Showing papers in "International Journal of Legal Medicine in 2018"

Developmental validation of GlobalFiler™ PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples

Abstract: The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.

DNA methylation in ELOVL2 and C1orf132 correctly predicted chronological age of individuals from three disease groups.

Abstract: Improving accuracy of the available predictive DNA methods is important for their wider use in routine forensic work. Information on age in the process of identification of an unknown individual may provide important hints that can speed up the process of investigation. DNA methylation markers have been demonstrated to provide accurate age estimation in forensics, but there is growing evidence that DNA methylation can be modified by various factors including diseases. We analyzed DNA methylation profile in five markers from five different genes (ELOVL2, C1orf132, KLF14, FHL2, and TRIM59) used for forensic age prediction in three groups of individuals with diagnosed medical conditions. The obtained results showed that the selected age-related CpG sites have unchanged age prediction capacity in the group of late onset Alzheimer's disease patients. Aberrant hypermethylation and decreased prediction accuracy were found for TRIM59 and KLF14 markers in the group of early onset Alzheimer's disease suggesting accelerated aging of patients. In the Graves' disease patients, altered DNA methylation profile and modified age prediction accuracy were noted for TRIM59 and FHL2 with aberrant hypermethylation observed for the former and aberrant hypomethylation for the latter. Our work emphasizes high utility of the ELOVL2 and C1orf132 markers for prediction of chronological age in forensics by showing unchanged prediction accuracy in individuals affected by three diseases. The study also demonstrates that artificial neural networks could be a convenient alternative for the forensic predictive DNA analyses.

Ancestry inference of 96 population samples using microhaplotypes.

Abstract: Microhaplotypes have become a new type of forensic marker with a great ability to identify and deconvolute mixtures because massively parallel sequencing (MPS) allows the alleles (haplotypes) of the multi-SNP loci to be determined directly for an individual. As originally defined, a microhaplotype locus is a short segment of DNA with two or more SNPs defining three or more haplotypes. The length is short enough, less than about 300 bp, that the read length of current MPS technology can produce a phase-known sequence of each chromosome of an individual. As part of the discovery phase of our studies, data on 130 microhaplotype loci with estimates of haplotype frequency data on 83 populations have been published. To provide a better picture of global allele frequency variation, we have now tested 13 more populations for 65 of the microhaplotype loci from among those with higher levels of inter-population gene frequency variation, including 8 loci not previously published. These loci provide clear distinctions among 6 biogeographic regions and provide some information distinguishing up to 10 clusters of populations.

Assessment of the Precision ID Ancestry panel

Abstract: The ability to provide accurate DNA-based forensic intelligence requires analysis of multiple DNA markers to predict the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of biological evidence. Massively parallel sequencing (MPS) enables the analysis of hundreds of DNA markers in multiple samples simultaneously, increasing the value of the intelligence provided to forensic investigators while reducing the depletion of evidential material resulting from multiple analyses. The Precision ID Ancestry Panel (formerly the HID Ion AmpliSeq™ Ancestry Panel) (Thermo Fisher Scientific) (TFS)) consists of 165 autosomal SNPs selected to infer BGA. Forensic validation criteria were applied to 95 samples using this panel to assess sensitivity (1 ng-15 pg), reproducibility (inter- and intra-run variability) and effects of compromised and forensic casework type samples (artificially degraded and inhibited, mixed source and aged blood and bone samples). BGA prediction accuracy was assessed using samples from individuals who self-declared their ancestry as being from single populations of origin (n = 36) or from multiple populations of origin (n = 14). Sequencing was conducted on Ion 318™ chips (TFS) on the Ion PGM™ System (TFS). HID SNP Genotyper v4.3.1 software (TFS) was used to perform BGA predictions based on admixture proportions (continental level) and likelihood estimates (sub-population level). BGA prediction was accurate at DNA template amounts of 125pg and 30pg using 21 and 25 PCR cycles respectively. HID SNP Genotyper continental level BGA assignments were concordant with BGAs for self-declared East Asian, African, European and South Asian individuals. Compromised, mixed source and admixed samples, in addition to sub-population level prediction, requires more extensive analysis.

