- Email: [email protected]
Identification of victims from mass grave discovery near Benghazi, Libya
Accepted Manuscript Identification of victims from mass grave discovery near Benghazi, Libya S. Sherein Ghaleb, Dalia Abd Elwahab Hassan, Fadwa A. Elroby, Khaled R. Mogassabi, Amin AttiaAlemam PII:
S1752-928X(18)30651-6
DOI:
https://doi.org/10.1016/j.jflm.2019.07.012
Reference:
YJFLM 1856
To appear in:
Journal of Forensic and Legal Medicine
Received Date: 18 November 2018 Revised Date:
12 June 2019
Accepted Date: 22 July 2019
Please cite this article as: Sherein Ghaleb S, Elwahab Hassan DA, Elroby FA, Mogassabi KR, AttiaAlemam A, Identification of victims from mass grave discovery near Benghazi, Libya, Journal of Forensic and Legal Medicine, https://doi.org/10.1016/j.jflm.2019.07.012. 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.
ACCEPTED MANUSCRIPT
Identification of victims from mass grave discovery near Benghazi, Libya Sherein S.Ghaleba, Dalia Abd Elwahab Hassanb, Fadwa A. Elrobyc, Khaled R. Mogassabid,Amin AttiaAlemame a Forensic Medicine and Clinical ToxicologyDepartment, CairoUniversity,
c
Forensic Medicine and Clinical Toxicology Department, Beni-SuefUniversity,
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Forensic Medicine and Clinical Toxicology Department, Beni-Suef University,
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Forensic Medicine and Clinical Toxicology Department, Benghazi University, Forensic Odontology Department , Benghazi University.
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Dalia Abd-ElwahabHassan Tel: +2 01092373209
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Corresponding author:
E-mail address: [email protected] Sherein S.Ghaleb
Fadwa A. Elroby
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E-mail address: [email protected]
E-mail address: [email protected] Khaled R. Mogassabi
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E-mail address: [email protected] Amin AttiaAlemam
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E-mail address: [email protected]
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Identification of victims from mass grave discovery near Benghazi, Libya
Abstract
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Background: A mass grave is any site that containing two or more associated corpses, at random or on purpose placed, of people who have died as a result of extra-judicial or random executions, not including those people who have died from armed confrontations or known major catastrophes. Case presentation: The purpose of this paper is to explain how to reconstruct a biological profile of decomposed or skeletonized bodies and clarify the efforts done by the Libyan scientist after 2011 revolution and to set a reference for other researcher. The alleged location of the grave, as well as the alleged number and identities of the persons buried in the grave were obtained exclusively from witnesses’ and relatives’ testimonies. Conclusion: As the testimonies said, the grave was located at the alleged location and seven skeletons were exhumed. Also, the osteological and DNA study made investigators to identify the exhumed skeletons. And the dental analysis support the identification of a seven man alleged to have been buried in the grave, 7 victims were discovered.
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Keywords: Mass grave;DNA analysis, Identification,Libya, Benghazi and revolution.
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1-Introduction
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The last decade has seen a steady increase in the number and types of both professional and lay people assisting the various processes involved in revealing and communicating the social, political and cultural contexts presented by mass graves. As to thenumber of individuals present in the grave in order for it to qualify as a mass grave, Mant suggested in 1987 that it need only contain two or more bodies that are in contact with each other. Skinner published a conflicting definition in the same year based on his review of investigations in Korea, Vietnam and Argentina. He suggests that a mass grave is any single burial unit containing at least half a dozen tightly packed, yet indiscriminately placed bodies.1 There are several serious problems becoming discernible from the activities of internationally active forensic bio archaeologists, including a lack of internationally standardized archaeological field protocol, a lack of scientific research pertaining to the understanding and analysis of mass grave and mass grave-related sites and a lack of communication and publication of knowledge among experts in the discipline.
