Dental age estimation based on the radiographic visibility of the periodontal ligament in the lower third molars: application of a new stage classification.

The purpose of the present study was to test whether a new stage classification based on radiographic visibility of the periodontal ligament in lower third molars in a Chinese population can be used for the 18- and 21-year thresholds. A total of 1300 orthopantomograms, including equal numbers of northern Chinese males and females evenly distributed between the ages of 15 and 40 years, were analyzed. The stages were defined according to the visibility of periodontal ligament for the outer parts of lower third molar roots because the visibility status of the periodontal ligament between the roots of lower third molars is none valuable in many Chinese individuals. Stage 0 was first achieved at the age of 17.05 years in males and 17.46 years in females. The earliest appearance of stage 1 was 17.47 years in males and 17.86 years in females. Stage 2 was first observed in males at the age of 21.43 years and in females at the age of 21.96 years. The onset of stage 3 was first observed at the age of 25.83 years in males and 23.14 years in females. Compared with the stage classification of Olze et al., which also considers the mesial parts of the roots, the number of assessable cases could be significantly increased. Therefore, our novel approach is effective for age estimation in the Chinese population.

Age assessment in the living using modified Gustafson's criteria in a northern Chinese population.

Regressive dental changes appear to be suitable for age assessment in living adults. In 2012, Olze et al. showed that several criteria presented by Gustafson for extracted teeth can also be applied to orthopantomograms. The objective of this study was to test the applicability and reliability of this method in a Chinese population. For this purpose, 1300 orthopantomograms of 650 female and 650 male Chinese aged between 15 and 40 years were evaluated. The characteristics of secondary dentin formation, periodontal recession, attrition, and cementum apposition were reviewed in all the mandibular premolars. The sample was split into a training and test dataset. Based on the training set, the correlation of the individual characteristics with chronological age was studied with a stepwise multiple regression analysis, in which individual characteristics formed the independent variable. According to the results, the R values amounted to 0.80 to 0.83; the standard error of estimate was 4.29 to 4.75 years. By analyzing the test dataset, the accuracy of the present study, Olze's and Timme's formulas were determined by the difference between the estimated dental age (DA) and chronological age (CA). Taking both mean differences and mean absolute differences into account, the Chinese age estimation formula did not always perform better compared with Olze's and Timme's formulas for both males and females. It was concluded that this method can be used in Chinese individuals for age assessment. However, the applicability of the method is limited by the quality of the X-ray images, and the method should only be applied by experienced forensic odontologists.

Marker evaluation for differentiation of blood and menstrual fluid by methylation-sensitive SNaPshot analysis.

The differentiation of blood and menstrual fluid is especially important in cases of alleged sexual assault. While the identification of blood is relatively straightforward, the identification of menstrual fluid in trace evidence has been shown to be more challenging. This may be due to the complex nature of menstrual fluid that leads to intra- and inter-individual differences in composition. Nevertheless, recent advances in DNA methylation profiling have revealed promising markers for the differentiation of the two body fluids and furthermore, markers to distinguish menstrual fluid from vaginal fluid. A literature study was performed and in total, 11 markers were evaluated in this study of which seven could be validated for menstrual fluid and blood identification purposes. Marker "BLU2" (chr16:29757334) was identified as most suitable for differentiation of blood and menstrual fluid.

Studies on the radiographic visibility of the periodontal ligament in lower third molars: can the Olze method be used in the Chinese population?

The purpose of the present research is to study the radiographic visibility of the periodontal ligament in third molars from a northern Chinese population to determine if this methodology could be used for the 18-year threshold. A total of 1300 conventional orthopantomograms from 650 males and 650 females of northern Chinese subjects aged between 15 and 40 years were analyzed. The radiographic visibility of the periodontal membrane of fully mineralized third molars was assessed using the stages described by Olze et al. (2010). Stage 0 first appeared at the age of 17.05 years in males and at the age of 18.76 years in females. Stage 1 was first detected at 18.52 years in males and 19.59 years in females. Stage 2 was first achieved by males at the age of 22.33 years and by females at the age of 21.37 years. The earliest appearance of stage 3 was 26.85 years in males and 24.92 years in females. If stage 1 is determined, it is possible to prove that an individual has already attained the age of 18 years. Stages 2 and 3 can be used to determine that a person is over 21 years of age. However, it should also be noted that in 271 cases of tooth 38 and 255 cases of tooth 48, the visibility stage could not be reliably assessed because of fused roots or narrowed furcations. Therefore, this method seems to be of limited value for age estimation in the Chinese population.

