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An improved process for the collection and DNA analysis of fired cartridge cases
Forensic Science International: Genetics 46 (2020) 102238
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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
An improved process for the collection and DNA analysis of fired cartridge cases
T
Todd W. Bille*, Glenn Fahrig, Steven M. Weitz, Greg A. Peiffer United States Bureau of Alcohol, Tobacco, Firearms, and Explosives, National Laboratory Center, 6000 Ammendale Road, Beltsville, MD, 20705, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Fired cartridge cases Forensic science DNA Trace DNA
Improvements to the DNA analysis of fired cartridge cases have been made in recent years, yet successful analysis of this important evidence type remains difficult. In this study, we describe both a novel device for the collection and transport of fired cartridge cases and a new DNA recovery method that incorporates a rinse-andswab technique. This technique combines two different types of swabs and a rinse solution with additives that reduce the degradative effects that copper has on DNA. The new recovery method yielded approximately threefold more DNA than the traditional double swab method and reduced the evidence of degradation. After validation, we estimated the real-world success rate of obtaining DNA profiles suitable for comparison with the rinse-and-swab method by testing over 100 cartridge cases collected from crime scenes. Approximately 67 % of the time (8 of 12), at least one DNA profile suitable for comparison was obtained from fired cartridge cases assumed to be associated with a single firearm using the collection device and the rinse-and-swab method when the fired cartridge cases were collected within 24 h.
1. Introduction
surfaces. The historically low rate of success in obtaining DNA profiles suitable for comparison from FCCs may be in part due to the deleterious effects on DNA integrity from the copper contained in brass. Studies suggest that copper may inhibit downstream PCR and/or degrade DNA through oxidative damage [12–19]. It is hypothesized that DNA damage is the result of oxidative stress from free radicals due to the reduction of transition metals (i.e. copper). Previous research has shown that when Bovine Serum Albumin (BSA) is added to the DNA extraction it can increase yields for challenging samples by chelating contaminants [20]. Lau et al. identified the copper binding portion of BSA, a three amino acid peptide, Gly-Gly-His (GGH), and showed that this tripeptide had similar copper binding specificity as human serum albumin [21]. Since then, the chelating ability of GGH has been shown to reduce DNA damage due to hydroxyl radicals and oxidative stress in certain conditions by complexing with copper [22,23]. We developed a DNA recovery method, which incorporates BSA and GGH in the rinse solution along with a series of rinsing and swabbing steps to reduce the deleterious effects of copper contained in brass cartridge cases and to maximize the recovery of DNA. Experiment 1 was conducted to evaluate the effect of BSA and GGH, individually and combined (BTmix), on the quantity of DNA recovered from laboratory prepared FCCs using the conventional double swab
Fired cartridge cases (FCCs) are typically not processed for DNA analysis due to a lack of historical success in obtaining DNA profiles suitable for comparison [1]. This attitude is slowly changing as more research is performed and laboratories using novel approaches are reporting greater success rates [2–9]. In a recent review, Montpetit summarized the research performed over the last ten years and demonstrated that FCCs are viable sources for successful DNA typing [10]. The goal of this study was to increase the success rate for DNA analysis of FCCs by improving multiple aspects of the process that could easily be incorporated into a laboratory’s current process flow, starting at the crime scene. During evidence collection, recovered FCCs are typically placed in manila envelopes or plastic bags that allow the cartridge cases to make contact with interior surfaces of the container. This contact/rubbing may result in loss of some or all of the DNA that was originally present on the cartridge case [11]. Additionally, contamination can occur during evidence collection or when multiple FCCs are packaged together. To mitigate the potential contamination and loss of DNA during collection and transport of the FCCs, a novel collection device was developed. This device minimizes evidence handling and suspends each FCC within the container preventing it from contacting the inner
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Corresponding author. E-mail address: [email protected] (T.W. Bille).
https://doi.org/10.1016/j.fsigen.2020.102238 Received 11 October 2019; Received in revised form 6 January 2020; Accepted 8 January 2020 Available online 18 January 2020 1872-4973/ Published by Elsevier B.V.
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accommodate cartridge cases from most handguns, excluding smaller calibers such as .22 caliber. The cap is used to collect the FCC by holding the tab and scooping up the cartridge case on the post. The cup is then placed over the suspended cartridge case and snapped onto the cap of the collection device. The center post on the cap and a raised button inside the cup hold the cartridge case in place and prevent contact between the outer surface of the FCC and the inner surface of the device. The device is vented to allow for airflow and drying, if necessary. Prior to use, the components of the collection device were soaked in a 10 % bleach solution for at least 1 h, rinsed with 70 % ethanol, and allowed to dry.
method [24]. In Experiments 2 and 3, DNA quantity and quality from laboratory prepared FCC samples were compared with and without BTmix for both the rinse-and-swab and double swab methods. A Pilot Study using real-world samples was performed to evaluate the efficacy of the combination of the novel collection device and the rinse-andswab method with BTmix. 2. Materials and methods 2.1. Modified QIAamp® DNA Micro extraction method The QIAamp® DNA Investigator Kit with the QIAamp® DNA Micro manufacturer’s protocol (Qiagen, Germantown, MD) [25] with several modifications was used to perform the DNA extractions. Modifications to the protocol include: doubling the Buffer AL volume; increasing Buffer AW2 volume to 700 μL; and eluting in two separate 50 μL volumes of TE−4 (Tris 10 mM, EDTA 0.1 mM, pH 8.0) DNA Suspension Buffer (Teknova, Hollister, CA) with 5 min incubations at room temperature resulting, in a final volume of 100 μL. A Qiagen Investigator® Lyse&Spin basket was used for the lysis portion of the extraction protocol with an incubation time between 3 and 18 h. At least one negative control was included with each extraction set to monitor systemic contamination.
2.5. Preparation of BTmix The final formulation of BTmix used in this study incorporated a concentration of 2 mg/mL UltraPure™ BSA (Invitrogen, Carlsbad, CA) and 62.5 mg/mL GGH tripeptide (Millipore Sigma, Burlington, MA). Higher concentrations of the GGH tripeptide were evaluated, but a decrease in DNA recovery was observed (data not shown). To create a 125 mg/mL GGH precursor solution, 743 μL of sterile nuclease-free water was added to 100 mg solid GGH tripeptide resulting in approximately 800 μL of solution. An equivalent volume of 4 mg/mL BSA precursor solution was generated by adding 64 μL of 50 mg/mL BSA solution to 736 μL of sterile nuclease-free water. The two precursor solutions were combined to create 1.6 mL of the working BTmix.
2.2. DNA quantitation, concentration, and fragment analysis
2.6. Double swabbing method
The DNA concentration for all samples was determined using the Applied Biosystems™ Quantifiler™ HP DNA Quantification Kit following the manufacturer’s protocol (ThermoFisher, Waltham, MA) [26]. When the DNA concentration was less than 33 pg/μL the extracts were concentrated down to 15 μL for amplification using the Millipore Microcon® 30 kDa Centrifugal Filter Unit (Millipore Sigma, Burlington, MA). All negative control samples were concentrated and amplified in parallel. The manufacturer’s protocol [27] was modified by adding 1 μL of carrier RNA (1 μg/μL) to the extract prior to transfer to the Microcon® filter and a 100 μL TE−4 pH 8.0 buffer wash step was performed prior to final collection. Generally, all samples were concentrated and the entire volume was amplified with a maximum of 500 pg of template DNA using the Applied Biosystems™ GlobalFiler™ PCR Amplification Kit on the Applied Biosystems™ Veriti Thermal Cycler following the manufacturer’s suggested protocol [28] with 28 cycles. Fragment separation was performed on the Applied Biosystems™ 3130 Genetic Analyzer (3 kV for 5 s) for Experiment 1 and the Pilot Study samples and on the Applied Biosystems™ 3500xl Genetic Analyzer (1.2 kV for 12 s) for Experiments 2 and 3. Data analysis was performed using GeneMapper™ ID-X v1.4.