The development of a post-mortem interval estimation for human remains found on land in the Netherlands

Abstract: The decomposition process of human remains can be used to estimate the post-mortem interval (PMI), but decomposition varies due to many factors. Temperature is believed to be the most important and can be connected to decomposition by using the accumulated degree days (ADD). The aim of this research was to develop a decomposition scoring method and to develop a formula to estimate the PMI by using the developed decomposition scoring method and ADD. A decomposition scoring method and a Book of Reference (visual resource) were made. Ninety-one cases were used to develop a method to estimate the PMI. The photographs were scored using the decomposition scoring method. The temperature data was provided by the Royal Netherlands Meteorological Institute. The PMI was estimated using the total decomposition score (TDS) and using the TDS and ADD. The latter required an additional step, namely to calculate the ADD from the finding date back until the predicted day of death. The developed decomposition scoring method had a high interrater reliability. The TDS significantly estimates the PMI (R 2 = 0.67 and 0.80 for indoor and outdoor bodies, respectively). When using the ADD, the R 2 decreased to 0.66 and 0.56. The developed decomposition scoring method is a practical method to measure decomposition for human remains found on land. The PMI can be estimated using this method, but caution is advised in cases with a long PMI. The ADD does not account for all the heat present in a decomposing remain and is therefore a possible bias.

As solid as a rock-comparison of CE- and MPS-based analyses of the petrosal bone as a source of DNA for forensic identification of challenging cranial bones.

Abstract: Short tandem repeat (STR) typing from skeletal remains can be a difficult task. Dependent on the environmental conditions of the provenance of the bones, DNA can be degraded and STR typing inhibited. Generally, dense and compact bones are known to preserve DNA better. Several studies already proved that femora and teeth have high DNA typing success rates. Unfortunately, these elements are not present in all cases involving skeletal remains. Processing partial or singular skeletal elements, it is favorable to select bone areas where DNA preservation is comparably higher. Especially, cranial bones are often accidentally discovered during criminal investigations. The cranial bone is composed of multiple parts. In this examination, we evaluated the potential of the petrous bone for human identification of skeletal remains in forensic case work. Material from different sections of eight unknown cranial bones and—where available—additionally other skeletal elements, collected at the DNA department of the Institute of Legal Medicine in Ulm, Germany, from 2010 to 2017, were processed with an optimized DNA extraction and STR typing strategy. The results highlight that STR typing from the petrous bones leads to reportable profiles in all individuals, even in cases where the analysis of the parietal bone failed. Moreover, the comparison of capillary electrophorese (CE) typing to massively parallel sequencing (MPS) analysis shows that MPS has the potential to analyze degraded human remains and is even capable to provide additional information about phenotype and ancestry of unknown individuals.

Forensic features and phylogenetic analyses of Sichuan Han population via 23 autosomal STR loci included in the Huaxia Platinum System.

Abstract: Short tandem repeats (STRs) are DNA regions with a variable number of repeated units (2–6 bp) and attractive to genetic application like forensic, population genetics, and anthropological studies. The Huaxia Platinum System allows the multiplex amplification of 23 autosomal STR loci and two gender determination loci. In the present study, genetic polymorphisms of these 23 autosomal STR loci were first obtained from 309 Sichuan Han individuals, southwest China. No evidence of deviation from the Hardy-Weinberg equilibrium (HWE) was identified. The combined match probability (CMP), combined power of discrimination (CPD), and combined power of exclusion (CPE) are 1.087 × 10−27, 0.999999999999999999999999999, and 0.9999999996 in the Sichuan Han Chinese population, respectively. The inter-population differentiation, PCA, MDS, and phylogenetic analysis revealed that Sichuan Han had a closer genetic relationship with neighboring Han Chinese populations. Our findings indicated that the 23 autosomal STR loci are polymorphic and informative in studied southwestern Chinese Han population and can be utilized as a powerful tool for forensic applications. In addition, population comparisons demonstrated that the genetic similarities widely existed among Han Chinese populations distributed in different administrative divisions, and genetic differences had existed between minorities (most prominently for Uyghur and Kazakh) and Han groups.

