OsiriX (version 3.9). Centroid size and shape (first 20 PCs of the Procrustes coordinates) and the inter-landmark (ILD) distances between all possible pairs of landmarks are then calculated. Sex classification effectiveness of the 3D multivariate descriptors of size and shape and selected ILD measurements are assessed and compared; robustness of findings is explored using resampling statistics. Cranial shape and size and the ILD measurements are sexually dimorphic and explain 3.2 to 54.3 % of sample variance; sex classification accuracy is 83.5–88.0 %. Sex estimation using 3D shape appears to have some advantages compared to approaches using size measurements. We have, however, identified a simple and biologically meaningful single non-traditional linear measurement (glabella–zygion) that classifies Western Australian individuals according to sex with a high degree of expected accuracy (87.5–88 %).

Development and validation of a postmortem radiological alteration index: the RA-Index

Abstract: This study aimed to derive an index quantifying the state of alteration of cadavers by quantifying the presence of gas in the body using postmortem multidetector computed tomography (MDCT) imaging, and to validate the index by defining its sensitivity and specificity. The RA (radiological alteration)-index was derived from postmortem MDCT data from 118 nontraumatically deceased people. To validate the index, 100 additional scanned bodies (50 % traumatically deceased) were retrospectively examined by two independent observers. Presence of gas at 82 sites was assessed by a radiologist, whereas a forensic pathologist only investigated the seven sites used for the RA-index. The RA-index was highly correlated to the overall presence of gas in all 82 sites (R(2) = 0.98 in the derivation set and 0.85 in the validation set). Semiquantitative evaluation of gas presence in each site showed moderate reliability (Cohen's kappa range, 0.41-0.78); nevertheless, the overall RA-index was very reliable (ICC(2,1) = 0.95; 95 % CI 0.92-0.96). Examiner using the RA-index detected heart cavities full of gas with a sensitivity of 100 % (95 % CI 51.7-100) and a specificity of 98.8 % (92.6-99.9). We conclude that determining the presence of gas at seven sites is a valid means to measure the distribution of gas due to cadaveric alteration in the entire body. The RA-index is rapid, easy-to-use, and reliable for nonexperienced users, and it is a valid method to suspect the normal presence of gas from cadaveric alteration. MDCT can be used to screen for gas embolism and to give indications for gas composition analysis (gas chromatography).

Human age estimation combining third molar and skeletal development

Abstract: The wide prediction intervals obtained with age estimation methods based on third molar development could be reduced by combining these dental observations with age-related skeletal information. Therefore, on cephalometric radiographs, the most accurate age-estimating skeletal variable and related registration method were searched and added to a regression model, with age as response and third molar stages as explanatory variable. In a pilot set up on a dataset of 496 (283 M; 213 F) cephalometric radiographs, the techniques of Baccetti et al. (2005) (BA), Seedat et al. (2005) (SE), Caldas et al. (2007) and Rai et al. (2008) (RA) were verified. In the main study, data from 460 (208 F, 224 M) individuals in an age range between 3 and 26 years, for which at the same day an orthopantogram and a cephalogram were taken, were collected. On the orthopantomograms, the left third molar development was registered using the scoring system described by Gleiser and Hunt (1955) and modified by Kohler (1994) (GH). On the cephalograms, cervical vertebrae development was registered according to the BA and SE techniques. A regression model, with age as response and the GH scores as explanatory variable, was fitted to the data. Next, information of BA, SE and BA + SE was, respectively, added to this model. From all obtained models, the determination coefficients and the root mean squared errors were calculated. Inclusion of information from cephalograms based on the BA, as well as the SE, technique improved the amount of explained variance in age acquired from panoramic radiographs using the GH technique with 48%. Inclusion of cephalometric BA + SE information marginally improved the previous result (+1%). The RMSE decreased with 1.93, 1.85 and 2.03 years by adding, respectively, BA, SE and BA + SE information to the GH model. The SE technique allows clinically the fastest and easiest registration of the degree of development of the cervical vertebrae. Therefore, the choice of technique to classify cervical vertebrae development in addition to third molar development is preferably the SE technique.