The conventional double swab method was performed on the outer surface of the FCCs using two cotton swabs (Puritan Medical Products, Guilford, ME), one wet and one dry [24]. For all double swabbed samples, 30 μL of the swabbing solution was added to the wet swab. 2.7. Rinse-and-swab method The rinse-and-swab method, as the name implies, consists of a solution that is used to rinse the exterior of an FCC and two separate swabbing steps. The method is described below and further detailed in Fig. 2. Prepare the rinse solution by mixing 1 mL of Qiagen ATL Buffer with 30 μL of BTmix per sample for each sample to be tested. A master mix can be prepared ahead of time if sampling multiple cartridge cases. To begin the collection, pick up the cartridge case by placing the tips of a pair of rubber-tipped forceps inside the open end of the cartridge case and exerting pressure outward. Hold the cartridge case at an angle over a sterile 15 mL disposable beaker (Fisher Scientific, Pittsburgh, PA). Using a micropipette, slowly dispense 1 mL of rinse solution on the exterior surface of one side of the cartridge case. When dispensing the rinse solution, start near the opening of the cartridge case and let it flow down the side while ensuring that the runoff is collected in the disposable beaker. After the initial rinse, rotate the cartridge case a quarter turn and rinse the exterior surface a second time using 500 μL of the collected rinse solution. The reduced volume is used to minimize foaming. Repeat this process two additional times to rinse all the remaining sides of the cartridge case. Perform a fifth and final rinse on the cartridge’s headstamp. After rinsing, swab the surface of the cartridge case with a sterile foam swab (Puritan Medical Products Company LLC, reference number 25–1506 1 PF) and break off the swab head into a Qiagen Investigator® Lyse&Spin basket. Repeat the entire rinse process a second time using 500 μL of the solution collected from the first rinse process. After the final rinse, tap the cartridge case on the rim of the disposable beaker to collect all the excess fluid from the cartridge and swab the cartridge case using a single cotton swab. Cut the tip of the cotton swab into a second Lyse&Spin basket and divide the rinse solution from the collection beaker equally between the two baskets (approximately 500 μL in each basket). Following completion of this process, rinse the cartridge cases in 95 % EtOH and allow them to dry to
2.3. Sample preparation For all laboratory-prepared FCCs, 9 mm brass ammunition (multiple manufacturers) was fired from a 9 mm Beretta semi-automatic pistol. A volunteer handled multiple new 9 mm brass cartridges in the left or right palm for 10–15 seconds then switched hands for an additional 10–15 seconds. The volunteer then loaded the cartridges in an ammunition magazine. After firing, the cartridge cases were collected and divided randomly between the sample sets. Cartridges were not cleaned and volunteers’ activities were not controlled prior to handling the cartridges. Informed consent was provided by all volunteers prior to their participation. 2.4. Cartridge case collection device The cartridge case collection device was developed to be easily implemented with minimal training and prevent the FCCs from coming in to contact with each other and the inner surfaces of the container (see Fig. 1). It consists of a 3D-printed plastic cap and a cup that can 2
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Fig. 1. Images of cartridge case collector transport device (patent pending) with an FCC in place. A - an image of the device components. B - a cut-away view showing the post and raised button.
rinse-and-swab with BTmix). The lysis portion of the DNA extraction protocol was 18 h for the double swabbed samples and 3 h for the rinseand-swab samples. Previous internal research has shown that DNA results (quality and quantity) were similar for lysis incubation times between 3 and 18 h; therefore, the incubation times were not expected to impact the observed results. The quantity of DNA recovered and the percentage of the handler’s DNA profile detected were calculated for each set.
prevent any corrosion that may interfere with subsequent examinations. Perform the modified QIAamp® DNA Micro extraction as previously described, except combine the divided lysate in a single MinElute column after the Buffer AL incubation step (Fig. 3). 2.8. Experiment 1: effects of additives Four different swab solutions were compared using the double swab method: (1) water; (2) 2 mg/mL BSA in water; (3) 62.5 mg/mL GGH in water; and (4) BTmix. Forty cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 10, each). An 18 h incubation time was used for the lysis portion of the DNA extraction protocol. Samples were evaluated for total DNA recovery and Degradation Index as determined by Quantifiler™ HP (ratio of small autosomal target concentration to large autosomal target concentration).
2.10. Significance testing The results for Experiment 1 are not normally distributed as determined by the Kolmogorov-Smirnov test. Therefore, the KruskalWallis test, which does not require normally distributed data, was used to compare the variances for each set of data from Experiment 1. Where significant differences were indicated, the Dunn’s multiple comparison test and the Holm-Bonferroni correction were used with a significance value of 0.05. Significance testing for Experiments 2 and 3 was performed using the 2-Tailed Mann-Whitney U test and the Holm-Bonferroni correction with significance levels of 0.05. Based on the laboratory’s experience analyzing trace DNA evidence, occasionally an extraordinarily high DNA yield for one sample within a set of replicate samples will be observed. In Experiment 2, outlier data (> 3 times the Interquartile Range, IQR) was removed from the statistical analysis.
2.9. Experiments 2 and 3: mock sample comparisons Four collection methods were compared: (1) double swab method using water; (2) double swab method using BTmix; (3) rinse-and-swab method without BTmix; and (4) the rinse-and-swab method with BTmix. In Experiment 2, 40 cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 10, each). In Experiment 3, 44 cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 11 for both double swab protocols, N = 10 for the rinse-and-swab without BTmix, and N = 12 for the
2.11. Pilot study: real world samples To examine the effectiveness of the new collection device in combination with the rinse-and-swab method on real world samples, the U.S. Bureau of Alcohol, Tobacco, Firearms, and Explosives Laboratory Fig. 2. Illustration of the rinse-and-swab method. A – Rinse the outer surface of the cartridge case while holding it over the disposable beaker. B – After the initial rinse, perform four additional rinses re-using a portion of the solution collected in the beaker to coat the entire surface of the cartridge case. C – Swab the surface of the cartridge case using a foam swab, then repeat the process with a second round of rinses and a final swabbing of the cartridge case using a cotton swab.
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Fig. 3. An illustration of the lysis and combining of the lysates on the QIAamp® column. A – Place the foam and cotton swab heads in separate Qiagen Lyse&Spin baskets and equally divide the rinse solution. B – After lysis incubation, centrifuge the tubes to collect the lysate. C – Add the Buffer AL to each tube and incubate. D – Load and centrifuge the divided lysate through a single QIAamp® column (this will take several centrifugation steps) resulting in a single sample for the remaining steps.
3. Results
(ATF) partnered with the Kansas City Police Department (KCPD) and the Kansas City Police Crime Laboratory (KCPCL). FCCs were collected from crime scenes using the cartridge case collector and shipped to the ATF Laboratory for analysis. Six batches of evidence were submitted to the laboratory for a total of ninety cartridge cases containing fifteen sets of FCCs attributed to separate firearms. A set of FCCs was assumed to be attributed to a firearm based on case information, recovery location, caliber, and witness statements. DNA collection was typically performed the day after the evidence was received by the laboratory. Following DNA collection, the cartridge cases were immediately entered into the National Integrated Ballistics Information Network (NIBIN). The rinse-and-swab method was performed on the cartridge cases individually for the first 47 samples. For the remaining 43 cartridge cases, DNA collection was performed in pairs using the same rinse solution and swabs to determine if the total quantity of DNA per sample could be increased and the total number of samples could be reduced when analyzing large numbers of cartridge cases. All samples in the pilot study were incubated for 3 h during the lysis portion of the DNA extraction protocol. DNA profiles developed were evaluated for suitability using the ATF Laboratory’s validated interpretation protocols. The probabilistic genotyping software STRmix™ (v2.4, ESR, New Zealand) was used as a tool to assist with interpretation and statistical analysis. The ATF Laboratory’s interpretation protocol does not set a lower boundary on the number of loci required for a suitable DNA profile. For a profile to be suitable for comparison, sufficient data must be present to reasonably determine the number of contributors (NOC) and the NOC must be fewer than or equal to three individuals. Each suitable DNA profile in the pilot study was compared to the ATF Laboratory Staff DNA Index. In general, the profiles were not compared to the KCPD officers that collected the FCCs unless contamination was suspected. The DNA profiles from Experiments 1 through 3 were compared to the handler and analyst that processed the samples. Success is defined as obtaining a DNA profile deemed suitable for comparison. Therefore, the success rate for the FCCs is calculated per DNA sample generated and the success per firearm is defined as obtaining at least one DNA profile suitable for comparison from a group of FCCs assumed to have originated from the same firearm.
