DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of ?eri?i, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex^® Fusion and PowerPlex^®Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains.     

Time-lapse resistivity surveys over simulated clandestine graves

The aim of this study was to develop a better understanding of how electrical resistivity surveys can be used to locate clandestine graves. Resistivity surveys were conducted regularly over three simulated clandestine graves containing a pig cadaver, no cadaver and a pig cadaver wrapped in tarpaulin, respectively. Additionally, soil and groundwater samples were collected from two more simulated graves outside the survey area. The grave containing a pig cadaver was detectable from a low resistivity anomaly in the survey data. Groundwater data suggest that the resistivity anomaly associated with the surveyed pig grave was caused by a localised increase in groundwater conductivity. Wrapping a cadaver was found to initially change the resistivity response of a grave to a high resistivity anomaly. Resistivity surveys did not detect the disturbed soil in the grave that did not contain a cadaver. Although soil samples showed grave soil to be more porous than undisturbed soil, the lack of response from the grave that did not contain a cadaver suggests that disturbed soil was not responsible for the resistivity anomalies observed in this study. Resistivity surveys successfully detected all graves containing cadavers throughout the study, whilst also showing the potential to eliminate the need for mass excavation in a genuine search.     

Differences in morphological age-related dental changes depending on postmortem interval.

Morphological methods for age estimation from teeth have been developed and applied to samples without taking the postmortem interval into consideration. We studied differences in morphological age-related changes between fresh extracted teeth and teeth from human skeletal remains in order to develop appropriate dental age estimation methods according to the time after death. Forty-three permanent teeth from dental patients were compared to 37 teeth obtained from human skeletal remains with a postmortem interval from 21 to 37 years. Morphological age-related changes were investigated by measuring variables on intact and half-sectioned teeth. A new computer assisted image analysis procedure to avoid subjectivity was developed to measure variables in sectioned specimens. Dental color, translucency length, attrition, cementum apposition, and secondary dentin showed higher values in teeth from human skeletal remains than in fresh extracted teeth. Variables obtained by morphometric analysis of computer-generated images (tooth length, tooth width, root length, and root area) showed higher values in fresh extracted teeth than in teeth from skeletal remains. The postmortem interval affects age-related morphological changes, and therefore different methods should be used for teeth of unknown postmortem interval.     

Identification and analyses of female civilian victims of the 1991 war in Croatia from the Glina and Petrinja areas

Introduction

The process of recovering and identifying human remains from individual and mass graves has proven to be the most effective method of resolving the fate of missing individuals in the former Yugoslavia. These efforts have two primary objectives: medicolegal—to identify the cause of death, and humanitarian—to bring closure to living family members, thus supporting the human rights of both the living and the deceased. From 1996 to the present, the remains of 388 individuals killed during the 1991 War in Croatia were recovered from the Glina and Petrinja areas (Sisa?ko–Moslova?ka County). The purpose of this paper is to report on the demographic and taphonomic characteristics of the recovered remains, and identify factors responsible for the discrepancy in the identification ratios.

Methods

To identify factors potentially responsible for the noted discrepancy, data were collected on the taphonomic characteristics of the recovered remains, the types of burials, as well as the factors responsible for positive identification. These factors included: forensic DNA testing, dental criteria, special signs (healed fractures, etc.), clothing and personal artifacts.

Results

The age distributions between males and females in the sample are significantly different. The mean age-at-death for males is 51.6 years, for females 67.0 years. In both sexes the majority of identifications (62.9% in females and 45.9% in males) were established through a combination of biological and non-biological features. In terms of the types of graves from which the bodies were recovered, positive identification in both sexes was lowest in mass graves (61.2% for males, and 84.2% for females). Females from mass graves were, however, better preserved than males—36.6% of males recovered from mass graves were completely skeletonized, while the same was true for only 19.3% of females (χ² = 7.06, P < 0.029). Completely skeletonized remains were identified in only 65.1% of cases, while partially skeletonized and saponified bodies were identified in 80.1% of cases.

Conclusion

Since positive identification in both sexes is strongly correlated with preservation of the bodies, the reason why female identification frequencies in the Glina and Petrinja regions are higher than male frequencies lies in the fact that females recovered from mass graves were better preserved than males recovered from mass graves. Identification of the factors responsible for this is a challenge that needs to be resolved in future studies.     