Minimally, complete teams include core expertise from crime scene managers, archaeologists, anthropologists and pathologists. In addition, there is often a met, or felt, need for further expertise in the form of evidence officers, security personnel, logisticians, heavy equipment operators, photographers, surveyors, dentists, mortuary managers, etc. Each person in these various functions is ‘in charge’ of their duty and assistants, and has the authority to control the realization of their role within the nexus of functions being performed at the site.2 One type of mass grave-related site is the execution site (ES), where multiple individuals are executed. Evidence of this site type relates primarily to the mode of execution, such as bullet cartridges or shredded clothing as well as human blood and tissue fragments. It is important to distinguish surface execution sites (SES) from grave execution sites (GES) in which victims are killed in a previously dug grave. The temporary surface deposition site (TDS) may be recognizable once the bulk of the body mass is removed by the presence of residual clothing, personal effects, and blood and bone fragments. On the other hand, a site with significant amounts of taphonomically altered human
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sloughed soft tissue, such as upper and lower limbs and fingernails and hair, might be missing; presumably left behind in at the primary inhumation site.5, 6
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bone but lacking extraneous evidence pertaining to the method of execution, such as bullet casings, likely represents a permanent surface deposition site (PDS). It can be expected that at a PDS, the degree of scatter of bones and other evidence will be much greater than at a TDS. 3 It is not unlikely that in some instances the perpetrators may choose to use natural features, such as ravines or caves, to inter a body mass which can be most easily referred to as the primary inhumation site (PIS). In some instances, a primary inhumation site may also be a grave execution site. Conversely, the inhumation may occur in a location far from where the victims were killed. 4 The secondary inhumation site (SIS) has been previously termed a secondary mass grave. It is defined as a grave that is created, usually clandestinely, from a primary grave at a remote location. Severely disarticulated and commingled bodies are the most easily recognized indicators of a secondary inhumation site, and are the result of the use of heavy machinery to scoop up the partially decomposed remains, transport them to the secondary location and inter them again using heavy machinery. The use of heavy machinery will also result in the mixing of soil and artifacts from the primary inhumation site. Easily disarticulated elements and
2. Material and methods • Location and exhumation of the grave On January, 2012, one revolutionary's battalions in Libya indicated the exact location of the grave to the archeological team. The grave was found in the place indicated, and seven skeletons were exhumed from a 3
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in our case for determination of the sex through the pelvis bone. Next, age and stature were estimated, we examined long bones and the age was between (25-35) years and the stature was between (163-170) cm. The main finding was observed in skeleton 5, which presented dental work, (Fig. 3). The presence of this dental caries could be useful for the identification process. This finding could be indicative of the victim’s socioeconomic status, an inaccurate valuation but one that could be compared with the available ante mortem information. There was antemortem picture of dead No. 5 describes the presence of dental caries in front teeth this is beside the information from his mother. This indicate that dead was No. 5 which confirmed by DNA analysis.
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mass grave located near the city of Benghazi (140 km west Benghazi in desert). Seven skeletons were exposed and recovered from a single grave, with some clear post mortem displacements observed,(Fig. 1). The remains were delivered for study to Benghazi medical center, a teaching hospital where corpses were placed in equipped morgue, (Fig. 2). The personal belongings found associated to the skeletons were their clothes. Two of the six families had recognized the clothing of their loved one in the mass grave where the bodies were found.
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• Osteological studyand analyses The state of preservation of the remains was moderate to good. It was possible to apply most of the methods for sex, age and stature estimation as well as for the recording of pathological conditions and non-metric traits. The visual 7, 8 and metric 9 study of the pelvis unequivocally indicated that all the skeletons were male. There are Three techniques for the visual evaluation of traits of the hip bone are: 1) the method of Phenice (1969), which uses three traits on the pubis, 2) the method of Iscan and Derrick (1984) using the posterior pelvis, and 3) the method of Ferembach et al. (1980) of sexing the entire pelvis through an evaluation of eleven traits.8 And we used the third one
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Two brothers were presumed to have been buried in the grave, but no sound conclusion was reached regarding the identification. But because they were two brothers it was not important for their parents to differentiate between their bodies. The match between the ante mortem and osteological findings was partial, due to a lack the ante mortem data. The corroboration of the information from different sources was partial, but it was
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disposable and were initially crushed manually so coarse. One sample for each corpse and subsequently was pulverized to promote the reaction of softening and DNA extraction.
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considered enough to request a DNA study, with compatible identities. • Identification by DNA analysis - 7 Sachets for biological samples, perfectly sealed, each containing 2 teeth marked with numbers from 1 to 7. These teeth have been identified in the laboratory Genome as indicated in table (1).
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o DNA extraction It has been necessary to use different extraction techniques for the individual items because they were finds dating of old (over 16 years) and the poor state of preservation. 1. Chelex-100 It is a resinous substance that, when mixed with the eluate of biological trace and subjecting the obtained solution at elevated temperatures and strong centrifugation allows separating the DNA from the rest of the substances composing the track. 2. Prep filer The second method was the one that uses the "prep Filer TM Forensic DNA Extraction Kit". 3. EZ1 The third method used was the one using the kit of Qiagen EZ1 with EZ1 advanced automatic extractor.
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- 6 packs of yellow paper, perfectly sealed, containing some hair belonging to a family member. These biological samples were transcribed on the facade the name of the deceased relative and the degree of relationship between the family and the deceased. The samples were identified at a laboratory Genome in Libya as indicated in table (2).
