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.

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.

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.

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.

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.

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.

Intra- and inter-observer variation in histological criteria used in age at death determination based on femoral cortical bone.

The microscopic method of age at death determination was introduced by Kerley in 1965. The method, which relies on the quantification of selected elements in cortical bone tissue, has been widely used, and several other researchers have modified or added to the method. Yet, very few studies have been carried out dealing with the intra- and inter-observer error. Furthermore, when such studies have been completed, the statistical tools for assessing variability have not been adequate. This study presents the results of applying simple quantitative statistics on several counts of microscopic elements as observed on photographic images of cortical bone, in order to assess intra- and inter-observer error. Overall, substantial error was present at the level of identifying and counting secondary osteons, osteon fragments and Haversian canals. Only secondary osteons can be reliably identified, precluding the use of osteon fragments and Haversian canals. The observers in this study included experienced and inexperienced users of the microscopic method, yet the variability was uniformly large for all observers, suggesting fundamental problems in definition and identification of the structural elements. Until more rigorous definitions of such elements have been agreed upon, the use of microscopical methods must be discouraged as a sole or uncontrolled method of evaluating age at death.

Statistical model estimating the occurrence of otitis media from temporal bone pneumatization.

In order to investigate the relationship between the pneumatization of temporal bones and the occurrence of otitis media in Greenlandic Inuit, 36 Greenlandic Inuit were examined by radiography of the temporal bones. The pneumatized cell area was measured planimetrically. All subjects answered a questionnaire on infectious middle ear disease (IMED), and an objective otologic examination was performed. Nine persons of 34 (26%) reported IMED in childhood, and there was IMED reported in all pneumatized areas below 400 mm2. Based on bilateral area measures, a polychotomous logistic regression model was applied. The occurrence of IMED was shown to be associated with smaller areas, and unilateral IMED was associated with pronounced asymmetry. The model has enabled risk assessment, and 8 persons were designated by the model as having had IMED with a sensitivity of 67% (confidence interval 30% to 93%) and a specificity of 92% (confidence interval 74% to 99%). When the model was applied to a historical anthropological Inuit material from 1700 to 1800 AD, 6 of 56 crania were designated as having had IMED. This method has improved the accuracy of estimating the occurrence of IMED in ancient populations.

Pneumatization of the temporal bones in a Greenlandic Inuit anthropological material.

The degree of pneumatization of the temporal bones correlates with exposure during childhood and adolescence to infectious middle ear diseases (IMED), both acute and chronic. The pneumatized area as seen on cranial X-rays can be measured. This was applied to an anthropological material in order to develop methods for assessing ancient populations' exposure to IMED. Fifty-six Greenlandic Inuit (Eskimo) crania were examined. The crania were sexed and measured. X-rays were taken bilaterally, using the projection of Runström II, and the pneumatized area was measured planimetrically by computer. In blind trials it was found that the inter- and intraobserver variation was non-significant; that the degree of pneumatization of the temporal ossa did not reflect cranial size or sexual dimorphism; and that the pneumatized areas correlated bilaterally. Asymmetry in the size of the pneumatized areas, an indicator of exposure to IMED, was found in 5 crania (9%).

CT-scanning of ancient Greenlandic Inuit temporal bones.

Additional morphological evidence of former infectious middle ear disease (IMED) was found by CT-scanning in 5 of 6 Greenlandic Inuit crania strongly suspected for former IMED due to earlier examination revealing either bilateral hypocellularity or asymmetry of the pneumatized area of the temporal bones. The CT-scans showed sclerosing and obliteration of the air cells and even destruction of the cellular septae, and a high degree of irregularity of the cells. Sclerosing of the surrounding bone tissue was also found. The findings in one cranium were dubious and could both be regarded as a congenital malformation or an infection in infanthood. CT-scan confirms and even adds to the results of conventional X-ray of temporal bones making hypotheses of paleopathology more reliable. The findings also support the environmental theory of pneumatization of the air cell system in the temporal bones.

A computer program for the estimation of time of death.

In the 1960s Marshall and Hoare presented a "Standard Cooling Curve" based on their mathematical analyses on the postmortem cooling of bodies. Although fairly accurate under standard conditions, the "curve" or formula is based on the assumption that the ambience temperature is constant and that the temperature at death is known. Also, Marshall and Hoare's formula expresses the temperature as a function of time, and not vice versa, the latter being the problem most often encountered by forensic scientists. A simple BASIC program that enables solving of Marshall and Hoare's equation for the postmortem cooling of bodies is presented. It is proposed that by having a computer program that solves the equation, giving the length of the cooling period in response to a certain rectal temperature, and which allows easy comparison of multiple solutions, the uncertainties related to ambience temperature and temperature at death can be quantified, substantiating estimations of time of death.

