Inter-population variation of histomorphometric variables used in the estimation of age-at-death.

Population variation of several microscopic structures used in age-at-death estimation was assessed for three different population samples. The aim of the study was to determine if the need exists for population-specific standards when dealing with individuals of African and European origin. A total sample 223 bone sections from the anterior cortex of the femur (n = 99 black South Africans, n = 94 white South Africans and n = 30 Danish individuals) were analysed using a stereological protocol. Variables assessed included the average number of osteons per grid area (OPD), osteon size and Haversian canal size. ANCOVA was employed for assessment of statistically significant differences. The results indicated that OPD differed significantly between the three groups, but that osteon size was similar for all individuals. Haversian canal size showed unpredictable changes with age and high levels of variation, making it unsuitable to use for age estimation as a single factor. As there are conflicting opinions in the literature on whether to use population-specific equations for the estimation of age-at-death or not, this paper provided additional insight into the use of specific variables and its related variation between groups.

Histological age-at-death estimation in white South Africans using stereology.

Various methods are available for estimating age from skeletal remains, amongst them the use of histomorphometry. It is generally argued that age estimation standards are population specific, but this in itself creates problems as the reference samples used are often not large enough and/or lack substantial representation of all age cohorts. Traditional age methods have been shown to suffer from problems such as age mimicry. This paper aims at establishing histological age-at-death standards for the white South African population by supplementing the available sample (lacking an adequate number of young adults) with another sample of European descent to avoid over-estimation of age in younger individuals caused by age mimicry. Bone microstructures related to the number of osteons and fragments, osteon size and Haversian canal size that change with advancing age were used for the development of regression formulae. A histomorphometric assessment of the anterior cortex of the femur was done using stereology for the estimation of age at death. All sections were analysed using the optical fractionator and nucleator probes. A sample of 94 bone sections (n = 50 male, n = 44 females) of white South African individuals were used. A sample of Danish individuals (n = 14 males, n = 16 females) was combined with the South African sample to create a normal age distribution for the reference sample. Single and multiple regression equations were developed after randomly selecting a hold-out sample (n = 14) for validation. Osteon size (average length, surface area and volume) showed the highest correlation with age, followed by the number of osteons and fragments per grid area. Haversian canal size showed inconsistent changes with advancing age. Using the regression equations, predicted ages were obtained for the 14 individuals. RMSE values ranged between 14 and 17 years, which we deemed acceptable.

Post-mortem MRI-based volumetry of the hippocampus in forensic cases of decedents with severe mental illness.

A decrease in the volume of the hippocampus is associated with severe mental illness, especially schizophrenia, and has been studied extensively in the living using magnetic resonance imaging. Autopsy cohorts also represent a valuable data source for imaging studies. However, post-mortem magnetic resonance imaging (PMMRI) is subject to unique challenges, such as the lower core temperature of scanned subjects and the influence of decomposition processes. This study aimed to determine if results from in vivo studies could be replicated on a post-mortem cohort of decedents who suffered from severe mental illness. We included 96 decedents with either schizophrenia (n = 34), depressive disorder (n = 17), or no known psychiatric diagnosis (n = 45) from April 2015 to January 2017. All cases underwent a T2-weighted cerebral MRI less than 24 h before autopsy. We used a manual segmentation algorithm to define the hippocampus on coronal images and subsequently estimate the volume of the region. The group with schizophrenia had a statistically significant 9.5% decrease in mean hippocampal volume compared with control subjects, while the group with depression trended towards a reduced volume, but this difference was not statistically significant. Thus we were able to replicate previous results from in vivo studies. PMMRI has unique potential for research in that it can be combined with procedures possible only in the research fields of clinical pathology and forensic science, e.g. histopathological sampling.

Age estimation using bone mineral density in South Africans.