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o preparation of the teeth to the analysis of DNA
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This phase consisted of the mechanical cleaning of the teeth in order to remove surface contaminants, by scraping with a sterile scalpel blade and subsequent incubation to UV rays for a period of 5 minutes, to obtain the destruction of exogenous DNA.
o Quantification of DNA DNA extracted from these samples and was then quantified using the kit Quantifier Human DNA Quantification Kit using the amplifier 7300 Real Time PCR System. The amount of DNA in trucks
All the teeth were separately inserted into an envelope sterile 5
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was determined by comparison of the intensity of the signal obtained with that of standards with known concentrations.
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o Amplification of DNA The material obtained in the previous phase extraction was amplified by PCR. This technique allows isolating and amplifying specific fragments of DNA with primer sequences (primers) that, under the action of a heat-resistant enzyme (Taq-polimeras) and in the presence of suitable reagents, allow exponential amplification of the fragment identified. At the end of the reaction, the amplified DNA fragment will constitute the predominant species present in the reaction tube allowing the specific study.
population. The identification of a sequence of primer capable of binding with high specificity traits not variants "and downstream" of the piece variant (polymorphic), allows the amplification of this segment allowing the study. Part of the length polymorphisms of DNA the class, at the time, most used and that of STRs. The variability in these fragments consists mostly of the number of times a given fragment short (short) and repeated consecutively. This class of polymorphisms, which is the most represented in the human genome, provides forensic scientist of a huge number of points that you can compare to recognize two different biological samples. The STRs can be constituted by repeated fragments ranging from 2 to 7 base pairs (the pairs of nucleobases are the units making up the DNA, like the rungs of a ladder). Those used in this work are composed of a repeated sequence of four base pairs because this type of STR and certainly the most suitable for forensic as recognized by several validations work .The analysis of STRs is performed by capillary electrophoresis. NB:written informed consent were taken from the relevant. In the first phase analytical were analyzed 6 samples of hair, to make sure that it was possible to obtain a profile usable for
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Some regions of DNA are called polymorphic in that they have a lot of variation in the population, there are several types of polymorphic regions and their study allows distinguishing each individual from another. polymorphism the DNA used in this work were chosen in the class of length polymorphisms called STRs (Short Tandem Repeats), which are currently the method of choice for personal identification via biological markers. These are regions of DNA, ranging from specific regions not variants, which can be present in different lengths in the human
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comparison then the profiles were obtained with two amplification kit. The profiles from the teeth of the 7 victims were obtained using two amplification kits.
probability that it is the sister of the other findings.
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The probability that the woman identified as the number C68412 to be the sister of the person who indicated as C67360 is 160, 027, 58 times greater than the probability that it is the sister of the other findings. The probability that the woman identified with the number C68409 to be the sister of the person who indicated as C67358 is 5669 times greater than the probability that the two are not brother and sister.
3. Results
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Gave comparison of the profiles which were obtained from the hair with those were obtained from the teeth. It immediately became clear that the profiles of the teeth were no. 2, 5, 6 compatible with the profiles of three mothers analyzed.
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The teeth no.2 son of Case 3 indicated with code C68410 with a probability of maternity 99.89258988. The teeth no.5 son of Case 4 indicated with code C68411 with a probability of maternity 99.9995514. The teeth no.6 son of Case 6 indicated with code C68413 with a probability of maternity 99.89258988. Then the profile of each of the 4 teeth that have yet to be assigned in, were compared with the three sisters who had been involved. The probability that the woman identified as the number C68414 to be the sister of the person who indicated as C67364 is 475, 733, 36 times greater than the
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The probability that the woman identified with the number C68409 to be the sister of the person who indicated as C67361 is 21, 25 times greater than the probability that the two are not brother and sister. The results to which they come and allow us to attribute the findings examined women who have required to be recognized according to table (3).
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4. Discussion
Examination of skeletal remains of suspected skeletons coming from ancient sites may shed light on reasons of the crime.
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nucleotides in length. During our work we did confirm findings published earlier that DNA could be extracted and amplified without decalcification 11.
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The problems which forensic scientists most often face while working with DNA extracted from different samples are either DNA degradation or DNA contamination. High temperatures, salt, water or soil conditions can damage genomic DNA and therefore make the identification process extremely difficult. The genetic analysis of those killed and buried in the mass graves through DNA from remains of bone and teeth requires the use of short sized loci that can be amplified by polymerase chain reaction (PCR) for which the short tandem repeat (STR) loci are most suitable 10. Short tandem repeats markers are polymorphic DNA loci at noncoding regions of the gene containing a repeated nucleotide sequence. Usually STR repeat units are from two to seven
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Forensic science is a fastevolving cluster of applied disciplines that operate independently as well as interdisciplinary. Fields like anthropology and odontology develop and expand incorporating new methods and theories. As a result, the boundaries between forensic science disciplines become blurred allowing a multidisciplinary involvement and approach, especially when skulls are involved 12. From the case series presented here, we have to conclude that information recovered from testimonies is of crucial importance for obtaining information regarding the presumed number and identities of the people buried in those graves and to prove to our culture how it is important to preserve
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radiograph.