Forensic performance of two insertion–deletion marker assays

Abstract: Improving the amplification and analysis of highly degraded DNA extracts has been a longstanding area of research in forensic genetics. One of the most promising recent developments in analysis of degraded DNA is the availability of short, biallelic insertion-deletion length polymorphisms (InDels) in highly multiplexed assays. InDels share many of the favourable characteristics of singlenucleotide polymorphisms (SNPs) that make them ideal markers for analysis of degraded DNA, including: analysis in short amplicon size ranges, high multiplexing capability and low mutation rates. In addition, as length-based polymorphisms, InDels can be analysed with the same simple dye-labelled PCR primer methods as standard forensic short tandem repeats. Separation and detection of fluorescently dye-labelled PCR products by capillary electrophoresis eliminate the multiple step protocols required by SNP typing with single-base extension assays and provide a closer relationship between the input DNA and the profile peak height ratios. Therefore InDel genotyping represents an effective new approach for human identification that adds informative new loci to the existing battery of forensic markers. To assess the utility of InDels for forensic analysis, we characterised population variation with two InDel identification assays: the 30-plex Qiagen DIPplex panel and a 38-plex panel developed by Pereira et al. in 2009 [1]. Allele frequencies were generated for the 68 markers in US African American, Caucasian, East Asian and Hispanic samples. We made a thorough assessment of the individual and combined performance of the InDel sets, as well as characterising profile artifacts and other issues related to the routine use of these newly developed forensic assays based on artificially degraded DNA and mixed source samples. © 2012 Springer-Verlag.

A comparison of three established age estimation methods on an adult Spanish sample.

Abstract: Most current methods for adult skeletal age-at-death estimation are based on American samples comprising individuals of European and African ancestry. Our limited understanding of population variability hampers our efforts to apply these techniques to various skeletal populations around the world, especially in global forensic contexts. Further, documented skeletal samples are rare, limiting our ability to test our techniques. The objective of this paper is to test three pelvic macroscopic methods [(1) Suchey-Brooks; (2) Lovejoy; and (3) Buckberry and Chamberlain] on a documented modern Spanish sample. These methods were selected because they are popular among Spanish anthropologists and because they never have been tested in a Spanish sample. The study sample consists of 80 individuals (55 males and 25 females) of known sex and age from the Valladolid collection. Results indicate that in all three methods, levels of bias and inaccuracy increase with age. The Lovejoy method performs poorly (27%) compared with Suchey-Brooks (71%) and Buckberry and Chamberlain (86%). However, the levels of correlation between phases and chronological ages are low and comparable in the three methods (<0.395). The apparent accuracy of the Suchey-Brooks and Buckberry and Chamberlain methods is largely based on the broad width of the methods’ estimated intervals. This study suggests that before systematic application of these three methodologies in Spanish populations, further statistical modeling and research into the covariance of chronological age with morphological change are necessary. Future methods should be developed specific to various world populations and should allow for both precision and flexibility in age estimation.

Diagnostic performance of ethyl glucuronide in hair for the investigation of alcohol drinking behavior: a comparison with traditional biomarkers.

Abstract: Background Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption.

Analysis of 12 X-STRs in Greenlanders, Danes and Somalis using Argus X-12

Abstract: X-chromosome markers have become a useful set of markers of choice when certain complex kinship cases need to be unravelled. The Argus X-12 kit allows the co-amplification in a single PCR reaction of 12 X-chromosome short tandem repeats located in four linkage groups. A number of 507 unrelated individuals from Greenland, Denmark and Somalia together with two generation families were typed using the Argus X-12 kit. Silent alleles for the DXS10148 and DXS10146 systems were observed in males, mostly from Somalia. High levels of intrapopulation variability and therefore high forensic parameter values were calculated for the three studied populations. The population in Greenland showed a significantly lower intrapopulation variability and a high genetic differentiation compared with 13 other populations. Significant levels of linkage disequilibrium were observed between markers belonging to the same linkage group, mainly in the populations in Greenland and Somalia. Family studies allowed the calculation of mutation and recombination frequencies. A higher male versus female mutation rate was obtained, with an average value of 3.3 × 10−3. Recombination fraction calculations performed on two generation families showed, as previously described, a not complete independence between X-chromosome linkage groups 3 and 4.