3.1. Experiment 1: effects of additives Results from Experiment 1 yielded quantitation values for all samples that were remarkably higher than those typically encountered in ATF casework. Recovery ranged from 200 pg to 14 ng total DNA (Fig. 4A). Complete handler profiles were obtained from all samples. These results may be attributable to using a volunteer who has historically been known to consistently deposit large amounts of DNA, excessive handling of laboratory prepared samples compared to realworld scenarios, and/or the short time between deposition and processing that was possible under research conditions, but that is not necessarily realistic for typical casework. All of the negative control samples were free of DNA with the exception of a single reagent blank that was consistent with the DNA analyst that performed the DNA extraction; however, this DNA profile was not detected in any of the test samples. Although BTmix and GGH both had a mean DNA recovery higher than that of water, significance testing was performed using the Kruskal-Wallis test and the results were not significant. The test was repeated after removing a 7.2 ng sample (approximately 2 times greater than the next highest sample) from the BTmix sample set and a 14.0 ng sample (approximately 7.5 times greater than the next highest sample) from the BSA sample set, but the significance did not change. A Degradation Index value of 1 indicates no degradation. Swabbing solutions containing additives had reduced mean Degradation Index values between 1.0 and 1.4 compared to samples swabbed with sterile water only with a mean of 1.9 (Fig. 4B). The Dunn’s multiple comparison test with the Holm-Bonferroni correction indicate that the difference in the Degradation Index between swabbing with water or GGH was statistically significant (p-value = 0.004, Supplemental Data, S1A). There is a large difference between swabbing with water (Average DI = 1.9) or BTmix (Average DI = 1.2), but it did not reach the level of significance (p-value = 0.068). The data indicate that using BSA or GGH alone as a swabbing or rinse-and-swab additive may have beneficial effects for FCCs, but the combined effect appeared to increase the DNA yield compared to each additive individually while still decreasing the Degradation Index. Therefore, all further experiments within this study were performed using both additives in concert as the BTmix.
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calculated using the combined data from Experiments 2 and 3 and compared for each of the recovery methods to ensure that the increased DNA recovery translated to an increase in genetic data detected. The average RFU per locus for each recovery method is summarized in Fig. 6 and as a percentage of the handler’s alleles (Supplemental Data, S4). The two-tailed Mann-Whitney U test pairwise comparisons with the Holm-Bonferroni correction indicate that the recovery of DNA as measured by the percentage of handler’s alleles detected (pvalue = 0.0004) and the average RFU per locus (p-value = 0.0003) are significantly higher using the rinse-and-swab method with BTmix when compared to the double swab method with water (Supplemental Data, S1C and S1D). 3.3. Real-world samples FCCs from crime scenes were collected with the cartridge case collector and DNA was recovered using the rinse-and-swab method with BTmix. FCCs were collected at one or more shooting scenes over a weekend and submitted to the laboratory as a batch the following week. Over the course of this study, six separate batches of FCCs were submitted and assumed to represent fifteen different firearms. The batches were separated into two groups (Group 1 consists of batches 1–3; Group 2 consists of batches 4–6). FCCs from the first group (Group 1, n = 47) were analyzed individually. For these samples, the rinse-and-swab method was performed on a single cartridge case and the rest of the analysis proceeded without combining the extract with any other sample. To simulate processing of samples in a typical casework flow, the second group of FCCs (Group 2, n = 43) was analyzed in pairs. For these samples, DNA was collected from both cartridge cases using the same rinse solution. When deemed necessary, the DNA extracts were combined into a single sample prior to PCR amplification to reflect normal laboratory processes. For example, if two different DNA extracts assumed to have originated from the same firearm contained levels of DNA that would not be expected to produce an interpretable DNA profile, the two samples were combined into a single sample at the concentration step. The 43 paired cartridge cases resulted in a total of 20 DNA samples (21 analyzed as pairs, 1 analyzed individually due to the odd number of cartridge cases in the batch, and two sets of DNA extracts combined prior to PCR amplification). The negative control samples from each extraction batch were free of DNA. On-scene contamination was observed in the second batch. Both of the suitable DNA profiles in Batch 2 were identified as consistent single source DNA profiles that were attributable to the officer that collected the FCCs. A summary of these results not including the two FCCs and one firearm associated with contamination are listed in Tables 1 and 2. A more detailed summary of the results for each FCC including the caliber, metal composition, damage, time between incident and collection, total DNA recovered, DNA typing success, and number of contributors can be found in the Supplementary Data (S5). The DNA profiles determined to be suitable for comparison were divided into the following categories and summarized in Table 3: single source, two person mixtures, and three person mixtures. Three single source DNA profiles were obtained from the FCCs, however two of these were confirmed to be contamination during collection and are not included in the table or calculated success rates.
Fig. 4. Total DNA recovered (A) and the Degradation Index per sample (B) determined by Quantifiler™ HP analysis for cartridge cases swabbed using the following swabbing solutions: water, BTmix, BSA, and GGH. Two data points for the DNA recovery (A) using BTmix and BSA (approximately 7 ng and 14 ng, respectively) were removed from the figure and calculated means.
3.2. Experiments 2 and 3: comparison of collection methods DNA was recovered from FCCs by either double swabbing with water, double swabbing with water and BTmix, rinse-and-swab method with Buffer ATL, or rinse-and-swab method with Buffer ATL and BTmix. All DNA samples were concentrated to a final volume of 15 μL and the entire volume was amplified with the exception of one sample in the rinse-and-swab with BTmix that was considered to be outlier data and not used in the calculations as noted below. The mean of the total recovered DNA per sample type was 49 pg, 85 pg, 153 pg, and 143 pg, respectively (Supplemental Data, S2). One outlier data point was removed from the mean calculation for the rinse-and-swab with BTmix (approximately 1.7 ng, > 3 times IQR). Duplication of the study yielded similar mean total DNA recoveries: 54 pg, 78 pg, 134 pg, and 171 pg, respectively (Supplemental Data, S3). Statistical significance testing was performed on the combined data set. The difference in the total DNA recovered between the double swab method using water as the wetting solution and rinse-and-swab method with the BTmix additives was statistically significant (Fig. 5) using a two-tailed Mann-Whitney U test with the Holm-Bonferroni correction p-value = 0.0009). Complete pairwise comparisons can be seen in Supplemental Data (S1B). The negative control samples in both replicates were free of DNA. The average peak heights of the handler’s alleles were also
3.4. Discussion FCCs are not routinely processed by the forensic DNA community due to the perception that informative results are too rare for the effort required. However, novel DNA recovery methods, increased sensitivity in amplification kits, more efficient DNA extraction methods, and improvements in the interpretation of low level and mixed DNA profiles have demonstrated increased success rates for FCCs [10]. In Experiment 1, our study showed that adding BSA and/or the copper-binding 5
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Fig. 5. Combined results of the collection method comparisons. Total DNA (pg) recovered from FCCs using four different methods: double swabbing with water, double swabbing with water and BTmix, rinse-and-swab method with Buffer ATL, or rinse-and-swab method with Buffer ATL and BTmix.
