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Identification by isotopes of human provenancing

Final Report Summary - IDIS (Identification by isotopes of human provenancing)

Final publishable summary report

Executive summary
Stable (δ2H, δ18O, δ13C, δ15N) and radiogenic (Sr and Pb) isotopes are becoming used in human provenancing studies in forensic sciences to track the origin and movements of humans. The isotopic composition in human tissues, acquired through dietary intake has been shown to have a general correlation with the isotopic composition of the environment (i.e. drinking water, soil, pollution). In forensic sciences, isotopic methods, mostly on stable isotopes, have been recently applied to track the place of origin and recent movements of present-day deceased unidentified individuals sometimes related to murder cases. Used as forensic intelligence, the isotopic data has been combined with DNA to aid identification. Despite the potential of the isotopic methods their application is still limited and the huge potential of combining multiple isotopic systems to provide provenance information is almost unrecognized. This project is designed to undertake some of the basic validatory studies required to assess the actual potential of the methodology.
The results derived from this project have demonstrated the potential of the multi-isotope method in accurately identifying regions of origin of unknown individuals. The results have been used for forensic intelligence in investigations by the Dutch police in The Netherlands and by the Dutch Army. This project has demonstrated that the multi-isotope method provides robust information and proves very valuable when other methodologies such as DNA are not useful or have failed.

Project context and objectives
Stable (δ2H, δ18O, δ13C, δ15N) and radiogenic (Sr and Pb) isotopes are used in human provenancing studies in forensic sciences to track the origin and movements of humans. The isotopic composition in human tissues, acquired through dietary intake has been shown to have a general correlation with the isotopic composition of the environment (i.e. drinking water, soil, pollution). In forensic sciences, isotopic methods, mostly on stable isotopes, have been recently applied to track the place of origin and recent movements of present-day deceased unidentified individuals sometimes related to murder cases. Used as forensic intelligence, the isotopic data has been combined with DNA to aid identification.
Despite the potential of the isotopic methods their application is still limited and the huge potential of combining multiple isotopic systems to provide provenance information is almost unrecognized. This study, therefore, aimed to rigorously evaluate the capabilities of a multi-isotopic approach by combining isotopic ratios of both light and heavy elements. In this study multi-isotope analysis δ2H-δ18O-δ13C-δ15N-Sr-Pb have been analysed in human tissues, such as hair and teeth from human volunteers to validate and further develop the multi-isotope method. The isotopic data analysed in hair and teeth from donors in The Netherlands has been used to build isotope databases of bio-available elements in the environment in The Netherlands. In addition, these data have been used to test correlations with environmental isotopic data, such as drinking water, soil, street dust, etc. available from data bases and from samples collected around The Netherlands and neighboring countries.
After the validation phase, one of the objectives of the project was to apply the multi-isotopic technique on 3-5 blind studies already solved by the NFI. However, in mid-2011 the project priorities changed, due to the initiation of a large project together with the Amsterdam-Amstelland Police in The Netherlands. The project consisted on applying the isotope method to a number of cold case investigations (n=12) to help identifying the geographical place of origin of unidentified individuals found dead in the Amsterdam area during the last 20-15 years and who had remained unidentified until the present time. This project was established in 2011 but was carried out during the second part of this project (July 2011-February 2013). One of the unidentified individuals from one of these cold cases was identified during the process of the isotope investigation by the police using “standard” investigative techniques. This case was therefore used for validation purposes.
Other cases in collaboration with the Dutch police and with the Recovery Unit of the Royal Dutch Army provided successful results that were used also for validation purposes of the method. More details about these cases are explained in section 4.1.3.
One objective of the initial project was to obtain different types of bones, hair and teeth from individuals from autopsies to test the homogeneity of isotope composition within individuals that always lived in The Netherlands and from individuals that moved from abroad to The Netherlands and some point in their lives. Despite having reached an agreement with the VU-Medical Center specialists and having had numerous meetings since the start of the project in 2010, it was not possible to obtain the autopsy samples for the research, because detailed information about the patient life could not be retrieved that was essential for our study. This part of the project could not be completed during the grant period but we are, however, still pursuing this study through other laboratories in The Netherlands.
Overall, this project has demonstrated that the multi-isotope method provides robust information and aids the police in their investigation to help identify the place or origin of unidentified individuals. This method is proving very valuable when other methodologies are not useful or have failed.

