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Persistence of immersed blood and hair DNA: A preliminary study based on casework
Journal of Forensic and Legal Medicine 51 (2017) 1e8
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j fl m
Persistence of immersed blood and hair DNA: A preliminary study based on casework verine Van Grunderbeeck 1 Christophe Frippiat*, Agathe Gastaldi 1, Se National Institute of Criminalistics and Criminology, Chauss ee de Vilvoorde 100, 1120 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2016 Received in revised form 2 May 2017 Accepted 4 July 2017 Available online 5 July 2017
In some cases, evidence is collected from rivers, canals, lakes or sink pipes. To determine the utility of analyzing these samples and for cases in which DNA was recovered from submerged bulletproof vest parts, we evaluated the time necessary to degrade the blood and, subsequently, DNA on bulletproof vests. In a second experiment, also based on cases, blood was diluted in water from a kitchen sink pipe and incubated at room temperature for different times. Subsequently, DNA quality was assessed. In a parallel experiment, hair roots were incubated in spring water for different time periods. This study demonstrates that after one week of immersion of the bulletproof vest parts in a canal only one sample from more than 100 samples gave a partial genetic profile. No genetic profile were obtained for the 99 other samples. After one month immersion and despite the finding that blood remained detectable on bulletproof vest parts, no genetic profile was obtained for all samples using the classical STR approach. For longer immersion times, no genetic profiles were obtained. In sink pipe water, an incubation time of 72 h (h) was necessary before significant blood degradation occurred. Nevertheless, high inter-sample variability was observed. This high variability may be explained by the variability of water composition coming from nine different sink pipes. For hair root cells incubated in water, we observed that more than 90% of the DNA was degraded after 72 h. © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Keywords: Hair Blood Waterway Trace persistence Water degradation DNA stability in water
1. Introduction Along with UV radiation and oxidation,1,2 water is an important factor affecting DNA integrity. In many cases, evidence is found in rivers, lakes, and sink pipes, and DNA experts must frequently process evidence collected from water. Based on several studies, despite the extreme sensitivity of STR (Small Tandem Repeat) profiling, and depending on the immersion duration, these types of evidence are considered unworkable due to the effects of water and microbial activity on cells and DNA.3,4 Given these considerations, DNA analysis is mainly used on immersed bodies to identify the victim.5 Despite this important feature, to our knowledge, the persistence of body fluid DNA has not been extensively studied. Jain et al. showed that blood remained detectable after at least 20 days of immersion in water.6 To investigate the persistence of various body fluids after water
* Corresponding author. E-mail address: [email protected] (C. Frippiat). 1 Equally contributed.
exposure, Borde et al. immersed various substrates stained with blood, semen and saliva in either a river or seawater. They showed that DNA degradation is directly related to the immersion time. In fresh river water, blood on fabrics persists for six weeks. Semen persists for three months under the same conditions.4 In the same study, the authors also observed that porous substrates help to protect body fluids. Several other studies showed the persistence of semen stains after multiple washes7 or immersion for up to 144 h.8 Raymond et al. described the persistence of touch trace DNA and demonstrated that the ability to recover DNA from cells on outdoor surfaces decreases significantly over two weeks.9 Flanagan and McAlister showed that water immersion (hand washing) was the main factor affecting DNA persistence under fingernails.10 Nevertheless, no study describes the persistence of nuclear hair root DNA in water or the persistence of blood DNA in sink pipes. The goal of this study was to determine the kinetics of DNA degradation of immersed biological traces, such as blood and hair, to determine the utility of DNA analysis from these types of evidence. To recreate common crime scenes, we reproduced three different scenarios.
http://dx.doi.org/10.1016/j.jflm.2017.07.009 1752-928X/© 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
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In the first scenario, we submerged parts of a bulletproof vest in which the Kevlar layers were stained with blood. The vest was submerged in an artificial waterway, and we followed the kinetics of DNA degradation. This situation may correspond to a robbery in which the perpetrator, despite the bulletproof jacket, is injured. Second, for current police procedures following a murder with blood evidence, police officers may withdraw liquid from sink pipes at crime scenes and/or at suspect houses. At that time, we would not know how much blood DNA is in the sink pipes. We also would not know whether we could extract sufficient blood DNA or whether its quality is sufficient to establish a genetic profile. To address these questions, fresh blood was incubated for different times in water from several kitchen sink pipes and degradation was followed according to the incubation times. This second scenario corresponds to a perpetrator washing, for example, a bloody knife. These two experiments truly represent forensic samples because, similar to true casework, the blood was exposed to a multitude of insults. Finally, in a third scenario, hair was immersed in spring water and the persistence of DNA was followed over time. 2. Materials and methods 2.1. Bulletproof vest experiment Fresh blood (1 ml or 5 ml) from 3 healthy volunteers was deposited on the internal Kevlar layers of bulletproof vest parts. Subsequently, the samples were dried for 24 h or 1 month at room temperature. Then, these bulletproof vest parts were placed in a plastic bag with 2 holes of 3 cm diameter. These samples were placed in a cage and submerged in an artificial canal for 1 week or 1 month. Immersion was performed using a crane. After retrieval, these bulletproof vest parts were completely dried at room temperature. Bulletproof parts were purchased from Seyntex (Tamise, Belgium). The parts are composed of 17 internal Kevlar layers and two external waterproof layers. To mimic a bulletproof vest perforated by a bullet, the extremity of the parts were not waterproof. 2.1.1. Comparison experiment 100 ml of fresh blood without additive were coagulated for approximately 4 h in microtubes. Spring water (500 ml) was added and the tube was incubated for periods ranging from 6 days to 6 months at 20 C protected from light. 2.2. Hair experiment Hairs were removed from volunteers and subsequently selected according to the number of cells on the root using DAPI staining, allowing nuclei visualization.11 This was performed to ensure that the test root hair and control root hair (not immersed) contained approximately the same number of nucleated cells. Subsequently, the roots were incubated in spring water for different times ranging from 2 h to 240 h. In all cases, control hairs and tested hairs for the same experimental conditions were from the same donor. 2.3. Sink pipe experiment Nine volunteers provided liquid from their kitchen sink pipes using clean syringes. Blood was diluted (100x and 200x) in sink pipe water. Thereafter, 1 ml of this mixture was incubated for various times, 0 h, 2 h, 24 h, 72 h or 7 days. Negative controls were taken at 0 h and 7 days.
