Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

The Black Death in Hereford, England: A demographic analysis of the Cathedral 14th-century plague mass graves and associated parish cemetery.

OBJECTIVES: This study explores the paleoepidemiology of the Black Death (1348-52 AD) mass graves from Hereford, England, via osteological analysis. Hereford plague mortality is evaluated in the local context of the medieval city and examined alongside other Black Death burials. METHODS: The Hereford Cathedral site includes mass graves relating to the Black Death and a 12th-16th century parish cemetery. In total, 177 adult skeletons were analyzed macroscopically: 73 from the mass graves and 104 from the parish cemetery. Skeletal age-at-death was assessed using transition analysis, and sex and stress markers were analyzed. RESULTS: The age-at-death distributions for the mass graves and parish cemetery were significantly different (p = 0.0496). Within the mass graves, young adults (15-24 years) were substantially over-represented, and mortality peaked at 25-34 years. From 35 years of age onwards, there was little variation in the mortality profiles for the mass graves and parish cemetery. Males and females had similar representation across burial types. Linear enamel hypoplasia was more prevalent within the mass graves (p = 0.0340) whereas cribra orbitalia and tibial periostitis were underrepresented. CONCLUSIONS: Mortality within the Hereford mass graves peaked at a slightly older age than is seen within plague burials from London, but the overall profiles are similar. This demonstrates that young adults were disproportionately at risk of dying from plague compared with other age groups. Our findings regarding stress markers may indicate that enamel hypoplasia is more strongly associated with vulnerability to plague than cribra orbitalia or tibial periostitis.

Skeletal evidence of the ethnic cleansing actions in the Free City of Danzig (1939-1942) based on the KL Stutthof victims analysis.

In the early days of World War II, many of the prominent and influential people of Polish nationality from the Free City of Danzig were arrested by the Germans and sent to the nearby concentration camp KL Stutthof. Nearly a hundred of them died within the next seven months upon their arrival, and were buried in a clandestine mass grave in a nearby forest. However, the exact nature of their death is unknown, as it is unclear what the attitude of the aggressors was toward the victims. We do not know whether there was only one executioner or there were several assassins, nor if the killing methodology was consistent with the other state-regulated executions. The studied material represents the commingled remains of a minimum thirty-four people, possibly all male, aged from under eighteen to over sixty at the time of death. Perimortem traumatic lesions are shown mainly on the skull bones. We asked whether the perimortem trauma lesions visible on the victims' skeletons could be informative on the cause and manner of their death. Our results show the prevalence of the perimortem trauma inflicted by a blunt object are on the parietal bones above the Hat Brim Line (HBL), which is commonly associated with a violent attack. The gunshot trauma was usually localized on the occipital bone or posterior parietal, which could indicate a shot to the back of the head, and this was commonly encountered during executions. No signs of defensive injuries can be explained either by restraining of the hands or by a surprise attack. The abundance and variability of the trauma type can be evident of multiple assailants. Moreover, the multiple impact points detected on several crania prove unnecessary overkill and brutality, which reflects the personal attitudes of the executioners towards the victims.

Analysis of skeletal trauma on the bodies found in a mass grave.

This study was based on the data from the casefiles of the Institute of Forensic Medicine (IFM) in Kosovo, to analyse and interpret trauma observed on individuals recovered from a mass grave in Rudnica, Serbia. The intention was to determine if there is a pattern of trauma characteristic of this mass grave that informs about the manner of death and whether this is consistent with witness testimonies. The study considers the limitations of such analysis and interpretation, with special consideration of the completeness of the remains. The casefiles of 54 individuals recovered from the Rudnica mass grave from April to June 2014 were examined. A descriptive analysis was undertaken of the demographic profile of the sample, primary site of burial, completeness of the bodies, type and distribution of trauma, and the documented cause of death. All the individuals identified from the Rudnica mass grave were male aged from 14 to 96 years at time of death originating from four separate primary events with two known primary burial sites. Overall, 56% of the bodies were almost complete, 35% incomplete, and 9% complete. Discussion of the determination of completeness is included herein. The only type of trauma documented on the remains was gunshot wound trauma with the distribution of injuries concentrated on the trunk, followed by the limbs and head/neck regions. The cause of death was established in 56% of the cases. A pattern of trauma on the skeletal remains from the Rudnica mass grave was established based on the distribution and type of trauma documented from the dataset of each individual. These findings can be used as a basis for future studies in this field of research by taking a similar approach on larger samples and addressing the limitations encountered here.

