Accepted Manuscript Title: Example of human individual identification from World War II gravesite Author: Andrzej Ossowski Marta Ku´s Piotr Brzezi´nski Jakub Pr¨uffer Jarosław Pi˛atek Gra˙zyna Zieli´nska Milena Bykowska Katarzyna Jałowi´nska Anton Torgaszev Antoliy Skoryukov Mirosław Parafiniuk PII: DOI: Reference:
S0379-0738(13)00412-X http://dx.doi.org/doi:10.1016/j.forsciint.2013.09.004 FSI 7346
To appear in:
FSI
Received date: Revised date: Accepted date:
4-2-2013 26-8-2013 2-9-2013
Please cite this article as: A. Ossowski, M. Ku´s, P. Brzezi´nski, J. Pr¨uffer, J. Pi˛atek, G. Zieli´nska, M. Bykowska, K. Jałowi´nska, A. Torgaszev, A. Skoryukov, M. Parafiniuk, Example of human individual identification from World War II gravesite., Forensic Science International (2013), http://dx.doi.org/10.1016/j.forsciint.2013.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page (with authors and addresses)
Example of human individual identification from World War II gravesite. Andrzej Ossowskia,b,c, Marta Kuśa, Piotr Brzezińskib, Jakub Prüfferd, Jarosław Piąteka, Grażyna Zielińskaa, Milena Bykowskac, Katarzyna Jałowińskaa, Anton Torgaszeve , Antoliy Skoryukovf, Mirosław Parafiniuka Pomeranian Medical University in Szczecin, Department of Forensic Medicine,
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Association „Pomorze1945”
Institute of National Remembrace, Branch Office in Szczecin
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Municipal Police Headquarters in Szczecin
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Union of Search Associations of the Tatarstan Republic
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Ministry of Defence of the Russian Federation
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Pomeranian Medical University in Szczecin, Department of Forensic Medicine, Powstańców Wielkopolskich Street 72, Szczecin, Poland
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Keywords: aDNA, DNA, STR, exhumation, war graves, DVI, prisoners of war
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Corresponding author: Mrs. Marta Barbara Kuś, M.Sc.Eng. Tel.: +48 0534095264 E-mail: [email protected]
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*Manuscript (without author identifiers)
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Abstract This paper presents the procedure elaborated by our team which was applied to the mode of identification of Red Army soldiers who were taken as prisoners by the German Army during World War II and deceased in captivity. In the course of our search the unmarked burial of ten Soviet prisoners of war was found. Historical, anthropological and genetic research conducted by us led to the personal identification of nine of them, including two by means of DNA analysis.
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1. Introduction The case presented in this paper is an example that modern methods applied for human personal identification and backed up by extensive archival research are effective in case of the World War II victims. Simultanoeusly to the civilising progress more and more attention was being paid to the fate of a single person, which led for instance to attempts of identifying soldiers being killed in action during numerous armed conflicts. Apart from wounded soldiers also killed ones were evacuated from the battlefield for the sake of their personal identification. Although the last war was raging over the area of the West Pomerania more than 67 years ago, the problem of killed and missing people during those events still exists. The next problem that has arisen recently is a huge number of unmarked and unregistered graves of prisoners that were taken by the German Army in the years 1939-1945. Huge gravesites can be found near former big prison camps, such as Czarne/Hammerstein, Borne Sulinowo/Gross-Born Linde or Stargard Szczeciński/Stargard. However, little separate gravesites existed in most localities of West Pomerania. As a matter of fact there was no possibility of personally identifying prisoners of war and accordingly no such attempts were made during exhumation works until now. In fairness German recognition marks for the prisoners of war were found with the remains from time to time, but the registers of such marks did not survive among documents preserved by the Deutsche Dienststelle archive in Berlin. By creating an electronic onlinbe database of the irrevocable manpower losses of the Red army during World War II the Ministry of Defence of the Russian Federation created for the first time the possibility of identifying Russian prisoners of war.
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Figure 1. Location of prisoners camp
According to the Register of Recommendations of the President of the Russian Federation No. 698 dated 23rd April 2003 on organization of activities for commemoration of the Great Patriotic War and to Decree No. 37 dated 22nd January 2006 „Issues of commemorating the fallen in the defense of the Fatherland” the Ministry of Defence of the Russian Federation created a computer database containing information about defenders of the Fatherland fallen and missing in the years of the Great Patriotic War as well as in the post-war period (OBD Memorial). (1). In identification research the key role is attributed to DNA analysis, both STR (short tandem repeats) and mitochondrial DNA (mtDNA) methods. DNA analysis with the use of STR markers have been widely used recently for personal identification both in ordinary criminal cases and mass-disasters victims’ identification (2,3,4). In case of totalitarian systems and war
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conflicts victims, especially World War II, such studies are conducted only incidentally. The reasons for this are various. Isolation of DNA of proper quality and quantity for DNA analysis with the use of STR markers causes many difficulties. Also a great number of victims’ remains discoveries as well as collecting the samples of comparative genetic material are a huge problem. In reality, conducting thorough search and identification works of the World War II victims is not possible without constraining the size of the group of persons in request. It is possible to conduct DNA analysis only after completion of preliminary identification research and thus finding supposed family members or relatives. The scale of this problem in Poland as well as in many other European countries is enormous, even though more than 60 years have elapsed since the war was ended.
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2. Purpose of the research The purpose of our research was to develop a universal procedure for prisoners of World War II gravesites enabling to conduct a full identification process with the use of DNA analysis in connection with historical and anthropological studies, according to the ISFG recommendations regarding DVI issues, modified to our case. (5).
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3. Materials and methods The gravesite from World War Two located in Linowno in the province Zachodniopomorskie was researched by our team. During the exploration of the site anthropological examination was performed and samples for further genetic tests were taken.
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According to recommendations #1 and #2 from ISFG (5), work is supposed to beingcoordinated the same way as it is done during mass disasters but because of the historical nature of the case the procedure was modified. The DVI team was responsible for the anthropological analysis and collecting material for DNA matter (historians, forensic anthropologists and forensic geneticists were present). According to recommendation #3, proper sample types were taken from each individual, namely healthy molars without fillings. Long compact bone samples were not taken for two reasons: Anthropological examination was done during the work and skeletons were not exhumed but buried once again, so due to possible contamination and technical problems, the DVI team did not take this kind of material. In addition, Russian diplomats were present during the work so cutting long bones would have been inappropriate and unethical. But having experience with extracting DNA from other bones the team decided to take phalanges. It was not a mass grave so there was no way to mistake the small bones. During most of the exhumations done by the team, complete remains were transported to the laboratory, and there samples were collected, namely teeth and long bones. Biological material was preserved in sterile packages and placed at once in cooling conditions and immediately transported to the laboratory where the samples were frozen in -20°C.
According to recommendation #5 from ISFG DNA testing should be performed by laboratories/a laboratory having experience with mass disaster samples. Our laboratory and
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the DVI team have done hundreds of exhumations so far, concerning burials from the World War II. In addition, we identified victims of two mass disasters in Poland. The first was the Mirosławiec air disaster. It occurred on 23.01.2008 when EADS CASA C-295 military transport plane crashed as it approached the Mirosławiec runway, killing all passengers and crew, a total of 20 victims, including high-ranking air force officers. Second was the Kamień Pomorski homeless hostel fire, which occurred on 13.04.2009. 23 people, including twelve children died. During the last few years we have performed identification processes of about 6,000 victims of World War II. Also, our team is now working on a project aiming to identify victims of totalitarian systems in Poland, killed by communists during the years 1944-1956. Our laboratory got a certificate from GEDNAP (German DNA Profiling) in 2009, 2010, 2011 and 2012; a certificate from the Polish Society of Forensic Medicine and Criminology (Polskie Towarzystwo Medycyny Sądowej i Kryminologii) in 2010/2011 and 2012/2013; our lab is working under PN-EN ISO 9001:2009, valid to 06.07.2013.
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Preparation of the material For the purpose of genetic best preserved teeth and good preserved phalanges were taken. Figure 2. Tooth used for the research
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Figure 3. Phalanges used for the research
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To avoid contamination, bone surfaces were cleaned mechanically with the use of a sterile scalpel and then washed with 5% sodium hypochlorite and distilled water and irradiated for 30 minutes under UV lamp (the teeth and phalanges were inverted during the process). The prepared samples were subsequently grinded (?) in a Freezer Mill 6770 (Spex SamplePrep) cryogenic grinder using liquid nitrogen following the manufacturer’s instructions. Powdered sample bone material was placed in sterile Falcon-type tubes.
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DNA extraction DNA from the powdered bone material was extracted by two methods. First, organic (phenol/chloroform), was based on the optimized protocol developed by the team of Prof. Dobosz (6, 7, 8, 9). Supernatant obtained this way was purified with QIAquick PCR Purification Kit (QiaGen) following the manufacturer’s instructions (10). Another extraction method used was the one of PrepFiler® Forensic DNA Extraction Kit (Applied Biosystems) according to optimized manufacturer’s protocol (11).
Real-Time PCR Real-Time PCR analysis was performed to measure concentration of extracted human DNA and to ascertain the presence of possible PCR reaction inhibitors. Quantifiler® Human DNA Quantification Kit (Applied Biosystems) was used following the manufacturer’s instructions (12). The analysis was performed with the use of an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) (13).
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Amplification DNA was amplified in PCR multiplex reaction using the commercial kit AmpFlSTR® NGM™ PCR Amplification Kit (Applied Biosystems) in accordance with the manufacturer’s protocol (14). 15 autosomal STR loci and an amelogenin gene marker (determination of sex of the individual) were amplified simultaneously. The obtained PCR products were separated in a capillary electrophoresis process with the use of ABI Prism 3130 Genetic Analyzer (Applied Biosystems). In a single case testing of STR markers located on the Y chromosome was carried out with the use of the commercial AmpFlSTR® Y-Filer™ PCR Amplification Kit (Applied Biosystems).
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In historical research archival documents of German Deutsche Dienststelle (WASt) from Berlin as well as Russian online database OBD-Memorial were utilized.