2.4. Blood detection 2.4.1. Lumiscene The detection of blood using Lumiscene (LUM001, LOCI Forensic, LE Nieuw-Vennep, The Netherlands) was performed following the manufacturer's recommendations. 2.4.2. Hexagon Obti Each sample was incubated in 200 ml of Hexagon Obti buffer overnight at 4 C. Subsequently, 80 ml of this mixture was deposited on an Hexagon Obti strip (Human GmbH, Germany) and the results were recorded 10 min and 1 h after deposition of the sample. 2.5. DNA extraction Samples were incubated in 240 ml of incubation buffer (DC920BC, Promega, USA) with 10 ml of 18 mg/ml protease K (Promega, Madison, USA) and incubated at 56 C overnight. Thereafter, solid samples were centrifuged using a spin basket. The elution was recovered. Next, 500 ml of lysis buffer from the DNA IQ™ Casework Sample Kit for Maxwell®16 (AS1210, Promega, Madison, USA) and 5 ml of 1 M DTT (V3151, Promega, Madison, USA) were added to the recovered elution. DNA was extracted from this mixture using the Maxwell®16 robot (Promega, Madison, USA) and the DNA IQ™ Casework Sample kit for Maxwell®16 (AS1210, Promega, Madison, USA) following the manufacturer's instructions.12 The extracted DNA was finally eluted in 80 ml of the elution buffer provided in the kit. 2.6. DNA quantification Quantification was performed using either the Quantifiler Trio system (Thermo Fisher, Waltham, Massachusetts, USA) or the Plexor HY kit (Promega, Madison, USA) on an Applied Biosystems 7500 Real-Time PCR machine according to the manufacturer's specifications. The quantification was performed in duplicate. The final quantification result was the mean of the duplicate. 2.7. PCR amplification The ESI17 Pro system (Promega, Madison, USA) was used according to the manufacturer's recommendations. A total of 500 pg of DNA was amplified per reaction (according Quantifiler quantification results) in a total reaction volume of 25 ml using a C1000 thermocycler (Biorad, Temse, Belgium). Each DNA sample was amplified once per experiment. If insufficient DNA was available to achieve 500 pg per PCR reaction, the volume of DNA was adjusted to approach 500 pg. The maximum volume of DNA used was 17.5 ml/ reaction. When specified, the ESX17 system (Promega, Madison, USA) was also used as described above for the ESI17 Pro system. 2.8. Capillary electrophoresis Amplified DNA samples were analyzed on a 3500 XL Genetic Analyzer (Thermo Fisher, Waltham, Massachusetts, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Thermo Fisher, Waltham, Massachusetts, USA), 1X genetic analysis buffer with EDTA (Thermo Fisher, Waltham, Massachusetts, USA), a 12 s injection and 15 kV electrophoresis. Two mL of PCR product were loaded with 10 ml of formamide (Thermo Fisher, Waltham, Massachusetts, USA). The Genemapper IDx 1.2 software (Thermo Fisher, Waltham, Massachusetts, USA) was used for analysis. The detection threshold was calculated based on the formula (average background þ 10 SD). The detection threshold is dependent on the fluorescent dyes, with 70 RFU for green, 60 RFU for yellow, 50 RFU
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for blue and 60 RFU for red. A stochastic threshold was calculated for the analysis on the 3500 XL Genetic Analyzer and fixed to 135 RFU. The formula used was ((average backgroundþ3STD)X3þ(average background þ 3STD)XDPHR)). DPHR was fixed at 0.6. 2.9. Waterway temperature recording Water temperature was recorded and data kindly provided by ne rale Agriculthe ‘Service public de Wallonie (SPW), Direction ge ture, Ressources Naturelles et Environnement - DGO3, partement de l'Environnement et de l'eau/Direction des eaux de De surface’. 3. Results 3.1. Scenario 1: blood on bulletproof vest Based on casework in which blood was discovered on the Kevlar layers of a bulletproof vest supposedly immersed for a long period of time, bulletproof vest parts were stained with 1 or 5 ml of fresh human blood, dried for 24 h or 1 month at room temperature to test the effect of two different drying periods prior to immersion. Subsequently the bulletproof vest parts were placed in a plastic bag with 2 holes of 3 cm of diameter a in a cage and finally immersed in an artificial waterway for periods ranging from 1 week to 6 months. Holes in the plastic bag were used to mimic a supermarket bag tied
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by its two handles. Fig. 1 shows one bulletproof vest part and the removal of one cage from the waterway. After removal from the waterway, bulletproof vest parts were dried at room temperature and submitted for daylight examination, Crime-lite examination and blood research. After immersion in an artificial waterway for one week and independently of the blood drying time before immersion, all bulletproof vest parts presented reddish stains and were positive using Lumiscene blood revelator. All parts were also positive using Hemastix and Hexagon Obti (human blood). After one month of immersion, the reddish stains disappeared, but the Lumiscene continued to react, showing the presence of latent blood traces. For the Hexagon Obti detection, the results after 1 month in water were similar to those observed after one week, except that the signal was weaker. Interestingly, we observed that negative control vest samples (without blood) gave a weak positive signal using Hemastix. We also noted that canal mud generates a false positive signal using Hemastix. Therefore, results obtained with this reagent should not be considered reliable. Hemastix is not suitable for this type of test, as previously described by Tobe et al.,13 and no genetic profile was obtained for negative control vest parts. DNA was extracted and quantified from more than 100 samples obtained by cutting Kevlar layers or swabbing waterproof layers. All obtained samples were below the detection threshold of the particular DNA quantification kit. No inhibition of real-time quantification was detected. Except for one sample (5 ml of blood, 1 month drying before immersion, 1 week in water), no STR profiles
Fig. 1. Photographs of the outside and inside of the bulletproof vest parts. A: Outside of a bulletproof vest part before immersion B: The arrow shows the reddish stain observed after 1 week of immersion. C: Removal of a cage containing bags after incubation.