The use of gelatine in wound ballistics research

Abstract: Blocks of gelatine are used in both lethality and survivability studies for broadly the same reason, i.e. comparison of ammunition effects using a material that it is assumed represents (some part of) the human body. The gelatine is used to visualise the temporary and permanent wound profiles; elements of which are recognised as providing a reasonable approximation to wounding in humans. One set of researchers aim to improve the lethality of the projectile, and the other to understand the effects of the projectile on the body to improve survivability. Research areas that use gelatine blocks are diverse and include ammunition designers, the medical and forensics communities and designers of ballistic protective equipment (including body armour). This paper aims to provide an overarching review of the use of gelatine for wound ballistics studies; it is not intended to provide an extensive review of wound ballistics as that already exists, e.g. Legal Med 23:21-29, 2016. Key messages are that test variables, projectile type (bullet, fragmentation), impact site on the body and intermediate layers (e.g. clothing, personal protective equipment (PPE)) can affect the resulting wound profiles.

Toward the adoption of cementochronology in forensic context.

Abstract: Because acellular dental cementum is considered to be formed continually throughout life and to not undergo remodeling processes, cementochronology is considered to be a method with the potential for directly assessing chronological age. Considering that most previous studies on humans have assumed the superior performance of this method, it is surprising that this technique is not more widely adopted in anthropology. To understand this controversy, we highlight that there is no standardized procedure for sample preparation. The numerous technical approaches that exist impact the reliability of the method, and the recent creation of an international work group (Cementochronology Research Program) demonstrates the need for researchers to share their experience to overcome these obstacles. This paper aims to address this paradox by debating the aspects that contribute to the limited use of this method and by illustrating its potential through an application on forensic cases. A protocol, which was recently certified according to the ISO-9001, was applied to nine anthropological cases from the Forensic Medicine Institute of Lille (northern France) and compared with routine osteological and dental methods. The results show that traditional methods matched the known age due to the wide extent of their range, while the accuracy and precision of cementochronological estimates was also notable. This paper establishes that cementochronology may serve as a particularly important tool for age estimation for forensic anthropologists and should, at least, be used in addition to other methods.

Acute phase response after fatal traumatic brain injury.

Abstract: An inflammatory response occurring after fatal traumatic brain injury (TBI) initiates time-dependent cascades of acute phase response. This may offer the potential to monitor postmortem biomarker levels of several pro-inflammatory cytokines to gain information about the cause of death and the trauma survival time. Cerebrospinal fluid (CSF) and serum samples were collected from forensic autopsies of 95 adult cadavers after postmortem intervals up to 6 days. The cases were divided according to their cause of death into fatal TBI (n = 46) with different survival times and age- and gender-matching non-TBI fatalities as controls (n = 49). Quantitative marker levels of interleukin-6 (IL-6), ferritin, soluble tumor necrosis factor receptor type 1, C-reactive protein, and lactate dehydrogenase were analyzed using immunoassays. Standardized statistical tests were performed to differentiate causes of death and survival time of TBI cases. The CSF IL-6, ferritin, and LDH levels after TBI were significantly higher than those in the controls (p < 0.001). Only serum IL-6 values showed comparable differences (p < 0.05). Both CSF and serum ferritin levels were discriminative between early and delayed death after TBI (p < 0.05). There were partly distinctive correlations between marker levels in both fluids with rising values after longer survival. There were up to moderate correlation between the marker levels and the postmortem interval due to postmortem hemolysis. However, neither CSF nor serum level ranges were affected by the age or gender of the subjects. This study is the first to measure all five proteins systematically in postmortem trauma cases. Ferritin and IL-6 proved themselves to be interesting postmortem biomarkers to provide specific information on the injury pattern and the survival time of traumatic fatalities. Such forensic investigations could serve as inexpensive and fast laboratory tests.

Exome analysis in 34 sudden unexplained death (SUD) victims mainly identified variants in channelopathy-associated genes.