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4. Results
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4.1. Gravesite analysis
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Figure 4. Location of Linowno now Figure 5. Location of Linowno before the World War Two
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During talks with the local people a theory emerged that on the cemetery soldiers of the Polish Army in the USSR killed in battle in 1945 were purportedly buried. Reviewing the activities of the Polish 4th Infantry Division on the 3rd of March 1945 in the area of Linowno it was assumed that the grave could contain the remains of lieutenant Arkadiusz Lange, corporal Emil Willman and six other soldiers who were killed nearby in vicinity of nonexistent little village of Lisówek. However, examination of other archival documents revealed that they were buried on the war cemeteries in Drawsko Pomorskie and Wałcz (15). We found some former German inhabitants of Linowno/Woltersdorf. In 2010 we were able to talk to Dietrich Strehlow. In the interview he accounted that the persons buried in an unidentified grave on the cemetery were most probably Soviet prisoners of war who had been directed to work from the Stalag in Stargard Szczeciński or Piła. The group of approx. 30 of them had to work in a big farm of August Otto. According to the witness’s account the prisoners’conditions were so poor that from time to time one of them died and was buried on the local cemetery. The cemetery in Linowno is situated in the north-eastern part of the village at a dirt road, approx. 300 m away from Gudowo-Lubieszewo road. It takes roughly a shape of arhombus with the sides 75-80 m long and an area of approx. 0,6 hectars. The plot is mostly sorrounded by spruce trees and dwarf plants. The cemetery is divided into northern and southern parts with the interior alley leading from the gate towards the east. A field reconnaissance let us establish in which part of the cemetery the grave is located. Our team got down to work on the 2nd December 2011 and 11th February 2012. In the appointed area search works commenced. Soon we found out there was no mass-grave in the area, but some single graves, whereas the bodies buried in them were without coffins what
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suggests that it was a war burial plot. During our work ten prisoners of war burials were found. The skeletons were laid along the axe east-west with their heads towards the west with the exception of the individual #1 who was buried in the opposite way. with his head towards the west (the burials numbering was conferred according to the order of excavation, in fact the individual #1 is situated as the ninth in a row). The depth of the graves was consistent and did not exceed 70 cm. The disposition of the skeletons and location of the gravesite with reference to the cemetery fence is shown on the sketch (Figure 6).
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4.2. Anthropological examination For each individual no perimortem traumas revealing the cause of death were found. Body height was determined by measuring femur bones and then comparing the results to Trotter and Glesser table. (16) Approximate age was determined based on changes in the surface of the pubic symphysis, obliteration of the cranial sutures and the degree of tooth crowns clash. (17,18,19)
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A half of a prisoner recognition mark was found at the neck of all individuals, except individual #2 and #10.
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Individual #1: individual aged 18-20, 177 cm tall, the remains were put in the grave with the face down, head towards the centre of the cemetery, hands arranged along the body. The inscription on the recognition mark was „STALAG II B 116281”.
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Individual #2: individual aged 20-25, 161 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The bones with traces of rickets. Individual #3: individual aged 25-30, 167 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „ KR. GEF. STALAG 315 23715”.
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Individual #4: individual aged 20-25, 158 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the chest just under the neck. Legs slightly bent suggesting that the prepared grave was too short to hold the body. The bones with traces of rickets. The inscription on the recognition mark was „STALAG IIB 113740”. Leather civilian belt found on the waist. Individual #5: individual aged 19-22, 171 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 24598”.
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Individual #6: individual aged 20-25, 161 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG IIB 24677”. Individual #7: individual aged 19-23, 167 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15100”.
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Individual #8: individual aged 22-26, 178 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15850”.
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Individual #9: individual aged 19-22, 169 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15759”, moreover the Soviet paper recognition mark was found in a bakelite tube. Some Soviet coins at face value of 3 kopyeiki and zinc parts of a German tent canvas were found.
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Individual #10: individual aged 19-22, 170 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis.
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The online database OBD-Memorial of the Ministry of Defence of the Russian Federation was searched with the numbers of recognition marks found with the bodies. At a first stage some hundreds results were obtained. Then these results were filtered with the numbers of the prisoners camps (only those in vicinity were taken into account) and finally the scanned cards were thoroughly examined to find the remarks about the place of death. The cards with data matching to eight found recognition marks were chosen.
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4.3. Archival query: The following was read out from the prisoners cards: Table 1. Archival query
Archival query enabled determining the identity of the next individual (excavation number 10) in spite of the fact that no recognition mark had been found with the body. Basing on data concerning dates of death of already identified prisoners it was established that they were buried starting with number 9, so the prisoner number 10 was buried on the same day as number 9 or earlier. So the database OBD-Memorial was searched for the STALAG 315 (first deceased prisoners came from that camp) and death date not later than October 23rd, 1941. For many prisoners the place of death was not given in the database because of difficulties in transcription of original German names to Russian alphabet. This is why the scans of original documents included in the database were thoroughly scrutinized. After searching through
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more than 800 sets of identification cards we came across the prisoner who was buried on 22nd October 1941 in Linowno/Woltersdorf in Drawsko Pomorskie district. Data obtained during anthropological examination of the skeleton matched those included in the prisoner identification card. Figure 7. Remains of prisoner of war B. Pyotr, excavation number 7
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Figure 9. Prisoner card of the POW Andrey L., excavation number 4
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Figure 8. Half of the POW recognition mark with personal number 15100 belonging to Pyotr B.
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Figure 10. Remains of prisoner of war Konstantin K., excavation number 8
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After archival query had been performed and personal data established based on recognition marks, the search for finding still alive close relatives was carried out. The next stage of identification process was the comparative DNA analysis.
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4.4. DNA Analysis According to recommendation #6 from ISFG (5), we used minimum required 12 independent loci, in this case – 16 loci from NGM system and 15 loci from Y-Filer system. According to recommendation #7 from ISFG (5), we reviewed thoroughly all allele calls and candidate matches. We generated composite DNA profiles after two isolations from teeth and two isolations from phalanges.
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Table 2. DNA concentration and PCR reaction inhibitors Table 3. STR profiles
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We have analyzed only these profiles in which peak heights were not less than 100 RFU (20,21). All the degraded profiles were rejected. To eliminate the loss of alleles, we repeated all the reactions and then compared the results, analyzing drop-outs and drop-ins. After analyzing all the results, no extra alleles were observed. Degradation was observed by the preferential amplification of shorter STR fragments. Our team working with the material has been profiled before, as all of us are working in a forensic lab. During the exhumation, we used sterile gloves, Tyvek full coveralls and protective masks. The material was taken and placed in sterile Falcons as quickly/soon as possible. It had no contact with people outside the team. Figure 11. An example of a profile obtained from sample 9. Figure 12. An example of a profile obtained from sample 8.
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Figure 13. An example of a profile obtained from sample 6. Table 4. Comparison Table 5. Comparison
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5. Results The presence of PCR reaction inhibitors in analyzed samples was not ascertained.
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Table 6. DNA concentration
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Table 7. DNA amplification
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According to recommendation #4 from ISFG (5), with help of Ministry of Defence of the Russian Federation, we collected material from the only living first-degree relatives. In the case of the individual #4 indicated on the basis of the recognition mark as Andryey Lvov the sample material was collected from his putative daughter. The performed DNA analysis confirmed the kinship between them and bio-statistical calculations determined its probability as high as 99,998%.
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In the case of the individual #7 indicated on the basis of the recognition mark as Pyotr B. the sample material was collected from his putative son. DNA typing with STR markers in NGM and MiniFiler system confirmed the kinship between them and bio-statistical calculations determined its probability as high as 99,9999994%. DNA analysis in Y-Filer system showed the same genetic profile of exhumed individual and of putative son, which additionally confirmed the identity of Pyotr B. Bio-statistical calculations determined its probability as high as 99,9999999994%.
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According to recommendation #10 from ISFG (5), we used bio-statistical tool used in our forensic cases, DNA STAT v.2.1. software
6. Discussion Nowadays, DVI teams have to gather many specialists from different disciplines. Depending on the case the team should be modified. The one analyzed by us has required geneticists and historians working together. In this case, without extensive historical research, identification of exhumed victims would have been impossible. Together, extremely various fields of science begin to give tremendous opportunities for future research. Considering the amount of still missing and unidentified victims of twentieth-century totalitarian regimes, a combination of current DVI procedures and historical research would be able to identify them in the future. Identification process of World War II victims is based on numerous elements depending on acquired historical data and accessibility of comparative genetic material. Continuous
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development of genetic methods offers new possibilities, but to explore them it is necessary to perform much other research to limit the number of persons involved. Up to now, creating a database of genetic profiles of the victims and comparative material acquired from close relatives is not possible due to the huge amount of missing and deceased persons. World War II victims’ identification process is really difficult because of the amount of victims as well as shortages of preserved archival documentation. The next problem is time factor and thus difficulties in finding relatives of the persons found during excavations. Similar problems have been encountered by our colleagues from Croatia. (22).
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Application of odontology in case of the World War Two victims killed on the Eastern Front is practically impossible (with exception of single USAF pilots shot down over Eastern Front areas) because of a total lack of dental documentation.
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In the case of prisoners of war it turned out in recent years that their captured prison cards were preserved in Russian archives. They contain data on age, sex, height, hair color, fingerprints, prisoner number stamped on the recognition mark and in many instances also a photograph. Such data is essential for comparative anthropological examination.
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Questionnaire system concerning missing persons used by research teams in Croatia (22) is useless in case of the World War II victims due to the huge amount of persons and time that elapsed since the war.
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Applying genetic methods in the identification process of World War II victims is actually only possible then when such analysis is preceded by other types of research, when it is possible to indicate a person or limit a group of persons involved on the basis of recognition marks, archival documents, etc. The same observations were made by Finnish teams identifying Finnish soldiers killed during the World War Two. Only in 20% of the cases it was possible to use DNA analysis (23). In our research the result is similar.