Fig. 6. Combined average RFU per locus from duplicate Experiments 2 and 3.
success rates per cartridge case and per firearm for Group 1 were 27 % (12 of 45) and 56 % (5 of 9), respectively. These samples were collected in three batches. The first and third batches were collected within hours of the shooting incidents and produced combined success rates per cartridge case and firearm of 35 % (12 of 34) and 71 % (5 of 7), respectively. The second batch was collected days to weeks after the shooting incidents, during which it had rained at least once. Additionally, the majority of the cartridge cases from the second batch (10 of 13) showed signs of damage, possibly from having been run over by at least one car. These cartridge cases did not produce DNA profiles suitable for comparison purposes. The only confirmed incident of contamination for all FCCs collected with the cartridge case collector occurred in Batch 2 when the FCCs had been damaged causing the opening to be significantly reduced. Although damaged, the collecting officer still attempted to use the collector by forcing the FCC onto the
tripeptide GGH to the swabbing solution decreases the DNA Degradation Index when compared to swabbing with water alone (Fig. 4B and Supplemental Data, S1A). In Experiments 2 and 3, the rinse-and-swab method with BTmix reproducibly demonstrated increases in total DNA recovered (approximately threefold; Fig. 5 and Supplemental Data, S1B) and average peak height per locus (over fourfold; Fig. 6 and Supplemental Data, S1D) compared to the traditional double swab method using water as the swabbing solution on FCCs handled and fired in the laboratory. Inhibition may still play a part in affecting the successful analysis of these samples, however, the actual electropherograms and quantification IPC data did not suggest inhibition was a major factor. While mock samples prepared in the laboratory provide valuable data, analyzing real-world samples is the true test of a new method. When the confirmed contamination samples in Batch 2 are removed the
Table 1 Summary of results for crime scene FCCs analyzed individually and in pairs. One cartridge case in Batch 5 was analyzed individually. *: Two DNA samples generated and the subsequent DNA profiles were removed from Batch 2 due to contamination during collection. Analyzed Individually (Group 1)
# of Cartridge Cases # of DNA Samples Generated # of Profiles Suitable % Success / Sample
Analyzed in Pairs (Group 2)
Batch 1
Batch 2
Batch 3
Total
Batch 4
Batch 5
Batch 6
Total
14 14 4 29 %
13 11* 0* 0%
20 20 8 40 %
47 45 12 27 %
20 9 1 11 %
9 5 0 0%
14 6 2 33 %
43 20 3 15 %
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Table 2 Summary of results per firearm when the FCCs were analyzed individually and in pairs. *: One firearm was removed from Batch 2 due to contamination during collection. Analyzed Individually (Group 1)
# of Firearms Successful Analysis % Success / Firearm
Analyzed in Pairs (Group 2)
Batch 1
Batch 2
Batch 3
Total
Batch 4
Batch 5
Batch 6
Total
4 3 75 %
2* 0 0%
3 2 67 %
9 5 56 %
2 1 50 %
1 0 0%
2 2 100 %
5 3 60 %
FCCs (18 cartridge cases total) were covered in dried mud and produced a single DNA profile suitable for comparison. A third set of five FCCs produced only one DNA extract with a detectable amount of DNA, but did not result in a DNA profile suitable for comparison purposes. The final two sets of FCCs, however, both produced at least one DNA profile suitable for comparison and resulted in quantifiable DNA in four of the seven DNA extracts. Both this study and Montpetit et al. [3] studies found that mixed DNA profiles greatly outnumber single source profiles at a ratio of at least 2 to 1 (Table 3). The cause of the mixtures can only be speculated, but some possible sources may be a shared ammunition magazine, the chamber of the firearm, secondary transfer from the handler, victim blood, or shared ammunition stored loose in a communal bag. These results could be used as an argument to analyze the FCCs individually to prevent the creation of uninterpretable mixtures where a DNA profile suitable for comparison might have existed. Additionally, total DNA quantities obtained from FCCs in this study are consistent with the results of Montpetit et al. [3]. This study demonstrated that 40 % of the FCCs from casework analyzed individually with the rinse-and-swab method using BTmix yielded at least 50 pg of total DNA that is similar to the 33 % described by Montpetit et al. During the course of this study, in addition to the collaboration with KCPD and KCPCL, FCCs from two other investigations involving a total of three firearms (a total of 30 FCCs) were analyzed using the rinse-andswab method with BTmix. These FCCs were submitted to the ATF Laboratory individually wrapped tightly in paper towels and placed in a small cardboard box several months earlier. In all three instances, at least one DNA profile suitable for comparison was developed from each group of FCCs assumed to have originated from the same firearm. One of these profiles was consistent with the person of interest and another resulted in a CODIS hit. This demonstrates that even with non-ideal circumstances, probative DNA profiles can be obtained from FCCs using the rinse-and-swab method. As with analysis of all evidence, the probative value and order of the examinations must be weighed against the timing and potential loss of valuable information. Efforts are currently underway to increase the percentage of recovered firearms and FCCs entered into the NIBIN database within 48 h to maximize their value for producing investigative leads. The process of entering FCCs into NIBIN, which includes cleaning the cartridge cases, does not allow for downstream DNA analysis. As part of this study, we wanted to determine how the rinse-and-swab method would affect the timing for entry of FCCs into NIBIN. DNA collection from individual cartridge cases took approximately five minutes each. Therefore, the DNA collection from twelve cartridge cases could be completed within an hour. For most of the samples
cap post. When used as designed there is no need to handle the FCC and this extra sample manipulation may be the cause of the contamination. This demonstrates the necessity to train end-users in both contamination prevention techniques during evidence collection and the use of this specific device for the collection of FCCs. Batch 2 demonstrates several important points. First, it is critical to collect the FCCs as soon as possible to avoid loss of DNA due to weather or other environmental factors that may occur in the intervening time. Second, the FCCs should be analyzed as soon as possible due to the loss of DNA over time when deposited on metal surfaces [29]. This same effect has been observed when cells were deposited directly on brass cartridge cases (data not shown). Finally, great care must be taken to avoid contaminating any evidence, and even more so when it is expected to contain trace quantities. The variability in the results reflects the nature of trace DNA analysis on any object. For example, a single firearm in this study was associated with nine FCCs, three of which produced quantifiable DNA and only one of those had greater than 100 pg of total DNA. A different firearm was associated with seven FCCs and produced detectable DNA in all the extracts, four of which contained greater than 100 pg total DNA. Many factors can affect the transfer and persistence of trace DNA. Additionally, the caliber of cartridge case, make and model of firearm, length of time between deposition and collection, and the metal composition may impact the results when analyzing FCCs. The vast majority of the FCCs collected from crime scenes in this study were composed of brass (80 brass, 9 nickel-plated, and 1 unknown). Given that brass substrates were previously demonstrated to be a challenge due to the possible degradative and inhibitory effects, it is encouraging that all of the DNA profiles suitable for comparison were obtained from brass FCCs. With respect to caliber, DNA analysis was successful on three of the four calibers observed (9 mm, .40 caliber, and .45 caliber). Only five .357 caliber cartridge cases were examined and no DNA profiles suitable for comparison were obtained. The surface areas for these calibers are not appreciably different and therefore, surface area would not be expected to be a major factor when comparing these results. Surface area may be more of a factor for smaller caliber FCCs such as .22 and .25, but these are less common. These FCC-specific factors warrant future studies. The analysis of the second group of crime scene FCCs (Group 2), which were analyzed in pairs, was less successful. The success rates per DNA sample and per firearm were 15 % (3 of 20) and 60 % (3 of 5), respectively. Due to the relatively low number of FCCs and assumed associated firearms, it was not possible to determine if the difference between Group 1 and Group 2 is statistically significant. As noted above, many factors can affect the success rate. In Group 2, two sets of
Table 3 Summary of single source, two person mixtures, and three person mixtures for DNA profiles determined to be suitable for comparison purposes. *: Two single source profiles were removed from Batch 2 due to contamination during collection. Types of Profiles
Single Source Profiles
Two Person Mix
Three Person Mix
Total Suitable Profiles
# of Each, Sampled Individually # of Each, Sampled in Pairs # of Each, Total % of Suitable Profiles
1* 0 1 7%
8 3 10 67 %
3 0 4 27 %
12 3 15
7
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collected in this study, DNA collection was performed within 24 h of the evidence being received by the laboratory and the cartridge cases were entered into NIBIN within the next 12 h. Samples in this study were not processed for latent prints, thus the effects of latent print chemical processing of FCCs on the effectiveness of the rinse-and-swab method with BTmix was not examined; however, traditional methods of latent print development on FCCs have typically resulted in low success rates [30,31]. This does not include the relatively new RECOVER Latent Fingerprint Technology process (Foster and Freeman, Sterling, VA) [32] that may be more effective. Future research should be performed to determine if the rinse-and-swab method with BTmix could be performed prior to the RECOVER:LFT process without affecting the ability to visualize latent prints. One of the aims of this research was to develop a method that was easily incorporated into a laboratory’s workflow. In its current form, the rinse-and-swab method with BTmix in conjunction with the modified QIAamp® DNA Micro extraction protocol requires splitting the lysate into two tubes that are combined onto the same silica column. Although viewed as a manageable burden given the increase in success rate, research is currently underway that indicates similar or better results when a reduced rinse volume is used during the DNA collection steps. Reducing the rinse volume will allow for the use of a single lysate tube, simplify the process, and allow for automation without sacrificing DNA recovery. This study, in addition to other studies examining the DNA analysis of FCCs, demonstrates that it is possible to obtain DNA profiles suitable for comparison from FCCs with a relatively high rate of success; therefore, the forensic science community should view FCCs as viable sources of DNA. The main goal of this study was to develop methods to improve various aspects of the collection and DNA analysis of FCCs that can be practically implemented in order to increase success rates. As discussed in the Montpetit review [10], comparing results among studies performed by multiple independent laboratories is difficult due to differing extraction methods, analysis parameters, amplification kit sensitivities, and interpretation thresholds. A strength of this study is that it offers methods that can be easily adapted into most forensic laboratory’s existing workflows, as it uses standard laboratory equipment and only a minor modification to the collection method and swabbing solution. The results demonstrated a high rate of success using the GlobalFiler™ PCR amplification kits without employing enhanced sensitivity techniques. Both the quantity (approximate threefold increase) and the quality (decreased Degradation Index) of the DNA recovered were improved using the rinse-and-swab method with BTmix compared to conventional double swab method with water. Finally, even the best DNA collection techniques will not overcome the loss of DNA at the crime scene or during collection and transport to the forensic laboratory. This study has shown that in order to maximize successful results, that the entire process must be considered. Proper collection and preservation starting at the scene followed by an improved DNA collection method can combine to significantly improve the success rate of DNA analysis from FCCs.