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were obtained using the ESIPro or ESX 17 kits. For the profile obtained (sample 5 ml of blood, 1 month drying before immersion, 1 week in water), with a combination of two STR PCRs (ESI17 Pro and ESX 17), only 17 of 28 alleles were detectable. The intensity of the combined profile was low and below or close to the stochastic threshold. Moreover, several drop-ins were observed. These observations render this profile useful but hard to interpret. Following these results, it was decided that samples corresponding to longer immersion times would not be submitted for analysis. As a comparison, 100 ml of fresh blood without additive was coagulated for approximately 4 h in microtubes. Thereafter, 500 ml of spring water was added and the tube was incubated for periods ranging from 6 days to 6 months at 20 C protected from light. Then, DNA was extracted, quantified and the STR profile generated. Our results revealed a significant decrease of the amount of DNA with incubation time. Compared to the 6-day incubation time, we observed an average decrease of 9.4-fold after 1 month of incubation and 224-fold after 6 months of incubation. Fig. 2 presents the amount of DNA obtained according to incubation time. However, the profile obtained after 6 months of incubation was nearly complete Only 6 alleles of the 180 expected (two PCR per samples, n ¼ 3) was not detected after the incubation time of 6 months.
significant and the average quantity reached 34.0% ± 52.9% of the 0 h incubation time. The standard deviation was high, showing high inter-sample variability. This high variability may be explained by the variability of water composition coming from nine different sink pipes. Notably, no inhibition of real-time quantification was detected. After 72 h and 7 days, the amount of recovered DNA reached 6.3% ± 13.5% and 0.5 ± 0.9%, respectively, of the amount corresponding to the 0 h incubation time. Similar results were obtained with blood diluted 200 times, except that the recovered blood DNA reached 6.0 ± 15.9% after 7 days. For STR profiling, the completeness (% of the expected amount of alleles) of the genetic profile reached 52.8% ± 33.1% and 39.8% ± 35.2% for blood diluted 100- and 200-fold, respectively, after 7 days of incubation. Fig. 4 summarizes results obtained for the various incubation times. For quantification, the standard deviation was high, again showing high inter-sample variability. For example, one sink pipe water sample had a very severe effect. Indeed, blood diluted in this water showed degradation of most of its DNA at 1 h and only 3 of 29 alleles could be amplified. Fig. 5 presents the % intensity in comparison to the mean intensity obtained for the 0 h incubation time. We observed a decrease of the intensity with increasing incubation times.
3.2. Scenario 2: Blood in sink pipe water 3.3. Immersed hair Nine volunteers provided liquid from their kitchen sink pipes using clean syringes. Immediately after sampling, fresh blood (from one volunteer) was diluted (100x and 200x) in sink pipe water, and thereafter 1 ml of the mixture was incubated for 0 h, 2 h, 24 h, 72 h or 7 days at room temperature (20.5 C ± 0.4 C). Using Hemastix and Hexagon Obti, the presence of blood was determined at 0 h and 7 days of incubation. All sink pipe water samples with added blood were positive after 7 days of incubation (n ¼ 17) using Hemastix and Hexagon Obti. As a control, we also tested sink pipe water samples without added blood. Except for one sample, all negative controls were slightly positive using Hemastix (n ¼ 5). With Hexagon Obti, all controls were negative. Using blood placed in sink pipe water and frozen immediately (0 h incubation time) as a reference, we observed a small decrease of the amount of recovered DNA from 100 diluted blood samples after 2 h of incubation (Fig. 3). After 24 h, the decrease was
Hairs were removed from healthy volunteers and subsequently DAPI stained to obtain approximately the same number of root cells in the control and test hairs. At least 90 cells were present on each hair root. After DAPI staining, hairs were rinsed with PBS and incubated in spring water for 2, 72, 168 or 240 h. Control hairs were not submitted to water immersion. Based on the microscopic examination of the hair after immersion and DAPI staining, we observed that the nuclei began to degrade after 72 h of incubation in water. This degradation increased to near completeness after 240 h. Fig. 6 shows images of the DAPI staining of hair roots before and after incubation. This degradation appeared to be hairdependent because some hair roots were not degraded after 240 h in water. This may be dependent on the hair stage (telogen, anagen). However, the quantity of DNA recovered from hairs after 72 h of water immersion was significantly decreased. Fig. 7 shows this decrease as a percentage of reference hair roots. This decrease
Fig. 2. DNA recovered from 100 ml of coagulated blood incubated in spring water. Quantification was performed using real-time PCR with the Plexor HY kit from Promega (n ¼ 3).
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Fig. 3. DNA recovered from blood incubated for 0e7 days in sink pipe water. The 100X and 200X correspond to blood diluted 100X or 200X in sink pipe water. The incubation temperature was 20.5 C ± 0.4 C. Quantification was performed using real-time PCR with the Quantifiler Trio kit from Applied Biosystems (n ¼ 9).
Fig. 4. Completeness percentage of STR profile obtained from blood incubated for 0e7 days in sink pipe water. Blood was diluted 100X or 200X in sink pipe water and incubated at 20.5 C ± 0.4 C. (n ¼ 9). The values above the columns give the average numbers of alleles detected. The value are rounded up or down to the nearest unit. A full profile contains 29 alleles.
was less for hairs incubated for 168 h. A possible explanation is a stochastic effect due to the small number of hairs used. A similar observation was recorded for STR profiling. After 72 h in water, all obtained STR profiles were unusable, but 3 of 5 were usable after 168 h. Fig. 8 shows the percentages of the reference profile intensity obtained after the different incubation times. The completeness of the genetic profile (% of the expected amount of alleles) reached 100% ± 0.0%, 17.5% ± 8.3%, 58.5% ± 38.1% and 7.7% ± 3.9% after 2 h, 72 h, 168 h and 240 h of incubation, respectively. Fig. 9 summarizes the results obtained for the various incubation times. 4. Discussion To determine the usefulness of DNA analysis from immersed evidence, we investigated the preservation of DNA under three different conditions corresponding to real casework: blood in bulletproof vest submerged in a waterway, blood in sink pipe water
and hair in water. For blood on Kevlar from a bulletproof vest submerged in a waterway, despite the finding that blood remained detectable after 1 month of immersion, only one partial STR profile (17 of 28 alleles were detectable) was obtained from more than 100 samples. This low quality STR profile was obtained when 5 ml of blood was deposited on Kevlar and dried for 1 month before immersion for 1 week. No STR profiles were obtained from the other samples, independently of the blood drying time before immersion. This study shows that degradation in waterways is rapid. Therefore, in future studies, shorter incubation times should be used to establish the kinetics of blood DNA degradation. By contrast, coagulated blood incubated in spring water for a long period (6 months) in a closed microtube is able to generate a full profile, despite very strong degradation. This suggests that oxidation may be responsible for the degradation observed with the bulletproof vest. In a similar study, Borde et al. showed that useful STR profiles continue to be obtained after immersion for 3 weeks
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Fig. 5. Percentages of the STR reference signal obtained from blood diluted 100X or 200X in sink pipe water and incubated at 20.5 C ± 0.4 C for 0e7 days (n ¼ 9).
Fig. 6. Representative pictures of control hair roots subjected to DAPI nuclear staining, or hair roots incubated in spring water for various time ranging from 2 h to 240 h.
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Fig. 7. Percentages of the expected amounts of DNA recovered from the immersed hair roots as a function of the immersion time. Quantification was performed using real-time PCR with the Plexor HY kit from Promega (2 h n ¼ 5, other incubation n ¼ 6).
