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

International Journal of Legal Medicine - In recent years, a lot of age prediction models based on different CpG motives in different cell types were published determining the biological age of a...     

Quantification of secondary dentine formation from orthopantomograms—a contribution to forensic age estimation methods in adults

The accuracy of most age determination methods in adults is unsatisfactory and therefore more precise methods are required. Teeth provide several useful points about an individuals age. With advancing age secondary dentine is deposited along the wall of the dental pulp chamber leading to a reduction in the size of the pulp cavity. These age-related changes can be determined and measured from dental radiographs. The aim of this study was to explore if measurements of the size of the pulp cavity performed on orthopantomograms can be used for individual age estimation. Measurements were made digitally for 6 types of teeth from orthopantomograms of 168 individuals aged between 14 and 81 years old. To compensate for errors due to the technique of radiography—for instance differences in angulation—ratios of distances were calculated: pulp/root length, pulp/tooth length and pulp/root width at three different root levels. The width ratios of the pulp cavity showed significant correlation to the chronological age and the coefficient of determination (r²) was highest in the upper lateral incisors (r²=0.913) when an exponential or a logistic regression model was constructed. At the same distance with a linear regression model the coefficient of determination (r²) reached 0.839.     

Comparison of different methods of DNA recovery and PCR amplification in STR profiling of casings—a retrospective study

International Journal of Legal Medicine - Casings represent common evidence in a forensic laboratory, due to high frequency of firearms usage during perpetration of criminal offenses. Possible DNA...     

Estimation of sex and age of “virtual skeletons”–a feasibility study

This article presents a feasibility study with the objective of investigating the potential of multi-detector computed tomography (MDCT) to estimate the bone age and sex of deceased persons. To obtain virtual skeletons, the bodies of 22 deceased persons with known age at death were scanned by MDCT using a special protocol that consisted of high-resolution imaging of the skull, shoulder girdle (including the upper half of the humeri), the symphysis pubis and the upper halves of the femora. Bone and soft-tissue reconstructions were performed in two and three dimensions. The resulting data were investigated by three anthropologists with different professional experience. Sex was determined by investigating three-dimensional models of the skull and pelvis. As a basic orientation for the age estimation, the complex method according to Nemeskéri and co-workers was applied. The final estimation was effected using additional parameters like the state of dentition, degeneration of the spine, etc., which where chosen individually by the three observers according to their experience. The results of the study show that the estimation of sex and age is possible by the use of MDCT. Virtual skeletons present an ideal collection for anthropological studies, because they are obtained in a non-invasive way and can be investigated ad infinitum.     

Magnetic resonance volumetric analysis of hippocampi in children in the age group of 6-to-12 years: a pilot study

Atrophy of the mesial temporal structures, especially the hippocampus, has been implicated in temporal lobe epilepsy. However, to date, there is very scant data regarding normal volumes of the hippocampus in the pediatric population. This is a pilot study to estimate the normal volumetric data for the Indian pediatric population between 6 and 12 years of age. We have also tried to understand whether age and gender have an effect on the hippocampal volumes in this age group. The study group comprised 20 children, 6–12-years old without history of epilepsy or other neurological deficits. There were nine boys and 11 girls. All scans were performed on a 1.5T GE echo speed scanner. 3D fast SPGR sequence was prescribed in the coronal plane. The images were post-processed on an Advantage Windows 3.1 workstation. Using an automated program, the same observer calculated the hippocampal area, in cubic centimeters, clockwise and anticlockwise. The clockwise/anticlockwise data were subjected to correlation analysis for detecting intra-observer agreement. The mean and SD for left and right hippocampal volumes were estimated. The lower and upper limits for normal hippocampal volumes were determined using 95% (± 2SD) limits on either side of the mean. In order to understand the effect of age on various hippocampal volumes we performed regression analysis. Mann–Whitneys test was used to test the significance of differences for gender variations. Correlation analysis established that there was intra-observer agreement. In the Indian pediatric population we have found the mean right hippocampal volume (RHV) to be 2.75 cm³ and mean left hippocampal volume (LHV) to be 2.49 cm³ . Mean hippocampal volume was found to be 2.67 cm³ (SD=0.42). The upper and lower limits for hippocampal volumes were 3.51 cm³ and 1.83 cm³, respectively, based on 95% (± 2SD) limits on either side of the mean. There was no effect of age or gender on the hippocampal volumes. In the Indian pediatric population we determined hippocampal volumes in a small series of healthy children. We found that hippocampal volumes 1.83 cm³ (2SD) can be considered to be abnormal. These findings can be used as normative data to evaluate cases of hippocampal sclerosis in the Indian population.     