[3] [4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12] [13] [14]
[15]
[16] [17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
[25] [26] [27]
Declaration of Competing Interest
[28]
None.
[29]
Appendix A. Supplementary data [30]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2020.102238.
[31]
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sensitive method to extract DNA from biological traces present on ammunition for the purpose of genetic profiling, Int. J. Legal Med. 125 (July 4) (2011) 597–602. S. Montpetit, P. O’Donnell, An optimized procedure for obtaining DNA from fired and unfired ammunition, Forensic Sci. Int. Genet. 17 (July) (2015) 70–74. V. Radojicic, M. Keckarevic Markovic, F. Puac, M. Kecmanovic, D. Keckarevic, Comparison of different methods of DNA recovery and PCR amplification in STR profiling of casings-a retrospective study, Int. J. Legal Med. 132 (November 6) (2018) 1575–1580. K.M. Horsman-Hall, Y. Orihuela, S.L. Karczynski, A.L. Davis, J.D. Ban, S.A. Greenspoon, Development of STR profiles from firearms and fired cartridge cases, Forensic Sci. Int. Genet. 3 (September 4) (2009) 242–250. M. Troy, C. Westring, P. Danielson, H. Mazzanti, Evaluation and optimization of DNA recovery from bullet cartridge cases, Proceedings of the American Academy of Forensic Sciences 67th Annual Scientific Meeting (2015). M. Matney, M. Powell, E. Salmon, K. Beatty, Method validation for DNA recovery from cartridge casings, Proceedings of the American Academy of Forensic Sciences 70th Annual Meeting (2018). T.C.R. Wan, L. MacDonald, Y. Perez, T. Bille, D. Podini, Recovering touch DNA from cartridge casings using a method of tape lifting, Proceedings of the American Academy of Forensic Sciences 67th Annual Meeting (2015). A. Mottar, D. Foran, Optimizing DNA recovery and extraction from spent cartridge, 66th Annual Proceedings of the American Academy of Forensic Sciences 20 (2014). S. Montpetit, Obtaining DNA from ammunition: a review, Wiley Interdiscip. Rev. Forensic Sci. (June) (2019) e1352. M. Goray, R.A.H. van Oorschot, J.R. Mitchell, DNA transfer within forensic exhibit packaging: potential for DNA loss and relocation, Forensic Sci. Int. Genet. 6 (March 2) (2012) 158–166. L.I. Moreno, B.R. McCord, Understanding metal inhibition: the effect of copper (Cu 2+) on DNA containing samples, Forensic Chem. 4 (June) (2017) 89–95. M. Vincent, P. Hartemann, M. Engels-Deutsch, Antimicrobial applications of copper, Int. J. Hyg. Environ. Health 219 (October 7) (2016) 585–591. M.P. Cervantes-Cervantes, J.V. Calderón-Salinas, A. Albores, J.L. Muñoz-Sánchez, Copper increases the damage to DNA and proteins caused by reactive oxygen species, Biol. Trace Elem. Res. 103 (March 3) (2005) 229–248. T. Bille, M. Grimes, D. Podini, Copper induced DNA damage on unfired brass cartridge casings, Proceedings of the 24th International Symposium on Human Identification (2013). G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (March 5) (2011) 1541–1547. C. Angelé-Martínez, K.V.T. Nguyen, F.S. Ameer, J.N. Anker, J.L. Brumaghim, Reactive oxygen species generation by copper(II) oxide nanoparticles determined by DNA damage assays and EPR spectroscopy, Nanotoxicology 11 (2) (2017) 278–288. M.G. Schmidt, et al., Sustained reduction of microbial burden on common hospital surfaces through introduction of copper, J. Clin. Microbiol. 50 (July 7) (2012) 2217–2223. M.M. Holland, R.M. Bonds, C.A. Holland, J.A. McElhoe, Recovery of mtDNA from unfired metallic ammunition components with an assessment of sequence profile quality and DNA damage through MPS analysis, Forensic Sci. Int. Genet. 39 (March) (2019) 86–96. T. Topală, A. Bodoki, L. Oprean, R. Oprean, Bovine serum albumin interactions with metal complexes, Med. Pharm. Rep. 87 (November 4) (2014) 215–219. S.J. Lau, T.P. Kruck, B. Sarkar, A peptide molecule mimicking the copper(II) transport site of human serum albumin. A comparative study between the synthetic site and albumin, J. Biol. Chem. 249 (September 18) (1974) 5878–5884. E. Kimoto, H. Tanaka, J. Gyotoku, F. Morishige, L. Pauling, Enhancement of antitumor activity of ascorbate against Ehrlich ascites tumor cells by the copper: glycylglycylhistidine complex, Cancer Res. 43 (February 2) (1983) 824–828. K. Yokawa, T. Kagenishi, T. Kawano, Prevention of oxidative DNA degradation by copper-binding peptides, Biosci. Biotechnol. Biochem. 75 (July 7) (2011) 1377–1379. D. Sweet, M. Lorente, J.A. Lorente, A. Valenzuela, E. Villanueva, An improved method to recover saliva from human skin: the double swab technique, J. Forensic Sci. 42 (March 2) (1997) 320–322. Qiagen, QIAamp® DNA Micro Handbook, Dec (2014). Applied Biosystems™, Quantifiler™ HP and Trio DNA Quantification Kits User Guide, Revision H (2020). Merck Millipore Ltd, Microcon® Centrifugal Filter Devices User Guide, Revision Jun (2018). Applied Biosystems™, GlobalFiler™ PCR Amplification Kit User Guide, Revision E (2020). T.W. Bille, C. Cromartie, M. Farr, Effects of cyanoacrylate fuming, time after recovery, and location of biological material on the recovery and analysis of DNA from post-blast pipe bomb fragments, J. Forensic Sci. 54 (September. 5) (2009) 1059–1067. S. Nunn, Touch DNA collection versus firearm fingerprinting: comparing evidence production and identification outcomes, J. Forensic Sci. 58 (May 3) (2013) 601–608. C.M.A. Girelli, B.J.M. Lobo, A.G. Cunha, J.C.C. Freitas, F.G. Emmerich, Comparison of practical techniques to develop latent fingermarks on fired and unfired cartridge cases, Forensic Sci. Int. 250 (May) (2015) 17–26. S.M. Bleay, P.F. Kelly, R.S.P. King, S.G. Thorngate, A comparative evaluation of the disulfur dinitride process for the visualisation of fingermarks on metal surfaces, Sci. Justice (July) (2019).