Fig. 8. Percentages of the reference STR profile signal obtained after 2 h (n ¼ 5) or other incubation times (n ¼ 6).
for fabrics stained with blood. In our experiments, the bag containing the bulletproof jacket is ballasted. In this condition, the bulletproof jacket is quickly covered by mud and then subjected to the adverse effect of microbial activities. Our results for the waterway and microtube, combined with the results of Borde et al., show that underwater DNA degradation is likely multifactorial. Our experiments conducted with blood incubated with sink pipe water samples showed that Hemastix reacts with sink pipe water, even in the absence of blood. None of the donors reported injury for 1 week or blood trace washing before the sampling of the water, which demonstrated that Hemastix is not suitable for this type of evidence. Conversely, no false positive reaction was observed using Hexagon Obti. Using Hexagon Obti, human blood remains detectable after 1 week of incubation in all sink pipe water samples. For the degradation of DNA and the establishment of a genetic profile, there is high variability between samples. Each water sample is different
from the others. Some sink pipe water samples have a very stringent effect compared to others. Nevertheless, we conclude that 72 h is the longest incubation time before significant degradation of blood occurs. For hairs incubated in spring water, although the number of hairs studied is small, incubation for 72 h is when degradation occurs, similar to blood in sink pipe water. In addition to highlighting the degradation of DNA after immersion in water, these results emphasize the need for better techniques to investigate highly degraded samples. When some alleles are observed below the detection threshold, simple techniques, such as post-PCR purification that improve the detection of amplified DNA, could be useful.14,15 MiniSTR may also be suitable for degraded samples. Nevertheless, the target of real-time PCR quantification used in this work is a short fragment of DNA (size between 75 and 99 bp, depending on the kit). This shows that when degradation is strong, classical approaches are not suitable. Reports
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Fig. 9. Completeness percentages of the STR profile obtained using DNA recovered from hair roots immersed in spring water for 2 he240 h (2 h n ¼ 5, other incubation times, n ¼ 6). The values above the columns give the average numbers of alleles detected. The value are rounded up or down to the nearest unit. The average full profile (several donors are used) contains 30 alleles.
about with preservation and recovery of ancient DNA suggest the use of mitochondrial DNA or cloning of the recovered DNA.16 Since the development of specific multiplex PCR systems, and depending on the future sensitivity of this technique, massive parallel sequencing may be a promising strategy.17 Acknowledgments This study was supported by the Federal Ministry of Justice of Belgium. References 1. Hall A, Sims LM, Ballantyne J. Assessment of DNA damage induced by terrestrial UV irradiation of dried bloodstains: forensic implications. Forensic Sci Int Genet. 2014;8(1):24e32. http://dx.doi.org/10.1016/j.fsigen.2013.06.010. 2. Gefrides L a, Powell MC, Donley M a, Kahn R. UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables. Forensic Sci Int Genet. 2010;4(2):89e94. http://dx.doi.org/10.1016/ j.fsigen.2009.06.008. 3. Nakanishi A, Moriya F, Hashimoto Y. Effects of environmental conditions to which nails are exposed on DNA analysis of them. Leg Med (Tokyo). 2003;5(Suppl 1):S194eS197. 4. Borde YM, Tonnany MB, Champod C. A study on the effects of immersion in river water and seawater on blood, saliva, and sperm placed on objects mimicking crime scene exhibits. Can Soc Forensic Sci J. 2008;41(3):149e163. 5. Armstrong EJ. Medocolegal Investigation of Deaths: Initial Processing. WaterRelated Death Investigation: Practical Methods and Forensic Applications. CRC Press Book. taylor and. Boca Raton: CRC Press; 2011:163e170. 6. Jain P, Singh HP. Detection and origin of blood stains on various types of cloth immersed in water for a prolonged period. Can Soc Forensic Sci J. 1984;17(2): 58e61. http://dx.doi.org/10.1080/00085030.1984.10757364.
7. Brayley-Morris H, Sorrell A, Revoir AP, Meakin GE, Court DS, Morgan RM. Persistence of DNA from laundered semen stains: implications for child sex trafficking cases. Forensic Sci Int Genet. 2015;19:165e171. http://dx.doi.org/ 10.1016/j.fsigen.2015.07.016. 8. Joshi UN., Subhedar SK., Saraf DK. Effect of water immersion on seminal stains on cotton cloth. Forensic Sci Int 17(1):9e11. 9. Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C. Trace evidence characteristics of DNA: a preliminary investigation of the persistence of DNA at crime scenes. Forensic Sci Int Genet. 2009;4(1):26e33. http://dx.doi.org/ 10.1016/j.fsigen.2009.04.002. 10. Flanagan N, McAlister C. The transfer and persistence of DNA under the fingernails following digital penetration of the vagina. Forensic Sci Int Genet. 2011;5(5):479e483. http://dx.doi.org/10.1016/j.fsigen.2010.10.008. 11. Bourguignon L, Hoste B, Boonen T, Vits K, Hubrecht F. A fluorescent microscopy-screening test for efficient STR-typing of telogen hair roots. Forensic Sci Int Genet. 2008;3(1):27e31. http://dx.doi.org/10.1016/ j.fsigen.2008.08.006. 12. Kit HE., Kit E. Tissue and Hair Extraction Kit (for Use with DNA IQ TM). Stand n.d.;(307):1e9. id NN. Evaluation of six presumptive tests for blood, 13. Tobe SS, Watson N, Dae their specificity, sensitivity, and effect on high molecular-weight DNA. J Forensic Sci. 2007;52(1):102e109. http://dx.doi.org/10.1111/j.15564029.2006.00324.x. 14. van Oorschot RA, Ballantyne KN, Mitchell RJ. Forensic trace DNA: a review. Investig Genet. 2010;1(1):14. http://dx.doi.org/10.1186/2041-2223-1-14. 15. Smith PJ, Ballantyne J. Simplified low-copy-number DNA analysis by post-PCR purification. J Forensic Sci. 2007;52(4):820e829. http://dx.doi.org/10.1111/ j.1556-4029.2007.00470.x. 16. Parsons TJ, Walter V. Preservation and recovery of DNA in postmortem specimens and. In: Sorg WH, M, eds. In Advances in Forensic Taphonomy: The Fate of Human Remains. New York: CPR Press; 1996:109e138. 17. Kim EH, Lee HY, Yang IS, Jung S-E, Yang WI, Shin K-J. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons. Forensic Sci Int Genet. 2016;22:1e7. http://dx.doi.org/ 10.1016/j.fsigen.2016.01.001.