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In the case shown above we had no doubts about connecting recognition marks to respective skeletons as it can frequently happen in case of mass-graves and hastily performed excavations as is shown by Finnish experiences (23). Taking samples of only phalanges and teeth during excavation is based on our earlier experiences that lead to the conclusion that presented extraction methods with the use of just such material is effective and there is no need of sampling bigger bones fragments during field works, which can be problematic and raises the risk of contamination of samples. Anthropological, historical, archival and genetic research proved to be effective in the case presented. Up to now it was possible to identify nine out of ten individuals. In the case of two of them the identity was confirmed by DNA tests. It should be emphasized that they are the first Soviet prisoners of war found in Poland and identified by means of genetic methods. Thanks to bringing interdisciplinary and international expert team into existence it was
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possible to carry out the identification process exceptionally effectively and fast: identification together with DNA analysis lasted only two months.
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Similar attitude to the subject and using interdisciplinary expert teams of historians, archeologists, anthropologists, criminology specialists and first of all geneticists, one can observe in the case of similar research performed all over the world (24). Similar observations were made by the teams working in Spain examining mass graves of the victims of the Civil War 1936-1939. They also place emphasis on the complexity of the research and its multidisciplinarity (25).
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In using both extraction methods and analyzing the results of Real-time PCR no substantial inhibition of PCR reaction was observed. Though exhumation material is one of the most troublesome one can encounter in profession of geneticist it can show the effectiveness of extraction methods in removal of inhibitors. No contamination was observed in the course of examination, which can be attributed to strict procedure obeyed from the commencement of excavation until the end of research work. Samples for genetic analysis was secured at once after exposure of the skeletons before any other examinations were performed and frozen in sterile and tight Falcon-type tubes. The material was purified before DNA extraction similarly like other exhumation teams do (26, 27) to avoid contamination and remove PCR reaction inhibitors. Our earlier experiences showed that the material after excavation should be immediately placed in tightly closed packages and frozen. Low temperature and humidity as well as protection against microorganisms’ growth are necessary for proper preservation of genetic material. Similar observations were done by Iwamura and his team (28).
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References:
1. http://www.obd-memorial.ru/
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2. L.G. Biesecker, J.E. Bailey-Wilson, J. Ballantyne, H. Baum, F.R. Bieber, C. Brenner, et al., DNA identification of victims from the September 11, 2001 World Trade Center attack: scientific challenges and policy implications, Science. 310 (2005) 1122-3. 3. A. Alonso, P. Martín, C. Albarrán, P. García, L. Fernández de Simón, M. Jesús Iturralde, et al., Challenges of DNA profiling in mass disaster investigations, CMJ 46 (2005) 540-8. 4. T.J. Parsons, R. Huel, J. Davoren, C. Katzmarzyk, A. Miloš, A. Selmanović, et al., Application of novel “mini-amplicon” STR multiplexes to high volume casework on degraded skeletal remains, Foren. Sci. Int. 1 (2007) 175-9. 5. M. Prinz et al., DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI), Foren. Sci. Int. Gen. 1 (2007) 3-12
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6. A. Lebioda, M. Żołędziewska, E. Kowalczyk, A. Jonkisz, T. Dobosz, Ultimate shortening of the PCR product in the STR system TH01 – a new perspective in testing of decay forensic samples. 19th International Congress International Society for Forensic Genetics, Münster (Germany), 28 August–1 September 2001, Programme and abstracts, (2001) 106.
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7. M. Żołędziewska, Badanie polimorfizmu mikrosatelitarnego w próbkach częściowo zdegradowanego DNA z użyciem własnej metody reakcji multipleksowej PCR, [Maszynopis powielany] Akad. Med. we Wrocławiu, Wrocław (2004).
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8. M. Żołędziewska, T. Dobosz, Comparison of various decalcificators in preparation of DNA from human rib bone. Anthropol. Rev. 65 (2002) 75–80.
10. QiaGen: QIAquick® Spin Handbook 03/2008.
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9. M. Żołędziewska, T. Dobosz, Gender determination in highly degraded DNA samples, Prog. Forensic. Genet. 9 (2003) 593–595.
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11. Applied Biosystems: PrepFiler® and PrepFiler® BTA Forensic DNA Extraction Kits User’s Manual, Foster City, CA., P/N 4463348 Rev. C, January 2012.
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12. Applied Biosystems: Quantifiler® Duo DNA Quantification Kit User’s Manual, Foster City, CA., P/N 4391294 Rev. A, January 2008. 13. Applied Biosystems: Applied Biosystems 7500 Real-Time PCR System, Manual.
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14. Applied Biosystems: AmpFlSTR® NGM™ PCR Amplification Kit User’s Manual, Foster City, CA., P/N 4425511 Rev. E, July 2011.
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15. www.pomorze1945.com/
16. M. Trotter, G.C. Gleser, A re-evaluation of estimation of stature based on measurements of stature taken during life and long bones after death, Am J Phys Anthropol 16 (1958) 79-123
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17. T.W. Todd, Age changes in the pubic bone: I. The white male pubis, Am J Phys Anthropol 3 (1920) 467-470 18. P.H. Stevenson, Age order of epiphyseal union in man, Am J Phys Anthropol 7 (1924) 59-93 19. D.H. Uberlaker, Estimating age at death from immature human skeletons: overview, J Forensic Sci 32 (1987) 1254-1263
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20. P. Gill et al., DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures, Foren. Sci. Int. 160 (2006) 90101
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21. P.M. Schneider et al., The German Stain Commission: recommendations for the interpretation of mixed stains, Int J Legal Med 123 (2009) 1-5 22. M. Slaus et al., Identification and analysis of human remains recovered from wells from the 1991 War in Croatia, Foren. Sci. Int. 171 (2007) 37-43. 23. J.U. Palo, M. Hedman, N. Söderholm, A. Sajantila, Repatriation and Identification of Finnish World War II Soldiers, CMJ 48 (2007) 528-535.
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24. C. Nunez et al., Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees, CMJ 52 (2011) 336-43.
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25. L. Rı´os, J.I. Casado Ovejero, J. Puente Prieto, Identification process in mass graves from the Spanish Civil War I, Foren. Sci. Int. 199 (2010) 27–36.
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26. S. Andelinovic, D. Sutlovic, I.E. Ivkosic, et. al., Twelve-year experience in identification of skeletal remains from mass graves, CMJ 46 (2005) 530-9.
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27. A.A. Westen, R.R.R. Gerretsen, G.J.R. Maat, Femur, rib, and tooth sample collection for DNA analysis in disaster victim identificztion (DVI), Foren. Sci. Med. Pathol. 4 (2008) 15-21.
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28. E.S. Miazato Iwamura, J.A. Soares-Vieira, D.R. Muñoz, human identification and analysis of DNA in bones, Rev. Hosp. Clín. Fac. Med. S. PAULO 59 (6) (2004) 383388.
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i
Figure5
Page 18 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure6
Page 19 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure7
Page 20 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure8
Page 21 of 35
Ac ce p
te
d
M
an
us
cr
ip t
Figure9
Page 22 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure10
Page 23 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure11
Page 24 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure12
Page 25 of 35
Ac
ce
pt
ed
M
an
us
cr
i
Figure13
Page 26 of 35
#5 Fedor K.
#6 Shakir F.
Logvin
Yakov
Akim
Aleksandr
Makey
Roman
Religion
orthodox
graecocatholic
orthodox
graecocatholic
graecocatholic
Place,date of birth
Timoshchevskaya 1923
Alma-Ata 05.06.1912
Tschesanino 1919
Oboymovo 1920
Rank
private
private
private
private
Unit
?
346 Construction Battalion
?
Profession
farm worker
farm worker
farm worker
Place,date of captivity
? 16.07.1942
Putkovitschy 22.06.1941
Camp
Stalag IIB Hammerstein
Prisoner’s number
116281
Height
172 cm
Hair
blond
Date and place of death
Andrey
Ivan
graeco-catholic
graecocatholic
Tschimkent 1915
Jeral 29.01.1920
Aleksandrovka 05.07.1917
graecocatholic PrimorskoAkhtyrskaya 17.10.1921
private
private
private
private
private
9 Armoured Regiment
?
201 Rifle Regiment
20 Rifle Regiment
5 Armoured Regiment
?
mechanic
farm worker
farm worker
farm worker
farm worker
tractor driver
Stalingrad 23.09.1942
Mokhylev 29.07.1941
Samborov 03.07.1941
Kovno 01.07.1941
Volma 07.07.1941
Desna 06.07.1941
Rudashkovitsy 17.07.1941
Stalag 315 Hammerstein
Stalag IIB Hammerstein
Stalag 315 Hammerstein
Stalag IIB Hammerstein
Stalag IIB Hammerstein
Stalag 315 Hammerstein
Stalag 315 Hammerstein
23715
113740
24598
24677
15100
15850
15759
Stalag 315 Hammerstein 14343 (not found)
166 cm
157 cm
170 cm
161 cm
161 cm
175 cm
167 cm
173 cm
blond
blond
dark
black
blond
dark blonde
black
blond
none
none
none
none
none
none
none
none
none
typhoid
none
none
none
none
none
none
none
none
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
23.03.1943 Linowno/ Woltersdorf
04.12.1941 Linowno/ Woltersdorf
22.05.1943 Linowno/ Woltersdorf
21.11.1941 Linowno/ Woltersdorf
14.11.1941 Linowno/ Woltersdorf
27.10.1941 Linowno/ Woltersdorf
24.10.1941 Linowno/ Woltersdorf
23.10.1941 Linowno/ Woltersorf
22.10.1941 Linowno/ Woltersdorf
Ac c
Condition of health
Vasiliy
us
Father’s name
Vaccinations
#10 Konstantin K.
an
Leonid D.
Distinguishing marks
#9 Yosif S.
M
Aleksey M.