Deceleration in maturation of bone during adolescent age in achondroplasia—a retrospective study using RUS scoring system

Objectives  Knowledge of bone age in achondroplasia is required for the prediction of adult height, timings of limb lengthening, and epiphysiodesis procedures. The purpose of this investigation was to determine the differences in skeletal age in achondroplasia and a control population with the Tanner–Whitehouse 3 method using the RUS score and to determine the right age for the interventional procedure for limb lengthening procedure or deformity correction in these patients. Materials and methods  Left hand radiographs of 34 patients (age range, 5–18 years) with achondroplasia were evaluated for skeletal age using the RUS scoring system, which were compared with the left hand radiographs of 41 patients (age range, 5–18 years) without achondroplasia measuring skeletal age. The difference in chronological age and RUS bone age were evaluated statistically according to gender and age group. Results  In the achondroplasia group, chronological age were 10.5 ± 4.3 years for males and 10.1 ± 3.6 years for females and RUS bone age were 9.2 ± 4.0 years for males and 8.9 ± 3.4 years for females, which showed statistically significantly difference (males p = 0.0003 and females p < 0.0001), while in the control group, chronological age were 11.1 ± 2.9 years for males and 10.7 ± 3.4 years for females and RUS bone age were 11.2 ± 3.4 years for males and 10.7 ± 3.3 years for females, which did not show statistically significantly difference (males p = 0.54 and females p = 0.76). Our finding suggested a delay of 1.4 years for males and 1.2 years for females in the maturation of bone in achondroplasia patients. Difference between chronological age and RUS bone age was 0.9 ± 1.1 for <10 years and 1.6 ± 0.9 for >10 years in the study group, while 0.1 ± 1.1 for <10 years and −0.2 ± 0.6 for >10 years in the control group, which also showed >statistically significant difference (<10 years p = 0.04 and >10 years p < 0.0001). These differences indicate that there was a delay in the maturation of bones by 1 year in the group <10 years and 1.8 years in the group >10 years in achondroplasia patients compared to nonachondroplasia patients. Conclusion  We recommend the use of the Tanner–Whitehouse 3 method especially the radius, ulna, short bone score to measure the skeletal age and to wait for a longer time before interventional procedures in achondroplasia patients. Each author certifies that he has no commercial associations (e.g., consultancies, stock ownership, equity interests, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article.     

Sensitive DIP-STR markers for the analysis of unbalanced mixtures from “touch” DNA samples

Casework samples collected for forensic DNA analysis can produce genomic mixtures in which the DNA of the alleged offender is masked by high quantities of DNA coming from the victim. DIP-STRs are novel genetic markers specifically developed to enable the target analysis of a DNA of interest in the presence of exceeding quantities of a second DNA (up to 1000-fold). The genotyping system, which is based on allele-specific amplifications of haplotypes formed by a deletion/insertion polymorphism (DIP) and a short tandem repeat (STR), combines the capacity of targeting the DNA of an individual with a strong identification power. Finally, DIP-STRs are autosomal markers therefore they can be applied to any combination of major and minor DNA.In this study we aimed to assess the ability of DIP-STRs to detect the minor contributor on challenging “touch” DNA samples simulated with representative crime-associated substrates and to compare their performance to commonly used male-specific markers (Y-STRs). As part of a comprehensive study on the relative DNA contribution of two persons handling the same object, we selected 71 unbalanced contact traces of which 14 comprised a male minor DNA contributor mixed to a female major DNA contributor. Using a set of six DIP-STRs, one to four markers were found to be informative for the minor DNA detection across traces. When compared to Y-STRs (14 traces), the DIP-STRs showed similar sensitivity in detecting the minor DNA across substrate materials with a similar occurrence of allele drop-out. Conversely, because of the sex combination of the two users of the object, 57 remaining traces could only be investigated by DIP-STRs. Of these, 30 minor DNA contributors could be detected by all informative markers while 12 traces showed events of allele drop-out. Finally, 15 traces showed no amplification of the minor DNA. These last 15 samples were mostly characterized by a combination of short handling time of the object, low DNA recovery and/or one single informative DIP-STR.In conclusion, the DIP-STRs represent alternative markers to help solving unbalanced two-source DNA mixtures, and also those produced from contact stains. These markers, in addition to a novel set of 10 DIP-STRs specifically developed according to forensic technical standards, will offer a valuable tool complementary to Y-STR markers.     