Analysis of pathological and non-pathological human skeletal remains by FT-IR spectroscopy

In this study, we report the chemical analyses of various non-pathological, tuberculosis and syphilis infected bone samples from different burial environments by Fourier transform infrared spectroscopy (FT-IR), in the framework of a general study of diagenesis. Dating human skeletal remains is one of the most important and yet unreliable aspects of forensic anthropology. In this paper, a new method has been suggested, using the crystallinity index and carbonate-phosphate index as a means of distinction between recent and archaeological, anthropological bone samples. Pathological bone samples were analyzed with the same method to see if changes in crystallinity interfere with the process of dating.     

Anthropological and radiographic comparison of vertebrae for identification of decomposed human remains

This case study demonstrates the importance of involving an anthropologist in forensic situations with decomposed remains. Anthropological consultation was used in conjunction with the comparison of antemortem and postmortem radiographs to establish positive identification of unknown, decomposed remains. The remains had no traditional identifying features such as fingerprints or dental. Through anthropological analysis, it was determined the decedent was male, between 20 and 23 years at time of death and c. 5'2' tall. This information allowed for a presumptive identification and a request for antemortem radiographs. The missing person was identified comparing the spinous processes of the cervical and thoracic vertebrae between ante- and postmortem radiographs.     

Controlled GPR grave research: comparison of reflection profiles between 500 and 250 MHz antennae

Since ground-penetrating radar (GPR) has become a popular search option for clandestine graves, controlled research is essential to determine the numerous variables that affect grave detection. The purpose of this study was to compare GPR reflection profiles of a controlled grave containing a large pig carcass and a blank control grave at 6 months interment in a Spodosol, which is a common soil type in Florida. Data collection was performed in perpendicular orientations over the graves using both 500 and 250 MHz antennae. Since reflection profiles are used to make initial in-field assessments during a forensic search, it is important for controlled research to evaluate this imagery option. Overall, it was possible to detect the grave containing a pig carcass at 6 months interment that was buried in a Spodosol using both the 500 and the 250 MHz antennae. While the 500 MHz antenna provided more detail within the grave containing a pig carcass, including detecting a soil disturbance and the pig carcass, the 250 MHz antenna also provided excellent imagery. Either antenna would provide optimal results for the type of soil that was sampled. Furthermore, it may be possible to locate actual forensic graves in this soil type when no response from the body is noted, as there may be a discernable response from the disturbed soil within the grave shaft and a noticeable disruption of the spodic horizon. Finally, survey orientation may also affect detection. Since data collection performed in two perpendicular directions detected the pig carcass and the grave floor of the control grave, data collection for an actual search involving a body interred for a long postmortem interval should be performed in both directions when time permits.     

Keeping the pieces together: comparison of mass grave excavation methodology

Mass graves are a complex and confusing mix of bodies, body parts, soils, artifacts, and other feature evidence. Forensic investigations of these complex crime scenes should attempt to maximize the collection of evidence, which includes the mortal remains, in their best possible condition as they were deposited within the graves. Two standard methods of mass grave excavation were examined with the aim of identifying the better approach. Two experienced teams using different methodologies excavated two separate but very comparable mass graves located in the same area. Single disarticulated skeletal elements not associated with a body at the time of removal from the grave were categorized and their counts analyzed to evaluate the efficiency of the differing excavation methodologies. The methodologies used were the 'pedestal' method, which focuses on the body mass, and the 'stratigraphic' method, in which the grave feature and contents are conjointly excavated. The first grave (Grave A), excavated using the 'pedestal' method, was observed to have a disproportionately larger amount of unassociated bones than did the second (Grave B), which used the 'stratigraphic' method. Chi-square (chi2) goodness-of-fit and contingency tests were performed on the total numbers of recorded elements and different categorical groups of bones, based on size and shape, in each grave. Results demonstrate that significantly greater numbers of unassociated elements resulted from the excavation of Grave A using the pedestal method, both in total number of disarticulated bones as well as within 'large' and 'medium' categories. Conversely 'small' skeletal elements were recovered at a higher rate in Grave B. The lower 'large' and 'medium' bone production rates from Grave B indicate that the stratigraphic method better maintained the provenience and articulation of remains than did Grave A, while the higher 'small' bone recovery rate may point to better recovery techniques of Grave B's excavation team.     