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.

Automatic data acquisition of anthropological measurements.

A computer program in BASIC is presented which enables the input of measurement data from a caliper directly into specific records in a dBASE IV or PARADOX database. The program circumvents the tedious procedure of first recording measurement data manually and then entering the data into a computer. Thus much time can be saved and the risk of wrong data entry is lowered. The program was easy to use, and no significant problems were encountered. Necessary hardware is a standard IBM compatible desktop computer, Mitotoyu Digimatic (TM) calipers and a Mitotoyu Digimatic MUX-10 Multiplexer (TM).

A computer program for planimetric analysis of digitized images.

Planimetrical measurements are made to calculate the area of an entity. By digitizing the entity the planimetrical measurements may be done by computer. This computer program was developed in conjunction with a research project involving measurement of the pneumatized cell system of the temporal bones as seen on X-rays. By placing the X-rays on a digitizer tablet and tracing the outline of the cell system, the area was calculated by the program. The calculated data and traced images could be stored and printed. The program is written in BASIC; necessary hardware is an IBM-compatible personal computer, a digitizer tablet and a printer.

Estimation of otitis media in ancient populations. A study of past and present Greenlandic Inuit.

Examination of disease patterns in the past has often been difficult due to lack of morphological evidence. This study presents a new unbiased method for estimation of occurrence of infectious middle ear disease (IMED) in childhood. The method is based on the relation between IMED in childhood and small or asymmetric pneumatized cell areas in the temporal bones as seen on standardised X-rays. A polychotomous logistic regression model was applied on 434 pneumatized cell areas in temporal bones from 34 adult living Greenlandic Inuit, 56 adult crania from the 18th to the 19th century, A.D. and 127 adult Inuit crania from the pre-European colonization period (before A.D. 1721) of Greenland. The occurrence of IMED as designated by the model was eight out of 34 (23.5 per cent) in living Inuit, 10 out of 56 (17.9 per cent) in crania from the 18th to 19th century and six out of 127 (4.7 per cent) in crania from the pre-colonization period. These frequencies differed significantly (p < 0.002). The mean area size also differed significantly, thus indicating a change in occurrence of IMED and a decrease in area sizes from past to present in Greenland.

A non-invasive technique for age at death determination.

Several methods for age at death determination of human skeletal remains have been developed. These methods have, especially in combination, proved to be useful in making individual identification of skeletal material in forensic cases. This study is based on the known correlation between actual age and structural changes in trabecular bone tissue. Using X-rays taken from live individuals, it provides a useful non-invasive ageing technique for the forensic examiner. An initial pilot study defined five phases of age-related changes in the trabecular tissue of the proximal end of the femur. A total of 60 X-rays, randomly selected, and covering an age span from 14 years to 94 years, were subsequently scored in blind trials. The results demonstrate a clear relationship between age and changes in the trabecular structures. Based on this relationship it is possible to obtain an age estimate by identification of one of the five phases. An exact age determination of a single individual was not possible, but could be approximated to within 20 years. In forensic cases, however, where the removal of soft tissue is not always possible, this method can contribute to the final age determination when used in conjunction with other well-known methods, and thereby strengthen the final age estimate.

Routine use of ultraviolet light in medicolegal examinations to evaluate stains and skin trauma.

The use of ultraviolet light induced fluorescence as an aid in forensic medical examinations of rape victims was evaluated preliminarily in a retrospective, non-consecutive study. In a four-month period, 17 cases were referred by the police for examinations at the Institute of Forensic Pathology. Ultraviolet light illumination (UVI) was used in seven cases, and in six cases fluorescent skin areas were observed. The fluorescence was due to lesions in four cases and stainings with saliva and semen in other two cases. In at least two cases, skin trauma detected with UVI were unobserved in ordinary light. It is concluded that UVI should be a routine part of forensic medical examinations. It may assist the forensic medical examiner in finding skin trauma and in locating stains, thus enabling retrieval of material for serological analyses. UVI is simple to carry out, requiring only a small, portable ultraviolet light source.