Evaluation of an automatic method for forensic age estimation by magnetic resonance imaging of the distal tibial epiphysis—a preliminary study focusing on the 18-year threshold

The contribution of magnetic resonance imaging to forensic age estimation of living individuals is a subject of ongoing research. Several studies have focused on the wrist, clavicle, knee, and foot, and shown interesting results regarding the 18-year threshold. Authors have developed various staging systems for epiphyseal growth plate maturation. However, the procedure is observer-dependent and requires experience and a certain time-learning process. To reduce these pitfalls, we have developed an automatic method based on the analysis of variations of gray levels within the epiphyseal–metaphyseal junction. This method was tested on 160 MRI scans of the distal tibial epiphysis in a sample of individuals aged from 8 to 25 years old, after intensity non-uniformity correction of all images. Results showed that in our sample, 97.4 % of males and 93.9 % of females aged 18 years or more would be correctly classified using this method. To our knowledge, automatic methods for MRI analysis have not been used in the field of age estimation yet. Further studies should be performed to assess the validity of this procedure.     

Evaluation of the significance of “redistribution” in I-123 IMP SPECT in cerebrovascular disorders—a comparative study with PET

We evaluated the significance of redistribution in¹²³I-IMP SPECT study using PET. Twelve lesions in ten patients were selected. These lesions were classified into the following three groups; ischemia, infarction of subacute phase and infarction of chronic phase. All lesions with ischemia or subacute infarction showed good or moderate redistribution. Three of four lesions with chronic infarction showed no redistribution. The mean values of rCBF and rCMRO₂ were highest in the lesions with good redistribution, but there was no differences in rOEF with the degree of redistribution. In conclusion, the tissue with good redistribution is not always viable and it was impossible to predict a region with increased rOEF by redistribution alone.This article was presented at the 1st EEC workshop on accuracy determination in PET, January 19–20th. 1989 Pisa, Italy (COMAC-BME Concerted Project Characterization and Standardization of PET Instrumentation)     

Influence of additional breast ultrasound on cancer detection in a cohort study for quality assurance in breast diagnosis—analysis of 102,577 diagnostic procedures

Purpose  

To determine the value of a breast ultrasound (US) examination in addition to mammography in cases of American College of Radiology (ACR) tissue pattern III and IV in symptomatic women and women at risk.     

“Black Bone” MRI: a potential non-ionizing method for three-dimensional cephalometric analysis—a preliminary feasibility study

Objectives:

CT offers a three-dimensional solution to the inaccuracies associated with lateral cephalogram-based cephalometric analysis. However, it is associated with significant concerns regarding ionizing radiation exposure. MRI offers a non-ionizing alternative, but this has been less well investigated. We present a novel gradient echo MRI sequence (“Black Bone”) and highlight the potential of this sequence in cephalometric analysis.

Methods:

After regional ethics approval, “Black Bone” imaging was obtained in eight patients in whom lateral cephalograms were available. “Black Bone”, T₁ and T₂ weighted spin echo imaging were obtained in the mid-sagittal plane, and measurements were compared with those obtained on the lateral cephalogram using both the Advantage Windows Workstation (GE Medical Systems, Buckinghamshire, UK) and the Dolphin^® cephalometric software (v. 11.5.04.23, Premium; Dolphin Imaging, Chatsworth, CA) by one assessor. Further assessment was made by scoring the ease of landmark identification on a ten-point scale.

Results:

“Black Bone” imaging surpassed T₁ and T₂ weighted imaging in terms of cephalometric landmark identification. A number of mid-sagittal cephalometric landmarks could not be clearly identified on T₂ weighted imaging, making analysis impossible. Measurements on “Black Bone” demonstrated the smallest discrepancy when compared with those obtained on the lateral cephalogram. The discrepancy seen between measurements completed on mid-sagittal MRI and the lateral cephalogram was compounded by inherent inaccuracies of the lateral cephalogram. The overall mean discrepancy between distance measurements on “Black Bone” imaging and those on the lateral cephalogram was 1–2 mm.