Autopsy and its role in Franco's dictatorship: a case of the last Republican mayor of the town Calera y Chozas (Toledo, Spain).

In the town of Calera y Chozas (Spain), five mass graves containing the remains of 28 individuals were discovered during a 2012 excavation. The witnesses and historical evidence indicated that the body of the last Republican mayor of the town, Felipe Fernández Varela, who had died in September 1939, was located in the mass grave designated as no. 1. Within this particular grave, only two bodies were found. Anthropological analysis showed that the first individual was significantly younger than 50 years, being the mayor's age at the time of death, while the age of the second individual was closer to 50. This second individual had a fractured skull, with a depression on the left parietal bone, and there were unmistakable signs of autopsy, which consisted of cut marks on the frontal bone and the sternal extremity of the right clavicle. Further historical research revealed documents concerning the autopsy performed on this individual. Although, according to the report, the cause of death was a stroke - the consequence of atherosclerosis and alcoholism - no reference was made to the forceful impact to the skull or intracranial bleeding. Considering the size of the fracture on the skull and the fact that there were no signs of bone healing, we believe that this impact, and not the stroke, was the direct cause of the death of the last Republican mayor. The mayor's case is a clear example of the role forensic medicine performed at the beginning of Franco's dictatorship. The task was not only to conceal the crime but also to tarnish the victim's name.

Geophysical imaging of buried human remains in simulated mass and single graves: Experiment design and results from pre-burial to six months after burial.

In this study, we present an experiment design and assess the capability of multiple geophysical techniques to image buried human remains in mass and individual graves using human cadavers willingly donated for scientific research. The study is part of a novel, interdisciplinary mass grave experiment established in May 2021 which consists of a mass grave with 6 human remains, 3 individual graves and 2 empty control graves dug to the same size as the mass grave and individual graves. Prior to establishing the graves, we conducted background measurements of electrical resistivity tomography (ERT), electromagnetics (EM), and ground penetrating radar (GPR) while soil profiles were analyzed in situ after excavating the graves. All graves were also instrumented with soil sensors for monitoring temporal changes in soil moisture, temperature, and electrical conductivity in situ. Measurements of ERT, EM and GPR were repeated 3, 37, 71 and 185 days after burial with further repeated measurements planned for another twelve months. ERT results show an initial increase in resistivity in all graves including the control graves at 3 days after burial and a continuous decrease thereafter in the mass and individual graves with the strongest decrease in the mass grave. Conductivity distribution from the EM shows a similar trend to the ERT with an initial decrease in the first 3 days after burial. Distortion in linear reflectors, presence of small hyperbolas and isolated strong amplitude reflectors in the GPR profiles across the graves is associated with known locations of the graves. These initial results validate the capability of geoelectrical methods in detecting anomalies associated with disturbed ground and human decay while GPR though show some success is limited by the geology of the site.

Applicability of pulp/tooth ratio method for age estimation.

The purpose of this study was to evaluate the reliability of the tooth/pulp ratio method in the process of age estimation at the moment of death in a forensic context and compare it with standard anthropological methods. After the exhumation of mass graves located in sites in Batajnica, Serbia, in 2002 and 2003, skeletal material was thoroughly analyzed by a group of anthropologists and pathologists. As a part of the investigation, orthopantomography (OPG) was performed for each individual. During 2018 these OPGs were reexamined for scientific purposes. Age-at-death was assessed by means of the pulp/tooth ratio method applied to all available lower premolars. Estimated age following standard anthropological methods and chronological age (obtained after DNA identification of victims) was taken from the records. Age estimation using the pulp/tooth ratio and standard methods was compared with chronological age. The pulp/tooth ratio method was accurate in 81.25% of all cases and the standard method was accurate in 56.25% of all cases. The pulp/tooth ratio method of age estimation was found to be applicable and accurate. However, age estimation should be based on all available methods.

Inter and intra-individual variation in skeletal DNA preservation in buried remains.