Abstract: Sudden cardiac death (SCD) is one of the major causes of mortality worldwide, mostly involving coronary artery disease in the elderly. In contrary, sudden death events in young victims often represent the first manifestation of undetected genetic cardiac diseases, which remained without any symptoms during lifetime. Approximately 30% of these sudden death cases have no definite cardiac etiology after a comprehensive medicolegal investigation and are therefore termed as sudden unexplained death (SUD) cases. Advances in high-throughput sequencing approaches have provided an efficient diagnostic tool to identify likely pathogenic variants in cardiovascular disease-associated genes in otherwise autopsy-negative SUD cases. The aim of this study was to genetically investigate a cohort of 34 unexplained death cases by focusing on candidate genes associated with cardiomyopathies and channelopathies. Exome analysis identified potentially disease-causing sequence alterations in 29.4% of the 34 SUD cases. Six (17.6%) individuals had variants with likely functional effects in the channelopathy-associated genes AKAP9, KCNE5, RYR2, and SEMA3A. Interestingly, four of these six SUD individuals were younger than 18 years of age. Since the total SUD cohort of this study included five children and adolescents, post-mortem molecular autopsy screening indicates a high diagnostic yield within this age group. Molecular genetic testing represents a valuable approach to uncover the cause of death in some of the SUD victims; however, 70–80% of the cases still remain elusive, emphasizing the importance of additional research to better understand the pathological mechanisms leading to a sudden death event.

Massively parallel sequencing-enabled mixture analysis of mitochondrial DNA samples.

Abstract: The mitochondrial genome has a number of characteristics that provide useful information to forensic investigations. Massively parallel sequencing (MPS) technologies offer improvements to the quantitative analysis of the mitochondrial genome, specifically the interpretation of mixed mitochondrial samples. Two-person mixtures with nuclear DNA ratios of 1:1, 5:1, 10:1, and 20:1 of individuals from different and similar phylogenetic backgrounds and three-person mixtures with nuclear DNA ratios of 1:1:1 and 5:1:1 were prepared using the Precision ID mtDNA Whole Genome Panel and Ion Chef, and sequenced on the Ion PGM or Ion S5 sequencer (Thermo Fisher Scientific, Waltham, MA, USA). These data were used to evaluate whether and to what degree MPS mixtures could be deconvolved. Analysis was effective in identifying the major contributor in each instance, while SNPs from the minor contributor’s haplotype only were identified in the 1:1, 5:1, and 10:1 two-person mixtures. While the major contributor was identified from the 5:1:1 mixture, analysis of the three-person mixtures was more complex, and the mixed haplotypes could not be completely parsed. These results indicate that mixed mitochondrial DNA samples may be interpreted with the use of MPS technologies.

Accuracy of the third molar maturity index in assessing the legal age of 18 years: a systematic review and meta-analysis.

Abstract: The age estimation is a complex procedure required in the daily practice of legal medicine. The maturity of third molars stands out by the age of 18 because these teeth are still in development. This systematic review aimed to assess the accuracy of the third molar maturity index (I 3M), proposed by Cameriere et al. (2008), in discriminating whether an individual is under or over 18 years. Seven electronic databases were screened: PubMed, Scopus, ISI Web of Science, Cochrane Library, LILACS, SIGLE, and CAPES. Eligible studies included an assessment of I 3M accuracy at the 0.08 cut-off value. The quality assessment was performed by using QUADAS 2. Three meta-analyses (MA) were accomplished: overall, one for males and another for females. From 2397 articles identified, 16 met the eligibility criteria. Of these, two showed high risk of bias, one in the reference standard domain and the other in the flow and timing domain. The percentage of individuals correctly classified ranged from 72.4 to 96.0%. The overall MA showed pooled sensitivity of 0.86 (0.84 to 0.87; p = 0.0000) and pooled specificity of 0.93 (0.92 to 0.94; p = 0.0000). The AUC (area under the summary receiver operator characteristics curve) and DOR (diagnostic odds ratio) values were, respectively, 0.9652 and 104.68, indicating an overall high discrimination effect. Separately, better results of accuracy were found for males. High heterogeneity was achieved for both sensibility (94.6%) and specificity (88.8%). We conclude that the I 3M is a suitable and useful method for estimating adulthood regarding forensic purposes, regardless of gender.

Forensic application of epidermal AQP3 expression to determination of wound vitality in human compressed neck skin

Abstract: In forensic practices, it is often difficult to determine wound vitality in compression marks of the neck with naked eyes. AQP1 and AQP3 are the major water channels associated with skin. Thus, we immunohistochemically examined the expression of AQP1 and AQP3 in neck skin samples to discuss their forensic applicability to determination of the wound vitality. Skin samples were obtained from 56 neck compression cases (hanging, 35 cases; strangulation, 21 cases). The intact skin from the same individual was taken as a control. Although AQP1 was immnunostained in dermal capillaries in both the neck compression marks and intact skin samples, there was no significant difference in the magnitude of AQP1 expression between both groups. On the contrary, AQP3-positive signals could be faintly detected in uninjured skin samples, and the positive signals seemed more intense in the keratinocytes in compression regions. Morphometrical analyses revealed that the ratio of AQP3-expressed keratinocytes was significantly enhanced in neck compression regions, compared with control groups. From the viewpoints of forensic pathology, immunohistochemical detection of AQP3 in the neck skin can be considered a valuable marker to diagnose the trace of antemortem compression.

Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification

Abstract: The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

Species identification of adult African blowflies (Diptera: Calliphoridae) of forensic importance.

Abstract: Necrophagous blowflies can provide an excellent source of evidence for forensic entomologists and are also relevant to problems in public health, medicine, and animal health. However, access to useful information about these blowflies is constrained by the need to correctly identify the flies, and the poor availability of reliable, accessible identification tools is a serious obstacle to the development of forensic entomology in the majority of African countries. In response to this need, a high-quality key to the adults of all species of forensically relevant blowflies of Africa has been prepared, drawing on high-quality entomological materials and modern focus-stacking photomicroscopy. This new key can be easily applied by investigators inexperienced in the taxonomy of blowflies and is made available through a highly accessible online platform. Problematic diagnostic characters used in previous keys are discussed.

Age estimation combining radiographic information of two dental and four skeletal predictors in children and subadults

Abstract: Improved age estimates may result from combining different age predictors. This study aimed to validate age estimation performances combining the radiographic development of teeth, cervical vertebrae, hand and wrist bones, skull, and mandible. The sampled consisted of 256 individuals aged between 4 and 20 years. Bayes’ rule with a multivariate continuation ratio model was applied for the distribution of the dental scores. The additional age information of the skeletal variables was assessed extending the dental model separately and combining the variables. The performances of all models were quantified and compared using mean error (ME), mean absolute error (MAE), and root mean squared error (RMSE). The best performance resulted combining all variables (ME − 0.04 for F and M; MAE 0.91 for F and 0.95 for M; RMSE 1.14 for F and 1.19 for M). To improve performances and minimize radiation doses, the combination of teeth and hand and wrist bones information is recommended.

Genomic portrait of population of Jharkhand, India, drawn with 15 autosomal STRs and 17 Y-STRs

Abstract: We analysed 15 autosomal STRs in 200 unrelated individuals (102 males and 98 females) and 17 Y-STRs in 102 unrelated males living in Jharkhand, India, to establish parameters of forensic interest. The examined autosomal STRs revealed high combined power of exclusion (CPE) and combined power of discrimination (CPD) as equal to 0.9999 and greater than 0.99999, respectively. The combined probability of match (CPm) and combined paternity index (CPI) for all 15 autosomal STR loci were found to be 5.15 × 10-18 and 6.83 × 105, respectively. A total of 97 unique haplotypes were obtained, of which 93 were observed only once. The haplotype diversity, discrimination capacity and matching probability for 17 Y-STR loci were 0.999, 0.951 and 1.09 × 10-2, respectively. The highest gene diversity values at the single copy locus DYS635 and multi-copy locus DYS385 a/b were 0.785 and 0.823, respectively.

Molecular tissue changes in early myocardial ischemia: from pathophysiology to the identification of new diagnostic markers

Abstract: Diagnosing early myocardial ischemia (the initial 4 to 6 h after interruption of blood flow to part of the myocardium) remains a challenge for clinical and forensic pathologists. Several immunohistochemical markers have been proposed for improving postmortem detection of early myocardial ischemia; however, no single marker appears to be both sufficiently specific as well as sensitive. This review summarizes the diverse categories of molecular tissue markers that have been investigated in human autopsy samples with acute myocardial infarction as well as in the well-established and widely used in vivo animal model of early myocardial ischemia (permanent ligation of the coronary artery). Recently identified markers appearing during the initial 2 h of myocardial ischemia are highlighted. Among them, only six were tested for specificity (C5b-9, hypoxia-inducible factor 1-alpha, vascular endothelial growth factor, heart fatty acid binding protein, connexin 43, and JunB). Despite the discovery of several potentially promising markers (in terms of early expression and specificity), many of them remain to be tested and validated for application in routine diagnostics in clinical and forensic pathology. In particular, research investigating the postmortem stability of these markers is required before any might be implemented into routine diagnostics. Establishing a standardized panel of immunohistochemical markers may be more useful for improving sensitivity and specificity than searching for a single marker.