The use of bone mineral density (BMD) to predict age-at-death in skeletal remains provides a usable alternative to other methods because the values obtained are not observer-dependent. The aim of this study was to investigate the usability of BMD to estimate age in South African populations, and to assess inter-population variation and sex-specific differences in BMD values from the proximal end of the femur. In order to estimate age, regression analysis was done for the construction of population dependent formulae. The sample comprised of a total of 123 femora of black and white South Africans. DXA scans were performed using the Hologic Discovery system. Data analysis was done by employing independent-samples t-tests and correlation/regression analyses. The results indicated a statistically significant difference between black and white South Africans. Male groups were also significantly different from one another, but black and white females showed no significant differences. Linear regression analysis revealed a significant correlation between BMD values and age for the white population and the combined sample, but not for the black population. Bootstrapping were employed to confirm validity of the results. In conclusion, this study showed that the use of DXA measurements of the femur for estimating age may be used for the estimation of age-at-death in white South Africans, but more research is needed to better understand the relationship between bone mineral density and age in black South Africans.

The use of stereological methods in the histomorphometric assessment of bone for age-at-death estimation.

Stereological examination of the anterior femur was done for the estimation of age-at-death. The aim of this study was to assess particular bone microstructures that change with advancing age and use these variables to create revised regression formulae applicable to the black population of South Africa. A sample of 99 bone sections (n=60 males and n=39 females) that had previously been analysed using 2D methods, were re-analysed using the optical fractionator and nucleator sampling methods. Single and multiple regression analyses were performed to assess the strength of the relationship between known age and all independent variables. For sex-pooled data, the average number of osteons per grid area (Avg_OPD) showed the highest correlation with age (r=0.528; r2=0.278), followed by average osteon volume (r=-0.383; r2=0.146). The remaining variables reflected a low correlation with age. Pooled, as well as sex-specific single regression formulae were constructed. Multiple regression formulae were constructed for pooled sexes only, as there were no significant difference between males and females overall. Although the employment of stereological methods ensured that the results are accurate and unbiased, the outcome was on par with previously reported SEE's and SD's for this population.

Childhood health as reflected in adult urban and rural samples from medieval Denmark.

This study examines the evidence of three skeletal markers relating to childhood health that leave permanent observable changes in the adult skeleton. Two are well known to paleopathology, namely Harris lines (HL) and linear enamel hypoplasia (LEH). The third skeletal marker is less commonly used; the permanent changes in the temporal bones, induced by chronic or recurrent infectious middle ear disease (IMED) in childhood. A total of 291 adult skeletons from an urban (n = 109) and a rural (n = 182) cemetery, from the Danish medieval period (1050-1536 CE) were included. The markers were examined for their co-occurrence, and differences between the two samples. No statistically significant difference for the three skeletal markers between the two samples was found. A trend was nevertheless apparent, with greater frequencies for all three skeletal markers for the urban population. A statistically significant relationship was found only between IMED and HL. This positive relation was very low (rɸ = 0.307, 0.275) and may be considered non-existent. The lack of co-occurrence is interpreted as if an individual was exposed to conditions that could cause the osteological expression of all three markers this could be a life-threatening health condition, during developing years.

Direct evidence of milk consumption from ancient human dental calculus.

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein ß-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.

A small skull from Flores dated to the 20th century.

A human skull with mandible from the Ngada District on the island of Flores, Indonesia, is described in order to contribute to the knowledge of variation in cranial architecture, which is important in interpretations of evolutionary cerebralisation. The skull was excavated in 1924 and sent to the National Museum in Copenhagen. The "Copenhagen Flores" (CF) male skull is radiocarbon-dated and of modern age. The cranium is small, but larger than e.g. Liang Bua skull (LB1) in every measurement. The (CT-scan based) cranial capacity of 1258 ml is normal for modern humans, but somewhat lower than values from the middle or upper Palaeolithics. The metric cranial data analysed in FORDISC, characterize the skull as a male Vietnamese rather than a Chinese or White individual. Tooth morphology shows the sundadont pattern and tooth size corresponds to that of teeth from Bali, Java and Malayan Orang Asli. Remarkable are the marked asymmetries in the dentition with rotation of an upper premolar and congenital absence of a third molar. In these respects the CF skull is similar to dentitions belonging to the pygmoid villagers of Rampasasa, a village not far from the Liang Bua cave, and to LB1.