Contents lists available at ScienceDirect
Journal of Forensic and Legal Medicine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j fl m
Persistence of immersed blood and hair DNA: A preliminary study based on casework verine Van Grunderbeeck 1 Christophe Frippiat*, Agathe Gastaldi 1, Se National Institute of Criminalistics and Criminology, Chauss ee de Vilvoorde 100, 1120 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2016 Received in revised form 2 May 2017 Accepted 4 July 2017 Available online 5 July 2017
In some cases, evidence is collected from rivers, canals, lakes or sink pipes. To determine the utility of analyzing these samples and for cases in which DNA was recovered from submerged bulletproof vest parts, we evaluated the time necessary to degrade the blood and, subsequently, DNA on bulletproof vests. In a second experiment, also based on cases, blood was diluted in water from a kitchen sink pipe and incubated at room temperature for different times. Subsequently, DNA quality was assessed. In a parallel experiment, hair roots were incubated in spring water for different time periods. This study demonstrates that after one week of immersion of the bulletproof vest parts in a canal only one sample from more than 100 samples gave a partial genetic profile. No genetic profile were obtained for the 99 other samples. After one month immersion and despite the finding that blood remained detectable on bulletproof vest parts, no genetic profile was obtained for all samples using the classical STR approach. For longer immersion times, no genetic profiles were obtained. In sink pipe water, an incubation time of 72 h (h) was necessary before significant blood degradation occurred. Nevertheless, high inter-sample variability was observed. This high variability may be explained by the variability of water composition coming from nine different sink pipes. For hair root cells incubated in water, we observed that more than 90% of the DNA was degraded after 72 h. © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
Keywords: Hair Blood Waterway Trace persistence Water degradation DNA stability in water
1. Introduction Along with UV radiation and oxidation,1,2 water is an important factor affecting DNA integrity. In many cases, evidence is found in rivers, lakes, and sink pipes, and DNA experts must frequently process evidence collected from water. Based on several studies, despite the extreme sensitivity of STR (Small Tandem Repeat) profiling, and depending on the immersion duration, these types of evidence are considered unworkable due to the effects of water and microbial activity on cells and DNA.3,4 Given these considerations, DNA analysis is mainly used on immersed bodies to identify the victim.5 Despite this important feature, to our knowledge, the persistence of body fluid DNA has not been extensively studied. Jain et al. showed that blood remained detectable after at least 20 days of immersion in water.6 To investigate the persistence of various body fluids after water
* Corresponding author. E-mail address: [email protected] (C. Frippiat). 1 Equally contributed.
exposure, Borde et al. immersed various substrates stained with blood, semen and saliva in either a river or seawater. They showed that DNA degradation is directly related to the immersion time. In fresh river water, blood on fabrics persists for six weeks. Semen persists for three months under the same conditions.4 In the same study, the authors also observed that porous substrates help to protect body fluids. Several other studies showed the persistence of semen stains after multiple washes7 or immersion for up to 144 h.8 Raymond et al. described the persistence of touch trace DNA and demonstrated that the ability to recover DNA from cells on outdoor surfaces decreases significantly over two weeks.9 Flanagan and McAlister showed that water immersion (hand washing) was the main factor affecting DNA persistence under fingernails.10 Nevertheless, no study describes the persistence of nuclear hair root DNA in water or the persistence of blood DNA in sink pipes. The goal of this study was to determine the kinetics of DNA degradation of immersed biological traces, such as blood and hair, to determine the utility of DNA analysis from these types of evidence. To recreate common crime scenes, we reproduced three different scenarios.
http://dx.doi.org/10.1016/j.jflm.2017.07.009 1752-928X/© 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.
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In the first scenario, we submerged parts of a bulletproof vest in which the Kevlar layers were stained with blood. The vest was submerged in an artificial waterway, and we followed the kinetics of DNA degradation. This situation may correspond to a robbery in which the perpetrator, despite the bulletproof jacket, is injured. Second, for current police procedures following a murder with blood evidence, police officers may withdraw liquid from sink pipes at crime scenes and/or at suspect houses. At that time, we would not know how much blood DNA is in the sink pipes. We also would not know whether we could extract sufficient blood DNA or whether its quality is sufficient to establish a genetic profile. To address these questions, fresh blood was incubated for different times in water from several kitchen sink pipes and degradation was followed according to the incubation times. This second scenario corresponds to a perpetrator washing, for example, a bloody knife. These two experiments truly represent forensic samples because, similar to true casework, the blood was exposed to a multitude of insults. Finally, in a third scenario, hair was immersed in spring water and the persistence of DNA was followed over time. 2. Materials and methods 2.1. Bulletproof vest experiment Fresh blood (1 ml or 5 ml) from 3 healthy volunteers was deposited on the internal Kevlar layers of bulletproof vest parts. Subsequently, the samples were dried for 24 h or 1 month at room temperature. Then, these bulletproof vest parts were placed in a plastic bag with 2 holes of 3 cm diameter. These samples were placed in a cage and submerged in an artificial canal for 1 week or 1 month. Immersion was performed using a crane. After retrieval, these bulletproof vest parts were completely dried at room temperature. Bulletproof parts were purchased from Seyntex (Tamise, Belgium). The parts are composed of 17 internal Kevlar layers and two external waterproof layers. To mimic a bulletproof vest perforated by a bullet, the extremity of the parts were not waterproof. 2.1.1. Comparison experiment 100 ml of fresh blood without additive were coagulated for approximately 4 h in microtubes. Spring water (500 ml) was added and the tube was incubated for periods ranging from 6 days to 6 months at 20 C protected from light. 2.2. Hair experiment Hairs were removed from volunteers and subsequently selected according to the number of cells on the root using DAPI staining, allowing nuclei visualization.11 This was performed to ensure that the test root hair and control root hair (not immersed) contained approximately the same number of nucleated cells. Subsequently, the roots were incubated in spring water for different times ranging from 2 h to 240 h. In all cases, control hairs and tested hairs for the same experimental conditions were from the same donor. 2.3. Sink pipe experiment Nine volunteers provided liquid from their kitchen sink pipes using clean syringes. Blood was diluted (100x and 200x) in sink pipe water. Thereafter, 1 ml of this mixture was incubated for various times, 0 h, 2 h, 24 h, 72 h or 7 days. Negative controls were taken at 0 h and 7 days.