Our ability to identify skeletal remains often relies on the quality and quantity of DNA extracted from bone and teeth. Current research on buried remains has been retrospective, and no study to our knowledge has comprehensively assessed both intra-individual and inter-individual variation in human skeletal DNA from all representative skeletal element types recovered from a burial. Three individuals were interred together in a single grave for four years. Following disinterment, skeletal DNA was extracted, quantified, and GlobalFiler™ results were produced from 49 bones per skeleton, representing all bone types. Multiple sites per bone were also tested to determine intra-bone variability. Co-extracted bacterial and fungal DNA were quantified to determine microbial loads in the bones. Results show that the small, cancellous bones of the feet outperformed other bones in terms of DNA yield, measured as nanograms per gram of bone powder, and short tandem repeat (STR) profile completeness. The cuneiforms, in particular, had consistently high human DNA yields for all three individuals. DNA yield varied by individual and depth within the grave, with the shallowest individual demonstrating the highest DNA yields While the feet exhibited the greatest variation in DNA yield across bone type and sampling site, they also demonstrated some of the highest DNA yields and the most complete STR profiles, evoking a re-evaluation of their use for skeletal DNA sampling and analysis.

Identification of victims from mass grave discovery near Benghazi, Libya.

BACKGROUND: A mass grave is any site that containing two or more associated corpses, at random or on purpose placed, of people who have died as a result of extra-judicial or random executions, not including those people who have died from armed confrontations or known major catastrophes. CASE PRESENTATION: The purpose of this paper is to explain how to reconstruct a biological profile of decomposed or skeletonized bodies and clarify the efforts done by the Libyan scientist after 2011 revolution and to set a reference for other researcher. The alleged location of the grave, as well as the alleged number and identities of the persons buried in the grave were obtained exclusively from witnesses' and relatives' testimonies. CONCLUSION: As the testimonies said, the grave was located at the alleged location and seven skeletons were exhumed. Also, the osteological and DNA study made investigators to identify the exhumed skeletons. And the dental analysis support the identification of a seven man alleged to have been buried in the grave, 7 victims were discovered.

Challenges associated with investigating a mass grave at the Koricani cliffs in central Bosnia.

More than 200 men were killed at the Koricani Cliffs on Mount Vlasic in central Bosnia during the Bosnian war. The location of this mass grave remained unknown for a long time following the war, until 2008, when the Missing Persons Institute discovered a site containing the remains of approximately 60 individuals. Later, in September 2017, a new mass grave was identified at this location that had not been robbed and skeletal remains remained close to the location where the victims had died. This grave was also unique, by definition, as it was a primary inhumation site, but with a high degree of commingling and disarticulation, typical of secondary inhumation locations. The exhumation team found the first remains in this grave approximately one and a half meters beneath the rocks, while the extent of the commingling resulted in necessary modifications to standardized exhumation protocols. The search and recovery process primarily focused on skulls, groups of bones that remained in clothing, and any bones that were still connected to each other. In total, 86 skulls, 137 groups of bones that had at least 2 bones connected, and a couple of hundred small bones that could not be appointed to individuals, were retrieved. The material was taken to the Sejkovaca Identification Centre where the team took over 1,300 DNA samples for analysis and are now awaiting the results.

Analysis of male specific region of the human Y chromosome sheds light on historical events in Nazi occupied eastern Poland.

In Poland, during the World War II, almost 3 million people were killed during the Nazi occupation, and about 570,000 during the Soviet occupation. Furthermore, historians have estimated that after the World War II at least 30,000 people were killed during the Stalinist regime in Poland (1944-1956). The exact number is unknown, because both executions and burials were kept secret. Thousands of people just vanished. As a response to those events, forensic scientists from the Pomeranian Medical University in Szczecin in cooperation with historians from the Institute of National Remembrance started the project of the Polish Genetic Database of Victims of Totalitarianism, which aim is to identify victims killed in the years 1939-1956. Several exhumations were done under the project, with the biggest one done in Bialystok. According to the information gathered by local historians, a detention centre in Bialystok was the place of the secret burials in late 1940s and 1950s. Surprisingly, except few graves from the post-war period, most of the burials found in Bialystok indicated that majority the victims were probably local civilians who died during the Nazi occupation. Unfortunately, data concerning what happened in the detention ward during that period of time is not very detailed. What was known is that people who got incarcerated were "political prisoners" what, according to Nazi politics, was based on their nationality, religion and activity against the Third Reich. The aim of this research was to test genetically the remains found in Bialystok to determine their possible ethnic background, in order to shed new light on the victims and what happened in the Bialystok detention centre during the Nazi occupation. The analysis of male specific region of the human Y chromosome shows that including phylogenetic analysis into the complex process led by the Polish Genetic Database of Victims of Totalitarianism may help with the final identification of hundreds of anonymous victims.

Wounded to the bone: Digital microscopic analysis of traumas in a medieval mass grave assemblage (Sandbjerget, Denmark, AD 1300-1350).