Vaginal microbial flora analysis by next generation sequencing and microarrays; can microbes indicate vaginal origin in a forensic context?

Abstract: Forensic analysis of biological traces generally encompasses the investigation of both the person who contributed to the trace and the body site(s) from which the trace originates. For instance, for sexual assault cases, it can be beneficial to distinguish vaginal samples from skin or saliva samples. In this study, we explored the use of microbial flora to indicate vaginal origin. First, we explored the vaginal microbiome for a large set of clinical vaginal samples (n = 240) by next generation sequencing (n = 338,184 sequence reads) and found 1,619 different sequences. Next, we selected 389 candidate probes targeting genera or species and designed a microarray, with which we analysed a diverse set of samples; 43 DNA extracts from vaginal samples and 25 DNA extracts from samples from other body sites, including sites in close proximity of or in contact with the vagina. Finally, we used the microarray results and next generation sequencing dataset to assess the potential for a future approach that uses microbial markers to indicate vaginal origin. Since no candidate genera/species were found to positively identify all vaginal DNA extracts on their own, while excluding all non-vaginal DNA extracts, we deduce that a reliable statement about the cellular origin of a biological trace should be based on the detection of multiple species within various genera. Microarray analysis of a sample will then render a microbial flora pattern that is probably best analysed in a probabilistic approach.

Haplogrouping mitochondrial DNA sequences in Legal Medicine/Forensic Genetics

Abstract: Haplogrouping refers to the classification of (partial) mitochondrial DNA (mtDNA) sequences into haplogroups using the current knowledge of the worldwide mtDNA phylogeny. Haplogroup assignment of mtDNA control-region sequences assists in the focused comparison with closely related complete mtDNA sequences and thus serves two main goals in forensic genetics: first is the a posteriori quality analysis of sequencing results and second is the prediction of relevant coding-region sites for confirmation or further refinement of haplogroup status. The latter may be important in forensic casework where discrimination power needs to be as high as possible. However, most articles published in forensic genetics perform haplogrouping only in a rudimentary or incorrect way. The present study features PhyloTree as the key tool for assigning control-region sequences to haplogroups and elaborates on additional Web-based searches for finding near-matches with complete mtDNA genomes in the databases. In contrast, none of the automated haplogrouping tools available can yet compete with manual haplogrouping using PhyloTree plus additional Web-based searches, especially when confronted with artificial recombinants still present in forensic mtDNA datasets. We review and classify the various attempts at haplogrouping by using a multiplex approach or relying on automated haplogrouping. Furthermore, we re-examine a few articles in forensic journals providing mtDNA population data where appropriate haplogrouping following PhyloTree immediately highlights several kinds of sequence errors.

Reliability of Schmeling’s stages of ossification of medial clavicular epiphyses and its validity to assess 18 years of age in living subjects

Abstract: Nowadays, due to the global increase in migration movements, forensic age estimation of living young adults has become an important focus of interest. Minors often have no identification documents providing their correct birth dates. Establishing the age of majority is therefore fundamental in order to determine whether juvenile penal systems or penal systems in force for adults are to be applied. Radiological examination of the clavicles is one of the methods recommended by the Study Group on Forensic Age Diagnostics. In this retrospective study, a sample of chest radiographs of 274 subjects, aged between 12 and 25 years, was studied according to Schmeling’s method in order to examine the ossification of both medial clavicular epiphyses. All stage classifications were evaluated by five examiners. Intra- and inter-examiner reliability was analysed by Cohen’s K statistic. Intra-examiner agreement was insufficient for two of the experts. Inter-examiner agreement, among the other three operators, was moderate (K = 0.509). Study of reliability highlighted difficulties in interpretation, the need to select qualified personnel and choice of the best radiographic image in order to reduce any anatomic overlaps. Although ossification of the medial clavicular epiphyses is recommended to assess whether an individual has already reached the age of majority or not, these results suggested that it is very difficult to clearly identify the five stages of ossification by using conventional chest radiography.

