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Evaluating the prevalence of DNA mixtures found in fingernail samples from victims and suspects in homicide cases
Forensic Science International: Genetics 5 (2011) 532–537
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Evaluating the prevalence of DNA mixtures found in fingernail samples from victims and suspects in homicide cases Bublil Nurit *, Gast Anat, Shenfeld Michal, Front Lilach, Freund Maya Forensic Biology Laboratory, The Israeli National Center of Forensic Medicine, The National Center of Forensic Medicine, 67 Ben Zvi Rd., P.O. Box 8495, 61085 Tel-Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2010 Received in revised form 18 November 2010 Accepted 10 December 2010
An important aspect of homicide investigations is the identification of the persons that had the last contact with the victim prior to death. Violent crimes are frequently characterized by a struggle between the victim and the perpetrator where biological material can be expected to be exchanged between them. Forensic DNA typing enables the generation of genetic profiles by extraction and amplification of cellular material found under fingernails. The evidential value of these samples may be critical if the secondary contributor found in a DNA mixture, can be matched with a potential suspect, or through a DNA database search. The amount of biological material transferred under the fingernails during ‘‘casual’’ activities is not sufficient to genotype reportable mixtures. This may not be the case with homicide victims that may have struggled and died under violent circumstances. The aim of this study was to evaluate the prevalence of DNA mixtures found under the fingernails of both victims and suspected perpetrators of violent deaths. We present a retrospective study of 137 DNA profiles genotyped from fingernail samples of homicide victims and suspects, collected at the Israeli National Center of Forensic Medicine. The majority of the samples produced single source profiles (n = 107, 78%) that matched those of the donor’s. DNA mixtures (n = 30, 22%) were found in increased frequency among victims (n = 25/100, 25%) compared to suspects (n = 5/37, 13.5%). Mixtures were sub-divided into high level (n = 15, 50%), low level (n = 9, 30%) and residual (n = 6, 20%), according to the number of the foreign contributors’ alleles. Thus, this distinctive group of homicide victims was found to express both elevated frequency of DNA mixtures together with highly informative value of the secondary foreign profiles, as compared to other studied populations. These findings support an important aspect for the criminal investigation in murder cases, where a struggle may have ensued and the identification of an additional profile found in a mixture from a fingernail sample may point to a possible perpetrator of the crime. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic DNA typing Mixture Fingernail Homicide
1. Introduction Finding foreign biological material under the fingernails of a victim and/or a suspect in a homicide case can be a valuable evidence in a criminal investigation [1,2]. A DNA mixture originating from the donor’s cells combined with a secondary source implies that physical contact occurred, in which biological material was transferred between these individuals [3–7]. Recent studies have shown that even though casual physical activities may leave traces of biological material under the fingernails, the background level of this cellular debris is not usually sufficient to produce detectable DNA mixtures in the
* Corresponding author. Tel.: +972 3 5127838; fax: +972 3 5127835. E-mail addresses: [email protected], [email protected] (B. Nurit). 1872-4973/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2010.12.003
majority of samples [8,9]. Most DNA profiles (80%) originating from swab samples taken from under the fingernails of individuals of the general population are characterized as a single source, typed only by the donors’ cells [8]. In some cases low level DNA mixtures can be found under the fingernails of volunteers of the general population. Similar results are presented by Cerri et al. [9] after characterization of fingernail swabs collected from individuals who died in non-violent death. The study of Dowlman et al. [10] which tested the effect of cohabiting interactions on DNA mixtures, presented higher prevalence of DNA mixtures from fingernail samples of volunteers compared to general population volunteers. These studies demonstrate that the foreign biological material accumulated under the fingernails during every day activities, is usually not sufficient to allow detection of high level DNA mixtures. In fact, finding a high level of DNA mixture under one’s fingernails is a relatively rare occurrence and is not correlated with ‘‘casual’’ everyday activities [10].
B. Nurit et al. / Forensic Science International: Genetics 5 (2011) 532–537
In contrast to daily activity, violent crimes can often be associated with intense physical contact between victim and perpetrator, due to struggling at the scene of the crime. The assumed struggle is expected to facilitate the transference of biological materials between two (or more), individuals [1–7]. As a result of the criminal act, blood, semen, epithelial cells or any other biological material is transferred from the perpetrator to the victim’s body. This material would be expected to remain on the victim’s body, since the fatal consequences of the event precluded him from disposing of this evidence. Hence, fingernail samples are routinely collected during autopsies of homicide victims at the Israel National Center of Forensic Medicine. Suspects may also be sampled if their arrest takes place a short time after the assault, usually, within 24 h. In criminal cases investigated between the years 2005 and 2010, dozens of fingernail swab samples were routinely collected from murder victims and/or suspects for further DNA analysis by the Forensic Biology Laboratory of the National Center of Forensic Medicine. The aim of this study is to evaluate the occurrence of DNA mixtures from under the fingernails of murder victims, a distinctive population in which DNA mixtures would be expected to be more prevalent. 2. Materials and methods 2.1. Data collection The data presented in this study was retrospectively analyzed from 137 DNA profiles genotyped from fingernail samples collected at the Israeli National Center of Forensic Medicine between 2005 and 2010. The majority of the profiles originated from homicide victims (n = 100), the remainder from suspects (n = 37) (Fig. 1). The cases included in the study were only those where reportable DNA profiles were achieved, being either single source or a mixture. Those containing DNA mixtures were limited