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsigen
An improved process for the collection and DNA analysis of fired cartridge cases
T
Todd W. Bille*, Glenn Fahrig, Steven M. Weitz, Greg A. Peiffer United States Bureau of Alcohol, Tobacco, Firearms, and Explosives, National Laboratory Center, 6000 Ammendale Road, Beltsville, MD, 20705, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Fired cartridge cases Forensic science DNA Trace DNA
Improvements to the DNA analysis of fired cartridge cases have been made in recent years, yet successful analysis of this important evidence type remains difficult. In this study, we describe both a novel device for the collection and transport of fired cartridge cases and a new DNA recovery method that incorporates a rinse-andswab technique. This technique combines two different types of swabs and a rinse solution with additives that reduce the degradative effects that copper has on DNA. The new recovery method yielded approximately threefold more DNA than the traditional double swab method and reduced the evidence of degradation. After validation, we estimated the real-world success rate of obtaining DNA profiles suitable for comparison with the rinse-and-swab method by testing over 100 cartridge cases collected from crime scenes. Approximately 67 % of the time (8 of 12), at least one DNA profile suitable for comparison was obtained from fired cartridge cases assumed to be associated with a single firearm using the collection device and the rinse-and-swab method when the fired cartridge cases were collected within 24 h.
1. Introduction
surfaces. The historically low rate of success in obtaining DNA profiles suitable for comparison from FCCs may be in part due to the deleterious effects on DNA integrity from the copper contained in brass. Studies suggest that copper may inhibit downstream PCR and/or degrade DNA through oxidative damage [12–19]. It is hypothesized that DNA damage is the result of oxidative stress from free radicals due to the reduction of transition metals (i.e. copper). Previous research has shown that when Bovine Serum Albumin (BSA) is added to the DNA extraction it can increase yields for challenging samples by chelating contaminants [20]. Lau et al. identified the copper binding portion of BSA, a three amino acid peptide, Gly-Gly-His (GGH), and showed that this tripeptide had similar copper binding specificity as human serum albumin [21]. Since then, the chelating ability of GGH has been shown to reduce DNA damage due to hydroxyl radicals and oxidative stress in certain conditions by complexing with copper [22,23]. We developed a DNA recovery method, which incorporates BSA and GGH in the rinse solution along with a series of rinsing and swabbing steps to reduce the deleterious effects of copper contained in brass cartridge cases and to maximize the recovery of DNA. Experiment 1 was conducted to evaluate the effect of BSA and GGH, individually and combined (BTmix), on the quantity of DNA recovered from laboratory prepared FCCs using the conventional double swab
Fired cartridge cases (FCCs) are typically not processed for DNA analysis due to a lack of historical success in obtaining DNA profiles suitable for comparison [1]. This attitude is slowly changing as more research is performed and laboratories using novel approaches are reporting greater success rates [2–9]. In a recent review, Montpetit summarized the research performed over the last ten years and demonstrated that FCCs are viable sources for successful DNA typing [10]. The goal of this study was to increase the success rate for DNA analysis of FCCs by improving multiple aspects of the process that could easily be incorporated into a laboratory’s current process flow, starting at the crime scene. During evidence collection, recovered FCCs are typically placed in manila envelopes or plastic bags that allow the cartridge cases to make contact with interior surfaces of the container. This contact/rubbing may result in loss of some or all of the DNA that was originally present on the cartridge case [11]. Additionally, contamination can occur during evidence collection or when multiple FCCs are packaged together. To mitigate the potential contamination and loss of DNA during collection and transport of the FCCs, a novel collection device was developed. This device minimizes evidence handling and suspends each FCC within the container preventing it from contacting the inner
⁎
Corresponding author. E-mail address: [email protected] (T.W. Bille).
https://doi.org/10.1016/j.fsigen.2020.102238 Received 11 October 2019; Received in revised form 6 January 2020; Accepted 8 January 2020 Available online 18 January 2020 1872-4973/ Published by Elsevier B.V.
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accommodate cartridge cases from most handguns, excluding smaller calibers such as .22 caliber. The cap is used to collect the FCC by holding the tab and scooping up the cartridge case on the post. The cup is then placed over the suspended cartridge case and snapped onto the cap of the collection device. The center post on the cap and a raised button inside the cup hold the cartridge case in place and prevent contact between the outer surface of the FCC and the inner surface of the device. The device is vented to allow for airflow and drying, if necessary. Prior to use, the components of the collection device were soaked in a 10 % bleach solution for at least 1 h, rinsed with 70 % ethanol, and allowed to dry.
method [24]. In Experiments 2 and 3, DNA quantity and quality from laboratory prepared FCC samples were compared with and without BTmix for both the rinse-and-swab and double swab methods. A Pilot Study using real-world samples was performed to evaluate the efficacy of the combination of the novel collection device and the rinse-andswab method with BTmix. 2. Materials and methods 2.1. Modified QIAamp® DNA Micro extraction method The QIAamp® DNA Investigator Kit with the QIAamp® DNA Micro manufacturer’s protocol (Qiagen, Germantown, MD) [25] with several modifications was used to perform the DNA extractions. Modifications to the protocol include: doubling the Buffer AL volume; increasing Buffer AW2 volume to 700 μL; and eluting in two separate 50 μL volumes of TE−4 (Tris 10 mM, EDTA 0.1 mM, pH 8.0) DNA Suspension Buffer (Teknova, Hollister, CA) with 5 min incubations at room temperature resulting, in a final volume of 100 μL. A Qiagen Investigator® Lyse&Spin basket was used for the lysis portion of the extraction protocol with an incubation time between 3 and 18 h. At least one negative control was included with each extraction set to monitor systemic contamination.
2.5. Preparation of BTmix The final formulation of BTmix used in this study incorporated a concentration of 2 mg/mL UltraPure™ BSA (Invitrogen, Carlsbad, CA) and 62.5 mg/mL GGH tripeptide (Millipore Sigma, Burlington, MA). Higher concentrations of the GGH tripeptide were evaluated, but a decrease in DNA recovery was observed (data not shown). To create a 125 mg/mL GGH precursor solution, 743 μL of sterile nuclease-free water was added to 100 mg solid GGH tripeptide resulting in approximately 800 μL of solution. An equivalent volume of 4 mg/mL BSA precursor solution was generated by adding 64 μL of 50 mg/mL BSA solution to 736 μL of sterile nuclease-free water. The two precursor solutions were combined to create 1.6 mL of the working BTmix.
2.2. DNA quantitation, concentration, and fragment analysis
2.6. Double swabbing method
The DNA concentration for all samples was determined using the Applied Biosystems™ Quantifiler™ HP DNA Quantification Kit following the manufacturer’s protocol (ThermoFisher, Waltham, MA) [26]. When the DNA concentration was less than 33 pg/μL the extracts were concentrated down to 15 μL for amplification using the Millipore Microcon® 30 kDa Centrifugal Filter Unit (Millipore Sigma, Burlington, MA). All negative control samples were concentrated and amplified in parallel. The manufacturer’s protocol [27] was modified by adding 1 μL of carrier RNA (1 μg/μL) to the extract prior to transfer to the Microcon® filter and a 100 μL TE−4 pH 8.0 buffer wash step was performed prior to final collection. Generally, all samples were concentrated and the entire volume was amplified with a maximum of 500 pg of template DNA using the Applied Biosystems™ GlobalFiler™ PCR Amplification Kit on the Applied Biosystems™ Veriti Thermal Cycler following the manufacturer’s suggested protocol [28] with 28 cycles. Fragment separation was performed on the Applied Biosystems™ 3130 Genetic Analyzer (3 kV for 5 s) for Experiment 1 and the Pilot Study samples and on the Applied Biosystems™ 3500xl Genetic Analyzer (1.2 kV for 12 s) for Experiments 2 and 3. Data analysis was performed using GeneMapper™ ID-X v1.4.