#7 Petr B.
cr
#4 Andrey L.
d
#3
ep te
#1
ip t
Table 1
#8 Konstantin K.
orthodox Kamyshyno 18.08.1920
Page 27 of 35
Table 2 extr. method & lysis time
0,000273
1
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,000243
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,005682
teeth phalanx bone phalanx bone
2
teeth teeth phalanx bone phalanx bone
3
teeth teeth 4
M
phalanx bone phalanx bone
Ac
0,000352 0,000755 0,000777 0,000644 0,002298 0,010266
0,000190
7
0,037331
8
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H
0,004402
teeth teeth
teeth teeth phalanx bone
0,002895
PrepFiler 2H phenol/chloroform 24H
6
teeth
phalanx bone phalanx bone
0,000644
0,000035
teeth
teeth
0,000513
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
5
teeth
phalanx bone phalanx bone
0,000256
0,001372
ce pt
phalanx bone phalanx bone
0,000018
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
ed
teeth teeth
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,000405
9
ip t
teeth
0,000200
cr
teeth
us
teeth
phalanx bone phalanx bone
DNA concentration [ng/µl]
name
an
material
0,000119 0,000020 0,000511
0,000628 0,000117 0,008953
0,000505
0,013776 0,001175 0,001734
0,004525 0,000894
Page 28 of 35
phalanx bone
phenol/chloroform 24H
0,000534
teeth
PrepFiler 2H phenol/chloroform 24H
0,000101
10
0,000218
Ac
ce pt
ed
M
an
us
cr
ip t
teeth
Page 29 of 35
phalanx bone teeth teeth teeth phalanx bone
2
phalanx bone teeth
teeth 3
phalanx bone teeth
teeth 4
phalanx bone teeth
teeth
phalanx bone teeth
D18 S51
D22 S1045
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
5
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H Mini-STR
D19 S433
TH01
FGA
D2 S441
D3 S1358
D1 S1656
D12 S391
13
17,19
12
-
XY
13,14
28,31.2
17
15,16
14
6
21
11,14
16,18
11
18
13
17,19
-
21
XY
13,14
28,31.2
17
15,16
14,16.2
6,9
21
11,14
16,18
11
-
13
-
-
-
-
13
-
-
16
-
-
-
11,14
-
13
18
13,16
19
-
-
XY
13,14
31.2
17
15
-
-
-
11
16
-
18
12
17,21
XY
28,31.2
17
cr
D21 S11
21,24
14,17
17,20
-
-
XY
12,13
28,30
16
11,17
13
-
-
11,14
14,15
-
-
17
17
11,13
-
XY
12,13
-
17
11
13,17.2
6,9
19
11
15
-
23
14,17
17,20
11,13
25
XY
12,13
28,30
16,17
11,17
13
6,9
19,23
11
15
14,15
23
14,17
17,20
11,13
17,25
XY
28,30
16
11,17
13,17.2
6,9
19,23
11,14
14,15
14
23
11,13
17,25
XY
28,30
16,17
XY
14
28
16
16
13,14
9
-
14
17,18
12,13
17,21
14
28,33.2
16
16
13,14
7,9
21,22
14
17,18
12,13
17,21
13,14
18
13
-
13,14
18
11,13
16,17
13,14 13,14
13,16 13,16
XY
12,12
19,23
18
-
17
XY
-
28
20
16
14
-
21
14
17,18
13
-
18
11
-
XY
14
28,33.2
-
16
13,14
7,9
21,22
14
17,18
12,13
17
11,13
16,17
XY
28,33.2
16
17
9
-
XY
14
31
16
12,16
13,13.2
8,9.3
24
14
17
17.3
20
17
9,12
21
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20
21,22
16
17
9,12
21
XY
14
30
16
12,16
13,13.2
8,9.3
-
14
15,17
-
20
13,16
17
9,12
21,25
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20,21
9,12
21,25
XY
30,31
15,16
Mini-STR
teeth
phalanx bone
D8 S1179
Mini-STR
teeth
phalanx bone
AMG
Mini-STR
teeth
phalanx bone
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
D2 S1338
us
1
D16 S539
ep te
phalanx bone
vWA
an
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H Mini-STR
D10 S1248
M
teeth
extr. method & lysis time
d
name
Ac c
material
ip t
Table3
20,24
13,15
16
11
-
XY
12
30,31.2
-
11,15
13,14
6,8
-
11,11.3
15,16
13
20
-
17
-
-
XY
12
31.2
12
11
13
-
-
11,11.3
15,16
17.3
18
-
-
-
-
XY
-
-
-
11
13,14
-
-
11,11.3
16
-
-
13,15
16
11,14
20,24
XY
12
30,31.2
12
11,17
13,14
6,8
21,24
11
15,16
13,17.3
18,20
11
20,24
XY
31.2
12,14
D13 S317
D7 S820
CS F1PO
11
10,12
9,11
12,13
11,12
10,12
11,14
10,12
11,12
8,9
9,10
11
12
9,10
10,11
Page 30 of 35
teeth
7
teeth 8
phalanx bone
14,15
16,18
11,12
19,24
XY
13
32.2
13,14
11,15
14
16,18
12
-
XY
13
30
-
14,15
16,18
11,12
19,24
XY
13
30,32.2
13,14
11,12
19,24
XY
PrepFiler 2H phenol/chlorofom 24H
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
teeth 9
phalanx bone teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
teeth 10
-
9.3
23,24
-
15,17
-
17
12,13
9.3
23,14
11,12
15
19.3
15,17
11,15
12
9.3
-
11,12
15
17.3
-
11,15
12,13
9.3
23,24
11,12
15
17.3,19.3
15,17
30,32.2
13,14
23,24
17
12,16
13,16
6,7
24
11.3,15
16,19
17,17.3
19
12,16
13,16
6,7
24
11.3,15
16,19
17,17.3
18,19
14,16
17
11
-
XY
10
28
14,16
17,18
11
-
XY
10,13
28,29
17,19
11
19,23
XY
28,29
17,19
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
14
11,13
16,26
XY
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
11,13
16,26
XY
27,30
19
18
12,13
20,23
XY
12,14
29
15,16
11
12,16.2
7,9.3
21
11,11.3
16
15,16
22,23
18
12,13
20,23
XY
12,14
29
15,16
11
12,16.2
9.3
21
11,11.3
16
15,16
22,23
18
-
23
XY
12,14
29
-
11
12,16.2
9.3
21
11,11.3
16
15,16
23
18
12,13
20
XY
12,14
29
15,16
11
12,16.2
9.3
21
11,11.3
16
15,16
23
12,13
20,23
XY
29
15,16
12,13 12,13 12,13 12,13
10,13
24
16
Mini-STR PrepFiler 2H phenol/chlorofom 24H Mini-STR
ip t
11,15
Mini-STR
teeth
teeth
-
ep te
teeth
teeth
-
Mini-STR
teeth
phalanx bone
-
M
teeth
phalanx bone
-
Mini-STR
teeth teeth
-
cr
phalanx bone
12
us
6
16
Ac c
phalanx bone
15
an
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
d
teeth
18,21
21
14
17
-
24
-
13
-
14
16,17
15
-
19,24
14
17
-
18
14
17,18
11
20
XY
13
30.2,31.2
13
16,17
13,15
8,9.3
19,24
10,14
16,17
13
-
11
24
XY
31.2
13,14
19,24
10,11
9
11,13
9,11
9,10
12
11,13
10,11
12
13,14
9,11
11,12
11
-
11
Page 31 of 35
4
D2 S1338
AMG
D8 S1179
D21 S11
D18 S51
D22 S1045
D19 S433
TH01
FGA
D2 S441
D3 S1358
D1 S1656
D12 S391
13,16
17
9
-
XY
14
31
16
12,16
13,13.2
8,9.3
24
14
17
17.3
20
13,16
17
9,12
21
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20
16
17
9,12
21
XY
14
30
16
12,16
13,13.2
8,9.3
-
14
15,17
-
20
13,16
17
9,12
21,25
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20,21
9,12
21,25
XY
30,31
15,16
9
21,25
XX
phenol/chlorofom 24H - MiniFiler PrepFiler 2H
13,15
16,17
10,14
30,32
13,15
12,15
13,15
7,9.3
20,22
11,14
17,18
16,17.3
19,21
D7S820
CSF1PO
8,9
9,10
11
8
10
11
M
p.54
20,24
D13 S317
d
teeth swab
D16 S539
ep te
phalanx bone phalanx bone
vWA
cr
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
D10 S1248
us
teeth
extr. method & lysis time
an
no
Ac c
material
ip t
Table4
Page 32 of 35
10
28
17
12,16
14,16
17,18
11
-
XY
10,13
28,29
17,19
11
19,23
XY
28,29
17,19
11
18,23
XY
24H PrepFiler 2H
D12 S391
13,16
6,7
24
11.3,15
16,19
17,17.3
19
13,16
6,7
24
11.3,15
16,19
17,17.3
18,19
16
Y_DYS391
12,15
7,9.3
Y_DYS439
us 16,17
Y_DYS393
phenol/chlorofom
28,33.2
12,16
an
7
10,12
G_DYS385
teeth
18
G_DYS19
extr. method & lysis time
14,15
D1 S1656
24 24,25
10,11.3
15,19
16,17
18,19
D13 S317
D7 S820
CSF1PO
9,11
9,10
12
11,12
10
11,12
R_DYS448
XY
D3 S1358
R_DYS438
-
D2 S441
R_DYS437
11
FGA
R_Y_GATA _H4
17
TH01
Y_DYS392
14,16
D19 S433
Y_DYS635
D22 S1045
cr
D18 S51
M
PrepFiler 2H
name
p.91
D21 S11
phenol/chlorofom 24H - MiniFiler
material
swab
D8 S1179
14
12
23
28
15
14
14
13
10
10
21
11
11
16
10
20
14
12
23
28
15
14
14
13
10
10
21
11
11
16
10
20
d
p.91
AMG
G_DYS458
swab
D2 S1338
B_DYS389II
teeth
D16 S539
ep te
7
vWA
B_DYS390
teeth
PrepFiler 2H phenol/chlorofom 24H
D10 S1248
B_DYS389I
teeth
extr. method & lysis time
Ac c
no
B_DYS456
material
ip t
Table5
Page 33 of 35
Table 6
Teeth organic method
Teeth PrepFiler
Phalanges organic method
Phalanges Prepfiler
lowest
0,0001
0,00003
0,00001
0,00001
highest
0,0138
0,0373
0,01
0,00229
average
0,003
0,005
0,0029
0,0007
Ac
ce pt
ed
M
an
us
cr
ip t
DNA concentration [ng/µl]
Page 34 of 35
Table 7
Teeth Organic method
Teeth PrepFiler
Phalanges Organic method
Phalanges PrepFiler
Allele per individual (average)
24,2
20,4
25,1
17,6
Ac
ce pt
ed
M
an
us
cr
ip t
DNA amplification
Page 35 of 35
S0379-0738(13)00412-X http://dx.doi.org/doi:10.1016/j.forsciint.2013.09.004 FSI 7346
To appear in:
FSI
Received date: Revised date: Accepted date:
4-2-2013 26-8-2013 2-9-2013
Please cite this article as: A. Ossowski, M. Ku´s, P. Brzezi´nski, J. Pr¨uffer, J. Pi˛atek, G. Zieli´nska, M. Bykowska, K. Jałowi´nska, A. Torgaszev, A. Skoryukov, M. Parafiniuk, Example of human individual identification from World War II gravesite., Forensic Science International (2013), http://dx.doi.org/10.1016/j.forsciint.2013.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page (with authors and addresses)
Example of human individual identification from World War II gravesite. Andrzej Ossowskia,b,c, Marta Kuśa, Piotr Brzezińskib, Jakub Prüfferd, Jarosław Piąteka, Grażyna Zielińskaa, Milena Bykowskac, Katarzyna Jałowińskaa, Anton Torgaszeve , Antoliy Skoryukovf, Mirosław Parafiniuka Pomeranian Medical University in Szczecin, Department of Forensic Medicine,
c
Association „Pomorze1945”
Institute of National Remembrace, Branch Office in Szczecin
d
Municipal Police Headquarters in Szczecin
e
Union of Search Associations of the Tatarstan Republic
us
Ministry of Defence of the Russian Federation
f
ip t
b
cr
a
an
Pomeranian Medical University in Szczecin, Department of Forensic Medicine, Powstańców Wielkopolskich Street 72, Szczecin, Poland
M
Keywords: aDNA, DNA, STR, exhumation, war graves, DVI, prisoners of war
Ac
ce pt
ed
Corresponding author: Mrs. Marta Barbara Kuś, M.Sc.Eng. Tel.: +48 0534095264 E-mail: [email protected]
Page 1 of 35
*Manuscript (without author identifiers)
ip t
Abstract This paper presents the procedure elaborated by our team which was applied to the mode of identification of Red Army soldiers who were taken as prisoners by the German Army during World War II and deceased in captivity. In the course of our search the unmarked burial of ten Soviet prisoners of war was found. Historical, anthropological and genetic research conducted by us led to the personal identification of nine of them, including two by means of DNA analysis.