The third molar maturity index in indicating the legal adult age in Kosovar population.

Abstract: The third molar tooth, known as the “wisdom tooth,” is the only tooth that can be used to determine legal adult age. This study aimed to test the accuracy of Cameriere’s third molar maturity index (I3M) in assessing the legal adult age of 18 years in the Kosovar population. Orthopantomographs (OPTs) of 1221 healthy living Kosovar individuals, aged between 12 and 23 years and with no congenital or developmental anomalies, were analyzed. Intra-class correlation coefficients were 0.858 (95% CI, 0.786 to 0.906) and 0.852 (95% CI, 0.779 to 0.903) for intra-rater and for inter-rater agreement, respectively. The sample was divided into training dataset (800 OPTs) and test dataset (421 OPTs). The training dataset was used to generate the logistic regression model, while the test dataset was used to study the performance of the model. I3M and gender as independent variables and adult age (≥ 18 years) or minor age (< 18 years) as the dependent variable were used for logistic regression analysis. The receiver operating curve (ROC) analysis was used to determine the specific cut-off value of I3M for predicting adult age. The results showed that only I3M statistically significantly contributed to discriminating adults and minors. ROC analysis showed that the cut-off value of I3M < 0.08 was the best in discriminating adults and minors. An analysis of the test dataset (421 OPTs) showed that as I3M decreased the age gradually increased. The performance of the cut-off value of I3M < 0.08, to discriminate between adults and minors, was analyzed by contingency tables for both sexes. In males, the accurate classification (Acc) was 0.968 (95% CI, 0.926 to 0.985), the sensitivity (Se) was 0.962 (95% CI, 0.925 to 0.978), and the specificity (Sp) was 0.976 (95% CI, 0.929 to 0.995). The Bayes post-test probability (Bayes PTP) was 0.975 (95% CI, 0.905 to 1.00). In females, Acc was 0.909 (95% CI, 0.870 to 0.917), Se and Sp were 0.826 (95% CI, 0.787 to 0.834) and 0.991 (95% CI, 0.953 to 1.00) respectively, while Bayes PTP was 0.989 (95% CI, 0.926 to 1.00). Our data support the usefulness of I3M < 0.08 to indicate legal adult age in a Kosovar population.

Hair analysis does not allow to discriminate between acute and chronic administrations of a drug in young children.

Abstract: There are many differences between the hair from children and that of adult subjects, the hair being thinner, more porous with a different growth rate from the usual 1 cm/month observed in adults. In order to determine whether hair analysis could discriminate between chronic use and acute administration of a drug in children like in adults, we analyzed hair from 18 children aged between 1 day and 15 years in whom the administration of different drugs was known (single therapeutic administration or acute intoxication). A strand of hair was sampled within 1 to 45 days after treatment or intoxication. Analysis was conducted using LC/MS/MS. In the 10 youngest children, aged between 1 day and 29 months, the compounds administered in hospital or responsible for intoxication (lidocaine, ropivacaine, diazepam, midazolam, levetiracetam, morphine, ketamine, methadone, buprenorphine, THC, MDMA) were found in all segments of the hair independently of the time of sampling (1–45 days after ingestion). The concentrations detected were similar along the hair shaft, showing a radial diffusion and incorporation of the analytes in the hair of young children from the sebum. Concentrations could be very high when sampled shortly after administration (72 ng/mg for methadone, 75 ng/mg for MDMA after 3 days) and lower when sampling later (1.2 ng/mg for MDMA after 45 days). In these cases, hair analysis allowed to highlight the compounds responsible for intoxication even when they had disappeared from the blood or urine but should not be used to discriminate long-term exposure to a drug. In the eight remaining children aged from 34 months to 15 years, the drugs used in hospital (lidocaine, diazepam, morphine) or responsible for intoxication (THC, codeine, buprenorphine) were not found in any analyzed segments sampled 1 to 5 days after administration of the drugs, in agreement with the non-incorporation of the drugs from the sebum into the hair. For those children aged over 34 months, hair analysis allows to determine the chronic administration of a drug, like in adults.