Application of age assessment based on the radiographic visibility of the root pulp of lower third molars in a northern Chinese population.

The purpose of the present study was to explore the potential application of radiographic visibility of the root pulp in lower third molars in a northern Chinese population, in order to determine if this methodology can be used to prove whether a person has surpassed the thresholds of 18 and 21 years of age. A total of 1300 orthopantomograms comprising equal numbers of females and males evenly distributed between the ages of 15 and 40 years were analyzed. The radiographic visibility of the root pulp of the lower third molars was assessed using the stages described by Olze et al. (2010). Stage 1 first appeared at 19.25 years in males and at 20.73 years in females. The earliest appearance of stage 2 happened at 22.33 years in males and at 22.41 years in females. Stage 3 was achieved first at 26.45 years in males and at 27.66 years in females. It was concluded that stages 1, 2, and 3 can be used to show that a person is over 18 years of age. If stages 2 or 3 are determined, it is possible to prove that an individual has already attained the age of 21 years.

Age estimation of Chinese children based on second molar maturity.

In forensic medicine and many other fields, age estimation by the use of teeth is of great importance for the purpose of individual identification. In the past, however, age estimation based on the second molar mineralization was scarcely performed. In this study, a total of 1657 panoramic radiographs taken from 834 males and 823 females of northern Chinese origin in the age bracket 5 to 25 years were assessed. The mineralization status of the second molars was determined using the classification described by Demirjian et al. Results showed that the left and right, as well as maxillary and mandibular second molars were generally at similar stages of mineralization. The maxillary left second molars (27) at stage D, mandibular left second molars (37) at stages C, D, F, and G, and mandibular right second molars (47) at stages D, F, and G showed a significantly lower average age in female subjects than in male subjects. In males, fully developed second molars first appeared with 12 years of age; in females, stage H occurred with 11 years at the earliest. One male individual and one female individual with second molars showing stage G were 23 years old. It was concluded that second molars showing stage H do not exclude an age under 14 years and that second molars showing stage G do not exclude an age above 18 years.

[Forensic age estimation in juveniles and young adults: Reducing the range of scatter in age diagnosis by combining different methods]. / Forensische Altersschätzung bei Jugendlichen und jungen Erwachsenen: Streubreitenreduktion der Altersdiagnose durch Kombination der Methoden.

The dramatic rise in the number of refugees entering Germany means that age estimation for juveniles and young adults whose age is unclear but relevant to legal and official procedures has become more important than ever. Until now, whether and to what extent the combination of methods recommended by the Study Group on Forensic Age Diagnostics has resulted in a reduction of the range of scatter of the summarized age diagnosis has been unclear. Hand skeletal age, third molar mineralization stage and ossification stage of the medial clavicular epiphyses were determined for 307 individuals aged between 10 and 29 at time of death on whom autopsies were performed at the Institutes of Legal Medicine in Berlin, Frankfurt am Main and Hamburg between 2001 and 2011. To measure the range of scatter, linear regression analysis was used to calculate the standard error of estimate for each of the above methods individually and in combination. It was found that combining the above methods led to a reduction in the range of scatter. Due to various limitations of the study, the statistical parameters determined cannot, however, be used for age estimation practice.

Validation of an immunochromatographic D-dimer test to presumptively identify menstrual fluid in forensic exhibits.

Identifying the biological source of a crime scene stain can be crucial for police investigations in many scenarios. Blood is one of the most common fluids found, and accurate differentiation between peripheral blood and menstrual fluid could provide valuable information regarding the issue of consent in sexual assault cases. For the detection of menstrual fluid, no easy-to-use presumptive test is available to date. Therefore, this study aimed to validate a simple immunochromatographic test for the indication of menstrual fluid, focusing on a D-dimer assay. The Clearview® rapid D-dimer test provides a diagnostic assay for the detection of fibrin degradation products. We validated the sensitivity and robustness of the assay using fresh and dried menstrual fluid samples, body fluid mixtures, diluted samples, and casework swabs. Cross reactivity was tested for saliva, semen, vaginal fluid, and blood. No false positive results were obtained; it was possible to successfully analyze mixtures, highly diluted samples, and casework swabs. The results of this study indicate that the D-dimer assay reliably detects menstrual fluid in forensic exhibits and is easy to implement into the current workflow of body fluid identification.

Dental age estimation in living individuals using 3.0 T MRI of lower third molars.