ce pt
ed
M
an
us
cr
1. Introduction The case presented in this paper is an example that modern methods applied for human personal identification and backed up by extensive archival research are effective in case of the World War II victims. Simultanoeusly to the civilising progress more and more attention was being paid to the fate of a single person, which led for instance to attempts of identifying soldiers being killed in action during numerous armed conflicts. Apart from wounded soldiers also killed ones were evacuated from the battlefield for the sake of their personal identification. Although the last war was raging over the area of the West Pomerania more than 67 years ago, the problem of killed and missing people during those events still exists. The next problem that has arisen recently is a huge number of unmarked and unregistered graves of prisoners that were taken by the German Army in the years 1939-1945. Huge gravesites can be found near former big prison camps, such as Czarne/Hammerstein, Borne Sulinowo/Gross-Born Linde or Stargard Szczeciński/Stargard. However, little separate gravesites existed in most localities of West Pomerania. As a matter of fact there was no possibility of personally identifying prisoners of war and accordingly no such attempts were made during exhumation works until now. In fairness German recognition marks for the prisoners of war were found with the remains from time to time, but the registers of such marks did not survive among documents preserved by the Deutsche Dienststelle archive in Berlin. By creating an electronic onlinbe database of the irrevocable manpower losses of the Red army during World War II the Ministry of Defence of the Russian Federation created for the first time the possibility of identifying Russian prisoners of war.
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Figure 1. Location of prisoners camp
According to the Register of Recommendations of the President of the Russian Federation No. 698 dated 23rd April 2003 on organization of activities for commemoration of the Great Patriotic War and to Decree No. 37 dated 22nd January 2006 „Issues of commemorating the fallen in the defense of the Fatherland” the Ministry of Defence of the Russian Federation created a computer database containing information about defenders of the Fatherland fallen and missing in the years of the Great Patriotic War as well as in the post-war period (OBD Memorial). (1). In identification research the key role is attributed to DNA analysis, both STR (short tandem repeats) and mitochondrial DNA (mtDNA) methods. DNA analysis with the use of STR markers have been widely used recently for personal identification both in ordinary criminal cases and mass-disasters victims’ identification (2,3,4). In case of totalitarian systems and war
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conflicts victims, especially World War II, such studies are conducted only incidentally. The reasons for this are various. Isolation of DNA of proper quality and quantity for DNA analysis with the use of STR markers causes many difficulties. Also a great number of victims’ remains discoveries as well as collecting the samples of comparative genetic material are a huge problem. In reality, conducting thorough search and identification works of the World War II victims is not possible without constraining the size of the group of persons in request. It is possible to conduct DNA analysis only after completion of preliminary identification research and thus finding supposed family members or relatives. The scale of this problem in Poland as well as in many other European countries is enormous, even though more than 60 years have elapsed since the war was ended.
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2. Purpose of the research The purpose of our research was to develop a universal procedure for prisoners of World War II gravesites enabling to conduct a full identification process with the use of DNA analysis in connection with historical and anthropological studies, according to the ISFG recommendations regarding DVI issues, modified to our case. (5).
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3. Materials and methods The gravesite from World War Two located in Linowno in the province Zachodniopomorskie was researched by our team. During the exploration of the site anthropological examination was performed and samples for further genetic tests were taken.
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According to recommendations #1 and #2 from ISFG (5), work is supposed to beingcoordinated the same way as it is done during mass disasters but because of the historical nature of the case the procedure was modified. The DVI team was responsible for the anthropological analysis and collecting material for DNA matter (historians, forensic anthropologists and forensic geneticists were present). According to recommendation #3, proper sample types were taken from each individual, namely healthy molars without fillings. Long compact bone samples were not taken for two reasons: Anthropological examination was done during the work and skeletons were not exhumed but buried once again, so due to possible contamination and technical problems, the DVI team did not take this kind of material. In addition, Russian diplomats were present during the work so cutting long bones would have been inappropriate and unethical. But having experience with extracting DNA from other bones the team decided to take phalanges. It was not a mass grave so there was no way to mistake the small bones. During most of the exhumations done by the team, complete remains were transported to the laboratory, and there samples were collected, namely teeth and long bones. Biological material was preserved in sterile packages and placed at once in cooling conditions and immediately transported to the laboratory where the samples were frozen in -20°C.
According to recommendation #5 from ISFG DNA testing should be performed by laboratories/a laboratory having experience with mass disaster samples. Our laboratory and
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the DVI team have done hundreds of exhumations so far, concerning burials from the World War II. In addition, we identified victims of two mass disasters in Poland. The first was the Mirosławiec air disaster. It occurred on 23.01.2008 when EADS CASA C-295 military transport plane crashed as it approached the Mirosławiec runway, killing all passengers and crew, a total of 20 victims, including high-ranking air force officers. Second was the Kamień Pomorski homeless hostel fire, which occurred on 13.04.2009. 23 people, including twelve children died. During the last few years we have performed identification processes of about 6,000 victims of World War II. Also, our team is now working on a project aiming to identify victims of totalitarian systems in Poland, killed by communists during the years 1944-1956. Our laboratory got a certificate from GEDNAP (German DNA Profiling) in 2009, 2010, 2011 and 2012; a certificate from the Polish Society of Forensic Medicine and Criminology (Polskie Towarzystwo Medycyny Sądowej i Kryminologii) in 2010/2011 and 2012/2013; our lab is working under PN-EN ISO 9001:2009, valid to 06.07.2013.
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Preparation of the material For the purpose of genetic best preserved teeth and good preserved phalanges were taken. Figure 2. Tooth used for the research
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Figure 3. Phalanges used for the research
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To avoid contamination, bone surfaces were cleaned mechanically with the use of a sterile scalpel and then washed with 5% sodium hypochlorite and distilled water and irradiated for 30 minutes under UV lamp (the teeth and phalanges were inverted during the process). The prepared samples were subsequently grinded (?) in a Freezer Mill 6770 (Spex SamplePrep) cryogenic grinder using liquid nitrogen following the manufacturer’s instructions. Powdered sample bone material was placed in sterile Falcon-type tubes.
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DNA extraction DNA from the powdered bone material was extracted by two methods. First, organic (phenol/chloroform), was based on the optimized protocol developed by the team of Prof. Dobosz (6, 7, 8, 9). Supernatant obtained this way was purified with QIAquick PCR Purification Kit (QiaGen) following the manufacturer’s instructions (10). Another extraction method used was the one of PrepFiler® Forensic DNA Extraction Kit (Applied Biosystems) according to optimized manufacturer’s protocol (11).
Real-Time PCR Real-Time PCR analysis was performed to measure concentration of extracted human DNA and to ascertain the presence of possible PCR reaction inhibitors. Quantifiler® Human DNA Quantification Kit (Applied Biosystems) was used following the manufacturer’s instructions (12). The analysis was performed with the use of an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) (13).
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Amplification DNA was amplified in PCR multiplex reaction using the commercial kit AmpFlSTR® NGM™ PCR Amplification Kit (Applied Biosystems) in accordance with the manufacturer’s protocol (14). 15 autosomal STR loci and an amelogenin gene marker (determination of sex of the individual) were amplified simultaneously. The obtained PCR products were separated in a capillary electrophoresis process with the use of ABI Prism 3130 Genetic Analyzer (Applied Biosystems). In a single case testing of STR markers located on the Y chromosome was carried out with the use of the commercial AmpFlSTR® Y-Filer™ PCR Amplification Kit (Applied Biosystems).
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In historical research archival documents of German Deutsche Dienststelle (WASt) from Berlin as well as Russian online database OBD-Memorial were utilized.