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to those with two contributors since peak height ratio (PHR) analysis is not possible for three or more contributors. The primary criteria of which victim’s fingernails were sampled was determined by the medical examiner, based on the circumstances known at the time of the autopsy. In general, gunshot victims were the only type of homicide cases that were disqualified for fingernail examination, under the assumption that a physical struggle may not have taken place before the victim’s death. 2.2. Sample collection The majority of the bodies submitted to the mortuary had their hands protected by bags. Samples from a victim’s fingernails were taken at the beginning of the external examination of the body by one of the following techniques (according to the methods in use at the time): clipping was primarily used from 2005 to 2007, substituted later by fingernail swabbing with a slightly wet cotton swab due to the practical aspects: easier collection of the debris from all 5 fingers on a single swab promote an easier DNA extraction, without the need of pre-wash stage which is carried out on nail clippings. According to the Biology Lab procedures, the swabs were dried for 24 h prior to storage (20 8C). Blood samples were routinely taken from all victims during the autopsy to use as a reference sample. The debris collected from under five fingernails from each hand was referred to as a single, independent sample. Samples from suspects were collected, either as clippings, or by swabbing of the fingernails (as described above). For suspects, buccal swabs were taken to serve as a reference sample. 2.3. DNA extraction and quantification Both types of fingernail samples, clippings or swabs were extracted using Chelex-100 procedure [11]. The primary wash of the fingernail clippings was carried out with double distilled, UVirradiated water, for 30 min at 37 8C. Only washing liquid served for the DNA extraction. For DNA extractions performed between the years 2005 and 2008 quantification was carried out using the QuantiFiler Human DNA Quantification kit (Applied Biosystems, USA). DUO QuantiFiler was employed on samples extracted from 2009 onwards. Real-time PCR was performed on an ABI 7500 Sequence Detection System according to the manufacturer’s protocols. 2.4. SGM-Plus PCR amplification kit Samples were amplified using the AMPFlSTR SGM Plus kit (cat no. 4307133 Applied Biosystems, USA). The optimal DNA amount for samples was 1.5 ng, according to our lab’s internal validation sensitivity test. Each DNA sample was amplified in a total reaction volume of 25 ml, on a 9700 thermal cycler (Applied Biosystems, USA) following standard PCR conditions as recommended by the manufacturer. A positive PCR control (AMPFlSTR control DNA 007) and a negative control (no DNA) were included in each set of amplifications. 2.5. Capillary electrophoresis
Fig. 1. Distribution of samples and genetic profile types generated from victims and suspects of homicide cases fingernails.
A loading mix was prepared with 10 ml Hi-Di formamide (Applied Biosystems, USA) and 0.5 ml GeneScan 500 ROX size standard (P/N 401734 Applied Biosystems, USA). Loading mix was added to 1.1 ml of PCR product and analyzed on 96 well reaction plates (Applied Biosystems, USA). Duplicate allelic ladder samples (1.1 ml) were included in each run. Samples were denatured at 95 8C for 3 min before being run on either ABI’s 3100 or 3130xl Genetic Analyzers (Applied Biosystems, USA).
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2.6. Data analysis
3.2. DNA mixture classification
GeneMapper ID software v3.2 was used to analyze all data, regardless of which model Genetic Analyzer was used for capillary electrophoresis. ROX 500 was employed for the size standard threshold for allele designation was set to 60 rfu for heterozygote, and 200 rfu for homozygote, according to our laboratory’s internal validation study. Stutter percentage was carried out according to manufacturer recommendation.
The type of each of the 30 DNA mixtures recovered from under the victim’s and suspect’s fingernails, was defined in both primary and secondary classifications (Fig. 3). The profile’s analysis revealed an unambiguous asymmetrical pattern of the DNA mixture expression, between the right and left hands (Fig. 3). High level DNA mixtures were found in 15 fingernail samples, which are 50% of all mixtures analyzed (Fig. 3). The remaining samples presented low level DNA mixtures (30%), or were characterized as residual profiles (20%) (Figs. 1, 3). Four cases which were typed as low level DNA mixtures, presented 11 alleles of the foreign contributor [case nos. 1, 5, 7, and 12]. Peak height ratio analysis of these mixtures allowed the designation of additional alleles, thus, upgrading the classification of the ‘‘interpreted’’ profile from a ‘low’ to ‘high’ level DNA mixture [case nos. 1, 5, 7, 12, and 15] (Fig. 3). As mentioned before, only primary classifications served in downstream statistical calculations. PHR analysis may also assist in determination of major and minor contributors within the DNA mixture. An interesting observation was recognized within a number of DNA mixtures (n = 8/30, 26.6%), seen mainly in victim samples (n = 7) but also in one suspect sample (n = 1): although these fingernail samples were collected from the donor, the major constituent of the DNA mixture was not that of the donor, as would be expected, but rather originated from the foreign contributor (Fig. 4). These cases were classified mainly as high level DNA mixtures [case nos. 2, 6, 10, 16, 20, and 22], even though two cases of low level DNA mixtures were also noted [case nos. 5 and 7].
2.7. DNA profile classification Single profiles, either partial or full, were characterized by one or two alleles at each locus throughout the profile (Table 1). The presence of more than two alleles at a locus was used to identify a possible mixture. Comparison between the DNA profiles originating from the fingernails and the blood or buccal reference samples allowed identifying the donor’s alleles from those from a foreign contributor. Thus, recognition and counting of the alleles attributed to the secondary, foreign contributor could be made. Stutter peak positions were not regarded as additional alleles if stutter peak heights matched manufacturer recommendation for SGM-Plus at each locus. Any peaks below threshold of 60 rfu were not called. The classification of the DNA mixture types was carried out according to the number of the foreign obligatory alleles presented in the profile: obligatory alleles were defined as those whose heights were above threshold, those not overlapping donor alleles, and could not be considered as stutters. These alleles were added to the alleles originating from the donor. The criteria for each of the mixture types: ‘‘residual’’; ‘‘low level’’ and ‘‘high level’’ mixtures are detailed in Table 1. Mixture interpretation allows additional information to be inferred based on differences in PHR. If an intimate sample was analyzed, and a two person mixture was assumed, then analysis of the donor alleles along with PHR differences, will allow recognition of additional alleles of the foreign contributor (Fig. 2). In this case, classification of the profile will be implemented twice: first, to type the profile as a ‘residual’, ‘low’ or ‘high’ level mixture, and second to use the additional information generated by the interpretation of the peak heights to depict the ‘‘interpreted’’ profile (Fig. 2). The final classification of the mixture for statistical calculation: ‘residual’, ‘low’ or ‘high’ was determined based only on the primary classification without the PHR interpretation. 3. Results 3.1. DNA profiling Single source profiles originating from the donor’s fingernails were found in the majority of samples, either victims or suspects (n = 107, 78%) (Fig. 1). DNA mixtures were observed in 30 samples (25 victims [cases 2, 5, 7–8, and 10–30] and five suspects [cases 1, 3, 4, 6, and 9]); which comprised 22% of all the cases tested (Figs. 1, 3).