Distribution of artifactual gas on post-mortem multidetector computed tomography (MDCT)

Abstract: Purpose We investigated the incidence and distribution of post-mortem gas detected with multidetector computed tomography (MDCT) to identify factors that could distinguish artifactual gas from cardiac air embolism.

Good shedder or bad shedder--the influence of skin diseases on forensic DNA analysis from epithelial abrasions.

Abstract: The successful analysis of weak biological stains by means of highly sensitive short tandem repeat (STR) amplification has been increased significantly over the recent years. Nevertheless, the percentage of reliably analysable samples varies considerably between different crime scene investigations even if the nature of the stains appears to be the same. It has been proposed that the amount and quality of DNA left at a crime scene may be due to individual skin conditions (among other factors). Therefore, we investigated DNA from handprints from 30 patients acutely suffering from skin diseases like atopic dermatitis, psoriasis or skin ulcer before and after therapy by STR amplification using the new and highly sensitive Powerplex® ESX17 kit in comparison to 22 healthy controls. Handprints from atopic dermatitis patients showed a correct and reliable DNA profile in 90% and 40% of patients before and after therapy, respectively. Regarding psoriasis patients, we detected full DNA profiles in only 64% and 55% of handprints before and after therapy. In contrast, in ulcus patients and controls, full DNA profiles were obtained in much lower numbers. We conclude that active skin diseases like atopic dermatitis or psoriasis have a considerable impact on the amplificable DNA left by skin contact with surfaces. Since up to 7% of adults in European countries suffer from one of these diseases, this could explain at least partially the varying quality of DNA from weak stains.

Medical examination of fitness for police custody in two large German towns

Abstract: Worldwide, there is a high risk of medical complications or death in police custody. This risk is often increased by unclear legislation, a lack of clearly defined responsibility and medical examination standards. Any solution to these problems requires as a very basis the systematic analysis of the medical examinations that determine whether a person is fit to be detained in custody. We analysed a total of 3,674 medical records on fitness for custody, taken from two large German towns (Halle/S and Bremen). The examined individuals were predominantly males or of a younger age. The indication in the majority of cases was acute alcoholic intoxication or drug withdrawal syndromes. Traumata and internal or mental diseases were also quite frequent. For approximately 50% of all cases, fitness for custody was declared on certain conditions. Only 39.8% were found to be unconditionally fit for detention in custody. In just under 10% of the cases, the person was found unfit for custody. These cases concerned mainly persons with psychological symptoms and advanced alcohol or drug withdrawal syndromes. We were able to show that the recent introduction of new police custody regulations in Halle/S had a significant influence on the medical decision on fitness for custody. Our detailed assessment has provided us with the basis to develop solutions for the improvement of medical care in police custody. The focus lies here on the organisation and legal regulation of the medical aspects of custody but also on policing and medical work.

A method for the analysis of 32 X chromosome insertion deletion polymorphisms in a single PCR

Abstract: Studies of human genetic variation predominantly use short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) but Insertion deletion polymorphisms (Indels) are being increasingly explored. They combine desirable characteristics of other genetic markers, especially the possibility of being analysed using short amplicon strategies, which increases the ease of analysis, contributing to justify their interest in population and forensic genetics. After the advent of autosomal and uniparental genomes (mtDNA and Y chromosome), these fields of research are also focusing on the X chromosome, given its special transmission pattern. The X chromosome markers brought new insights into the history of modern human populations and also proved useful in forensic kinship investigations, namely in deficient relationship cases and in cases where autosomes are uninformative. This work describes an X-Indel multiplexsystemamplifying32biallelicmarkersinonesingle PCR. The multiplex includes X-Indels shown to be poly- morphic in the major human population groups and follows a short amplicon strategy. The set was applied in the genetic characterization of sub-Saharan African, European and East Asian population samples and revealed high forensic efficiency, as measured by the accumulated power of discrimination (0.9999990 was the lowest value in males and 0.999999999998 was the highest in females) and mean exclusion chance varied between 0.998 and 0.9996 in duos and between 0.99997 and 0.999998 in trios. Finally, a segregation analysis was performed using trio constellations of father-mother-daughters in order to address the transmis- sion pattern and assess mutation rates of this type of markers.