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the ante mortem documentation of any medical report like teeth
5. Conclusion
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Testimonies are especially important when the presence of a grave containing a low number of victims is suspected, as in the case presented here. The decision was made to request a DNA study that made it possible to propose the identification of all seven skeletons exhumed. However, we have to be cautious about any Statement regarding the identification of a group of people presumably buried together once the identification for one or more of the persons
from the group have been proposed. Various events may have transpired between the kidnappings of people from towns and their assassination in the countryside, precluding in some cases a complete coincidence between testimonies and archeological and osteological findings. In that case, our criterion would have been to indicate that a positive identification could not be proposed, although in light of the accumulated evidence it could have been reasonable to point to a compatible identity awaiting a DNA study.
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6. References
[6] D. Manning, Srebrenica investigation, summary of forensicevidence. IC TY Evidence Report, Unpublished documentProperty of the United Nations, International Criminal Tribunal for the Former Yugoslavia, 2000.
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[1] M.F. Skinner, D. Alempijevic and M. Djuric-Srejic, Guidelines for international forensic bioarchaeology monitors of mass grave exhumations, Forensic Sci. Int. 134 (2003) 79–90. [2] M. Skinner, J. Sterenberg, Turf wars: Authority and responsibility for the investigation of mass graves, Forensic Sci. Int. 151 (2005) 221–232. [3] E. Jessee, M. Skinner, A typology of mass grave and mass grave-related sites, Forensic Sci. Int. 152 (2005) 55–59.
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[4] J. Sterenberg, Archaeological Techniques and Methods for theExcavation and Recording of Contemporary rimary andSecondary Mass Graves. Unpublished M.Sc. Thesis fromBournemouth University, UK, 2002 [5] T. Simmons, Taphonomy of a karstic cave execution site atHrgar, Bosnia-Herzegovina, in: William D. Haglund, MarcellaH. Sorg (Eds.), Advances in Forensic Taphonomy: Method,Theory and Archaeologic al Perspectives, CRC Press, USA,2001, pp. 263–308.
[7] J.E. Buikstra, D.H. Ubelaker, Standards for Data Collection from Human Skeletal Remains, Arkansas Archaeological Survey, Fayetteville, 1994. [8] J. Bruzek, A method for visual determination of sex using the human hip bone, Am. J. Phys. Anthropol. 117 (2002) 157–168. [9] P. Murail, J. Bruzek, F. Houe¨ t, E. Cunha, DSP: a tool for probabilistic sex diagnosis using worldwide variability in hip-bone measurements, Bull. Mem. Soc. Anthropol. Paris (2005) 167–176. [10] Clayton TM, Whitaker JP, Fisher DL, LeDA, Holland MM, Weedn VW, Maguire CN, Di Zinno JA, Kimpton CP, Gill P. Further validation of a quadruplex STR DNA Typing system: a collaborative effort to identify victims of a mass
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Journal of Forensic Sciences 1993; 38:60-68.
disaster. Forensic Sci International 1995; 76:17-25. [11] Fisher LD, Holland MM, Mitchell L, Sledzik SP, Wilcox WA, Wadhams M, Weedn WV. Extraction, Evaluation and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone.
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[12] Nuzzolese E, Borrini M, Forensic approach to an archaeological casework of "vampire" skeletal remains in Venice: odontologicaland anthropological prospectus. Journal of Forensic Science. 2010 Nov;55(6):1634-7.
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Teethsamplen.1
Idgenome C67358
Teethsamplen.2
C67359
Teethsamplen.3
C67360
Teethsamplen.4
C67361
Teethsamplen.5
C67362
Teethsamplen.6
C67363
Teethsamplen.7
C67364
Degree of relationship Sister Mother Mother Sister Mother Sister
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Name of deceased Case 1, case 2 Case 3 Case 4 Case 5 Case 6 Case 7
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Table (2):Biological samples of family members with Id Genome
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IdLibyan
Id Genome C68409 C68410 C68411 C68412 C68413 C68414
Table (3): The probable relationships of examined women to discovered skeletons
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C67358 C67359 C67360 C67361 C67362 C67363 C67364
Name given to the finding Case 1, case 2 Case 3 Case5 Case 1, Case 2 Case 4 Case 6 Case7
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NO. findings
Relationship to
brother son brother brother son son brother
NO. Identification mother / sister C68409 C68410 C68412 C68409 C68411 C68413 C68414
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Fig.(1): Exhumation of remains from a single grave in Benghazi desert
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Fig. (2): Study of remains in Benghazi medical center.