4. Discussion This study tried to expand the understanding of the significance of DNA mixtures found from fingernail samples in criminal investigations, especially among homicide victims. The rapid changes in methodologies implemented in forensic DNA typing during the last decade required a re-assessment of the prevalence of DNA mixtures in these cases, particularly, after background level of DNA mixtures in the general population has been described in recent publications [8–10,12]. The manifestation of a DNA mixture under the fingernails of a murder victim, or a suspect, is a rare occurrence: 78% of the samples, either swabs or fingernail clippings, produced a single source profile which could be matched to the donor. This result is supported by previous findings [8,9] which have shown that most fingernail samples do not generate DNA mixtures: the study by Cook and Dixon [8] found that the majority of fingernail swabs collected from volunteers from the general population provided a single source profile, and only 6% produced DNA mixtures. No high level DNA profile types could be found among these cases. Similar results were found by Cerri et al. [9] in fingernail samples collected from non-violent crimes deceased, where only 5% of the samples allowed the designation of a two person mixtures, but the
Table 1 Classification of DNA profiles according to the number and type of alleles designation.
Primary classification
Classification
Number of alleles
Criteria
Single DNA profile
Equal to or less than 2 (per locus)
–
Less than 4 4–11 Equal to or more than 12
Number of foreign alleles added to the self donor profile; stutters were not included; alleles with peak height <60 rfu were not included
[Primary classification result + ‘‘interpreted’’ alleles]
Detection of additional foreign contributors’ alleles is applicable if PHR allows
DNA mixtures: Residual profile Low level DNA mixture High level DNA mixture Secondary classification
PHR ‘‘interpreted’’ profiles
B. Nurit et al. / Forensic Science International: Genetics 5 (2011) 532–537
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Fig. 2. Second contributors’ alleles detection by peak heights ratio analysis. The self donor alleles are filled; foreign obligatory alleles are present (#). PHR analysis implies that both contributors share the same allele at the locus (*). Homozygote alleles of the second contributor are noted (~).
remaining 95% genotyped a single source profile [9]. These studies provided the evidential value of the low background level of DNA mixtures within the general population, leading to the inevitable conclusion that the cellular debris accumulated under one’s fingernails throughout routine casual activities, is not consistent with DNA mixtures, and particularly not high level or reportable type of mixtures. Results of this study show that the prevalence of DNA mixtures found among murder victims fingernail samples, is much higher: 25% of the victims’ samples genotyped provided additional alleles other than the self component. Moreover, the primary classification of approximately half of the samples revealed high level DNA mixtures, increasing the prospect for a positive identification of the second contributor. Similarly, few of the samples that were characterized as ‘low’ level DNA (n = 5/9), still presented a high informative value due to the designation of 10 or 11 alleles of the second contributor. This study has also illustrated an asymmetric pattern of mixtures between the right and left hand of the donors (see Fig. 3). The circumstances of a violent crime are varied and may affect the persistence of a foreign material found under one’s fingernails, thus DNA analysis should be carried out from both donor hands.
The study of Malsom et al. [12] presented elevated frequency of DNA mixtures in fingernail samples collected from cohabitating couples: 17% of the samples resulted in reportable mixtures. The common theme for all of these cases was that intimate contact took place 48 h prior to sample collection. It was also found that not all intimate contact led to the designation of high level mixtures, but all high level mixtures were associated with intimate contact [12]. It was also found that by itself, cohabitation found to increase the likelihood of gaining only a residual profile (less than 5 alleles) of the foreign contributor, and not a high level mixture. Various activities which were considered as the source for the transfer of small amount of DNA from one person to another, such as hand shaking, hugging, dressing a child, sharing a bed, sharing working surrounding, etc., were found insufficient for generating high level DNA mixtures [10,12]. On the other hand, homicide victims presented an increased prevalence of both ‘high’ and ‘low’ types of DNA mixtures. These sorts of mixtures are a rare entity within the general population, even among cohabitating couples [12]. Consequently, the appearances of high level DNA mixtures among murder victims should be associated with an unusual type of contact rather than a casual activity. The mixture could be the result of a close, violent, physical
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B. Nurit et al. / Forensic Science International: Genetics 5 (2011) 532–537
Fig. 3. Classification of 30 DNA mixtures typed from fingernail samples of homicide cases’ victims and suspects. If PHR analysis reveals that a donors’ allele is shared with the foreign contributor then this allele is added to generate the ‘‘interpreted’’ profile of the second contributor (max. alleles no = 22).
contact that enabled an accelerated transfer of cellular material between the victim and attacker. Considering that these victims died in violent circumstances; the origin for this foreign material could be the perpetrator. The main difference we found between the victims group and the suspects was that the DNA mixtures were recognized twice as much from victims’ fingernails (25%) as compared with the
suspects (13.5%). This difference may be explained by the lethal outcome of the attack: if a struggle took place and biological material was exchanged, then the lack of activity ensures that the death of the victim will preserve all cellular material under the fingernails. Given that the deceased no longer had the ability to wash away evidence, then unless washed away by environmental conditions, or by the perpetrator’s manipulations, the foreign
Fig. 4. A DNA mixture characterized by the foreign contributor being the major component that overcomes the donor self profile.