Battle-related mass burials are considered the most unequivocal evidence of past violence. However, most published studies involve only macroscopic analysis of skeletal remains, commonly arriving only at broad conclusions regarding trauma interpretation. The current study considers a possible avenue for achieving both greater detail and accuracy through digital microscopy. Patterns of injury were investigated among 45 individuals from a Medieval Danish mass grave (Sandbjerget, AD 1300-1350). Injuries were recorded on every anatomical element, except hand and foot bones. Each was photographed and cast, facilitating remote evaluations. Macroscopic analysis was compared with digital microscopy in order to test the relative utility of the latter in characterizing skeletal injuries (mechanism, weapon class, direction, timing of injury). The location of 201 observed injuries, mainly sharp force defects, suggested that many lesions were probably not inflicted by face-to-face opponents. Some microscopic features were indicative of a specific lesion type and weapon class. Digital microscopy was therefore demonstrated to be a complementary tool to macroscopic assessment, enhancing feature observation and quantification and serving to compensate for many of the limitations of macroscopic assessment.

In search for the grave of 100 Poles executed on March 20, 1942 in Zgierz, Poland - research by SIGO (Network for Genetic Identification of Victims).

It can be reasonably assumed that remains exhumed in 2012 and 2013 during archaeological explorations conducted in the Lucmierz Forest, an important area on the map of the German Nazi terror in the region of Lodz (Poland), are in fact the remains of a hundred Poles murdered by the Nazis in Zgierz on March 20, 1942. By virtue of a decision of the Polish Institute of National Remembrance's Commission for the Prosecution of Crimes Against the Polish Nation, the verification of this research hypothesis was entrusted to SIGO (Network for Genetic Identification of Victims) Consortium appointed by virtue of an agreement of December 11, 2015. The Consortium is an extension of the PBGOT (Polish Genetic Database of Totalitarianisms Victims). So far, the researchers have retrieved 14 DNA profiles from among the examined remains, including 12 male and 2 female profiles. Furthermore, 12 DNA profiles of the victims' family members have been collected. Due to the fact that next-of-kin relatives of the victims of the Zgierz massacre are of advanced age, it is of key importance to collect genetic material as soon as possible from the other surviving family members, identified on the basis of a list of victims that has been nearly completely compiled by the Polish Institute of National Remembrance (IPN) and is presented in this paper.

A case study of an unknown mass grave - Hostages killed 70 years ago by a Nazi firing squad identified thanks to genetics.

Almost 6 million people died in Poland during the Nazi occupation and about 570 thousand during the Soviet occupation. But the end of the war was not the end of the trauma. Historians estimate that at least 30 thousand people were killed during the Stalinist regime in Poland. In 2012 the Institute of National Remembrance started to search for hidden burials of victims of communism. Many exhumations were carried out under the project. One of them took place in Bialystok, eastern Poland. According to information gathered by local historians, a detention centre in the heart of city was the place of secret burials of victims of the communist regime. During the exhumation work a burial pit with the remains of 24 victims was found. It's characteristics supported the hypothesis that these people were shot on the spot, in a mass execution during the Nazi occupation. Historians knew of only one such execution, but its victims - according to the available records - were supposed to have been exhumed at the end of the war. Exhumation works and the discovery of the discussed mass grave put in question the events of 1944, which would have been impossible without the field work. The first identifications confirmed the doubts of historians, since both the results of genetic profiling and the conducted anthropological analysis revealed that at the end of the war a mistake was made, and bodies other than those suspected had been exhumed. Having established this fact, the mass grave created at that time should be investigated to reveal the identity of the remains uncovered then.

Nitrous oxide, methane and carbon dioxide patterns and dynamics from an experimental pig mass grave.