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4. Results
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4.1. Gravesite analysis
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Figure 4. Location of Linowno now Figure 5. Location of Linowno before the World War Two
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During talks with the local people a theory emerged that on the cemetery soldiers of the Polish Army in the USSR killed in battle in 1945 were purportedly buried. Reviewing the activities of the Polish 4th Infantry Division on the 3rd of March 1945 in the area of Linowno it was assumed that the grave could contain the remains of lieutenant Arkadiusz Lange, corporal Emil Willman and six other soldiers who were killed nearby in vicinity of nonexistent little village of Lisówek. However, examination of other archival documents revealed that they were buried on the war cemeteries in Drawsko Pomorskie and Wałcz (15). We found some former German inhabitants of Linowno/Woltersdorf. In 2010 we were able to talk to Dietrich Strehlow. In the interview he accounted that the persons buried in an unidentified grave on the cemetery were most probably Soviet prisoners of war who had been directed to work from the Stalag in Stargard Szczeciński or Piła. The group of approx. 30 of them had to work in a big farm of August Otto. According to the witness’s account the prisoners’conditions were so poor that from time to time one of them died and was buried on the local cemetery. The cemetery in Linowno is situated in the north-eastern part of the village at a dirt road, approx. 300 m away from Gudowo-Lubieszewo road. It takes roughly a shape of arhombus with the sides 75-80 m long and an area of approx. 0,6 hectars. The plot is mostly sorrounded by spruce trees and dwarf plants. The cemetery is divided into northern and southern parts with the interior alley leading from the gate towards the east. A field reconnaissance let us establish in which part of the cemetery the grave is located. Our team got down to work on the 2nd December 2011 and 11th February 2012. In the appointed area search works commenced. Soon we found out there was no mass-grave in the area, but some single graves, whereas the bodies buried in them were without coffins what
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suggests that it was a war burial plot. During our work ten prisoners of war burials were found. The skeletons were laid along the axe east-west with their heads towards the west with the exception of the individual #1 who was buried in the opposite way. with his head towards the west (the burials numbering was conferred according to the order of excavation, in fact the individual #1 is situated as the ninth in a row). The depth of the graves was consistent and did not exceed 70 cm. The disposition of the skeletons and location of the gravesite with reference to the cemetery fence is shown on the sketch (Figure 6).
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4.2. Anthropological examination For each individual no perimortem traumas revealing the cause of death were found. Body height was determined by measuring femur bones and then comparing the results to Trotter and Glesser table. (16) Approximate age was determined based on changes in the surface of the pubic symphysis, obliteration of the cranial sutures and the degree of tooth crowns clash. (17,18,19)
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A half of a prisoner recognition mark was found at the neck of all individuals, except individual #2 and #10.
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Individual #1: individual aged 18-20, 177 cm tall, the remains were put in the grave with the face down, head towards the centre of the cemetery, hands arranged along the body. The inscription on the recognition mark was „STALAG II B 116281”.
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Individual #2: individual aged 20-25, 161 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The bones with traces of rickets. Individual #3: individual aged 25-30, 167 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „ KR. GEF. STALAG 315 23715”.
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Individual #4: individual aged 20-25, 158 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the chest just under the neck. Legs slightly bent suggesting that the prepared grave was too short to hold the body. The bones with traces of rickets. The inscription on the recognition mark was „STALAG IIB 113740”. Leather civilian belt found on the waist. Individual #5: individual aged 19-22, 171 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 24598”.
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Individual #6: individual aged 20-25, 161 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG IIB 24677”. Individual #7: individual aged 19-23, 167 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15100”.
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Individual #8: individual aged 22-26, 178 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15850”.
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Individual #9: individual aged 19-22, 169 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis. The inscription on the recognition mark was „STALAG 315 15759”, moreover the Soviet paper recognition mark was found in a bakelite tube. Some Soviet coins at face value of 3 kopyeiki and zinc parts of a German tent canvas were found.
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Individual #10: individual aged 19-22, 170 cm tall, the remains were put in the grave with the head in direction of the cemetery gate, palms put on the pelvis.
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The online database OBD-Memorial of the Ministry of Defence of the Russian Federation was searched with the numbers of recognition marks found with the bodies. At a first stage some hundreds results were obtained. Then these results were filtered with the numbers of the prisoners camps (only those in vicinity were taken into account) and finally the scanned cards were thoroughly examined to find the remarks about the place of death. The cards with data matching to eight found recognition marks were chosen.
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4.3. Archival query: The following was read out from the prisoners cards: Table 1. Archival query
Archival query enabled determining the identity of the next individual (excavation number 10) in spite of the fact that no recognition mark had been found with the body. Basing on data concerning dates of death of already identified prisoners it was established that they were buried starting with number 9, so the prisoner number 10 was buried on the same day as number 9 or earlier. So the database OBD-Memorial was searched for the STALAG 315 (first deceased prisoners came from that camp) and death date not later than October 23rd, 1941. For many prisoners the place of death was not given in the database because of difficulties in transcription of original German names to Russian alphabet. This is why the scans of original documents included in the database were thoroughly scrutinized. After searching through
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more than 800 sets of identification cards we came across the prisoner who was buried on 22nd October 1941 in Linowno/Woltersdorf in Drawsko Pomorskie district. Data obtained during anthropological examination of the skeleton matched those included in the prisoner identification card. Figure 7. Remains of prisoner of war B. Pyotr, excavation number 7
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Figure 9. Prisoner card of the POW Andrey L., excavation number 4
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Figure 8. Half of the POW recognition mark with personal number 15100 belonging to Pyotr B.
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Figure 10. Remains of prisoner of war Konstantin K., excavation number 8
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After archival query had been performed and personal data established based on recognition marks, the search for finding still alive close relatives was carried out. The next stage of identification process was the comparative DNA analysis.
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4.4. DNA Analysis According to recommendation #6 from ISFG (5), we used minimum required 12 independent loci, in this case – 16 loci from NGM system and 15 loci from Y-Filer system. According to recommendation #7 from ISFG (5), we reviewed thoroughly all allele calls and candidate matches. We generated composite DNA profiles after two isolations from teeth and two isolations from phalanges.
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Table 2. DNA concentration and PCR reaction inhibitors Table 3. STR profiles
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We have analyzed only these profiles in which peak heights were not less than 100 RFU (20,21). All the degraded profiles were rejected. To eliminate the loss of alleles, we repeated all the reactions and then compared the results, analyzing drop-outs and drop-ins. After analyzing all the results, no extra alleles were observed. Degradation was observed by the preferential amplification of shorter STR fragments. Our team working with the material has been profiled before, as all of us are working in a forensic lab. During the exhumation, we used sterile gloves, Tyvek full coveralls and protective masks. The material was taken and placed in sterile Falcons as quickly/soon as possible. It had no contact with people outside the team. Figure 11. An example of a profile obtained from sample 9. Figure 12. An example of a profile obtained from sample 8.
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Figure 13. An example of a profile obtained from sample 6. Table 4. Comparison Table 5. Comparison
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5. Results The presence of PCR reaction inhibitors in analyzed samples was not ascertained.
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Table 6. DNA concentration
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Table 7. DNA amplification
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According to recommendation #4 from ISFG (5), with help of Ministry of Defence of the Russian Federation, we collected material from the only living first-degree relatives. In the case of the individual #4 indicated on the basis of the recognition mark as Andryey Lvov the sample material was collected from his putative daughter. The performed DNA analysis confirmed the kinship between them and bio-statistical calculations determined its probability as high as 99,998%.
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In the case of the individual #7 indicated on the basis of the recognition mark as Pyotr B. the sample material was collected from his putative son. DNA typing with STR markers in NGM and MiniFiler system confirmed the kinship between them and bio-statistical calculations determined its probability as high as 99,9999994%. DNA analysis in Y-Filer system showed the same genetic profile of exhumed individual and of putative son, which additionally confirmed the identity of Pyotr B. Bio-statistical calculations determined its probability as high as 99,9999999994%.
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According to recommendation #10 from ISFG (5), we used bio-statistical tool used in our forensic cases, DNA STAT v.2.1. software
6. Discussion Nowadays, DVI teams have to gather many specialists from different disciplines. Depending on the case the team should be modified. The one analyzed by us has required geneticists and historians working together. In this case, without extensive historical research, identification of exhumed victims would have been impossible. Together, extremely various fields of science begin to give tremendous opportunities for future research. Considering the amount of still missing and unidentified victims of twentieth-century totalitarian regimes, a combination of current DVI procedures and historical research would be able to identify them in the future. Identification process of World War II victims is based on numerous elements depending on acquired historical data and accessibility of comparative genetic material. Continuous
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development of genetic methods offers new possibilities, but to explore them it is necessary to perform much other research to limit the number of persons involved. Up to now, creating a database of genetic profiles of the victims and comparative material acquired from close relatives is not possible due to the huge amount of missing and deceased persons. World War II victims’ identification process is really difficult because of the amount of victims as well as shortages of preserved archival documentation. The next problem is time factor and thus difficulties in finding relatives of the persons found during excavations. Similar problems have been encountered by our colleagues from Croatia. (22).
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Application of odontology in case of the World War Two victims killed on the Eastern Front is practically impossible (with exception of single USAF pilots shot down over Eastern Front areas) because of a total lack of dental documentation.
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In the case of prisoners of war it turned out in recent years that their captured prison cards were preserved in Russian archives. They contain data on age, sex, height, hair color, fingerprints, prisoner number stamped on the recognition mark and in many instances also a photograph. Such data is essential for comparative anthropological examination.
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Questionnaire system concerning missing persons used by research teams in Croatia (22) is useless in case of the World War II victims due to the huge amount of persons and time that elapsed since the war.
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Applying genetic methods in the identification process of World War II victims is actually only possible then when such analysis is preceded by other types of research, when it is possible to indicate a person or limit a group of persons involved on the basis of recognition marks, archival documents, etc. The same observations were made by Finnish teams identifying Finnish soldiers killed during the World War Two. Only in 20% of the cases it was possible to use DNA analysis (23). In our research the result is similar.
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In the case shown above we had no doubts about connecting recognition marks to respective skeletons as it can frequently happen in case of mass-graves and hastily performed excavations as is shown by Finnish experiences (23). Taking samples of only phalanges and teeth during excavation is based on our earlier experiences that lead to the conclusion that presented extraction methods with the use of just such material is effective and there is no need of sampling bigger bones fragments during field works, which can be problematic and raises the risk of contamination of samples. Anthropological, historical, archival and genetic research proved to be effective in the case presented. Up to now it was possible to identify nine out of ten individuals. In the case of two of them the identity was confirmed by DNA tests. It should be emphasized that they are the first Soviet prisoners of war found in Poland and identified by means of genetic methods. Thanks to bringing interdisciplinary and international expert team into existence it was
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possible to carry out the identification process exceptionally effectively and fast: identification together with DNA analysis lasted only two months.