B. Nurit et al. / Forensic Science International: Genetics 5 (2011) 532–537
material would most likely remain under the victim’s fingernails. If the amount is sufficient, a reportable DNA mixture, consisting of the victim himself and the perpetrator, may be expected to be found. The perpetrator on the other hand, will probably make any effort to eliminate all evidence related to the criminal act. Thus, hand washing, bathing, fingernail clipping or any other washing acts are expected to be taken. Even if not deliberate, but rather as a result of routine hygiene, these acts will lead to decreased chance of finding a DNA mixture under the perpetrator’s fingernails. The prevalence of DNA mixtures found among suspect’s samples was higher (13.5%), compared with the published incidence found among volunteers from the general population (6%) [8]. In addition, both ‘high’ and ‘low’ level mixtures were noted among suspect’s fingernail samples, compared with ‘drop in’ or residual profiles within the general population [8]. The differences found between these two populations, the general population and murder suspects, are not unexpected due to fact that the declaration of an individual as a suspect should be based upon reliable, preliminary information that connect them to the criminal act, rather than any randomly picked individual. The informative value of a secondary contributor within ‘high’ or even ‘low’ level DNA mixture is considerable. Malsom et al. [12] found that the majority of DNA mixtures (23/ 24) that were collected from cohabitating couples enabled the identification of the second contributor as the legitimate partner of the donor [12]. Thus, the presence of the second contributor had a rational explanation and it was not left unreciprocated. The positive identification of a person as the secondary contributor within a ‘high’ level DNA mixture that was generated from fingernail samples from homicide victims require an explanation: the mixture cannot be considered as a background level, or the result of a ‘casual’ activity, including casual touch between two contributors. Consequently, unless that person is the victim’s partner and intimate contact took place between them less than 48 h prior to the victim’s death, then one of the most reasonable explanations for the finding is that this person may be considered the perpetrator involved in the assault. In this study we have found that out of the 23 homicide cases tested, in 12 cases the non-self, foreign component was found to match the suspect, or the victim’s reference profile. These cases resulted in charges against the alleged suspect. An important finding in this study was the appearance of DNA mixtures characterized by the major component originating from the foreign contributor, rather than the donor himself, as might be expected: in seven cases, the suspect’s full profile was expressed more strongly than that of the victim himself, and in one case it was the full profile of the victim that overcame the suspect’s selfalleles. These cases may indicate an aggressive type of physical contact that had allegedly taken place between the victim and the perpetrator, to allow such large amount of biological material to be transferred. Dowlman et al. [10] describe only one case, out of 80 samples of cohabitating couples, in which the female partner’s profile had overcome the donor male profile. In that case the authors assumed that this finding must be correlated with the type
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of the intimate contact that took place between the partners. No such mixtures, in which the foreign contributor became the major component was found among volunteers from the general population. 5. Conclusion The prevalence of DNA mixtures found under fingernail samples from murder victims and/or suspects is relatively high, compared with other populations studied so far [8–10,12]. Both of these populations present not only higher incidence but also an exceptional type of DNA mixture, in which the secondary contributor can often be well characterized, therefore, increasing the chance for a positive match to a reference sample. If a high level DNA mixture is genotyped, especially if the foreign contributor is the major component, there are limited scenarios that can explain such large amount of foreign material from under one’s fingernail. No casual activity, cohabitating or sharing work environment can be considered as a relevant explanation. A violent, brutal struggle, taking place in proximity to the time of the victim’s death may account for it. Thus, the evidential value of DNA mixtures, originating from fingernail samples of a victim, or a suspect, may be a powerful tool in a homicide investigation. Acknowledgments The authors wish to thank Dr. Tzipi Kahana and Mrs. Carla Oz for reviewing this manuscript. References [1] A. Piccinini, F. Betti, M. Capra, A. Comino, A 5-year study on DNA recovered from fingernail clippings in homicide cases Milan, Prog. Forensic Genet. 1239 (2003) 929–932. [2] A. Ferna´ndez-Rodrı´guez, M.J. Iturralde, L. Ferna´ndez de Simo´n, J. Capilla, M. Sancho, Genetic analysis of fingernail debris: application to forensic casework, Prog. Forensic Genet. 1239 (2003) 921–924. [3] P. Wiegand, T. Bajanowski, B. Brinkmann, DNA typing of debris from fingernails, Int. J. Legal Med. 106 (2) (1993) 81–83. [4] M. Sanchez-Hanke, K. Puschel, C. Augustine, P. Wiegand, B. Brinkmann, PCR typing of DNA extracted from epidermal particles by scratching, Adv. Forensic HaemoGenet. 6 (1996) 316–318. [5] R.A. Wickenheiser, Trace DNA: a review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact, J. Forensic Sci. 47 (2002) 442–450. [6] T. Lederer, P. Betz, S. Seidl, DNA analysis of fingernail debris using different multiplex systems: a case report, Int. J. Legal Med. 114 (2001) 263–266. [7] C. Oz, A. Zamir, An evaluation of the relevance of routine DNA typing of fingernails clippings for forensic casework, J. Forensic Sci. 45 (2000) 158–160. [8] O. Cook, L. Dixon, The prevalence of mixed DNA profiles in fingernail samples taken from individuals in the general population, Forensic Sci. Int. Genet. 1 (2007) 62–68. [9] N. Cerri, A. Verzeletti, V. Cortellini, A. Cincotta, F. De Ferrari, Prevalence of mixed DNA profiles in fingernail swabs from autoptoc cases, Forensic Sci. Int.: Genet. Suppl. Ser. 2 (2009) 163–164. [10] E.A. Dowlman, N.C. Martin, M.J. Foy, T. Lochner, T. Neocleous, The prevalence of mixed DNA profiles on fingernail swabs, Sci. Justice 50 (2010) 64–71. [11] P.S. Walsh, D.A. Metzger, R.H. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques 10 (1991) 506–513. [12] S. Malsom, N. Flanagan, C. McAlister, L. Dixon, The prevalence of mixed DNA profiles in fingernail samples taken from couples who co-habit using autosomal and Y-STR, Forensic Sci. Int. Genet. 3 (2009) 57–62.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Evaluating the prevalence of DNA mixtures found in fingernail samples from victims and suspects in homicide cases Bublil Nurit *, Gast Anat, Shenfeld Michal, Front Lilach, Freund Maya Forensic Biology Laboratory, The Israeli National Center of Forensic Medicine, The National Center of Forensic Medicine, 67 Ben Zvi Rd., P.O. Box 8495, 61085 Tel-Aviv, Israel