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Fig. (3): Skeleton 5 with dental caries.
S1752-928X(18)30651-6
DOI:
https://doi.org/10.1016/j.jflm.2019.07.012
Reference:
YJFLM 1856
To appear in:
Journal of Forensic and Legal Medicine
Received Date: 18 November 2018 Revised Date:
12 June 2019
Accepted Date: 22 July 2019
Please cite this article as: Sherein Ghaleb S, Elwahab Hassan DA, Elroby FA, Mogassabi KR, AttiaAlemam A, Identification of victims from mass grave discovery near Benghazi, Libya, Journal of Forensic and Legal Medicine, https://doi.org/10.1016/j.jflm.2019.07.012. 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.
ACCEPTED MANUSCRIPT
Identification of victims from mass grave discovery near Benghazi, Libya Sherein S.Ghaleba, Dalia Abd Elwahab Hassanb, Fadwa A. Elrobyc, Khaled R. Mogassabid,Amin AttiaAlemame a Forensic Medicine and Clinical ToxicologyDepartment, CairoUniversity,
c
Forensic Medicine and Clinical Toxicology Department, Beni-SuefUniversity,
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b
Forensic Medicine and Clinical Toxicology Department, Beni-Suef University,
d
Forensic Medicine and Clinical Toxicology Department, Benghazi University, Forensic Odontology Department , Benghazi University.
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Dalia Abd-ElwahabHassan Tel: +2 01092373209
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Corresponding author:
E-mail address: [email protected] Sherein S.Ghaleb
Fadwa A. Elroby
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E-mail address: [email protected]
E-mail address: [email protected] Khaled R. Mogassabi
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E-mail address: [email protected] Amin AttiaAlemam
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E-mail address: [email protected]
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Identification of victims from mass grave discovery near Benghazi, Libya
Abstract
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Background: A mass grave is any site that containing two or more associated corpses, at random or on purpose placed, of people who have died as a result of extra-judicial or random executions, not including those people who have died from armed confrontations or known major catastrophes. Case presentation: The purpose of this paper is to explain how to reconstruct a biological profile of decomposed or skeletonized bodies and clarify the efforts done by the Libyan scientist after 2011 revolution and to set a reference for other researcher. The alleged location of the grave, as well as the alleged number and identities of the persons buried in the grave were obtained exclusively from witnesses’ and relatives’ testimonies. Conclusion: As the testimonies said, the grave was located at the alleged location and seven skeletons were exhumed. Also, the osteological and DNA study made investigators to identify the exhumed skeletons. And the dental analysis support the identification of a seven man alleged to have been buried in the grave, 7 victims were discovered.
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Keywords: Mass grave;DNA analysis, Identification,Libya, Benghazi and revolution.
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1-Introduction
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The last decade has seen a steady increase in the number and types of both professional and lay people assisting the various processes involved in revealing and communicating the social, political and cultural contexts presented by mass graves. As to thenumber of individuals present in the grave in order for it to qualify as a mass grave, Mant suggested in 1987 that it need only contain two or more bodies that are in contact with each other. Skinner published a conflicting definition in the same year based on his review of investigations in Korea, Vietnam and Argentina. He suggests that a mass grave is any single burial unit containing at least half a dozen tightly packed, yet indiscriminately placed bodies.1 There are several serious problems becoming discernible from the activities of internationally active forensic bio archaeologists, including a lack of internationally standardized archaeological field protocol, a lack of scientific research pertaining to the understanding and analysis of mass grave and mass grave-related sites and a lack of communication and publication of knowledge among experts in the discipline.