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Similar attitude to the subject and using interdisciplinary expert teams of historians, archeologists, anthropologists, criminology specialists and first of all geneticists, one can observe in the case of similar research performed all over the world (24). Similar observations were made by the teams working in Spain examining mass graves of the victims of the Civil War 1936-1939. They also place emphasis on the complexity of the research and its multidisciplinarity (25).
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In using both extraction methods and analyzing the results of Real-time PCR no substantial inhibition of PCR reaction was observed. Though exhumation material is one of the most troublesome one can encounter in profession of geneticist it can show the effectiveness of extraction methods in removal of inhibitors. No contamination was observed in the course of examination, which can be attributed to strict procedure obeyed from the commencement of excavation until the end of research work. Samples for genetic analysis was secured at once after exposure of the skeletons before any other examinations were performed and frozen in sterile and tight Falcon-type tubes. The material was purified before DNA extraction similarly like other exhumation teams do (26, 27) to avoid contamination and remove PCR reaction inhibitors. Our earlier experiences showed that the material after excavation should be immediately placed in tightly closed packages and frozen. Low temperature and humidity as well as protection against microorganisms’ growth are necessary for proper preservation of genetic material. Similar observations were done by Iwamura and his team (28).
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References:
1. http://www.obd-memorial.ru/
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2. L.G. Biesecker, J.E. Bailey-Wilson, J. Ballantyne, H. Baum, F.R. Bieber, C. Brenner, et al., DNA identification of victims from the September 11, 2001 World Trade Center attack: scientific challenges and policy implications, Science. 310 (2005) 1122-3. 3. A. Alonso, P. Martín, C. Albarrán, P. García, L. Fernández de Simón, M. Jesús Iturralde, et al., Challenges of DNA profiling in mass disaster investigations, CMJ 46 (2005) 540-8. 4. T.J. Parsons, R. Huel, J. Davoren, C. Katzmarzyk, A. Miloš, A. Selmanović, et al., Application of novel “mini-amplicon” STR multiplexes to high volume casework on degraded skeletal remains, Foren. Sci. Int. 1 (2007) 175-9. 5. M. Prinz et al., DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI), Foren. Sci. Int. Gen. 1 (2007) 3-12
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6. A. Lebioda, M. Żołędziewska, E. Kowalczyk, A. Jonkisz, T. Dobosz, Ultimate shortening of the PCR product in the STR system TH01 – a new perspective in testing of decay forensic samples. 19th International Congress International Society for Forensic Genetics, Münster (Germany), 28 August–1 September 2001, Programme and abstracts, (2001) 106.
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7. M. Żołędziewska, Badanie polimorfizmu mikrosatelitarnego w próbkach częściowo zdegradowanego DNA z użyciem własnej metody reakcji multipleksowej PCR, [Maszynopis powielany] Akad. Med. we Wrocławiu, Wrocław (2004).
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8. M. Żołędziewska, T. Dobosz, Comparison of various decalcificators in preparation of DNA from human rib bone. Anthropol. Rev. 65 (2002) 75–80.
10. QiaGen: QIAquick® Spin Handbook 03/2008.
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9. M. Żołędziewska, T. Dobosz, Gender determination in highly degraded DNA samples, Prog. Forensic. Genet. 9 (2003) 593–595.
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11. Applied Biosystems: PrepFiler® and PrepFiler® BTA Forensic DNA Extraction Kits User’s Manual, Foster City, CA., P/N 4463348 Rev. C, January 2012.
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12. Applied Biosystems: Quantifiler® Duo DNA Quantification Kit User’s Manual, Foster City, CA., P/N 4391294 Rev. A, January 2008. 13. Applied Biosystems: Applied Biosystems 7500 Real-Time PCR System, Manual.
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14. Applied Biosystems: AmpFlSTR® NGM™ PCR Amplification Kit User’s Manual, Foster City, CA., P/N 4425511 Rev. E, July 2011.
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15. www.pomorze1945.com/
16. M. Trotter, G.C. Gleser, A re-evaluation of estimation of stature based on measurements of stature taken during life and long bones after death, Am J Phys Anthropol 16 (1958) 79-123
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17. T.W. Todd, Age changes in the pubic bone: I. The white male pubis, Am J Phys Anthropol 3 (1920) 467-470 18. P.H. Stevenson, Age order of epiphyseal union in man, Am J Phys Anthropol 7 (1924) 59-93 19. D.H. Uberlaker, Estimating age at death from immature human skeletons: overview, J Forensic Sci 32 (1987) 1254-1263
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20. P. Gill et al., DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures, Foren. Sci. Int. 160 (2006) 90101
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21. P.M. Schneider et al., The German Stain Commission: recommendations for the interpretation of mixed stains, Int J Legal Med 123 (2009) 1-5 22. M. Slaus et al., Identification and analysis of human remains recovered from wells from the 1991 War in Croatia, Foren. Sci. Int. 171 (2007) 37-43. 23. J.U. Palo, M. Hedman, N. Söderholm, A. Sajantila, Repatriation and Identification of Finnish World War II Soldiers, CMJ 48 (2007) 528-535.
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24. C. Nunez et al., Genetic analysis of 7 medieval skeletons from the Aragonese Pyrenees, CMJ 52 (2011) 336-43.
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25. L. Rı´os, J.I. Casado Ovejero, J. Puente Prieto, Identification process in mass graves from the Spanish Civil War I, Foren. Sci. Int. 199 (2010) 27–36.
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26. S. Andelinovic, D. Sutlovic, I.E. Ivkosic, et. al., Twelve-year experience in identification of skeletal remains from mass graves, CMJ 46 (2005) 530-9.
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27. A.A. Westen, R.R.R. Gerretsen, G.J.R. Maat, Femur, rib, and tooth sample collection for DNA analysis in disaster victim identificztion (DVI), Foren. Sci. Med. Pathol. 4 (2008) 15-21.
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28. E.S. Miazato Iwamura, J.A. Soares-Vieira, D.R. Muñoz, human identification and analysis of DNA in bones, Rev. Hosp. Clín. Fac. Med. S. PAULO 59 (6) (2004) 383388.
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#5 Fedor K.
#6 Shakir F.
Logvin
Yakov
Akim
Aleksandr
Makey
Roman
Religion
orthodox
graecocatholic
orthodox
graecocatholic
graecocatholic
Place,date of birth
Timoshchevskaya 1923
Alma-Ata 05.06.1912
Tschesanino 1919
Oboymovo 1920
Rank
private
private
private
private
Unit
?
346 Construction Battalion
?
Profession
farm worker
farm worker
farm worker
Place,date of captivity
? 16.07.1942
Putkovitschy 22.06.1941
Camp
Stalag IIB Hammerstein
Prisoner’s number
116281
Height
172 cm
Hair
blond
Date and place of death
Andrey
Ivan
graeco-catholic
graecocatholic
Tschimkent 1915
Jeral 29.01.1920
Aleksandrovka 05.07.1917
graecocatholic PrimorskoAkhtyrskaya 17.10.1921
private
private
private
private
private
9 Armoured Regiment
?
201 Rifle Regiment
20 Rifle Regiment
5 Armoured Regiment
?
mechanic
farm worker
farm worker
farm worker
farm worker
tractor driver
Stalingrad 23.09.1942
Mokhylev 29.07.1941
Samborov 03.07.1941
Kovno 01.07.1941
Volma 07.07.1941
Desna 06.07.1941
Rudashkovitsy 17.07.1941
Stalag 315 Hammerstein
Stalag IIB Hammerstein
Stalag 315 Hammerstein
Stalag IIB Hammerstein
Stalag IIB Hammerstein
Stalag 315 Hammerstein
Stalag 315 Hammerstein
23715
113740
24598
24677
15100
15850
15759
Stalag 315 Hammerstein 14343 (not found)
166 cm
157 cm
170 cm
161 cm
161 cm
175 cm
167 cm
173 cm
blond
blond
dark
black
blond
dark blonde
black
blond
none
none
none
none
none
none
none
none
none
typhoid
none
none
none
none
none
none
none
none
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
healthy
23.03.1943 Linowno/ Woltersdorf
04.12.1941 Linowno/ Woltersdorf
22.05.1943 Linowno/ Woltersdorf
21.11.1941 Linowno/ Woltersdorf
14.11.1941 Linowno/ Woltersdorf
27.10.1941 Linowno/ Woltersdorf
24.10.1941 Linowno/ Woltersdorf
23.10.1941 Linowno/ Woltersorf
22.10.1941 Linowno/ Woltersdorf
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Condition of health
Vasiliy
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Father’s name
Vaccinations
#10 Konstantin K.
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Leonid D.
Distinguishing marks
#9 Yosif S.
M
Aleksey M.
#7 Petr B.