Radiological age estimation: based on third molar mineralization and eruption in Turkish children and young adults

Abstract: Radiographic evaluation of mineralization and eruption stages of third molars using dental panoramic radiographies can be an efficient tool for chronological age estimation in both forensic sciences and legal medicine. The third molar tooth is utilized for dental age estimation about the age span of 15–23 years because it represents the only tooth still in development. The aim of this study is to obtain and analyze data regarding third molar development and eruption in Turkish population for dental age estimation. A total of 744 dental panoramic radiographies of 394 female and 350 male subjects aged between 8 and 22 years were examined. Third molar development was determined according to the Nolla classification system, and eruption was assessed relative to the alveolar bone level. Mandibular and maxillary third molars were generally found at similar stages of development on both sides. Nolla stage 6 (completed crown calcification) was reached at around the age of 15 in both maxillary and mandibular third molars in both sexes. Alveolar emergence was at around the age of 16 in males and around age of 17 in females. Although third molars’ eruption shows greater variability than development of third molars, data which were obtained from this study about eruption of these teeth can be supportive to development data for age estimation.

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice

Abstract: Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h-21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h-3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was 4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.

The examination and identification of bite marks in foods using 3D scanning and 3D comparison methods

Abstract: Bite mark analysis offers the opportunity to identify the biter based on the individual characteristics of the dentitions. Normally, the main focus is on analysing bite mark injuries on human bodies, but also, bite marks in food may play an important role in the forensic investigation of a crime. This study presents a comparison of simulated bite marks in different kinds of food with the dentitions of the presumed biter. Bite marks were produced by six adults in slices of buttered bread, apples, different kinds of Swiss chocolate and Swiss cheese. The time-lapse influence of the bite mark in food, under room temperature conditions, was also examined. For the documentation of the bite marks and the dentitions of the biters, 3D optical surface scanning technology was used. The comparison was performed using two different software packages: the ATOS modelling and analysing software and the 3D studio max animation software. The ATOS software enables an automatic computation of the deviation between the two meshes. In the present study, the bite marks and the dentitions were compared, as well as the meshes of each bite mark which were recorded in the different stages of time lapse. In the 3D studio max software, the act of biting was animated to compare the dentitions with the bite mark. The examined food recorded the individual characteristics of the dentitions very well. In all cases, the biter could be identified, and the dentitions of the other presumed biters could be excluded. The influence of the time lapse on the food depends on the kind of food and is shown on the diagrams. However, the identification of the biter could still be performed after a period of time, based on the recorded individual characteristics of the dentitions.

Hair analysis of drugs involved in drug-facilitated sexual assault and detection of zolpidem in a suspected case

Abstract: In drug-facilitated crimes, victims are subjected to nonconsensual acts while they are incapacitated by the effects of a drug. A specific LC-MS/MS protocol for determining benzodiazepines and hypnotics at low concentration in hair specimens was developed and validated in order to target the allegedly administered drugs on a chronological basis. In the case hereby reported, a 26-year-old woman claimed to have been sexually assaulted after being administered an allegedly drugged coffee, but toxicological analysis of urine and blood provided no evidence of any drug intake. Subsequently, a second woman accused the same man of sexual abuse. Hence, the suspect was prosecuted. Specimens were collected from four subjects (two alleged victims, the suspect and his wife) and segmental hair analysis was performed. The results revealed that zolpidem was present at low picogram per milligram concentration in three out of eleven segments of hair specimen obtained from the first of the alleged victims, offering plain evidence of single or sporadic exposure, whereas the agent was detected in the high picogram per milligram range in the hair collected from suspect's wife, coherently with therapeutic administration. The presence of interfering signals typical of the keratin-containing matrix was found and possible hair degradation by cosmetic treatments was investigated by electron microscopy, so as to obtain a judicious interpretation of the analytical findings.