Conclusions:

Overall, “Black Bone” MRI offered an improved method of cephalometric landmark identification over routine MRI sequences, and provides a potential non-ionizing alternative to CT for three-dimensional cephalometrics.     

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

International Journal of Legal Medicine - DNA traces on clothes of drowned bodies can provide important evidence for police investigations, especially in cases of suspected suicides or homicides....     

Carbon-11 labelling of eticlopride in two different positions—a selective high-affinity ligand for the study of dopamine D-2 receptors using PET

A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((−)-(S)-5-chloro-3-ethyl-N-((1-ethyl-2-pyrrolidinyl)methyl)-6-methoxysalicylamide), was labelled with ¹¹C in two different positions ([N-ethyl-¹¹C]eticlopride (I)) and ([methyl-¹¹C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [¹¹C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [¹¹C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15–20% (EOB) with an overall synthesis time of 45–60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases >99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors.     

Determination of AB-FUBINACA and 5F-NPB-22 in rats exposed to “Bonsai” via inhalation and analysis of seized product

Synthetic cannabinoids (SCs) are the most rapidly growing class of recreational designer drugs. Illicit drug manufacturers began to produce herbal smoking materials under a variety of brands names, e.g. “Spice, K2, Bonsai, Yucatan Fire”. They were appeared on the European market in 2008. In this study, types of SCs in the herbal product sold as “Bonsai” in Turkey were determined and the identification of these substances in biological samples collected from rats depending on the inhalation of different amounts of plant material were aimed.To determine the SC species in the content of the plant product, analysis was performed via gas chromatography-mass spectrometry. Liquid-liquid extraction methods were utilized for blood and organ samples, while solid-phase extraction with β-glucuronidase enzyme treatment was applied for urine sample preparation. The relationship between the amount of burned plant and the amount of SCs accumulated in the blood, urine and organ samples of rats exposed to the plant product by inhalation was examined by liquid chromatography-tandem mass spectrometry.AB-FUBINACA and 5F-NPB-22 were detected in the herbal product. A significant correlation was found between the amount of herbal product inhaled and the prevalence of SCs, especially in lung tissues while no SCs were detected in the blood and urine samples of rats.There is currently no study on biological samples of individuals exposed to herbal products containing SCs by inhalation. Regarding the findings obtained in this study, the overall increase in the amounts of herbal product inhaled was demonstrated to pose a potential risk to humans.     

Temporal profiles of axonal injury following impact acceleration traumatic brain injury in rats—a comparative study with diffusion tensor imaging and morphological analysis

Traumatic axonal injury (TAI) plays a major role in the development of neurological impairments after traumatic brain injury (TBI), but it is commonly difficult to evaluate it precisely and early with conventional histological biomarkers, especially when the patients experience short-term survival after TBI. Diffusion tensor imaging (DTI) has shown some promise in detecting TAI, but longitudinal studies on the compromised white matter with DTI at early time points (≤72 h) following impact acceleration TBI are still absent. In the present study, rats were subjected to the Marmarou model and imaged with DTI at 3, 12, 24, and 72 h (n?=?5 each) post-injury. Using a region-of-interest-based approach, the regions of interest including the corpus callosum, bilateral external capsule, internal capsule, and pyramidal tract were studied. Two DTI parameters, fraction anisotropy and axial diffusivity, were significantly reduced from 3 to 72 h in each region after trauma, corresponding to the gradient of axonal damage demonstrated by immunohistochemical staining of β-amyloid precursor protein and neurofilament light chain. Remarkably, DTI changes predicted the approximate time in the acute phase following TBI. These results indicate that the temporal profiles of diffusion parameters in DTI may be able to provide a tool for early diagnosis of TAI following impact acceleration TBI.     

“Rust stain”: a rare mark in firearm suicide—a case report and review of the literature

International Journal of Legal Medicine - Rust stains are marks left by firearms in case of prolonged contact with the cutaneous surfaces. These peculiar signs along with other well-documented...     

Development and validation of a novel method “SpermX™” for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory’s ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows.Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1–18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.