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples

Abstract: DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.

The comparative performance of PMI estimation in skeletal remains by three methods (C-14, luminol test and OHI): analysis of 20 cases

Abstract: When estimating post-mortem interval (PMI) in forensic anthropology, the only method able to give an unambiguous result is the analysis of C-14, although the procedure is expensive. Other methods, such as luminol tests and histological analysis, can be performed as preliminary investigations and may allow the operators to gain a preliminary indication concerning PMI, but they lack scientific verification, although luminol testing has been somewhat more accredited in the past few years. Such methods in fact may provide some help as they are inexpensive and can give a fast response, especially in the phase of preliminary investigations. In this study, 20 court cases of human skeletonized remains were dated by the C-14 method. For two cases, results were chronologically set after the 1950s; for one case, the analysis was not possible technically. The remaining 17 cases showed an archaeological or historical collocation. The same bone samples were also screened with histological examination and with the luminol test. Results showed that only four cases gave a positivity to luminol and a high Oxford Histology Index (OHI) score at the same time: among these, two cases were dated as recent by the radiocarbon analysis. Thus, only two false-positive results were given by the combination of these methods and no false negatives. Thus, the combination of two qualitative methods (luminol test and microscopic analysis) may represent a promising solution to cases where many fragments need to be quickly tested.

Investigation of DNA transfer onto clothing during regular daily activities

Abstract: Low levels of DNA from an unidentified human source, often referred to as trace DNA, are ubiquitous, can be transferred onto objects by either direct or indirect methods and have an unknown longevity in situ. Clothing items from crime scenes are often submitted for trace DNA analysis, usually in attempt to identify a person of interest. This study examined the transfer of DNA onto three 10 × 10 cm areas located on the front, back and shoulder of an individual’s external clothing (n = 300) during a regular day’s activity. After wearing for a day, the DNA quantity on all three areas increased approximately 8-fold, which usually corresponded with an increase in the endogenous DNA from the wearer on the front area of the shirt. However, the back area of the shirt was more likely to demonstrate mixtures of endogenous and extraneous DNA. An additional study was also carried out to examine whether domestic laundering is a possible mechanism for the transfer of foreign DNA onto freshly laundered items and revealed that 74% of UV-treated cotton swatch samples produced DNA profiles after laundry with household garments. In summary, this study highlights the ease of DNA transfer onto an individual’s external clothing during a regular day, and that extraneous DNA may be already on the clothing item prior to it being worn. The study provides empirical data to assist in the interpretation of trace DNA profiles and support a Bayesian approach to estimate statistical likelihoods for the transfer of foreign DNA.

Application of 3D models of palatal rugae to personal identification: hints at identification from 3D-3D superimposition techniques.

Abstract: Palatal rugae are known in literature as individualizing anatomical structures with a strong potential for personal identification. However, a 3D assessment of their uniqueness has not yet been performed. The present study aims at verifying the uniqueness of 3D models of the palate. Twenty-six subjects were recruited among the orthodontic patients of a private dental office; from every patient, at least two dental casts were taken in different time periods, for a total of 62 casts. Dental casts were digitized by a 3D laser scanner (iSeries, Dental Wings©, Montreal, Canada). The palatal area was identified, and a series of 250 superimpositions was then performed automatically through VAM©software in order to reach the minimum point-to point distance between two models. In 36 matches the models belonged to the same individual, whereas in 214 mismatches they came from different subjects. The RMS (root mean square) of point-to-point distances was then calculated by 3D software. Possible statistically significant differences were assessed through Mann-Whitney test (p < 0.05). Results showed a statistically significant difference in RMS mean point-to-point distance between matches (mean 0.26 mm; SD 0.12) and mismatches (mean 1.30; SD 0.44) (p < 0.0001).All matches reached an RMS value below 0.50 mm. This study first provided an assessment of uniqueness of palatal rugae, based on their anatomical 3D conformations, with consequent applications to personal identification.

Improved Y-STR typing for disaster victim identification, missing persons investigations, and historical human skeletal remains

Abstract: Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861-1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874-1877), the Seven Years' War (1756-1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.

Forensic age estimation using computed tomography of the medial clavicular epiphysis: a systematic review.