Minimally, complete teams include core expertise from crime scene managers, archaeologists, anthropologists and pathologists. In addition, there is often a met, or felt, need for further expertise in the form of evidence officers, security personnel, logisticians, heavy equipment operators, photographers, surveyors, dentists, mortuary managers, etc. Each person in these various functions is ‘in charge’ of their duty and assistants, and has the authority to control the realization of their role within the nexus of functions being performed at the site.2 One type of mass grave-related site is the execution site (ES), where multiple individuals are executed. Evidence of this site type relates primarily to the mode of execution, such as bullet cartridges or shredded clothing as well as human blood and tissue fragments. It is important to distinguish surface execution sites (SES) from grave execution sites (GES) in which victims are killed in a previously dug grave. The temporary surface deposition site (TDS) may be recognizable once the bulk of the body mass is removed by the presence of residual clothing, personal effects, and blood and bone fragments. On the other hand, a site with significant amounts of taphonomically altered human
2
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sloughed soft tissue, such as upper and lower limbs and fingernails and hair, might be missing; presumably left behind in at the primary inhumation site.5, 6
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bone but lacking extraneous evidence pertaining to the method of execution, such as bullet casings, likely represents a permanent surface deposition site (PDS). It can be expected that at a PDS, the degree of scatter of bones and other evidence will be much greater than at a TDS. 3 It is not unlikely that in some instances the perpetrators may choose to use natural features, such as ravines or caves, to inter a body mass which can be most easily referred to as the primary inhumation site (PIS). In some instances, a primary inhumation site may also be a grave execution site. Conversely, the inhumation may occur in a location far from where the victims were killed. 4 The secondary inhumation site (SIS) has been previously termed a secondary mass grave. It is defined as a grave that is created, usually clandestinely, from a primary grave at a remote location. Severely disarticulated and commingled bodies are the most easily recognized indicators of a secondary inhumation site, and are the result of the use of heavy machinery to scoop up the partially decomposed remains, transport them to the secondary location and inter them again using heavy machinery. The use of heavy machinery will also result in the mixing of soil and artifacts from the primary inhumation site. Easily disarticulated elements and
2. Material and methods • Location and exhumation of the grave On January, 2012, one revolutionary's battalions in Libya indicated the exact location of the grave to the archeological team. The grave was found in the place indicated, and seven skeletons were exhumed from a 3
ACCEPTED MANUSCRIPT
in our case for determination of the sex through the pelvis bone. Next, age and stature were estimated, we examined long bones and the age was between (25-35) years and the stature was between (163-170) cm. The main finding was observed in skeleton 5, which presented dental work, (Fig. 3). The presence of this dental caries could be useful for the identification process. This finding could be indicative of the victim’s socioeconomic status, an inaccurate valuation but one that could be compared with the available ante mortem information. There was antemortem picture of dead No. 5 describes the presence of dental caries in front teeth this is beside the information from his mother. This indicate that dead was No. 5 which confirmed by DNA analysis.
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mass grave located near the city of Benghazi (140 km west Benghazi in desert). Seven skeletons were exposed and recovered from a single grave, with some clear post mortem displacements observed,(Fig. 1). The remains were delivered for study to Benghazi medical center, a teaching hospital where corpses were placed in equipped morgue, (Fig. 2). The personal belongings found associated to the skeletons were their clothes. Two of the six families had recognized the clothing of their loved one in the mass grave where the bodies were found.
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• Osteological studyand analyses The state of preservation of the remains was moderate to good. It was possible to apply most of the methods for sex, age and stature estimation as well as for the recording of pathological conditions and non-metric traits. The visual 7, 8 and metric 9 study of the pelvis unequivocally indicated that all the skeletons were male. There are Three techniques for the visual evaluation of traits of the hip bone are: 1) the method of Phenice (1969), which uses three traits on the pubis, 2) the method of Iscan and Derrick (1984) using the posterior pelvis, and 3) the method of Ferembach et al. (1980) of sexing the entire pelvis through an evaluation of eleven traits.8 And we used the third one
4
Two brothers were presumed to have been buried in the grave, but no sound conclusion was reached regarding the identification. But because they were two brothers it was not important for their parents to differentiate between their bodies. The match between the ante mortem and osteological findings was partial, due to a lack the ante mortem data. The corroboration of the information from different sources was partial, but it was
ACCEPTED MANUSCRIPT
disposable and were initially crushed manually so coarse. One sample for each corpse and subsequently was pulverized to promote the reaction of softening and DNA extraction.
RI PT
considered enough to request a DNA study, with compatible identities. • Identification by DNA analysis - 7 Sachets for biological samples, perfectly sealed, each containing 2 teeth marked with numbers from 1 to 7. These teeth have been identified in the laboratory Genome as indicated in table (1).
SC
o DNA extraction It has been necessary to use different extraction techniques for the individual items because they were finds dating of old (over 16 years) and the poor state of preservation. 1. Chelex-100 It is a resinous substance that, when mixed with the eluate of biological trace and subjecting the obtained solution at elevated temperatures and strong centrifugation allows separating the DNA from the rest of the substances composing the track. 2. Prep filer The second method was the one that uses the "prep Filer TM Forensic DNA Extraction Kit". 3. EZ1 The third method used was the one using the kit of Qiagen EZ1 with EZ1 advanced automatic extractor.
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- 6 packs of yellow paper, perfectly sealed, containing some hair belonging to a family member. These biological samples were transcribed on the facade the name of the deceased relative and the degree of relationship between the family and the deceased. The samples were identified at a laboratory Genome in Libya as indicated in table (2).
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o preparation of the teeth to the analysis of DNA
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This phase consisted of the mechanical cleaning of the teeth in order to remove surface contaminants, by scraping with a sterile scalpel blade and subsequent incubation to UV rays for a period of 5 minutes, to obtain the destruction of exogenous DNA.
o Quantification of DNA DNA extracted from these samples and was then quantified using the kit Quantifier Human DNA Quantification Kit using the amplifier 7300 Real Time PCR System. The amount of DNA in trucks
All the teeth were separately inserted into an envelope sterile 5
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was determined by comparison of the intensity of the signal obtained with that of standards with known concentrations.