The problem of aging human remains and living individuals: a review.

Forensic anthropology is affected by the unavoidable limits concerning difficulties in standardization of methods and procedures; age estimation is one of the main tasks of forensic anthropology and odontology, both on the dead and the living: literature has shown several methods of age estimation, and although they may be thought of as equivalent, every procedure has its limits, mean error, practical situation and age range where it gives the best results; the lack of standardization and consensus concerning which method can be used, as well as the lack of a practical approach in different cases is the main limit in a correct age estimation process. This review aims at exposing the experience of the authors working in the FASE (Forensic Anthropology Society of Europe) subsection of IALM (International Academy of Legal Medicine) in the field of age estimation both on the dead and the living, at highlighting advantages and limits of each method, and suggesting practical solutions concerning the age estimation process for adults and subadults, dead and living, and pedopornographic material.

Intra- and interobserver error of the Greulich-Pyle method as used on a Danish forensic sample.

The Section of Forensic Medicine at the University of Copenhagen performs age evaluations of the living at the request of the police. The established procedure in these cases involves: (1) a physical examination; (2) an odontological examination based on evaluation of an orthopantomogram and intraoral dental radiographs. Different methods are used depending on the maturity of the individual examined; and (3) a carpal X-ray examination, using the Greulich and Pyle Atlas (GPA) method. We present the results of intra- and interobserver tests of carpal X-rays in blind trials, and a comparison of the age estimations by carpal X-rays and odontological age estimation. We retrieved 159 cases from the years 2000-2002 (inclusive). The intra- and interobserver errors are overall small. We found full agreement in 126/159 cases, and this was between experienced users and novices. Overall, the mean difference was 0.053 years, with a standard deviation of +/-0.567 years. Our results show that the direct and naïve use of the GPA is simple and reproducible, even when applied by non-experienced users. This is probably also why the method is still widely applied, even though other authors have stated that atlas-based techniques are obsolete and ought to be replaced by other methods. Specifically, the GPA test sample consisted of American "white" children "above average in economic and educational status", leading to the question as to how comparable subjects being scored by the GPA method today are to this original sample. Indeed, we found a minor difference in the age estimations for older juveniles between odontologists and radiologists, which may indicate that more studies on comparative dental and skeletal aging for geographically different populations would be beneficial. Finally, if using the GPA method in legal cases, we would advise that one should closely adhere to the standard deviations given, and intermediary scores should be noted and not just a resultant age. The final age statement should also address the problems of reference populations.

Rare mtDNA haplogroups and genetic differences in rich and poor Danish Iron-Age villages.

The Roman Iron-Age (0-400 AD) in Southern Scandinavia was a formative period, where the society changed from archaic chiefdoms to a true state formation, and the population composition has likely changed in this period due to immigrants from Middle Scandinavia. We have analyzed mtDNA from 22 individuals from two different types of settlements, Bøgebjerggård and Skovgaarde, in Southern Denmark. Bøgebjerggård (ca. 0 AD) represents the lowest level of free, but poor farmers, whereas Skovgaarde 8 km to the east (ca. 200-270 AD) represents the highest level of the society. Reproducible results were obtained for 18 subjects harboring 17 different haplotypes all compatible (in their character states) with the phylogenetic tree drawn from present day populations of Europe. This indicates that the South Scandinavian Roman Iron-Age population was as diverse as Europeans are today. Several of the haplogroups (R0a, U2, I) observed in Bøgebjerggård are rare in present day Scandinavians. Most significantly, R0a, harbored by a male, is a haplogroup frequent in East Africa and Arabia but virtually absent among modern Northern Europeans. We suggest that this subject was a soldier or a slave, or a descendant of a female slave, from Roman Legions stationed a few hundred kilometers to the south. In contrast, the haplotype distribution in the rich Skovgaarde shows similarity to that observed for modern Scandinavians, and the Bøgebjerggård and Skovgaarde population samples differ significantly (P approximately 0.01). Skovgaarde may represent a new upper-class formed by migrants from Middle Scandinavia bringing with them Scandinavian haplogroups.