Main results
(1) Testing and validating the optimal cleaning protocols of human hair to remove external surface contamination for subsequent isotope analysis, specifically for Sr and Pb, of hair keratin. The methods used (a) chloroform, methanol and milli-q water and (b) leaching the hair for few seconds with 0.2M HNO3 acid. The cleaning protocols were tested using hair from individuals (donors) that lived in The Netherlands and did not travel. When the most efficient cleaning protocol was established, Sr and Pb isotope analysis were carried out along hair of a women who moved from India to The Netherlands. This test was carried out to study the potential of using Sr and Pb isotope ratios in human hair to determine mobility. The main result was that such elements are useful if the Sr and Pb isotope composition of the origin and destination locations have contrasting isotope composition. The same experiments were carried out using facial hair of a man traveller that moved from The Netherlands to the USA and back. This study was presented as a poster presentation in the 19th IAFS – International Association of Forensic Sciences Conference in Madeira, Portugal, September 2011. This work was also published in the Journal of Analytical Atomic Spectrometry in 2012.
(2) Testing existing cleaning protocols of hair samples for stable isotope analysis (δ2H, δ18O, δ13C, δ15N) of human hair. A full validation report was written establishing factors such as the weight range of hair sample ideal for an accurate and precise isotope analysis for either δ13C-δ15N, and δ2H-δ18O, gas flows, GC column, linearity, etc. In the case of δ18O analysis, it was established that interferences with N2 were taking place and improvement of the analytical method was necessary. This development work is now planned for after the end of the project.
(3) Establishing the best technique for separation of collagen protein from bone was also tested and validated using existing methods. The existing published methods were used for archaeological bone, thus small modifications were applied for modern bone. Once the method was established, we continued validating the analysis of δ13C-δ15N values in bone collagen, optimising the weight range necessary for a precise and accurate analysis.
(4) Validation and development of δ18O analysis in phosphates. The objective was to develop a method to analyse δ18O values in the phosphate phase of bone bio-apatite and tooth enamel. The method was established and resulted in successful analysis of tooth enamel samples but further improvement is necessary in bone bio-apatite. The δ18O values in the phosphate phase in tooth
enamel was compared to δ18O values in the same samples analysed in the carbonate phase. The data showed a good correlation comparable to published results of other studies. The main conclusion was that δ18O analysis in enamel carbonate produced accurate and precise results. Moreover, this method is faster and more efficient than separating and analysing the phosphate phase from tooth enamel.
(5) Validation and testing sample preparation protocols of tooth enamel such as surface cleaning, drilling and sample dissolution for Sr-Pb isotope analysis. Sr and Pb fractions were separated from the same dissolved sample aliquot. Sample amounts were established to obtain adequate Sr and Pb concentrations for precise and accurate analysis. Tests to minimise the amount of samples required were validated in the second half of the project establishing that sub milligram sample sizes are viable.
(6) Collection of water samples, soil samples and hair samples from around the Netherlands and abroad was started by the researcher of the project and collaborators as well as with collaboration of the Dutch police around The Netherlands. Isotope analysis of the samples were carried out with the purpose of building an isotope database of bio-available elements in The Netherlands and neighbouring countries. Available isotope databases and published relevant data such as Pb isotopes in teeth from different countries, δ18O- δ2H isotope data of precipitation around the world and Sr isotope data of geology and bio-available Sr from different parts of the world, for instance, were collected and collated for building a database. This task started at the beginning of the project and continued through out and will be an ongoing action in the new employment of the researcher.
(7) The researcher (Dr. Laura Font), project coordinator (Prof. Gareth Davies) and collaborators of the project at the Netherlands Forensic Institute - NFI (Dr. Gerard van der Peijl, Dr. Els van Wetten and Dr. Wim Heijnen) met regularly, every 6 months, to evaluate project objectives during the following 6 months period and evaluate the progress reports and validation reports of the techniques that were written and submitted to NFI. Dr. Laura Font was validated as an expert for the application of the isotope method for human provenancing for police case investigations by the NFI and external expert Prof. Peter Horn from the Department of Earth and Environmental Sciences – Ludwig Maximilians University Munich, after all the validation of methods were completed.
(8) The researcher, Dr. Laura Font, worked on a police case investigation as a “blind study” and as part of the validation as an expert. The case was investigated by isotope experts Prof. Peter Horn (Munich), Prof. Gareth Davies (VU) and Dr. Gerard van der Peijl (NFI). The results of both parties were compared at the end of the investigation. The conclusions established by the experts and researcher were in agreement and the arguments and protocols used by Dr. Font were evaluated as optimal by the experts.
(9) A collaboration with dentists from the aural surgery department of the Vrije University Amsterdam Medical Centre (VUmc) was set up to collect third molar samples from donor patients. The objective was to obtain third molars from patients born and living always in The Netherlands and patients that moved during childhood from abroad into the Netherlands. Sr, Pb and δ18O isotopes were analysed to test the variations in isotope composition between Dutch and foreign born individuals and also to set up a database of bio-available Sr-Pb and oxygen in The Netherlands and abroad.
(10) Two bachelor thesis (Simone Mooibroek and Kay Vogel) were completed during this period as part of the validation and method development of δ13C and δ15N analysis in human hair. The thesis of Simone Mooibroek concentrated on establishing δ13C and δ15N variations in groups of people living in The Netherlands but potentially following different types of diet due to their different cultural background. Simone acquired samples from Dutch students of a school in Nijmegen and from
foreign students (mostly Asian) attending the same school. The results show that most of the students, Dutch and foreign, followed a standard omnivore diet with comparable δ13C and δ15N values and no major influence of different culture in diet were observed . The thesis of Kay Vogel focused on analysing δ13C and δ15N values in hair of individuals travelling and group of individuals from the same family following the same diet and consuming the same products. The main result was that travellers showed δ13C and δ15N changes during travel due to access of different food types (i.e. travelling to USA vs Europe – maize vs wheat) and also individuals from the same family and consuming the same food showed differences due to variations in the metabolism (i.e. men vs women).
(11) A project was established in collaboration with the Amsterdam-Amstelland Police (The Netherlands) – application of the isotope method to a number of unidentified individuals (n=12) found during the last 20-15 years in the Amsterdam area and remained unidentified until present time. This project was established in 2011 and the analytical and interpretation of results started in July 2011. From this project one of the unidentified individuals from one case became identified during the course of the isotope investigation. The cold case team and researchers at VU (Dr. Font and Prof Davies ) and NFI (Dr. Van der Peijl) decided to use this case for validation of the isotope method. The results of the multi-isotope study proved to be successful, and suggested a region in Eastern Europe including parts of Poland and The Carpathian region. The individual originated from South Poland. This study has been submitted to Science and Justice for a publication.
(12) A project in collaboration with the Dutch Army for the identification of two World War II soldiers was established. Since DNA, dental records and other methodologies did not provide the identity of these two casualties, isotope analysis were conducted to try to established the place of origin of the two casualties. The isotope results suggested a provenance from the United Kingdom. These results were later confirmed by some of the remains of equipment found with the bodies, which indicated the individuals belonged to the British Royal Marines. The results of this study will be published in Science and Justice in 2014 (proofs returned).
(13) A project in collaboration with the Dutch police (Utrecht Police) was arranged in 2012 to do a multi-isotope study on remains from a body of a girls found in 1976 near Utrecht – “The girl from Heul case”. The isotope results suggested a provenance from Germany. This is a still ongoing investigation. The isotope results for this case together with other results for this investigation have been broadcasted in a TV program on the Dutch television (http://www.uitzendinggemist.nl/afleveringen/1342176) and in numerous news papers (i.e. http://www.volkskrant.nl/vk/nl/2686/Binnenland/article/detail/3399331/2013/02/25/Nieuw-onderzoek-werpt-sprankje-licht-op-moordzaak-Heulmeisje-1976.dhtml)
(14) Other successful cases in collaboration with the Dutch police were: a) analysis of scalp hair from a lady found dead in the water. The isotope results suggested a provenance from Germany, which was later validated as the place where she was living before death; b) analysis of hair and umbilical cord from a baby foundling. The isotope analysis suggested a provenance of the baby and mother from southern Germany. Thanks to a match found of DNA with another baby abandoned three years previously, the identity of the mother could be found and also confirmed a provenance from South Germany, as suggested by the multi-isotope study.
(15) Another collaboration was set up with researchers at Naturalis museum in Leiden involving a case related to finding the geographical place of origin of a wolf found shot dead in The Netherlands. The question was if wolfs were living in The Netherlands or the wolf was shot elsewhere and transported to The Netherlands to hide the crime. The isotope results suggested that the wolf came from packs
living in Eastern Europe. This research was published in LUTRA Journal of the Dutch Mammal Society.