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o Amplification of DNA The material obtained in the previous phase extraction was amplified by PCR. This technique allows isolating and amplifying specific fragments of DNA with primer sequences (primers) that, under the action of a heat-resistant enzyme (Taq-polimeras) and in the presence of suitable reagents, allow exponential amplification of the fragment identified. At the end of the reaction, the amplified DNA fragment will constitute the predominant species present in the reaction tube allowing the specific study.
population. The identification of a sequence of primer capable of binding with high specificity traits not variants "and downstream" of the piece variant (polymorphic), allows the amplification of this segment allowing the study. Part of the length polymorphisms of DNA the class, at the time, most used and that of STRs. The variability in these fragments consists mostly of the number of times a given fragment short (short) and repeated consecutively. This class of polymorphisms, which is the most represented in the human genome, provides forensic scientist of a huge number of points that you can compare to recognize two different biological samples. The STRs can be constituted by repeated fragments ranging from 2 to 7 base pairs (the pairs of nucleobases are the units making up the DNA, like the rungs of a ladder). Those used in this work are composed of a repeated sequence of four base pairs because this type of STR and certainly the most suitable for forensic as recognized by several validations work .The analysis of STRs is performed by capillary electrophoresis. NB:written informed consent were taken from the relevant. In the first phase analytical were analyzed 6 samples of hair, to make sure that it was possible to obtain a profile usable for
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Some regions of DNA are called polymorphic in that they have a lot of variation in the population, there are several types of polymorphic regions and their study allows distinguishing each individual from another. polymorphism the DNA used in this work were chosen in the class of length polymorphisms called STRs (Short Tandem Repeats), which are currently the method of choice for personal identification via biological markers. These are regions of DNA, ranging from specific regions not variants, which can be present in different lengths in the human
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comparison then the profiles were obtained with two amplification kit. The profiles from the teeth of the 7 victims were obtained using two amplification kits.
probability that it is the sister of the other findings.
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The probability that the woman identified as the number C68412 to be the sister of the person who indicated as C67360 is 160, 027, 58 times greater than the probability that it is the sister of the other findings. The probability that the woman identified with the number C68409 to be the sister of the person who indicated as C67358 is 5669 times greater than the probability that the two are not brother and sister.
3. Results
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Gave comparison of the profiles which were obtained from the hair with those were obtained from the teeth. It immediately became clear that the profiles of the teeth were no. 2, 5, 6 compatible with the profiles of three mothers analyzed.
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The teeth no.2 son of Case 3 indicated with code C68410 with a probability of maternity 99.89258988. The teeth no.5 son of Case 4 indicated with code C68411 with a probability of maternity 99.9995514. The teeth no.6 son of Case 6 indicated with code C68413 with a probability of maternity 99.89258988. Then the profile of each of the 4 teeth that have yet to be assigned in, were compared with the three sisters who had been involved. The probability that the woman identified as the number C68414 to be the sister of the person who indicated as C67364 is 475, 733, 36 times greater than the
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The probability that the woman identified with the number C68409 to be the sister of the person who indicated as C67361 is 21, 25 times greater than the probability that the two are not brother and sister. The results to which they come and allow us to attribute the findings examined women who have required to be recognized according to table (3).
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4. Discussion
Examination of skeletal remains of suspected skeletons coming from ancient sites may shed light on reasons of the crime.
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nucleotides in length. During our work we did confirm findings published earlier that DNA could be extracted and amplified without decalcification 11.
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The problems which forensic scientists most often face while working with DNA extracted from different samples are either DNA degradation or DNA contamination. High temperatures, salt, water or soil conditions can damage genomic DNA and therefore make the identification process extremely difficult. The genetic analysis of those killed and buried in the mass graves through DNA from remains of bone and teeth requires the use of short sized loci that can be amplified by polymerase chain reaction (PCR) for which the short tandem repeat (STR) loci are most suitable 10. Short tandem repeats markers are polymorphic DNA loci at noncoding regions of the gene containing a repeated nucleotide sequence. Usually STR repeat units are from two to seven
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Forensic science is a fastevolving cluster of applied disciplines that operate independently as well as interdisciplinary. Fields like anthropology and odontology develop and expand incorporating new methods and theories. As a result, the boundaries between forensic science disciplines become blurred allowing a multidisciplinary involvement and approach, especially when skulls are involved 12. From the case series presented here, we have to conclude that information recovered from testimonies is of crucial importance for obtaining information regarding the presumed number and identities of the people buried in those graves and to prove to our culture how it is important to preserve
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radiograph.