2.4. Blood detection 2.4.1. Lumiscene The detection of blood using Lumiscene (LUM001, LOCI Forensic, LE Nieuw-Vennep, The Netherlands) was performed following the manufacturer's recommendations. 2.4.2. Hexagon Obti Each sample was incubated in 200 ml of Hexagon Obti buffer overnight at 4 C. Subsequently, 80 ml of this mixture was deposited on an Hexagon Obti strip (Human GmbH, Germany) and the results were recorded 10 min and 1 h after deposition of the sample. 2.5. DNA extraction Samples were incubated in 240 ml of incubation buffer (DC920BC, Promega, USA) with 10 ml of 18 mg/ml protease K (Promega, Madison, USA) and incubated at 56 C overnight. Thereafter, solid samples were centrifuged using a spin basket. The elution was recovered. Next, 500 ml of lysis buffer from the DNA IQ™ Casework Sample Kit for Maxwell®16 (AS1210, Promega, Madison, USA) and 5 ml of 1 M DTT (V3151, Promega, Madison, USA) were added to the recovered elution. DNA was extracted from this mixture using the Maxwell®16 robot (Promega, Madison, USA) and the DNA IQ™ Casework Sample kit for Maxwell®16 (AS1210, Promega, Madison, USA) following the manufacturer's instructions.12 The extracted DNA was finally eluted in 80 ml of the elution buffer provided in the kit. 2.6. DNA quantification Quantification was performed using either the Quantifiler Trio system (Thermo Fisher, Waltham, Massachusetts, USA) or the Plexor HY kit (Promega, Madison, USA) on an Applied Biosystems 7500 Real-Time PCR machine according to the manufacturer's specifications. The quantification was performed in duplicate. The final quantification result was the mean of the duplicate. 2.7. PCR amplification The ESI17 Pro system (Promega, Madison, USA) was used according to the manufacturer's recommendations. A total of 500 pg of DNA was amplified per reaction (according Quantifiler quantification results) in a total reaction volume of 25 ml using a C1000 thermocycler (Biorad, Temse, Belgium). Each DNA sample was amplified once per experiment. If insufficient DNA was available to achieve 500 pg per PCR reaction, the volume of DNA was adjusted to approach 500 pg. The maximum volume of DNA used was 17.5 ml/ reaction. When specified, the ESX17 system (Promega, Madison, USA) was also used as described above for the ESI17 Pro system. 2.8. Capillary electrophoresis Amplified DNA samples were analyzed on a 3500 XL Genetic Analyzer (Thermo Fisher, Waltham, Massachusetts, USA) using a 36 cm capillary array (Applied Biosystems, USA), POP-4 polymer (Thermo Fisher, Waltham, Massachusetts, USA), 1X genetic analysis buffer with EDTA (Thermo Fisher, Waltham, Massachusetts, USA), a 12 s injection and 15 kV electrophoresis. Two mL of PCR product were loaded with 10 ml of formamide (Thermo Fisher, Waltham, Massachusetts, USA). The Genemapper IDx 1.2 software (Thermo Fisher, Waltham, Massachusetts, USA) was used for analysis. The detection threshold was calculated based on the formula (average background þ 10 SD). The detection threshold is dependent on the fluorescent dyes, with 70 RFU for green, 60 RFU for yellow, 50 RFU
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for blue and 60 RFU for red. A stochastic threshold was calculated for the analysis on the 3500 XL Genetic Analyzer and fixed to 135 RFU. The formula used was ((average backgroundþ3STD)X3þ(average background þ 3STD)XDPHR)). DPHR was fixed at 0.6. 2.9. Waterway temperature recording Water temperature was recorded and data kindly provided by ne rale Agriculthe ‘Service public de Wallonie (SPW), Direction ge ture, Ressources Naturelles et Environnement - DGO3, partement de l'Environnement et de l'eau/Direction des eaux de De surface’. 3. Results 3.1. Scenario 1: blood on bulletproof vest Based on casework in which blood was discovered on the Kevlar layers of a bulletproof vest supposedly immersed for a long period of time, bulletproof vest parts were stained with 1 or 5 ml of fresh human blood, dried for 24 h or 1 month at room temperature to test the effect of two different drying periods prior to immersion. Subsequently the bulletproof vest parts were placed in a plastic bag with 2 holes of 3 cm of diameter a in a cage and finally immersed in an artificial waterway for periods ranging from 1 week to 6 months. Holes in the plastic bag were used to mimic a supermarket bag tied
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by its two handles. Fig. 1 shows one bulletproof vest part and the removal of one cage from the waterway. After removal from the waterway, bulletproof vest parts were dried at room temperature and submitted for daylight examination, Crime-lite examination and blood research. After immersion in an artificial waterway for one week and independently of the blood drying time before immersion, all bulletproof vest parts presented reddish stains and were positive using Lumiscene blood revelator. All parts were also positive using Hemastix and Hexagon Obti (human blood). After one month of immersion, the reddish stains disappeared, but the Lumiscene continued to react, showing the presence of latent blood traces. For the Hexagon Obti detection, the results after 1 month in water were similar to those observed after one week, except that the signal was weaker. Interestingly, we observed that negative control vest samples (without blood) gave a weak positive signal using Hemastix. We also noted that canal mud generates a false positive signal using Hemastix. Therefore, results obtained with this reagent should not be considered reliable. Hemastix is not suitable for this type of test, as previously described by Tobe et al.,13 and no genetic profile was obtained for negative control vest parts. DNA was extracted and quantified from more than 100 samples obtained by cutting Kevlar layers or swabbing waterproof layers. All obtained samples were below the detection threshold of the particular DNA quantification kit. No inhibition of real-time quantification was detected. Except for one sample (5 ml of blood, 1 month drying before immersion, 1 week in water), no STR profiles
Fig. 1. Photographs of the outside and inside of the bulletproof vest parts. A: Outside of a bulletproof vest part before immersion B: The arrow shows the reddish stain observed after 1 week of immersion. C: Removal of a cage containing bags after incubation.