Ethical issues/review
An ethical approval was awarded to the project through the medical centre of the Vrije Universiteit VUmc, ultimately approved by the national ethical commission. The review was required as the research involved donated human hair and teeth samples. Anonymous questionnaires approved by the VUmc ethical where completed by donors to provide information concerning diet and geographic movement. Sample storage in locked refrigerators and the ultimate destruction of all human tissue were a condition of granting the ethical approval. The project has complied with all terms and conditions stipulated associated with the ethical approval.

Potential impact and dissemination activities
The results of the multi-isotope studies have been published in scientific peer reviewed journals (JAAS and Science and Justice; see table A1) and international conferences (see table A2). The isotope results of police investigations have also been broadcasted in the Dutch television (http://www.uitzendinggemist.nl/afleveringen/1342176) and Dutch newspapers (see table A2).
(i.e. http://www.volkskrant.nl/vk/nl/2686/Binnenland/article/detail/3399331/2013/02/25/Nieuw-onderzoek-werpt-sprankje-licht-op-moordzaak-Heulmeisje-1976.dhtml)
Numerous presentations of the method have been given to the Dutch police and prosecutors in The Netherlands to disseminate the potential of the multi-isotope method for forensic intelligence in police investigations. The majority of presentations have been in open talks at the NFI and in workshops given by the NFI to police investigators. Other presentation have been in private as discussing the details of a particular case cannot occur in public.
A logo for this project was designed (by Joanne Davies), which was used in presentations and for promoting the project when samples were requested to the VUmc dentist from patients (see point 9 in section 4.1.3).