Insect Successional Pattern of a Corpse in Cooler Months of Subtropical Southeastern Texas

Abstract:  Here, we characterize the cool weather insect fauna found associated with partially skeletonized and desiccated human remains recovered from an abandoned house in an urban area of subtropical, coastal Galveston County, Texas, and use the information to conclude an approximate postmortem interval of 7–10 months. The predominant factors that allow for a confident assessment of the postmortem interval include climatological data, entomological data, and anthropological data. The documented insect fauna represents a unique assemblage present in a particular environment (an urban abandoned house in coastal Texas) at a particular time of year (winter) and includes expected forensically significant insects such as calliphorid flies, muscid flies, and dermestid beetles but also includes less commonly encountered insects such as an unusually dense population of live case-making clothes moths.     

Reanalysis of Korean War Anthropological Records to Support the Resolution of Cold Cases

Re‐investigation of previously unidentified remains from the Korean War has yielded 55 new identifications, each with corresponding records of prior anthropological analyses. This study compares biological assessments for age at death, stature, and ancestry across (i) anthropological analyses from the 1950s, (ii) recent anthropological analyses of those same sets of remains, and (iii) the reported antemortem biological information for the identified individual. A comparison of long bone measurements from both the 1950s and during reanalysis is also presented. These comparisons demonstrate commonalities and continuing patterns of errors that are useful in refining both research on Korean War cold case records and forensic anthropological analyses performed using methods developed from the 1950s identifications.     

Macroscopic observations of the effects of varying fresh water pH on bone

Little is known about the decomposition of remains in aquatic environments of varying pH, and even less is known about the specific effects of these environments on bone. Bovine bones were placed in solutions of pH 1, 4, 7, 10, and 14 and observed over a period of 1 year. All solutions eventually removed or dissolved the soft tissues from the external surface of the bone. The pH 7 and pH 10 solutions had little effect on the bone, but the other solutions affected the bone to varying degrees. Extreme pH levels were the most destructive, while more moderate pH levels had lesser but significant and interesting effects. Empirical data on postmortem aquatic changes may be extremely useful in forensic contexts for both improving time since death estimates and also for providing better information to underwater recovery experts thereby potentially increasing the quantity and quality of remains recovery.     

Next generation sequencing technology in Second World War victim identification

The killings during the Second World War (WWII), with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with Next Generation Sequencing (NGS) technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with capillary electrophoresis (CE) analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended posterior probability (PP) of 99.9% was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986%, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case, NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.     

Different skeletal elements as a source of DNA for genetic identification of Second World War victims

To this day process of identification of missing persons from skeletonized human remains with help of forensic genetics proves to be complex and challenging. The success rate of genetic identification in bones strongly depends on a combination of various factors, most importantly environmental factors and post-mortem interval. Furthermore, there are individual-specific factors that affect DNA preservation, such as race, gender, age and type of skeletal elements. The goal of our study was to optimize sampling process through determining which skeletal elements are superior in their preservation of DNA in 70-yearold skeletons belonging to victims of Second World War. We sampled different types of bones and teeth from three such skeletons found in Slovenian hidden mass grave Huda jama, 56 elements from each respective skeleton, together 168 elements. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Prior to powdering bones and teeth, we removed contaminants. We decalcified 0.5 g bone and tooth powder followed by extraction and purification of DNA using Biorobot EZ1 (Qiagen). Quantification of obtained nuclear DNA was carried out using PowerQuant kit (Promega) and autosomal STR typing using ESSplex SE QS kit (Qiagen). Best parameters to assess skeletal elements that are superior in their DNA preservation were quantity of DNA and number of successfully typed STR loci. Metacarpal and metatarsal bones proved to be the best, followed by intermediate cuneiform, first distal foot phalanx, talus, petrous bone and tibia. We also created elimination database for persons involved in exhumation, anthropological and genetic analyses and exclude potential contamination.     

The application of differential thermal analysis and thermogravimetric analysis to dating bone remains.

Thirty-four samples of bone remains of known time since death were studied by using DTA, TGA, and derivative-TGA techniques. The DTA patterns enable us to distinguish recent from old (more than 100 years) bones. The TGA curve is also significant for an extreme series. Both DTA and TGA curves show a correlation that allows us to obtain patterns with high significance for the extreme series. They also make evident the decomposition grade that bone organic material undergoes during the postmortem putrefactive process.     