An historical skull collection and its use in forensic odontology and anthropology.

The Institute of Forensic Medicine, Copenhagen, houses a collection of historical skulls of unclear origin, marked with a general geographic or "racial descriptor". Would these historical skulls be of any value for the forensic odontologist and anthropologist concerned with teaching and casework? We tried to clarify this question by recording non-metric dental traits and by performing craniometric analyses. A morphological and morphometric investigation of anatomical/dental traits in 80 adult skulls was performed. For each skull four non-metric dental traits using the ASU-System and three non-metric cranial traits were recorded. Nineteen cranial measures were also taken following the FORDISC programme manual. The non-metrical data were tabulated as frequencies, and the metric data were entered in the FORDISC programme. Observed non-metric trait frequencies were compared with published data. The FORDISC programme computed a discriminatory analysis for each skull and thereby assigned the skull to the most probable ethnic category. The results for the non-metric traits showed that the traits generally followed the expected frequencies in 80% of the cases. The FORDISC programme correctly assigned ethnicity based on skull measurements in overall 70% of the cases. It was found that this historical collection does show expected dental non-metric and craniometric traits and the collection may be of value in forensic casework in terms of comparison and for teaching purposes.

The effect of calcium and vitamin D3 supplementation on the healing of the proximal humerus fracture: a randomized placebo-controlled study.

The purpose of this study was to (1) quantify the healing process of the human osteoporotic proximal humerus fracture (PHF) expressed in terms of callus formation over the fracture region using BMD scanning, and (2) quantify the impact of medical intervention with vitamin D3 and calcium on the healing process of the human osteoporotic fracture. The conservatively treated PHF was chosen in order to follow the genuine fracture healing without influence of osteosynthetic materials or casts. Thirty women (mean age = 78 years; range = 58-88) with a PHF, osteoporosis or osteopenia (based on a hip scan, WHO criteria), and not taking any drugs related to bone formation, including calcium or vitamin D supplementation, were randomly assigned to either oral 800 IU vitamin D3 plus 1 g calcium or placebo, in a double-blind prospective study. We measured biochemical, radiographic, and bone mineral density effect parameters to evaluate the impact on the healing process. Scanning procedures of the fractured shoulder included use of a fixation device to obtain the highest possible precision. Double scans of the fractured shoulder revealed a coefficient of variation (CV) on BMD measurements that improved from 2.8% immediately after fracture occurrence to 1.7% at 12 weeks (P = 0.003) approaching the 1.2% levels observed over the healthy shoulder. BMD was similar in the two groups at baseline (active 0.534 g/cm2 vs. placebo 0.518 g/cm2), and both increased over the 12-week observation period, with peak levels in week 6. By week 6 BMD levels were higher in the active group (0.623 g/cm2) compared with the placebo group (0.570 g/cm2, P = 0.006). Thirty seven percent of the patients presented with vitamin D levels below 30 nmol/l, indicative of mild vitamin D insufficiency. In conclusion, we have demonstrated that it is possible to quantify callus formation of the PHF with sufficiently high precision to demonstrate the positive influence of vitamin D3 and calcium over the first 6 weeks after fracture. Whether this results in more stable fractures, extends to other fracture types, or applies to other osteogenic bone agents such as bisphosphonates remains to be examined.

Bone mineral density measurement over the shoulder region.