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were obtained using the ESIPro or ESX 17 kits. For the profile obtained (sample 5 ml of blood, 1 month drying before immersion, 1 week in water), with a combination of two STR PCRs (ESI17 Pro and ESX 17), only 17 of 28 alleles were detectable. The intensity of the combined profile was low and below or close to the stochastic threshold. Moreover, several drop-ins were observed. These observations render this profile useful but hard to interpret. Following these results, it was decided that samples corresponding to longer immersion times would not be submitted for analysis. As a comparison, 100 ml of fresh blood without additive was coagulated for approximately 4 h in microtubes. Thereafter, 500 ml of spring water was added and the tube was incubated for periods ranging from 6 days to 6 months at 20 C protected from light. Then, DNA was extracted, quantified and the STR profile generated. Our results revealed a significant decrease of the amount of DNA with incubation time. Compared to the 6-day incubation time, we observed an average decrease of 9.4-fold after 1 month of incubation and 224-fold after 6 months of incubation. Fig. 2 presents the amount of DNA obtained according to incubation time. However, the profile obtained after 6 months of incubation was nearly complete Only 6 alleles of the 180 expected (two PCR per samples, n ¼ 3) was not detected after the incubation time of 6 months.
significant and the average quantity reached 34.0% ± 52.9% of the 0 h incubation time. The standard deviation was high, showing high inter-sample variability. This high variability may be explained by the variability of water composition coming from nine different sink pipes. Notably, no inhibition of real-time quantification was detected. After 72 h and 7 days, the amount of recovered DNA reached 6.3% ± 13.5% and 0.5 ± 0.9%, respectively, of the amount corresponding to the 0 h incubation time. Similar results were obtained with blood diluted 200 times, except that the recovered blood DNA reached 6.0 ± 15.9% after 7 days. For STR profiling, the completeness (% of the expected amount of alleles) of the genetic profile reached 52.8% ± 33.1% and 39.8% ± 35.2% for blood diluted 100- and 200-fold, respectively, after 7 days of incubation. Fig. 4 summarizes results obtained for the various incubation times. For quantification, the standard deviation was high, again showing high inter-sample variability. For example, one sink pipe water sample had a very severe effect. Indeed, blood diluted in this water showed degradation of most of its DNA at 1 h and only 3 of 29 alleles could be amplified. Fig. 5 presents the % intensity in comparison to the mean intensity obtained for the 0 h incubation time. We observed a decrease of the intensity with increasing incubation times.
3.2. Scenario 2: Blood in sink pipe water 3.3. Immersed hair Nine volunteers provided liquid from their kitchen sink pipes using clean syringes. Immediately after sampling, fresh blood (from one volunteer) was diluted (100x and 200x) in sink pipe water, and thereafter 1 ml of the mixture was incubated for 0 h, 2 h, 24 h, 72 h or 7 days at room temperature (20.5 C ± 0.4 C). Using Hemastix and Hexagon Obti, the presence of blood was determined at 0 h and 7 days of incubation. All sink pipe water samples with added blood were positive after 7 days of incubation (n ¼ 17) using Hemastix and Hexagon Obti. As a control, we also tested sink pipe water samples without added blood. Except for one sample, all negative controls were slightly positive using Hemastix (n ¼ 5). With Hexagon Obti, all controls were negative. Using blood placed in sink pipe water and frozen immediately (0 h incubation time) as a reference, we observed a small decrease of the amount of recovered DNA from 100 diluted blood samples after 2 h of incubation (Fig. 3). After 24 h, the decrease was
Hairs were removed from healthy volunteers and subsequently DAPI stained to obtain approximately the same number of root cells in the control and test hairs. At least 90 cells were present on each hair root. After DAPI staining, hairs were rinsed with PBS and incubated in spring water for 2, 72, 168 or 240 h. Control hairs were not submitted to water immersion. Based on the microscopic examination of the hair after immersion and DAPI staining, we observed that the nuclei began to degrade after 72 h of incubation in water. This degradation increased to near completeness after 240 h. Fig. 6 shows images of the DAPI staining of hair roots before and after incubation. This degradation appeared to be hairdependent because some hair roots were not degraded after 240 h in water. This may be dependent on the hair stage (telogen, anagen). However, the quantity of DNA recovered from hairs after 72 h of water immersion was significantly decreased. Fig. 7 shows this decrease as a percentage of reference hair roots. This decrease
Fig. 2. DNA recovered from 100 ml of coagulated blood incubated in spring water. Quantification was performed using real-time PCR with the Plexor HY kit from Promega (n ¼ 3).
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Fig. 3. DNA recovered from blood incubated for 0e7 days in sink pipe water. The 100X and 200X correspond to blood diluted 100X or 200X in sink pipe water. The incubation temperature was 20.5 C ± 0.4 C. Quantification was performed using real-time PCR with the Quantifiler Trio kit from Applied Biosystems (n ¼ 9).
Fig. 4. Completeness percentage of STR profile obtained from blood incubated for 0e7 days in sink pipe water. Blood was diluted 100X or 200X in sink pipe water and incubated at 20.5 C ± 0.4 C. (n ¼ 9). The values above the columns give the average numbers of alleles detected. The value are rounded up or down to the nearest unit. A full profile contains 29 alleles.
was less for hairs incubated for 168 h. A possible explanation is a stochastic effect due to the small number of hairs used. A similar observation was recorded for STR profiling. After 72 h in water, all obtained STR profiles were unusable, but 3 of 5 were usable after 168 h. Fig. 8 shows the percentages of the reference profile intensity obtained after the different incubation times. The completeness of the genetic profile (% of the expected amount of alleles) reached 100% ± 0.0%, 17.5% ± 8.3%, 58.5% ± 38.1% and 7.7% ± 3.9% after 2 h, 72 h, 168 h and 240 h of incubation, respectively. Fig. 9 summarizes the results obtained for the various incubation times. 4. Discussion To determine the usefulness of DNA analysis from immersed evidence, we investigated the preservation of DNA under three different conditions corresponding to real casework: blood in bulletproof vest submerged in a waterway, blood in sink pipe water
and hair in water. For blood on Kevlar from a bulletproof vest submerged in a waterway, despite the finding that blood remained detectable after 1 month of immersion, only one partial STR profile (17 of 28 alleles were detectable) was obtained from more than 100 samples. This low quality STR profile was obtained when 5 ml of blood was deposited on Kevlar and dried for 1 month before immersion for 1 week. No STR profiles were obtained from the other samples, independently of the blood drying time before immersion. This study shows that degradation in waterways is rapid. Therefore, in future studies, shorter incubation times should be used to establish the kinetics of blood DNA degradation. By contrast, coagulated blood incubated in spring water for a long period (6 months) in a closed microtube is able to generate a full profile, despite very strong degradation. This suggests that oxidation may be responsible for the degradation observed with the bulletproof vest. In a similar study, Borde et al. showed that useful STR profiles continue to be obtained after immersion for 3 weeks
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Fig. 5. Percentages of the STR reference signal obtained from blood diluted 100X or 200X in sink pipe water and incubated at 20.5 C ± 0.4 C for 0e7 days (n ¼ 9).
Fig. 6. Representative pictures of control hair roots subjected to DAPI nuclear staining, or hair roots incubated in spring water for various time ranging from 2 h to 240 h.
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Fig. 7. Percentages of the expected amounts of DNA recovered from the immersed hair roots as a function of the immersion time. Quantification was performed using real-time PCR with the Plexor HY kit from Promega (2 h n ¼ 5, other incubation n ¼ 6).