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 August 2010 Received in revised form 18 November 2010 Accepted 10 December 2010
An important aspect of homicide investigations is the identification of the persons that had the last contact with the victim prior to death. Violent crimes are frequently characterized by a struggle between the victim and the perpetrator where biological material can be expected to be exchanged between them. Forensic DNA typing enables the generation of genetic profiles by extraction and amplification of cellular material found under fingernails. The evidential value of these samples may be critical if the secondary contributor found in a DNA mixture, can be matched with a potential suspect, or through a DNA database search. The amount of biological material transferred under the fingernails during ‘‘casual’’ activities is not sufficient to genotype reportable mixtures. This may not be the case with homicide victims that may have struggled and died under violent circumstances. The aim of this study was to evaluate the prevalence of DNA mixtures found under the fingernails of both victims and suspected perpetrators of violent deaths. We present a retrospective study of 137 DNA profiles genotyped from fingernail samples of homicide victims and suspects, collected at the Israeli National Center of Forensic Medicine. The majority of the samples produced single source profiles (n = 107, 78%) that matched those of the donor’s. DNA mixtures (n = 30, 22%) were found in increased frequency among victims (n = 25/100, 25%) compared to suspects (n = 5/37, 13.5%). Mixtures were sub-divided into high level (n = 15, 50%), low level (n = 9, 30%) and residual (n = 6, 20%), according to the number of the foreign contributors’ alleles. Thus, this distinctive group of homicide victims was found to express both elevated frequency of DNA mixtures together with highly informative value of the secondary foreign profiles, as compared to other studied populations. These findings support an important aspect for the criminal investigation in murder cases, where a struggle may have ensued and the identification of an additional profile found in a mixture from a fingernail sample may point to a possible perpetrator of the crime. ß 2010 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic DNA typing Mixture Fingernail Homicide
1. Introduction Finding foreign biological material under the fingernails of a victim and/or a suspect in a homicide case can be a valuable evidence in a criminal investigation [1,2]. A DNA mixture originating from the donor’s cells combined with a secondary source implies that physical contact occurred, in which biological material was transferred between these individuals [3–7]. Recent studies have shown that even though casual physical activities may leave traces of biological material under the fingernails, the background level of this cellular debris is not usually sufficient to produce detectable DNA mixtures in the
* Corresponding author. Tel.: +972 3 5127838; fax: +972 3 5127835. E-mail addresses: [email protected], [email protected] (B. Nurit). 1872-4973/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2010.12.003
majority of samples [8,9]. Most DNA profiles (80%) originating from swab samples taken from under the fingernails of individuals of the general population are characterized as a single source, typed only by the donors’ cells [8]. In some cases low level DNA mixtures can be found under the fingernails of volunteers of the general population. Similar results are presented by Cerri et al. [9] after characterization of fingernail swabs collected from individuals who died in non-violent death. The study of Dowlman et al. [10] which tested the effect of cohabiting interactions on DNA mixtures, presented higher prevalence of DNA mixtures from fingernail samples of volunteers compared to general population volunteers. These studies demonstrate that the foreign biological material accumulated under the fingernails during every day activities, is usually not sufficient to allow detection of high level DNA mixtures. In fact, finding a high level of DNA mixture under one’s fingernails is a relatively rare occurrence and is not correlated with ‘‘casual’’ everyday activities [10].
B. Nurit et al. / Forensic Science International: Genetics 5 (2011) 532–537
In contrast to daily activity, violent crimes can often be associated with intense physical contact between victim and perpetrator, due to struggling at the scene of the crime. The assumed struggle is expected to facilitate the transference of biological materials between two (or more), individuals [1–7]. As a result of the criminal act, blood, semen, epithelial cells or any other biological material is transferred from the perpetrator to the victim’s body. This material would be expected to remain on the victim’s body, since the fatal consequences of the event precluded him from disposing of this evidence. Hence, fingernail samples are routinely collected during autopsies of homicide victims at the Israel National Center of Forensic Medicine. Suspects may also be sampled if their arrest takes place a short time after the assault, usually, within 24 h. In criminal cases investigated between the years 2005 and 2010, dozens of fingernail swab samples were routinely collected from murder victims and/or suspects for further DNA analysis by the Forensic Biology Laboratory of the National Center of Forensic Medicine. The aim of this study is to evaluate the occurrence of DNA mixtures from under the fingernails of murder victims, a distinctive population in which DNA mixtures would be expected to be more prevalent. 2. Materials and methods 2.1. Data collection The data presented in this study was retrospectively analyzed from 137 DNA profiles genotyped from fingernail samples collected at the Israeli National Center of Forensic Medicine between 2005 and 2010. The majority of the profiles originated from homicide victims (n = 100), the remainder from suspects (n = 37) (Fig. 1). The cases included in the study were only those where reportable DNA profiles were achieved, being either single source or a mixture. Those containing DNA mixtures were limited