MicroCT detection of gunshot residue in fresh and decomposed firearm wounds.

Abstract: Gunshot residue (GSR) evidence may be altered or obscured by after-death events such as putrefaction, autolysis, and/or damage by animals. The present study aimed at evaluating and comparing the amount and differential distribution of GSR utilizing microcomputed tomography (microCT) analysis of fresh and decomposed gunshot wounds. A total of 60 experimental shootings at three different firing distances (5, 15, and 30 cm) were performed on human calves surgically amputated for medical reasons. Thirty specimens (10 for each tested distance) were immediately formalin-fixed, while the other 30 specimens were enclosed in a cowshed for 15 days, before formalin fixation (air temperature ranging from 11°C to 38°C). MicroCT analysis with three-dimensional image reconstruction detected GSR particles in all the investigated entrance wounds. In fresh specimens, GSR was concentrated on the skin surface around the entrance hole and in the epidermis and dermis layers around the cavity, while in decomposed specimens, the high density particles were detected only in the dermis layer. No GSR was detected in exit wounds of both fresh and decomposed specimens regardless of the tested firing distance. Statistical analysis demonstrated that also in decomposed wounds the amount of GSR roughly correlated with the distance from which the gun was fired, exhibiting, however, a higher variability than in fresh samples. The obtained results suggest that microCT analysis can be a valid screening tool for differentiating decomposed entrance and exit gunshot wounds.

Investigation of the force associated with the formation of lacerations and skull fractures

Abstract: Post-mortem examination is often relied upon in order to determine whether a suspicious death was natural, accidental, suicidal or homicidal. However, in many cases the mechanism by which a single injury has been inflicted cannot be determined with certainty based on pathological examination alone. Furthermore the current method of assessing applied force relating to injury is restricted to an arbitrary and subjective scale (mild, moderate, considerable, or severe). This study investigates the pathophysiological nature of head injuries caused by blunt force trauma, specifically in relation to the incidence and formation of a laceration. An experimental model was devised to assess the force required to cause damage to the scalp and underlying skull of porcine specimens following a single fronto-parietal impact. This was achieved using a drop tower equipped with adapted instrumentation for data acquisition. The applied force and implement used could be correlated with resultant injuries and as such aid pathological investigation in the differentiation between falls and blows. Experimentation revealed prevalent patterns of injury specific to the reconstructed mechanism involved. It was found that the minimum force for the occurrence of a laceration was 4,000 N.

Database of post-mortem rectal cooling cases under strictly controlled conditions: a useful tool in death time estimation.

Abstract: The most common method used in determining the estimated time since death in the early post-mortem phase is back-calculation based on rectal temperature decrease. Cooling experiments are essential for model generation and validation. Post-mortem temperature models are necessary to perform back-calculations. Thus far, cooling experiments have not been performed under controlled environmental conditions. The present study provides data on 84 post-mortem cooling experiments under strictly controlled environmental conditions. For a period of 5 years, starting in 2003, deceased persons with a known time of death and known environmental conditions at the death scene were transferred to a climatic chamber for the process of body cooling. The environmental temperature was programmed to the death scene temperature and kept constant throughout the process of body cooling. Rectal and ambient temperatures were measured every minute. Relevant case-specific information was summarized in a FileMaker database. The database serves as a reference tool for the comparison of real cases in forensic routine and to check the plausibility of model-derived estimates.