Effect of 1,2-indanedione on PCR-STR typing of fingerprints deposited on thermal and carbonless paper

The determination of the date of death from bone remains is of scientific interest but also has important legal implications. The establishment of the postmortem interval (PMI) is a very complex problem because of the great number of intrinsic factors that may alter the normal course of postmortem change, such as the age, sex, constitution and previous physiological and pathological states of the subject, and external factors. In order to evaluate the utility of X-ray diffraction and the measurement of some components in dating bone remains, a total of 69 long bones from 69 different cadavers (41 males, 28 females) with a mean age of 68 years (S.D.=17.6, range 12-97) were used. The bones were removed from cement tombs of Murcia Cemetery, where they had lain for documented times of between 7 and 54 years (S.D.=11.6, mean time 17.6 years). We have studied potassium, sulphur, nitrogen, urea, total protein, phosphorus, and some X-ray diffraction (XRD) parameters related to the degree of crystallinity of the mineral component in medullar and cortical bone zones to establish which of the two provides the most useful information for calculating the PMI. In the overall analysis of our data, we believe that the use of both XRD and biochemical analyses (especially of urea, potassium and sulphur) particularly in the cortical zone of the bone could be an alternative method for dating osseous remains.     

Guidelines for international forensic bio-archaeology monitors of mass grave exhumations

The systematic exhumation of mass graves is becoming a frequent occurrence globally. Historically, it has been mostly anthropologists with their particular expertise in osteology that have been engaged by non-governmental organizations (NGO) to monitor mass grave exhumations and postmortem examinations, conducted by a host country or international forensic team, to ensure competence and a concern for justice. The excavation of graves and examination of their contents for the purposes of personal identification of victims and/or collection of evidence for prosecution require the creation of standards that meet international concerns for forensic investigation of the highest quality. However, many anthropologists are not experienced with large sites; do not have much, if any, expertise in archaeology; and are not equipped by training to assess the quality of a forensic pathologist's autopsy. This contribution is directed to both the NGO and the bio-archaeologist who are involved in the exhumation of mass graves. The experience and skills appropriate for the bio-archaeological monitor are outlined; similarly, the policies and standard operating procedures (SOP's) of the NGO that will enable the bio-archaeologist to perform their task are detailed. It is becoming increasingly clear that how a grave site was created, filled, and concealed, along with subsequent processes of site formation (e.g. slumping, robbing, animal scavenging and taphonomic alteration), require the expertise of a forensic archaeologist to discover and record site complexity along with associated forensic evidence. Similarly, the bodies themselves which are often skeletonized, fragmented and commingled pose challenges for the anthropologists and pathologists whose postmortem examinations must be critically evaluated. A model protocol for observations to make at the site as well as at the mortuary facility is provided to guide the monitor and to provide a structure for reports which are of standardized content suitable for international agencies concerned with mass grave investigation.     

Bone Mineral Density Adult Age Estimation in Forensic Anthropology: A Test of the DXAGE Application

Estimating age‐at‐death of individuals represented only by skeletonized human remains is a fundamental aspect of forensic anthropological casework. Recently, several researchers have proposed that bone mineral density (BMD) is a useful predictor of age‐at‐death in forensic contexts. Navega et al. (JFS 63(2):497–503) developed an online application called DXAGE for calculating age‐at‐death from BMD parameters. This study tests the utility of DXAGE by utilizing data from the National Health and Nutrition Examination Survey (NHANES). BMD data from a female subsample (n = 470) of the NHANES 2007–2008 dataset were analyzed, and the relationship between predicted age and real age was examined. Inaccuracy was 14.25 years, and bias was ?7.20 years. Results show that there is a weak correlation between predicted and actual age (r = 0.47) using the DXAGE application. While BMD data are potentially useful for predicting age age‐at‐death, the DXAGE application should be used cautiously in forensic anthropological contexts.     

DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala

No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.     

Implant Bone Integration Importance in Forensic Identification

Odontological identification consists of the comparison of antemortem dental information regarding a missing person with postmortem data from an unidentified corpse or human remains. Usually, the comparison concerns morphologic features that the operator chooses among all the visible characteristics because of inter‐individual uniqueness; for this reason, implants can be of enormous assistance. A case concerning the recovery of a burnt oral implant, connected to a bone fragment, among 2780 charred bone fragments, suspected to have belonged to a victim of homicide, is presented to demonstrate that dental implants and their site of bone integration represent a very precious element for personal forensic identification. Because of their morphological invariability in time and because of their morphologic uniqueness, they were used as evidence to associate unidentified human charred remains to a missing person where DNA analysis failed to do so. The case illustrates the fundamental contribution, not yet described in literature, given by the clinical aspects of tooth replacement with dental implants to a forensic discipline. Clinical practitioners should therefore be aware of the great importance of their work and of dental records in a forensic identification scenario.