Fig. 8. Percentages of the reference STR profile signal obtained after 2 h (n ¼ 5) or other incubation times (n ¼ 6).
for fabrics stained with blood. In our experiments, the bag containing the bulletproof jacket is ballasted. In this condition, the bulletproof jacket is quickly covered by mud and then subjected to the adverse effect of microbial activities. Our results for the waterway and microtube, combined with the results of Borde et al., show that underwater DNA degradation is likely multifactorial. Our experiments conducted with blood incubated with sink pipe water samples showed that Hemastix reacts with sink pipe water, even in the absence of blood. None of the donors reported injury for 1 week or blood trace washing before the sampling of the water, which demonstrated that Hemastix is not suitable for this type of evidence. Conversely, no false positive reaction was observed using Hexagon Obti. Using Hexagon Obti, human blood remains detectable after 1 week of incubation in all sink pipe water samples. For the degradation of DNA and the establishment of a genetic profile, there is high variability between samples. Each water sample is different
from the others. Some sink pipe water samples have a very stringent effect compared to others. Nevertheless, we conclude that 72 h is the longest incubation time before significant degradation of blood occurs. For hairs incubated in spring water, although the number of hairs studied is small, incubation for 72 h is when degradation occurs, similar to blood in sink pipe water. In addition to highlighting the degradation of DNA after immersion in water, these results emphasize the need for better techniques to investigate highly degraded samples. When some alleles are observed below the detection threshold, simple techniques, such as post-PCR purification that improve the detection of amplified DNA, could be useful.14,15 MiniSTR may also be suitable for degraded samples. Nevertheless, the target of real-time PCR quantification used in this work is a short fragment of DNA (size between 75 and 99 bp, depending on the kit). This shows that when degradation is strong, classical approaches are not suitable. Reports
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Fig. 9. Completeness percentages of the STR profile obtained using DNA recovered from hair roots immersed in spring water for 2 he240 h (2 h n ¼ 5, other incubation times, n ¼ 6). The values above the columns give the average numbers of alleles detected. The value are rounded up or down to the nearest unit. The average full profile (several donors are used) contains 30 alleles.
about with preservation and recovery of ancient DNA suggest the use of mitochondrial DNA or cloning of the recovered DNA.16 Since the development of specific multiplex PCR systems, and depending on the future sensitivity of this technique, massive parallel sequencing may be a promising strategy.17 Acknowledgments This study was supported by the Federal Ministry of Justice of Belgium. References 1. Hall A, Sims LM, Ballantyne J. Assessment of DNA damage induced by terrestrial UV irradiation of dried bloodstains: forensic implications. Forensic Sci Int Genet. 2014;8(1):24e32. http://dx.doi.org/10.1016/j.fsigen.2013.06.010. 2. Gefrides L a, Powell MC, Donley M a, Kahn R. UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables. Forensic Sci Int Genet. 2010;4(2):89e94. http://dx.doi.org/10.1016/ j.fsigen.2009.06.008. 3. Nakanishi A, Moriya F, Hashimoto Y. Effects of environmental conditions to which nails are exposed on DNA analysis of them. Leg Med (Tokyo). 2003;5(Suppl 1):S194eS197. 4. Borde YM, Tonnany MB, Champod C. A study on the effects of immersion in river water and seawater on blood, saliva, and sperm placed on objects mimicking crime scene exhibits. Can Soc Forensic Sci J. 2008;41(3):149e163. 5. Armstrong EJ. Medocolegal Investigation of Deaths: Initial Processing. WaterRelated Death Investigation: Practical Methods and Forensic Applications. CRC Press Book. taylor and. Boca Raton: CRC Press; 2011:163e170. 6. Jain P, Singh HP. Detection and origin of blood stains on various types of cloth immersed in water for a prolonged period. Can Soc Forensic Sci J. 1984;17(2): 58e61. http://dx.doi.org/10.1080/00085030.1984.10757364.
7. Brayley-Morris H, Sorrell A, Revoir AP, Meakin GE, Court DS, Morgan RM. Persistence of DNA from laundered semen stains: implications for child sex trafficking cases. Forensic Sci Int Genet. 2015;19:165e171. http://dx.doi.org/ 10.1016/j.fsigen.2015.07.016. 8. Joshi UN., Subhedar SK., Saraf DK. Effect of water immersion on seminal stains on cotton cloth. Forensic Sci Int 17(1):9e11. 9. Raymond JJ, van Oorschot RAH, Gunn PR, Walsh SJ, Roux C. Trace evidence characteristics of DNA: a preliminary investigation of the persistence of DNA at crime scenes. Forensic Sci Int Genet. 2009;4(1):26e33. http://dx.doi.org/ 10.1016/j.fsigen.2009.04.002. 10. Flanagan N, McAlister C. The transfer and persistence of DNA under the fingernails following digital penetration of the vagina. Forensic Sci Int Genet. 2011;5(5):479e483. http://dx.doi.org/10.1016/j.fsigen.2010.10.008. 11. Bourguignon L, Hoste B, Boonen T, Vits K, Hubrecht F. A fluorescent microscopy-screening test for efficient STR-typing of telogen hair roots. Forensic Sci Int Genet. 2008;3(1):27e31. http://dx.doi.org/10.1016/ j.fsigen.2008.08.006. 12. Kit HE., Kit E. Tissue and Hair Extraction Kit (for Use with DNA IQ TM). Stand n.d.;(307):1e9. id NN. Evaluation of six presumptive tests for blood, 13. Tobe SS, Watson N, Dae their specificity, sensitivity, and effect on high molecular-weight DNA. J Forensic Sci. 2007;52(1):102e109. http://dx.doi.org/10.1111/j.15564029.2006.00324.x. 14. van Oorschot RA, Ballantyne KN, Mitchell RJ. Forensic trace DNA: a review. Investig Genet. 2010;1(1):14. http://dx.doi.org/10.1186/2041-2223-1-14. 15. Smith PJ, Ballantyne J. Simplified low-copy-number DNA analysis by post-PCR purification. J Forensic Sci. 2007;52(4):820e829. http://dx.doi.org/10.1111/ j.1556-4029.2007.00470.x. 16. Parsons TJ, Walter V. Preservation and recovery of DNA in postmortem specimens and. In: Sorg WH, M, eds. In Advances in Forensic Taphonomy: The Fate of Human Remains. New York: CPR Press; 1996:109e138. 17. Kim EH, Lee HY, Yang IS, Jung S-E, Yang WI, Shin K-J. Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons. Forensic Sci Int Genet. 2016;22:1e7. http://dx.doi.org/ 10.1016/j.fsigen.2016.01.001.