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#4 Andrey L.
d
#3
ep te
#1
ip t
Table 1
#8 Konstantin K.
orthodox Kamyshyno 18.08.1920
Page 27 of 35
Table 2 extr. method & lysis time
0,000273
1
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,000243
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,005682
teeth phalanx bone phalanx bone
2
teeth teeth phalanx bone phalanx bone
3
teeth teeth 4
M
phalanx bone phalanx bone
Ac
0,000352 0,000755 0,000777 0,000644 0,002298 0,010266
0,000190
7
0,037331
8
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H PrepFiler 2H
0,004402
teeth teeth
teeth teeth phalanx bone
0,002895
PrepFiler 2H phenol/chloroform 24H
6
teeth
phalanx bone phalanx bone
0,000644
0,000035
teeth
teeth
0,000513
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
5
teeth
phalanx bone phalanx bone
0,000256
0,001372
ce pt
phalanx bone phalanx bone
0,000018
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
ed
teeth teeth
PrepFiler 2H phenol/chloroform 24H PrepFiler 2H phenol/chloroform 24H
0,000405
9
ip t
teeth
0,000200
cr
teeth
us
teeth
phalanx bone phalanx bone
DNA concentration [ng/µl]
name
an
material
0,000119 0,000020 0,000511
0,000628 0,000117 0,008953
0,000505
0,013776 0,001175 0,001734
0,004525 0,000894
Page 28 of 35
phalanx bone
phenol/chloroform 24H
0,000534
teeth
PrepFiler 2H phenol/chloroform 24H
0,000101
10
0,000218
Ac
ce pt
ed
M
an
us
cr
ip t
teeth
Page 29 of 35
phalanx bone teeth teeth teeth phalanx bone
2
phalanx bone teeth
teeth 3
phalanx bone teeth
teeth 4
phalanx bone teeth
teeth
phalanx bone teeth
D18 S51
D22 S1045
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
5
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H Mini-STR
D19 S433
TH01
FGA
D2 S441
D3 S1358
D1 S1656
D12 S391
13
17,19
12
-
XY
13,14
28,31.2
17
15,16
14
6
21
11,14
16,18
11
18
13
17,19
-
21
XY
13,14
28,31.2
17
15,16
14,16.2
6,9
21
11,14
16,18
11
-
13
-
-
-
-
13
-
-
16
-
-
-
11,14
-
13
18
13,16
19
-
-
XY
13,14
31.2
17
15
-
-
-
11
16
-
18
12
17,21
XY
28,31.2
17
cr
D21 S11
21,24
14,17
17,20
-
-
XY
12,13
28,30
16
11,17
13
-
-
11,14
14,15
-
-
17
17
11,13
-
XY
12,13
-
17
11
13,17.2
6,9
19
11
15
-
23
14,17
17,20
11,13
25
XY
12,13
28,30
16,17
11,17
13
6,9
19,23
11
15
14,15
23
14,17
17,20
11,13
17,25
XY
28,30
16
11,17
13,17.2
6,9
19,23
11,14
14,15
14
23
11,13
17,25
XY
28,30
16,17
XY
14
28
16
16
13,14
9
-
14
17,18
12,13
17,21
14
28,33.2
16
16
13,14
7,9
21,22
14
17,18
12,13
17,21
13,14
18
13
-
13,14
18
11,13
16,17
13,14 13,14
13,16 13,16
XY
12,12
19,23
18
-
17
XY
-
28
20
16
14
-
21
14
17,18
13
-
18
11
-
XY
14
28,33.2
-
16
13,14
7,9
21,22
14
17,18
12,13
17
11,13
16,17
XY
28,33.2
16
17
9
-
XY
14
31
16
12,16
13,13.2
8,9.3
24
14
17
17.3
20
17
9,12
21
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20
21,22
16
17
9,12
21
XY
14
30
16
12,16
13,13.2
8,9.3
-
14
15,17
-
20
13,16
17
9,12
21,25
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20,21
9,12
21,25
XY
30,31
15,16
Mini-STR
teeth
phalanx bone
D8 S1179
Mini-STR
teeth
phalanx bone
AMG
Mini-STR
teeth
phalanx bone
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
D2 S1338
us
1
D16 S539
ep te
phalanx bone
vWA
an
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H Mini-STR
D10 S1248
M
teeth
extr. method & lysis time
d
name
Ac c
material
ip t
Table3
20,24
13,15
16
11
-
XY
12
30,31.2
-
11,15
13,14
6,8
-
11,11.3
15,16
13
20
-
17
-
-
XY
12
31.2
12
11
13
-
-
11,11.3
15,16
17.3
18
-
-
-
-
XY
-
-
-
11
13,14
-
-
11,11.3
16
-
-
13,15
16
11,14
20,24
XY
12
30,31.2
12
11,17
13,14
6,8
21,24
11
15,16
13,17.3
18,20
11
20,24
XY
31.2
12,14
D13 S317
D7 S820
CS F1PO
11
10,12
9,11
12,13
11,12
10,12
11,14
10,12
11,12
8,9
9,10
11
12
9,10
10,11
Page 30 of 35
teeth
7
teeth 8
phalanx bone
14,15
16,18
11,12
19,24
XY
13
32.2
13,14
11,15
14
16,18
12
-
XY
13
30
-
14,15
16,18
11,12
19,24
XY
13
30,32.2
13,14
11,12
19,24
XY
PrepFiler 2H phenol/chlorofom 24H
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
teeth 9
phalanx bone teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
teeth 10
-
9.3
23,24
-
15,17
-
17
12,13
9.3
23,14
11,12
15
19.3
15,17
11,15
12
9.3
-
11,12
15
17.3
-
11,15
12,13
9.3
23,24
11,12
15
17.3,19.3
15,17
30,32.2
13,14
23,24
17
12,16
13,16
6,7
24
11.3,15
16,19
17,17.3
19
12,16
13,16
6,7
24
11.3,15
16,19
17,17.3
18,19
14,16
17
11
-
XY
10
28
14,16
17,18
11
-
XY
10,13
28,29
17,19
11
19,23
XY
28,29
17,19
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
14
11,13
16,26
XY
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
16
14
11,13
16,26
XY
10,13
27,30
19
11,16
12,13
7,9
18,21
10,14
14,15
16,17
18,23
11,13
16,26
XY
27,30
19
18
12,13
20,23
XY
12,14
29
15,16
11
12,16.2
7,9.3
21
11,11.3
16
15,16
22,23
18
12,13
20,23
XY
12,14
29
15,16
11
12,16.2
9.3
21
11,11.3
16
15,16
22,23
18
-
23
XY
12,14
29
-
11
12,16.2
9.3
21
11,11.3
16
15,16
23
18
12,13
20
XY
12,14
29
15,16
11
12,16.2
9.3
21
11,11.3
16
15,16
23
12,13
20,23
XY
29
15,16
12,13 12,13 12,13 12,13
10,13
24
16
Mini-STR PrepFiler 2H phenol/chlorofom 24H Mini-STR
ip t
11,15
Mini-STR
teeth
teeth
-
ep te
teeth
teeth
-
Mini-STR
teeth
phalanx bone
-
M
teeth
phalanx bone
-
Mini-STR
teeth teeth
-
cr
phalanx bone
12
us
6
16
Ac c
phalanx bone
15
an
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
d
teeth
18,21
21
14
17
-
24
-
13
-
14
16,17
15
-
19,24
14
17
-
18
14
17,18
11
20
XY
13
30.2,31.2
13
16,17
13,15
8,9.3
19,24
10,14
16,17
13
-
11
24
XY
31.2
13,14
19,24
10,11
9
11,13
9,11
9,10
12
11,13
10,11
12
13,14
9,11
11,12
11
-
11
Page 31 of 35
4
D2 S1338
AMG
D8 S1179
D21 S11
D18 S51
D22 S1045
D19 S433
TH01
FGA
D2 S441
D3 S1358
D1 S1656
D12 S391
13,16
17
9
-
XY
14
31
16
12,16
13,13.2
8,9.3
24
14
17
17.3
20
13,16
17
9,12
21
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20
16
17
9,12
21
XY
14
30
16
12,16
13,13.2
8,9.3
-
14
15,17
-
20
13,16
17
9,12
21,25
XY
14
30,31
15,16
12,16
13,13.2
8,9.3
20,24
14
15,17
17.3
20,21
9,12
21,25
XY
30,31
15,16
9
21,25
XX
phenol/chlorofom 24H - MiniFiler PrepFiler 2H
13,15
16,17
10,14
30,32
13,15
12,15
13,15
7,9.3
20,22
11,14
17,18
16,17.3
19,21
D7S820
CSF1PO
8,9
9,10
11
8
10
11
M
p.54
20,24
D13 S317
d
teeth swab
D16 S539
ep te
phalanx bone phalanx bone
vWA
cr
teeth
PrepFiler 2H phenol/chlorofom 24H PrepFiler 2H phenol/chlorofom 24H
D10 S1248
us
teeth
extr. method & lysis time
an
no
Ac c
material
ip t
Table4
Page 32 of 35
10
28
17
12,16
14,16
17,18
11
-
XY
10,13
28,29
17,19
11
19,23
XY
28,29
17,19
11
18,23
XY
24H PrepFiler 2H
D12 S391
13,16
6,7
24
11.3,15
16,19
17,17.3
19
13,16
6,7
24
11.3,15
16,19
17,17.3
18,19
16
Y_DYS391
12,15
7,9.3
Y_DYS439
us 16,17
Y_DYS393
phenol/chlorofom
28,33.2
12,16
an
7
10,12
G_DYS385
teeth
18
G_DYS19
extr. method & lysis time
14,15
D1 S1656
24 24,25
10,11.3
15,19
16,17
18,19
D13 S317
D7 S820
CSF1PO
9,11
9,10
12
11,12
10
11,12
R_DYS448
XY
D3 S1358
R_DYS438
-
D2 S441
R_DYS437
11
FGA
R_Y_GATA _H4
17
TH01
Y_DYS392
14,16
D19 S433
Y_DYS635
D22 S1045
cr
D18 S51
M
PrepFiler 2H
name
p.91
D21 S11
phenol/chlorofom 24H - MiniFiler
material
swab
D8 S1179
14
12
23
28
15
14
14
13
10
10
21
11
11
16
10
20
14
12
23
28
15
14
14
13
10
10
21
11
11
16
10
20
d
p.91
AMG
G_DYS458
swab
D2 S1338
B_DYS389II
teeth
D16 S539
ep te
7
vWA
B_DYS390
teeth
PrepFiler 2H phenol/chlorofom 24H
D10 S1248
B_DYS389I
teeth
extr. method & lysis time
Ac c
no
B_DYS456
material
ip t
Table5
Page 33 of 35
Table 6
Teeth organic method
Teeth PrepFiler
Phalanges organic method
Phalanges Prepfiler
lowest
0,0001
0,00003
0,00001
0,00001
highest
0,0138
0,0373
0,01
0,00229
average
0,003
0,005
0,0029
0,0007
Ac
ce pt
ed
M
an
us
cr
ip t
DNA concentration [ng/µl]
Page 34 of 35
Table 7
Teeth Organic method
Teeth PrepFiler
Phalanges Organic method
Phalanges PrepFiler
Allele per individual (average)
24,2
20,4
25,1
17,6
Ac
ce pt
ed
M
an
us
cr
ip t
DNA amplification
Page 35 of 35