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to those with two contributors since peak height ratio (PHR) analysis is not possible for three or more contributors. The primary criteria of which victim’s fingernails were sampled was determined by the medical examiner, based on the circumstances known at the time of the autopsy. In general, gunshot victims were the only type of homicide cases that were disqualified for fingernail examination, under the assumption that a physical struggle may not have taken place before the victim’s death. 2.2. Sample collection The majority of the bodies submitted to the mortuary had their hands protected by bags. Samples from a victim’s fingernails were taken at the beginning of the external examination of the body by one of the following techniques (according to the methods in use at the time): clipping was primarily used from 2005 to 2007, substituted later by fingernail swabbing with a slightly wet cotton swab due to the practical aspects: easier collection of the debris from all 5 fingers on a single swab promote an easier DNA extraction, without the need of pre-wash stage which is carried out on nail clippings. According to the Biology Lab procedures, the swabs were dried for 24 h prior to storage (20 8C). Blood samples were routinely taken from all victims during the autopsy to use as a reference sample. The debris collected from under five fingernails from each hand was referred to as a single, independent sample. Samples from suspects were collected, either as clippings, or by swabbing of the fingernails (as described above). For suspects, buccal swabs were taken to serve as a reference sample. 2.3. DNA extraction and quantification Both types of fingernail samples, clippings or swabs were extracted using Chelex-100 procedure [11]. The primary wash of the fingernail clippings was carried out with double distilled, UVirradiated water, for 30 min at 37 8C. Only washing liquid served for the DNA extraction. For DNA extractions performed between the years 2005 and 2008 quantification was carried out using the QuantiFiler Human DNA Quantification kit (Applied Biosystems, USA). DUO QuantiFiler was employed on samples extracted from 2009 onwards. Real-time PCR was performed on an ABI 7500 Sequence Detection System according to the manufacturer’s protocols. 2.4. SGM-Plus PCR amplification kit Samples were amplified using the AMPFlSTR SGM Plus kit (cat no. 4307133 Applied Biosystems, USA). The optimal DNA amount for samples was 1.5 ng, according to our lab’s internal validation sensitivity test. Each DNA sample was amplified in a total reaction volume of 25 ml, on a 9700 thermal cycler (Applied Biosystems, USA) following standard PCR conditions as recommended by the manufacturer. A positive PCR control (AMPFlSTR control DNA 007) and a negative control (no DNA) were included in each set of amplifications. 2.5. Capillary electrophoresis
Fig. 1. Distribution of samples and genetic profile types generated from victims and suspects of homicide cases fingernails.
A loading mix was prepared with 10 ml Hi-Di formamide (Applied Biosystems, USA) and 0.5 ml GeneScan 500 ROX size standard (P/N 401734 Applied Biosystems, USA). Loading mix was added to 1.1 ml of PCR product and analyzed on 96 well reaction plates (Applied Biosystems, USA). Duplicate allelic ladder samples (1.1 ml) were included in each run. Samples were denatured at 95 8C for 3 min before being run on either ABI’s 3100 or 3130xl Genetic Analyzers (Applied Biosystems, USA).
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2.6. Data analysis
3.2. DNA mixture classification
GeneMapper ID software v3.2 was used to analyze all data, regardless of which model Genetic Analyzer was used for capillary electrophoresis. ROX 500 was employed for the size standard threshold for allele designation was set to 60 rfu for heterozygote, and 200 rfu for homozygote, according to our laboratory’s internal validation study. Stutter percentage was carried out according to manufacturer recommendation.
The type of each of the 30 DNA mixtures recovered from under the victim’s and suspect’s fingernails, was defined in both primary and secondary classifications (Fig. 3). The profile’s analysis revealed an unambiguous asymmetrical pattern of the DNA mixture expression, between the right and left hands (Fig. 3). High level DNA mixtures were found in 15 fingernail samples, which are 50% of all mixtures analyzed (Fig. 3). The remaining samples presented low level DNA mixtures (30%), or were characterized as residual profiles (20%) (Figs. 1, 3). Four cases which were typed as low level DNA mixtures, presented 11 alleles of the foreign contributor [case nos. 1, 5, 7, and 12]. Peak height ratio analysis of these mixtures allowed the designation of additional alleles, thus, upgrading the classification of the ‘‘interpreted’’ profile from a ‘low’ to ‘high’ level DNA mixture [case nos. 1, 5, 7, 12, and 15] (Fig. 3). As mentioned before, only primary classifications served in downstream statistical calculations. PHR analysis may also assist in determination of major and minor contributors within the DNA mixture. An interesting observation was recognized within a number of DNA mixtures (n = 8/30, 26.6%), seen mainly in victim samples (n = 7) but also in one suspect sample (n = 1): although these fingernail samples were collected from the donor, the major constituent of the DNA mixture was not that of the donor, as would be expected, but rather originated from the foreign contributor (Fig. 4). These cases were classified mainly as high level DNA mixtures [case nos. 2, 6, 10, 16, 20, and 22], even though two cases of low level DNA mixtures were also noted [case nos. 5 and 7].
2.7. DNA profile classification Single profiles, either partial or full, were characterized by one or two alleles at each locus throughout the profile (Table 1). The presence of more than two alleles at a locus was used to identify a possible mixture. Comparison between the DNA profiles originating from the fingernails and the blood or buccal reference samples allowed identifying the donor’s alleles from those from a foreign contributor. Thus, recognition and counting of the alleles attributed to the secondary, foreign contributor could be made. Stutter peak positions were not regarded as additional alleles if stutter peak heights matched manufacturer recommendation for SGM-Plus at each locus. Any peaks below threshold of 60 rfu were not called. The classification of the DNA mixture types was carried out according to the number of the foreign obligatory alleles presented in the profile: obligatory alleles were defined as those whose heights were above threshold, those not overlapping donor alleles, and could not be considered as stutters. These alleles were added to the alleles originating from the donor. The criteria for each of the mixture types: ‘‘residual’’; ‘‘low level’’ and ‘‘high level’’ mixtures are detailed in Table 1. Mixture interpretation allows additional information to be inferred based on differences in PHR. If an intimate sample was analyzed, and a two person mixture was assumed, then analysis of the donor alleles along with PHR differences, will allow recognition of additional alleles of the foreign contributor (Fig. 2). In this case, classification of the profile will be implemented twice: first, to type the profile as a ‘residual’, ‘low’ or ‘high’ level mixture, and second to use the additional information generated by the interpretation of the peak heights to depict the ‘‘interpreted’’ profile (Fig. 2). The final classification of the mixture for statistical calculation: ‘residual’, ‘low’ or ‘high’ was determined based only on the primary classification without the PHR interpretation. 3. Results 3.1. DNA profiling Single source profiles originating from the donor’s fingernails were found in the majority of samples, either victims or suspects (n = 107, 78%) (Fig. 1). DNA mixtures were observed in 30 samples (25 victims [cases 2, 5, 7–8, and 10–30] and five suspects [cases 1, 3, 4, 6, and 9]); which comprised 22% of all the cases tested (Figs. 1, 3).