The conventional double swab method was performed on the outer surface of the FCCs using two cotton swabs (Puritan Medical Products, Guilford, ME), one wet and one dry [24]. For all double swabbed samples, 30 μL of the swabbing solution was added to the wet swab. 2.7. Rinse-and-swab method The rinse-and-swab method, as the name implies, consists of a solution that is used to rinse the exterior of an FCC and two separate swabbing steps. The method is described below and further detailed in Fig. 2. Prepare the rinse solution by mixing 1 mL of Qiagen ATL Buffer with 30 μL of BTmix per sample for each sample to be tested. A master mix can be prepared ahead of time if sampling multiple cartridge cases. To begin the collection, pick up the cartridge case by placing the tips of a pair of rubber-tipped forceps inside the open end of the cartridge case and exerting pressure outward. Hold the cartridge case at an angle over a sterile 15 mL disposable beaker (Fisher Scientific, Pittsburgh, PA). Using a micropipette, slowly dispense 1 mL of rinse solution on the exterior surface of one side of the cartridge case. When dispensing the rinse solution, start near the opening of the cartridge case and let it flow down the side while ensuring that the runoff is collected in the disposable beaker. After the initial rinse, rotate the cartridge case a quarter turn and rinse the exterior surface a second time using 500 μL of the collected rinse solution. The reduced volume is used to minimize foaming. Repeat this process two additional times to rinse all the remaining sides of the cartridge case. Perform a fifth and final rinse on the cartridge’s headstamp. After rinsing, swab the surface of the cartridge case with a sterile foam swab (Puritan Medical Products Company LLC, reference number 25–1506 1 PF) and break off the swab head into a Qiagen Investigator® Lyse&Spin basket. Repeat the entire rinse process a second time using 500 μL of the solution collected from the first rinse process. After the final rinse, tap the cartridge case on the rim of the disposable beaker to collect all the excess fluid from the cartridge and swab the cartridge case using a single cotton swab. Cut the tip of the cotton swab into a second Lyse&Spin basket and divide the rinse solution from the collection beaker equally between the two baskets (approximately 500 μL in each basket). Following completion of this process, rinse the cartridge cases in 95 % EtOH and allow them to dry to
2.3. Sample preparation For all laboratory-prepared FCCs, 9 mm brass ammunition (multiple manufacturers) was fired from a 9 mm Beretta semi-automatic pistol. A volunteer handled multiple new 9 mm brass cartridges in the left or right palm for 10–15 seconds then switched hands for an additional 10–15 seconds. The volunteer then loaded the cartridges in an ammunition magazine. After firing, the cartridge cases were collected and divided randomly between the sample sets. Cartridges were not cleaned and volunteers’ activities were not controlled prior to handling the cartridges. Informed consent was provided by all volunteers prior to their participation. 2.4. Cartridge case collection device The cartridge case collection device was developed to be easily implemented with minimal training and prevent the FCCs from coming in to contact with each other and the inner surfaces of the container (see Fig. 1). It consists of a 3D-printed plastic cap and a cup that can 2
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Fig. 1. Images of cartridge case collector transport device (patent pending) with an FCC in place. A - an image of the device components. B - a cut-away view showing the post and raised button.
rinse-and-swab with BTmix). The lysis portion of the DNA extraction protocol was 18 h for the double swabbed samples and 3 h for the rinseand-swab samples. Previous internal research has shown that DNA results (quality and quantity) were similar for lysis incubation times between 3 and 18 h; therefore, the incubation times were not expected to impact the observed results. The quantity of DNA recovered and the percentage of the handler’s DNA profile detected were calculated for each set.
prevent any corrosion that may interfere with subsequent examinations. Perform the modified QIAamp® DNA Micro extraction as previously described, except combine the divided lysate in a single MinElute column after the Buffer AL incubation step (Fig. 3). 2.8. Experiment 1: effects of additives Four different swab solutions were compared using the double swab method: (1) water; (2) 2 mg/mL BSA in water; (3) 62.5 mg/mL GGH in water; and (4) BTmix. Forty cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 10, each). An 18 h incubation time was used for the lysis portion of the DNA extraction protocol. Samples were evaluated for total DNA recovery and Degradation Index as determined by Quantifiler™ HP (ratio of small autosomal target concentration to large autosomal target concentration).
2.10. Significance testing The results for Experiment 1 are not normally distributed as determined by the Kolmogorov-Smirnov test. Therefore, the KruskalWallis test, which does not require normally distributed data, was used to compare the variances for each set of data from Experiment 1. Where significant differences were indicated, the Dunn’s multiple comparison test and the Holm-Bonferroni correction were used with a significance value of 0.05. Significance testing for Experiments 2 and 3 was performed using the 2-Tailed Mann-Whitney U test and the Holm-Bonferroni correction with significance levels of 0.05. Based on the laboratory’s experience analyzing trace DNA evidence, occasionally an extraordinarily high DNA yield for one sample within a set of replicate samples will be observed. In Experiment 2, outlier data (> 3 times the Interquartile Range, IQR) was removed from the statistical analysis.
2.9. Experiments 2 and 3: mock sample comparisons Four collection methods were compared: (1) double swab method using water; (2) double swab method using BTmix; (3) rinse-and-swab method without BTmix; and (4) the rinse-and-swab method with BTmix. In Experiment 2, 40 cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 10, each). In Experiment 3, 44 cartridges were handled and loaded into four ammunition magazines. After firing, the cartridge cases were randomly divided into four sets of samples (N = 11 for both double swab protocols, N = 10 for the rinse-and-swab without BTmix, and N = 12 for the
2.11. Pilot study: real world samples To examine the effectiveness of the new collection device in combination with the rinse-and-swab method on real world samples, the U.S. Bureau of Alcohol, Tobacco, Firearms, and Explosives Laboratory Fig. 2. Illustration of the rinse-and-swab method. A – Rinse the outer surface of the cartridge case while holding it over the disposable beaker. B – After the initial rinse, perform four additional rinses re-using a portion of the solution collected in the beaker to coat the entire surface of the cartridge case. C – Swab the surface of the cartridge case using a foam swab, then repeat the process with a second round of rinses and a final swabbing of the cartridge case using a cotton swab.
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Fig. 3. An illustration of the lysis and combining of the lysates on the QIAamp® column. A – Place the foam and cotton swab heads in separate Qiagen Lyse&Spin baskets and equally divide the rinse solution. B – After lysis incubation, centrifuge the tubes to collect the lysate. C – Add the Buffer AL to each tube and incubate. D – Load and centrifuge the divided lysate through a single QIAamp® column (this will take several centrifugation steps) resulting in a single sample for the remaining steps.
3. Results
(ATF) partnered with the Kansas City Police Department (KCPD) and the Kansas City Police Crime Laboratory (KCPCL). FCCs were collected from crime scenes using the cartridge case collector and shipped to the ATF Laboratory for analysis. Six batches of evidence were submitted to the laboratory for a total of ninety cartridge cases containing fifteen sets of FCCs attributed to separate firearms. A set of FCCs was assumed to be attributed to a firearm based on case information, recovery location, caliber, and witness statements. DNA collection was typically performed the day after the evidence was received by the laboratory. Following DNA collection, the cartridge cases were immediately entered into the National Integrated Ballistics Information Network (NIBIN). The rinse-and-swab method was performed on the cartridge cases individually for the first 47 samples. For the remaining 43 cartridge cases, DNA collection was performed in pairs using the same rinse solution and swabs to determine if the total quantity of DNA per sample could be increased and the total number of samples could be reduced when analyzing large numbers of cartridge cases. All samples in the pilot study were incubated for 3 h during the lysis portion of the DNA extraction protocol. DNA profiles developed were evaluated for suitability using the ATF Laboratory’s validated interpretation protocols. The probabilistic genotyping software STRmix™ (v2.4, ESR, New Zealand) was used as a tool to assist with interpretation and statistical analysis. The ATF Laboratory’s interpretation protocol does not set a lower boundary on the number of loci required for a suitable DNA profile. For a profile to be suitable for comparison, sufficient data must be present to reasonably determine the number of contributors (NOC) and the NOC must be fewer than or equal to three individuals. Each suitable DNA profile in the pilot study was compared to the ATF Laboratory Staff DNA Index. In general, the profiles were not compared to the KCPD officers that collected the FCCs unless contamination was suspected. The DNA profiles from Experiments 1 through 3 were compared to the handler and analyst that processed the samples. Success is defined as obtaining a DNA profile deemed suitable for comparison. Therefore, the success rate for the FCCs is calculated per DNA sample generated and the success per firearm is defined as obtaining at least one DNA profile suitable for comparison from a group of FCCs assumed to have originated from the same firearm.