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the ante mortem documentation of any medical report like teeth
5. Conclusion
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Testimonies are especially important when the presence of a grave containing a low number of victims is suspected, as in the case presented here. The decision was made to request a DNA study that made it possible to propose the identification of all seven skeletons exhumed. However, we have to be cautious about any Statement regarding the identification of a group of people presumably buried together once the identification for one or more of the persons
from the group have been proposed. Various events may have transpired between the kidnappings of people from towns and their assassination in the countryside, precluding in some cases a complete coincidence between testimonies and archeological and osteological findings. In that case, our criterion would have been to indicate that a positive identification could not be proposed, although in light of the accumulated evidence it could have been reasonable to point to a compatible identity awaiting a DNA study.
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6. References
[6] D. Manning, Srebrenica investigation, summary of forensicevidence. IC TY Evidence Report, Unpublished documentProperty of the United Nations, International Criminal Tribunal for the Former Yugoslavia, 2000.
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[1] M.F. Skinner, D. Alempijevic and M. Djuric-Srejic, Guidelines for international forensic bioarchaeology monitors of mass grave exhumations, Forensic Sci. Int. 134 (2003) 79–90. [2] M. Skinner, J. Sterenberg, Turf wars: Authority and responsibility for the investigation of mass graves, Forensic Sci. Int. 151 (2005) 221–232. [3] E. Jessee, M. Skinner, A typology of mass grave and mass grave-related sites, Forensic Sci. Int. 152 (2005) 55–59.
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[4] J. Sterenberg, Archaeological Techniques and Methods for theExcavation and Recording of Contemporary rimary andSecondary Mass Graves. Unpublished M.Sc. Thesis fromBournemouth University, UK, 2002 [5] T. Simmons, Taphonomy of a karstic cave execution site atHrgar, Bosnia-Herzegovina, in: William D. Haglund, MarcellaH. Sorg (Eds.), Advances in Forensic Taphonomy: Method,Theory and Archaeologic al Perspectives, CRC Press, USA,2001, pp. 263–308.
[7] J.E. Buikstra, D.H. Ubelaker, Standards for Data Collection from Human Skeletal Remains, Arkansas Archaeological Survey, Fayetteville, 1994. [8] J. Bruzek, A method for visual determination of sex using the human hip bone, Am. J. Phys. Anthropol. 117 (2002) 157–168. [9] P. Murail, J. Bruzek, F. Houe¨ t, E. Cunha, DSP: a tool for probabilistic sex diagnosis using worldwide variability in hip-bone measurements, Bull. Mem. Soc. Anthropol. Paris (2005) 167–176. [10] Clayton TM, Whitaker JP, Fisher DL, LeDA, Holland MM, Weedn VW, Maguire CN, Di Zinno JA, Kimpton CP, Gill P. Further validation of a quadruplex STR DNA Typing system: a collaborative effort to identify victims of a mass
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Journal of Forensic Sciences 1993; 38:60-68.
disaster. Forensic Sci International 1995; 76:17-25. [11] Fisher LD, Holland MM, Mitchell L, Sledzik SP, Wilcox WA, Wadhams M, Weedn WV. Extraction, Evaluation and Amplification of DNA from Decalcified and Undecalcified United States Civil War Bone.
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[12] Nuzzolese E, Borrini M, Forensic approach to an archaeological casework of "vampire" skeletal remains in Venice: odontologicaland anthropological prospectus. Journal of Forensic Science. 2010 Nov;55(6):1634-7.
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Teethsamplen.1
Idgenome C67358
Teethsamplen.2
C67359
Teethsamplen.3
C67360
Teethsamplen.4
C67361
Teethsamplen.5
C67362
Teethsamplen.6
C67363
Teethsamplen.7
C67364
Degree of relationship Sister Mother Mother Sister Mother Sister
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Name of deceased Case 1, case 2 Case 3 Case 4 Case 5 Case 6 Case 7
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Table (2):Biological samples of family members with Id Genome
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IdLibyan
Id Genome C68409 C68410 C68411 C68412 C68413 C68414
Table (3): The probable relationships of examined women to discovered skeletons
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C67358 C67359 C67360 C67361 C67362 C67363 C67364
Name given to the finding Case 1, case 2 Case 3 Case5 Case 1, Case 2 Case 4 Case 6 Case7
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NO. findings
Relationship to
brother son brother brother son son brother
NO. Identification mother / sister C68409 C68410 C68412 C68409 C68411 C68413 C68414
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Fig.(1): Exhumation of remains from a single grave in Benghazi desert
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Fig. (2): Study of remains in Benghazi medical center.
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Fig. (3): Skeleton 5 with dental caries.