Abstract: Medicolegal physicians are increasingly called upon to aid in determining the administrative age group affiliation of refugees with questionable unaccompanied minor claims. According to guidelines for forensic age assessment, age differentiation along the 18-year-old cut-off relies on clavicular ossification. The thin-slice computed tomography scan (TSCTs) of the medial clavicular epiphysis (MCE) is one of the methods contributing to this assessment, though it is not yet universally accepted. The aim of this systematic review was to identify scientific papers where age was assessed using TSCTs of the MCE and to observe whether this examination was reproducible and reliable in estimating a person's age relative to the 18-year-old threshold. A search algorithm was applied to several databases to identify articles in accordance with the PRISMA (Preferred Reporting Items for Systematic-Reviews and Meta-Analyses) statement. One boxplot per article was constructed, separating by stage of maturation and sex. The 13 articles selected represented a sample of 5605 individuals (3396 males, 2209 females) aged 10 to 35 years. All individuals classified as stages 4 and 5 were aged 18 years or older. The same result was obtained concerning stage 3c, except in one article. The results thus appear reliable and reproducible, in particular, with respect to the 18-year-old threshold; medicolegal physicians should be able to estimate that all individuals in stages 4 and 5 are at least 18 years old. Additional studies applied to several other populations in the world should complement the selected studies.

Sex determination of a Tunisian population by CT scan analysis of the skull.

Abstract: It is widely accepted that the estimation of biological attributes in the human skeleton is more accurate when population-specific standards are applied. With the shortage of such data for contemporary North African populations, it is duly required to establish population-specific standards. We present here the first craniometric standards for sex determination of a contemporary Tunisian population. The aim of this study was to analyze the correlation between sex and metric parameters of the skull in this population using CT scan analysis and to generate proper reliable standards for sex determination of a complete or fragmented skull. The study sample comprised cranial multislice computed tomography scans of 510 individuals equally distributed by sex. ASIRTM software in a General ElectricTM workstation was used to position 37 landmarks along the volume-rendered images and the multiplanar slices, defining 27 inter-landmark distances. Frontal and parietal bone thickness was also measured for each case. The data were analyzed using basic descriptive statistics and logistic regression with cross-validation of classification results. All of the measurements were sexually dimorphic with male values being higher than female values. A nine-variable model achieved the maximum classification accuracy of 90% with −2.9% sex bias and a six-variable model yielded 85.9% sexing accuracy with −0.97% sex bias. We conclude that the skull is highly dimorphic and represents a reliable bone for sex determination in contemporary Tunisian individuals.

Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault-validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer.

Abstract: Sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. While reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. Especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. We report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the SERATEC PMB test, and its performance on real casework samples. The PMB test is a duplex test combining human hemoglobin and D-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. The results of this study showed that the duplex D-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of D-dimers. The method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. Results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases.

Validation and evaluation of measuring methods for the 3D documentation of external injuries in the field of forensic medicine

Abstract: Three-dimensional (3D) measurement techniques are gaining importance in many areas. The latest developments brought more cost-effective, user-friendly, and faster technologies onto the market. Which 3D techniques are suitable in the field of forensic medicine and what are their advantages and disadvantages? This wide-ranging study evaluated and validated various 3D measurement techniques for the forensic requirements. High-tech methods as well as low-budget systems have been tested and compared in terms of accuracy, ease of use, expenditure of time, mobility, cost, necessary knowhow, and their limitations. Within this study, various commercial measuring systems of the different techniques were tested. Based on the first results, one measuring system was selected for each technique, which appeared to be the most suitable for the forensic application or is already established in forensic medicine. A body of a deceased, a face and an injury of a living person, and a shoe sole were recorded by 11 people with different professions and previous knowledge using the selected systems. The results were assessed and the personal experiences were evaluated using a questionnaire. In addition, precision investigations were carried out using test objects. The study shows that the hand-held scanner and photogrammetry are very suitable for the 3D documentation of forensic medical findings. Their moderate acquisition costs and easy operation could lead to more frequent application in forensic medicine in the future. For special applications, the stripe-light scanner still has its justification due to its high precision, the flexible application area, and the high reliability. The results show that, thanks to the technological advances, the 3D measurement technology will have more and more impact on the routine of the forensic medical examination.