Radiographic evaluation of Gustafson’s criteria for the purpose of forensic age diagnostics

Abstract: The main criteria used in dental age estimation in living young individuals are mineralisation and eruption of the third molars. In order to further diversify the spectrum of characteristics after completion of third molar development, tests were undertaken to determine whether the characteristics studied by Gustafson can be ascertained with the required forensic certainty with the aid of the orthopantomogram and whether the evaluation of these could render forensic odontological age diagnoses possible beyond the 18th year of life. For this purpose, 1,299 conventionally produced orthopantomograms of 650 female and 649 male Germans aged from 15 to 40 years were studied. The characteristics of secondary dentin formation, cementum apposition, periodontal recession and attrition were evaluated in all the mandibular premolars. The correlation of the individual characteristics with chronological age was studied with the aid of a multiple regression analysis in which chronological age formed the dependent variable. Depending on the tooth studied, the R values amounted to 0.65 to 0.73; the standard error of estimate was 5.3 to 5.7 years. Basically, the regression equations calculated can be recommended for age estimation in living individuals, although it should be borne in mind that the applicability of the new method presented is limited by the quality of the X-ray images.

Can facial proportions taken from images be of use for ageing in cases of suspected child pornography? A pilot study

Abstract: The age of the victim plays a crucial role for the legal implications concerning pornography. Judges therefore often call on forensic experts to verify the age of individuals depicted on photographs or videos. However, there is no scientifically established protocol available for forensic practice in such cases. The conventional methods such as the evaluation of secondary sexual characteristics provide unsatisfactory results particularly when the legally relevant ages for child pornography (i.e. 14 and 18 years) are concerned. To overcome these limits, a European research group has explored the applicability of facial proportions as an age indicator on images. In this pilot study, standardized facial images of 353 females and 20 males from four age groups (6, 10, 14 and 18 years) were randomly selected for the metric analysis from a large data set including German, Italian and Lithuanian subjects. In this sample, several indices extracted from the frontal and lateral photographs were closely correlated to their respective indices taken from the living individuals. Furthermore, age-related changes were identified for indices taken from the photographs. The discriminant analysis showed that for the pooled sample, 60.3% of the cases were correctly classified into the respective age group. The percentage of correctly classified cases increased in the respective country samples as follows: 69.9% for Germany, 69.4% for Lithuania and 80.5% for Italy. The present study suggests that the metric assessment of the face may be used for age estimation on images. Nonetheless, more work needs to be done in order to verify the reliability of these findings on a large sample.

Evaluation of C-reactive protein, procalcitonin, tumor necrosis factor alpha, interleukin-6, and interleukin-8 as diagnostic parameters in sepsis-related fatalities

Abstract: The aims of this study were to investigate the usefulness of serum C-reactive protein, procalcitonin, tumor necrosis factor alpha, interleukin-6, and interleukin-8 as postmortem markers of sepsis and to compare C-reactive protein and procalcitonin values in serum, vitreous humor, and cerebrospinal fluid in a series of sepsis cases and control subjects, in order to determine whether these measurements may be employed for the postmortem diagnosis of sepsis. Two study groups were formed, a sepsis group (eight subjects coming from the intensive care unit of two university hospitals, with a clinical diagnosis of sepsis in vivo) and control group (ten autopsy cases admitted to two university medicolegal centers, deceased from natural and unnatural causes, without elements to presume an underlying sepsis as the cause of death). Serum C-reactive protein and procalcitonin concentrations were significantly different between sepsis cases and control cases, whereas serum tumor necrosis factor alpha, interleukin-6, and interleukin-8 values were not significantly different between the two groups, suggesting that measurement of interleukin-6, interleukin-8, and tumor necrosis factor alpha is non-optimal for postmortem discrimination of cases with sepsis. In the sepsis group, vitreous procalcitonin was detectable in seven out of eight cases. In the control group, vitreous procalcitonin was clearly detectable only in one case, which also showed an increase of all markers in serum and for which the cause of death was myocardial infarction associated with multi-organic failure. According to the results of this study, the determination of vitreous procalcitonin may be an alternative to the serum procalcitonin for the postmortem diagnosis of sepsis.