The purpose of this study was to (1). establish a method for measuring bone mineral density (BMD) over the shoulder region; (2). compare the relationship between shoulder BMD levels with hip BMD and body mass index (BMI); and (3). discuss the relevance of the shoulder scan as an early indicator of osteoporosis compared with hip scans, the latter representing a weight-bearing part of the skeleton. We developed a scanning procedure, including a shoulder fixation device, and determined the most appropriate software in order to establish a reference material with the highest possible precision. Duplicate scans of the dominant side shoulder on 80 healthy, non-osteoporotic Danish women revealed a coefficient of variation (CVSD) on BMD measurements of 1.7%, with no difference between young and old subjects. Shoulder BMD values were significantly lower than hip BMD values (P <0.00001). Both hip and shoulder values decreased with age (P <0.001). The difference between hip and shoulder BMD levels increased significantly with increased body mass index (BMI) (P <0.002). The positive relationship between the increased hip/shoulder BMD differential with BMI supports the conclusion that the shoulder is subject to the least relative influence of weight and stress loading because of migration of calcium to weight and stress-bearing areas. Since the effect of this migration could mask local osteoporotic bone loss, shoulder BMD measurement is likely to minimize false indicators of healthy bone in women with high BMI, and might therefore be a relevant early stage indicator of osteoporosis.

Mobile autopsy teams in the investigation of war crimes in Kosovo 1999.

On request of the International Criminal Tribunal for the former Yugoslavia (ICTY), the Danish-Swedish forensic teams worked in Kosovo during the summer and the fall of 1999. The teams worked mainly as "mobile teams" at sites with few graves. Only two larger sites were examined. Most of the bodies were buried separately. A few "multiple burial" graves were examined, but no mass graves were encountered. The main purpose of the autopsies was to establish the cause and manner of death. Identification was of less importance, but a majority of the bodies had been identified prior to the autopsy. A total of 308 bodies, mainly males, were examined. The age varied greatly with a mean age of 47 years. The most common cause of death was gun shot wounds and the most common manner of death was homicide.

Cranial thickness in relation to age, sex and general body build in a Danish forensic sample.

The cranial thickness was measured in 64 individuals (43 males, 21 females) autopsied at our institute. The thickness was measured by taking a biopsy with a trephine at four specific locations on the skull. Complete medical records and pathologic autopsy results were available. While none of the individuals had suffered from diseases affecting bone or bone metabolism as such, a large sub group consisted of individuals with a history of, and autopsy finds consistent with, chronic substance and alcohol abuse. There was no statistically significant difference in cranial thickness measures between this group and the rest of the material. Subsequent analyses failed to reveal any correlations between the cranial thickness and sex and age and height and weight of the individual. This is in accordance with most earlier studies, which likewise show no correlation, or only very faint trends, between cranial thickness and these parameters. This study, thus, adds to other studies showing that cranial thickness cannot be used in aging or sexing human remains. Likewise, in a forensic pathological setting, cranial thickness cannot be inferred from the individuals stature and build, which may be an issue in cases of interpersonal violence with cranial trauma.

mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion.

The Eskimo-Aleut language phylum is distributed from coastal Siberia across Alaska and Canada to Greenland and is well distinguished from the neighboring Na Dene languages. Genetically, however, the distinction between Na Dene and Eskimo-Aleut speakers is less clear. In order to improve the genetic characterization of Eskimos in general and Greenlanders in particular, we have sequenced hypervariable segment I (HVS-I) of the mitochondrial DNA (mtDNA) control region and typed relevant RFLP sites in the mtDNA of 82 Eskimos from Greenland. A comparison of our data with published sequences demonstrates major mtDNA types shared between Na Dene and Eskimo, indicating a common Beringian history within the Holocene. We further confirm the presence of an Eskimo-specific mtDNA subgroup characterized by nucleotide position 16265G within mtDNA group A2. This subgroup is found in all Eskimo groups analyzed so far and is estimated to have originated <3,000 years ago. A founder analysis of all Eskimo and Chukchi A2 types indicates that the Siberian and Greenland ancestral mtDNA pools separated around the time when the Neo-Eskimo culture emerged. The Greenland mtDNA types are a subset of the Alaskan mtDNA variation: they lack the groups D2 and D3 found in Siberia and Alaska and are exclusively A2 but at the same time lack the A2 root type. The data are in agreement with the view that the present Greenland Eskimos essentially descend from Alaskan Neo-Eskimos. European mtDNA types are absent in our Eskimo sample.