4. Discussion This study tried to expand the understanding of the significance of DNA mixtures found from fingernail samples in criminal investigations, especially among homicide victims. The rapid changes in methodologies implemented in forensic DNA typing during the last decade required a re-assessment of the prevalence of DNA mixtures in these cases, particularly, after background level of DNA mixtures in the general population has been described in recent publications [8–10,12]. The manifestation of a DNA mixture under the fingernails of a murder victim, or a suspect, is a rare occurrence: 78% of the samples, either swabs or fingernail clippings, produced a single source profile which could be matched to the donor. This result is supported by previous findings [8,9] which have shown that most fingernail samples do not generate DNA mixtures: the study by Cook and Dixon [8] found that the majority of fingernail swabs collected from volunteers from the general population provided a single source profile, and only 6% produced DNA mixtures. No high level DNA profile types could be found among these cases. Similar results were found by Cerri et al. [9] in fingernail samples collected from non-violent crimes deceased, where only 5% of the samples allowed the designation of a two person mixtures, but the
Table 1 Classification of DNA profiles according to the number and type of alleles designation.
Primary classification
Classification
Number of alleles
Criteria
Single DNA profile
Equal to or less than 2 (per locus)
–
Less than 4 4–11 Equal to or more than 12
Number of foreign alleles added to the self donor profile; stutters were not included; alleles with peak height <60 rfu were not included
[Primary classification result + ‘‘interpreted’’ alleles]
Detection of additional foreign contributors’ alleles is applicable if PHR allows
DNA mixtures: Residual profile Low level DNA mixture High level DNA mixture Secondary classification
PHR ‘‘interpreted’’ profiles
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Fig. 2. Second contributors’ alleles detection by peak heights ratio analysis. The self donor alleles are filled; foreign obligatory alleles are present (#). PHR analysis implies that both contributors share the same allele at the locus (*). Homozygote alleles of the second contributor are noted (~).
remaining 95% genotyped a single source profile [9]. These studies provided the evidential value of the low background level of DNA mixtures within the general population, leading to the inevitable conclusion that the cellular debris accumulated under one’s fingernails throughout routine casual activities, is not consistent with DNA mixtures, and particularly not high level or reportable type of mixtures. Results of this study show that the prevalence of DNA mixtures found among murder victims fingernail samples, is much higher: 25% of the victims’ samples genotyped provided additional alleles other than the self component. Moreover, the primary classification of approximately half of the samples revealed high level DNA mixtures, increasing the prospect for a positive identification of the second contributor. Similarly, few of the samples that were characterized as ‘low’ level DNA (n = 5/9), still presented a high informative value due to the designation of 10 or 11 alleles of the second contributor. This study has also illustrated an asymmetric pattern of mixtures between the right and left hand of the donors (see Fig. 3). The circumstances of a violent crime are varied and may affect the persistence of a foreign material found under one’s fingernails, thus DNA analysis should be carried out from both donor hands.
The study of Malsom et al. [12] presented elevated frequency of DNA mixtures in fingernail samples collected from cohabitating couples: 17% of the samples resulted in reportable mixtures. The common theme for all of these cases was that intimate contact took place 48 h prior to sample collection. It was also found that not all intimate contact led to the designation of high level mixtures, but all high level mixtures were associated with intimate contact [12]. It was also found that by itself, cohabitation found to increase the likelihood of gaining only a residual profile (less than 5 alleles) of the foreign contributor, and not a high level mixture. Various activities which were considered as the source for the transfer of small amount of DNA from one person to another, such as hand shaking, hugging, dressing a child, sharing a bed, sharing working surrounding, etc., were found insufficient for generating high level DNA mixtures [10,12]. On the other hand, homicide victims presented an increased prevalence of both ‘high’ and ‘low’ types of DNA mixtures. These sorts of mixtures are a rare entity within the general population, even among cohabitating couples [12]. Consequently, the appearances of high level DNA mixtures among murder victims should be associated with an unusual type of contact rather than a casual activity. The mixture could be the result of a close, violent, physical
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Fig. 3. Classification of 30 DNA mixtures typed from fingernail samples of homicide cases’ victims and suspects. If PHR analysis reveals that a donors’ allele is shared with the foreign contributor then this allele is added to generate the ‘‘interpreted’’ profile of the second contributor (max. alleles no = 22).
contact that enabled an accelerated transfer of cellular material between the victim and attacker. Considering that these victims died in violent circumstances; the origin for this foreign material could be the perpetrator. The main difference we found between the victims group and the suspects was that the DNA mixtures were recognized twice as much from victims’ fingernails (25%) as compared with the
suspects (13.5%). This difference may be explained by the lethal outcome of the attack: if a struggle took place and biological material was exchanged, then the lack of activity ensures that the death of the victim will preserve all cellular material under the fingernails. Given that the deceased no longer had the ability to wash away evidence, then unless washed away by environmental conditions, or by the perpetrator’s manipulations, the foreign
Fig. 4. A DNA mixture characterized by the foreign contributor being the major component that overcomes the donor self profile.