The objective of the three-year study was to examine spatial and temporal patterns of fluxes and soil pore air concentrations of methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O) from an experimental mass grave located in a temperate environment. The mass grave (5×10m) contained twenty pig carcasses at a depth of approximately 1m was compared to a plot of the same dimensions containing only disturbed soil, as well as an undisturbed plot. Soil pore air CH4 concentrations were sub-ambient (<1.8ppm) except at 75 and 100cm depths at the mass grave in years 1 and 2 but decreased in year 3. The consumption of CH4 within the aerobic soil resulted in small negative fluxes at the soil surface. Soil pore air CO2 concentration showed an increase with depth in all three plots, with the largest increase (>100,000ppm at 1m) in the mass grave, though there was a marked decrease from years 1 to 3. Surface fluxes of CO2 showed strong seasonal variations, peaking in summer. Soil pore air N2O concentration showed major increases in the mass grave, compared to the other two plots with the pattern maintained over the three years, resulting in larger surface fluxes of N2O. To establish the role of the carcasses in N2O dynamics, we incubated a soil sample containing carcass material which resulted in fast rates of N2O production and consumption. The maintenance of elevated pore air concentration and surface flux of N2O throughout the 3 years suggests that this is a long-term pattern and likely the best of the three gases to use to detect graves. Thus, we suggest that measurement of soil pore air concentrations, especially of N2O, could be a simple and effective approach to help determine the location of clandestine graves.

Analyzing and Interpreting Lime Burials from the Spanish Civil War (1936-1939): A Case Study from La Carcavilla Cemetery.

Over 500 victims of the Spanish Civil War (1936-1939) were buried in the cemetery of La Carcavilla (Palencia, Spain). White material, observed in several burials, was analyzed with Raman spectroscopy and powder XRD, and confirmed to be lime. Archaeological findings at La Carcavilla's cemetery show that the application of lime was used in an organized way, mostly associated with coffinless interments of victims of Francoist repression. In burials with a lime cast, observations made it possible to draw conclusions regarding the presence of soft tissue at the moment of deposition, the sequence of events, and the presence of clothing and other evidence. This study illustrates the importance of analyzing a burial within the depositional environment and taphonomic context.

Preserved brains from the Spanish Civil War mass grave (1936) at La Pedraja1, Burgos, Spain.

During the excavation of the Spanish Civil War mass grave at La Pedraja (Burgos, Spain), 104 individuals were found interred within it, 45 of which displayed brains that were preserved but dehydrated and reduced in size. This exceptional finding has resulted in the formation of a multidisciplinary team, with the aim of obtaining as much information as possible and to primarily understand the taphonomic phenomena that has led to the preservation of these brains. The following types of analyses were undertaken on three of these brains: macroscopy, histology, radiology, chemical-toxicology, genetics, chemical analysis of the soil and 3D modelling for stereolithography. The historical context was considered, plus all archaeological and other forensic data provided by the investigation of the mass grave. The results of the analyses on these morphologically identifiable human brains confirmed the presence of nerve structures, fatty acids, and in one case ante-mortem evidence for an intracranial haemorrhage. The fatty acid profile corresponds to the process of saponification. Therefore, the interpretation is that the preservation of these brains at the mass grave of La Pedraja was due to the saponification process, which was influenced by the manner and cause of death, the chemical composition of the brain, the physicochemical properties of the soil and the meteorological conditions at the time.

Developing the use of Structure-from-Motion in mass grave documentation.

Methods for mass-grave documentation have changed markedly since the first forensic investigations nearly 70 years ago. Recently, however, there has been little advancement in developing new and better methodology, especially when compared to other forensic disciplines and even within traditional archaeology. This paper proposes a new approach, using 3D modelling for the documentation and eventual analysis of mass-graves. Structure-from-Motion (SfM), which creates digital 3D models from a set of still photographs, was tested on a small, simulated mass grave. The results of this test suggest that the method offers resolution previously unavailable to mass-grave investigators, and facilitates stronger analytical potential than the more traditional methods. Further tests are needed to validate these methods, but these initial findings are promising and their application could enhance our knowledge of mass grave dynamics.

Forensic or archaeological issue: is chemical analysis of dental restorations helpful in assessing time since death and identification of skeletonized human remains?

In 2011, small mass grave with completely skeletonized remains was discovered in Belgrade suburb. An eyewitness claimed that skeletons belonged to German soldiers killed in WWII. Anthropologists were engaged to investigate whether the skeletal remains correspond to the indicated German group or represent more recent case requiring court trial. Numerous dental restorations were noticed. Owing to the fact that different dental materials were used in dental practice at certain times, the aim of this study was to explore whether analysis of dental restorations could help in identification and estimation of time since death. Inductively coupled plasma optical emission spectrometry revealed that dental fillings corresponded to copper amalgam, conventional silver amalgam, silicophosphate cement, and zinc phosphate cement. Chemical results combined with anthropological and historical facts suggest that the individuals lived before the 1960s in country with well-developed dental service at that time. Therefore, chemical analysis of dental fillings was useful to distinguish between skeletal remains that are too old to be of forensic interest and the remains relevant to legal investigations.