In order to increase the validity of age estimation in adolescents and young adults when there is no legitimation for X-ray examinations, it seems desirable to be able to assess the mineralization of third molars using X-ray-free imaging procedures. In the present study, the mineralization stages of lower third molars were determined prospectively in 269 male and 248 female individuals aged 12 to 24 years using 3.0 T MRI. The classification system of Demirjian et al. was used to determine the stages. This study presents the minima and maxima, means and standard deviations, median values, and lower and upper quartiles separately for both sexes, for the mineralization stages B-H. Statistically significant sex differences were observed for the mineralization stages C, E, F, and G, and a faster developmental rate was observed for males. It was concluded that magnetic resonance imaging is an X-ray-free alternative to orthopantomography when assessing mineralization of third molars.

Development of two age estimation models for buccal swab samples based on 3 CpG sites analyzed with pyrosequencing and minisequencing.

The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites.

Assessment of the radiographic visibility of the periodontal ligament in the lower third molars for the purpose of forensic age estimation in living individuals.

The main criterion for dental age estimation in living individuals is the mineralisation of third molars. However, the mineralisation of third molars can be completed before the forensically relevant age of 18 years has been attained. In a material of 1,198 orthopantomograms from 629 females and 569 males aged between 15 and 40 years, the radiographic visibility of the periodontal membrane of fully mineralised third molars was assessed according to stages 0, 1, 2 and 3. Stage 0 first appeared at the age of 17.2 years in females and at the age of 17.6 years in males. Stage 1 was first achieved by females between 18.9 and 20.0 years and by males between 20.1 and 20.2 years. The earliest appearance of stage 2 was between 22.5 and 23.1 years in females and at 22.3 years in males. The occurrence of stage 3 was first found between 24.6 and 25.2 years in females and between 25.4 and 26.2 years in males. If stage 1 is determined, it is, therefore, possible to prove that an individual has already attained the legally relevant age of 18 years. For stages 2 and 3, it can be stated beyond reasonable doubt that a person is over 21 years of age.

Studies on the chronology of third molar mineralization in First Nations people of Canada.

Forensic age estimation of living subjects has become increasingly important in recent years. One main criterion for dental age estimation in the relevant age group is the evaluation of third molar mineralization. In the present study, we determined the stages of third molar mineralization in 347 female and 258 male First Nations people of Canada aged 11 to 29 years based on radiological evidence from 605 conventional orthopantomograms. The results presented here provide useful data on the mineralization stages of third molars that can be used for forensic estimation of the minimum and most probable ages including the range of scatter of investigated persons.

Independent validation of body fluid-specific CpG markers and construction of a robust multiplex assay.

Potential forensic use of tissue-specific DNA methylation markers has recently been discussed for the identification of the biological source of a stain. In this study 13 promising markers were evaluated to identify suitable candidate markers for the development of a robust and reliable multiplex assay. The results of this study suggest that a combination of only four highly informative markers will be enough for clear body fluid identification. A multiplex assay was developed for the identification of menstrual blood, saliva, semen, and venous blood. This assay was successfully applied to the identification of these body fluids in mixtures and crime scene stains. The multiplex assay aids in the identification of not only single source body fluids but also of body fluid mixtures. The main advantage of using DNA methylation assays over alternative tests is that it can be applied at a later time point in the investigative process since testing is possible even after DNA analysis.

Application of mtDNA SNP analysis in forensic casework.

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain. To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.

Mitochondrial DNA control region diversity in hairs and body fluids of monozygotic triplets.

Length heteroplasmy of the homopolymeric cytosine stretch in the hypervariable region II of the mitochondrial D-loop was investigated in blood, buccal cells and hair shafts of monozygotic triplets. The proportions of length heteroplasmy were determined by cloning and sequencing of multiple independent clones. Blood and buccal cells showed an accumulation of molecules with one and two insertions of cytosine residues in relation to the Cambridge Reference Sequence (CRS). The results did not show statistically significant differences between blood and buccal cells of one and the same individual and also not between the three monozygotic brothers. In the hair samples a loss of cytosine residues was established in all three monozygotic individuals compared to blood and buccal cells, suggesting that this must be a regular process. Furthermore, the hair shaft samples showed significant differences between the frequencies of 7, 8 or 9 Cs in the poly C region comparing the three individuals (p<0.008) and in addition there were highly significant differences (p<0.0001) when comparing the results for six different hairs of each individual separately. From these results it can be assumed that besides a common genetic bottleneck during embryonic development, a post-embryonic bottleneck seems to exist in each hair follicle.