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material would most likely remain under the victim’s fingernails. If the amount is sufficient, a reportable DNA mixture, consisting of the victim himself and the perpetrator, may be expected to be found. The perpetrator on the other hand, will probably make any effort to eliminate all evidence related to the criminal act. Thus, hand washing, bathing, fingernail clipping or any other washing acts are expected to be taken. Even if not deliberate, but rather as a result of routine hygiene, these acts will lead to decreased chance of finding a DNA mixture under the perpetrator’s fingernails. The prevalence of DNA mixtures found among suspect’s samples was higher (13.5%), compared with the published incidence found among volunteers from the general population (6%) [8]. In addition, both ‘high’ and ‘low’ level mixtures were noted among suspect’s fingernail samples, compared with ‘drop in’ or residual profiles within the general population [8]. The differences found between these two populations, the general population and murder suspects, are not unexpected due to fact that the declaration of an individual as a suspect should be based upon reliable, preliminary information that connect them to the criminal act, rather than any randomly picked individual. The informative value of a secondary contributor within ‘high’ or even ‘low’ level DNA mixture is considerable. Malsom et al. [12] found that the majority of DNA mixtures (23/ 24) that were collected from cohabitating couples enabled the identification of the second contributor as the legitimate partner of the donor [12]. Thus, the presence of the second contributor had a rational explanation and it was not left unreciprocated. The positive identification of a person as the secondary contributor within a ‘high’ level DNA mixture that was generated from fingernail samples from homicide victims require an explanation: the mixture cannot be considered as a background level, or the result of a ‘casual’ activity, including casual touch between two contributors. Consequently, unless that person is the victim’s partner and intimate contact took place between them less than 48 h prior to the victim’s death, then one of the most reasonable explanations for the finding is that this person may be considered the perpetrator involved in the assault. In this study we have found that out of the 23 homicide cases tested, in 12 cases the non-self, foreign component was found to match the suspect, or the victim’s reference profile. These cases resulted in charges against the alleged suspect. An important finding in this study was the appearance of DNA mixtures characterized by the major component originating from the foreign contributor, rather than the donor himself, as might be expected: in seven cases, the suspect’s full profile was expressed more strongly than that of the victim himself, and in one case it was the full profile of the victim that overcame the suspect’s selfalleles. These cases may indicate an aggressive type of physical contact that had allegedly taken place between the victim and the perpetrator, to allow such large amount of biological material to be transferred. Dowlman et al. [10] describe only one case, out of 80 samples of cohabitating couples, in which the female partner’s profile had overcome the donor male profile. In that case the authors assumed that this finding must be correlated with the type
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of the intimate contact that took place between the partners. No such mixtures, in which the foreign contributor became the major component was found among volunteers from the general population. 5. Conclusion The prevalence of DNA mixtures found under fingernail samples from murder victims and/or suspects is relatively high, compared with other populations studied so far [8–10,12]. Both of these populations present not only higher incidence but also an exceptional type of DNA mixture, in which the secondary contributor can often be well characterized, therefore, increasing the chance for a positive match to a reference sample. If a high level DNA mixture is genotyped, especially if the foreign contributor is the major component, there are limited scenarios that can explain such large amount of foreign material from under one’s fingernail. No casual activity, cohabitating or sharing work environment can be considered as a relevant explanation. A violent, brutal struggle, taking place in proximity to the time of the victim’s death may account for it. Thus, the evidential value of DNA mixtures, originating from fingernail samples of a victim, or a suspect, may be a powerful tool in a homicide investigation. Acknowledgments The authors wish to thank Dr. Tzipi Kahana and Mrs. Carla Oz for reviewing this manuscript. References [1] A. Piccinini, F. Betti, M. Capra, A. Comino, A 5-year study on DNA recovered from fingernail clippings in homicide cases Milan, Prog. Forensic Genet. 1239 (2003) 929–932. [2] A. Ferna´ndez-Rodrı´guez, M.J. Iturralde, L. Ferna´ndez de Simo´n, J. Capilla, M. Sancho, Genetic analysis of fingernail debris: application to forensic casework, Prog. Forensic Genet. 1239 (2003) 921–924. [3] P. Wiegand, T. Bajanowski, B. Brinkmann, DNA typing of debris from fingernails, Int. J. Legal Med. 106 (2) (1993) 81–83. [4] M. Sanchez-Hanke, K. Puschel, C. Augustine, P. Wiegand, B. Brinkmann, PCR typing of DNA extracted from epidermal particles by scratching, Adv. Forensic HaemoGenet. 6 (1996) 316–318. [5] R.A. Wickenheiser, Trace DNA: a review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact, J. Forensic Sci. 47 (2002) 442–450. [6] T. Lederer, P. Betz, S. Seidl, DNA analysis of fingernail debris using different multiplex systems: a case report, Int. J. Legal Med. 114 (2001) 263–266. [7] C. Oz, A. Zamir, An evaluation of the relevance of routine DNA typing of fingernails clippings for forensic casework, J. Forensic Sci. 45 (2000) 158–160. [8] O. Cook, L. Dixon, The prevalence of mixed DNA profiles in fingernail samples taken from individuals in the general population, Forensic Sci. Int. Genet. 1 (2007) 62–68. [9] N. Cerri, A. Verzeletti, V. Cortellini, A. Cincotta, F. De Ferrari, Prevalence of mixed DNA profiles in fingernail swabs from autoptoc cases, Forensic Sci. Int.: Genet. Suppl. Ser. 2 (2009) 163–164. [10] E.A. Dowlman, N.C. Martin, M.J. Foy, T. Lochner, T. Neocleous, The prevalence of mixed DNA profiles on fingernail swabs, Sci. Justice 50 (2010) 64–71. [11] P.S. Walsh, D.A. Metzger, R.H. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques 10 (1991) 506–513. [12] S. Malsom, N. Flanagan, C. McAlister, L. Dixon, The prevalence of mixed DNA profiles in fingernail samples taken from couples who co-habit using autosomal and Y-STR, Forensic Sci. Int. Genet. 3 (2009) 57–62.