3.1. Experiment 1: effects of additives Results from Experiment 1 yielded quantitation values for all samples that were remarkably higher than those typically encountered in ATF casework. Recovery ranged from 200 pg to 14 ng total DNA (Fig. 4A). Complete handler profiles were obtained from all samples. These results may be attributable to using a volunteer who has historically been known to consistently deposit large amounts of DNA, excessive handling of laboratory prepared samples compared to realworld scenarios, and/or the short time between deposition and processing that was possible under research conditions, but that is not necessarily realistic for typical casework. All of the negative control samples were free of DNA with the exception of a single reagent blank that was consistent with the DNA analyst that performed the DNA extraction; however, this DNA profile was not detected in any of the test samples. Although BTmix and GGH both had a mean DNA recovery higher than that of water, significance testing was performed using the Kruskal-Wallis test and the results were not significant. The test was repeated after removing a 7.2 ng sample (approximately 2 times greater than the next highest sample) from the BTmix sample set and a 14.0 ng sample (approximately 7.5 times greater than the next highest sample) from the BSA sample set, but the significance did not change. A Degradation Index value of 1 indicates no degradation. Swabbing solutions containing additives had reduced mean Degradation Index values between 1.0 and 1.4 compared to samples swabbed with sterile water only with a mean of 1.9 (Fig. 4B). The Dunn’s multiple comparison test with the Holm-Bonferroni correction indicate that the difference in the Degradation Index between swabbing with water or GGH was statistically significant (p-value = 0.004, Supplemental Data, S1A). There is a large difference between swabbing with water (Average DI = 1.9) or BTmix (Average DI = 1.2), but it did not reach the level of significance (p-value = 0.068). The data indicate that using BSA or GGH alone as a swabbing or rinse-and-swab additive may have beneficial effects for FCCs, but the combined effect appeared to increase the DNA yield compared to each additive individually while still decreasing the Degradation Index. Therefore, all further experiments within this study were performed using both additives in concert as the BTmix.
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calculated using the combined data from Experiments 2 and 3 and compared for each of the recovery methods to ensure that the increased DNA recovery translated to an increase in genetic data detected. The average RFU per locus for each recovery method is summarized in Fig. 6 and as a percentage of the handler’s alleles (Supplemental Data, S4). The two-tailed Mann-Whitney U test pairwise comparisons with the Holm-Bonferroni correction indicate that the recovery of DNA as measured by the percentage of handler’s alleles detected (pvalue = 0.0004) and the average RFU per locus (p-value = 0.0003) are significantly higher using the rinse-and-swab method with BTmix when compared to the double swab method with water (Supplemental Data, S1C and S1D). 3.3. Real-world samples FCCs from crime scenes were collected with the cartridge case collector and DNA was recovered using the rinse-and-swab method with BTmix. FCCs were collected at one or more shooting scenes over a weekend and submitted to the laboratory as a batch the following week. Over the course of this study, six separate batches of FCCs were submitted and assumed to represent fifteen different firearms. The batches were separated into two groups (Group 1 consists of batches 1–3; Group 2 consists of batches 4–6). FCCs from the first group (Group 1, n = 47) were analyzed individually. For these samples, the rinse-and-swab method was performed on a single cartridge case and the rest of the analysis proceeded without combining the extract with any other sample. To simulate processing of samples in a typical casework flow, the second group of FCCs (Group 2, n = 43) was analyzed in pairs. For these samples, DNA was collected from both cartridge cases using the same rinse solution. When deemed necessary, the DNA extracts were combined into a single sample prior to PCR amplification to reflect normal laboratory processes. For example, if two different DNA extracts assumed to have originated from the same firearm contained levels of DNA that would not be expected to produce an interpretable DNA profile, the two samples were combined into a single sample at the concentration step. The 43 paired cartridge cases resulted in a total of 20 DNA samples (21 analyzed as pairs, 1 analyzed individually due to the odd number of cartridge cases in the batch, and two sets of DNA extracts combined prior to PCR amplification). The negative control samples from each extraction batch were free of DNA. On-scene contamination was observed in the second batch. Both of the suitable DNA profiles in Batch 2 were identified as consistent single source DNA profiles that were attributable to the officer that collected the FCCs. A summary of these results not including the two FCCs and one firearm associated with contamination are listed in Tables 1 and 2. A more detailed summary of the results for each FCC including the caliber, metal composition, damage, time between incident and collection, total DNA recovered, DNA typing success, and number of contributors can be found in the Supplementary Data (S5). The DNA profiles determined to be suitable for comparison were divided into the following categories and summarized in Table 3: single source, two person mixtures, and three person mixtures. Three single source DNA profiles were obtained from the FCCs, however two of these were confirmed to be contamination during collection and are not included in the table or calculated success rates.
Fig. 4. Total DNA recovered (A) and the Degradation Index per sample (B) determined by Quantifiler™ HP analysis for cartridge cases swabbed using the following swabbing solutions: water, BTmix, BSA, and GGH. Two data points for the DNA recovery (A) using BTmix and BSA (approximately 7 ng and 14 ng, respectively) were removed from the figure and calculated means.
3.2. Experiments 2 and 3: comparison of collection methods DNA was recovered from FCCs by either double swabbing with water, double swabbing with water and BTmix, rinse-and-swab method with Buffer ATL, or rinse-and-swab method with Buffer ATL and BTmix. All DNA samples were concentrated to a final volume of 15 μL and the entire volume was amplified with the exception of one sample in the rinse-and-swab with BTmix that was considered to be outlier data and not used in the calculations as noted below. The mean of the total recovered DNA per sample type was 49 pg, 85 pg, 153 pg, and 143 pg, respectively (Supplemental Data, S2). One outlier data point was removed from the mean calculation for the rinse-and-swab with BTmix (approximately 1.7 ng, > 3 times IQR). Duplication of the study yielded similar mean total DNA recoveries: 54 pg, 78 pg, 134 pg, and 171 pg, respectively (Supplemental Data, S3). Statistical significance testing was performed on the combined data set. The difference in the total DNA recovered between the double swab method using water as the wetting solution and rinse-and-swab method with the BTmix additives was statistically significant (Fig. 5) using a two-tailed Mann-Whitney U test with the Holm-Bonferroni correction p-value = 0.0009). Complete pairwise comparisons can be seen in Supplemental Data (S1B). The negative control samples in both replicates were free of DNA. The average peak heights of the handler’s alleles were also
3.4. Discussion FCCs are not routinely processed by the forensic DNA community due to the perception that informative results are too rare for the effort required. However, novel DNA recovery methods, increased sensitivity in amplification kits, more efficient DNA extraction methods, and improvements in the interpretation of low level and mixed DNA profiles have demonstrated increased success rates for FCCs [10]. In Experiment 1, our study showed that adding BSA and/or the copper-binding 5
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Fig. 5. Combined results of the collection method comparisons. Total DNA (pg) recovered from FCCs using four different methods: double swabbing with water, double swabbing with water and BTmix, rinse-and-swab method with Buffer ATL, or rinse-and-swab method with Buffer ATL and BTmix.
Fig. 6. Combined average RFU per locus from duplicate Experiments 2 and 3.
success rates per cartridge case and per firearm for Group 1 were 27 % (12 of 45) and 56 % (5 of 9), respectively. These samples were collected in three batches. The first and third batches were collected within hours of the shooting incidents and produced combined success rates per cartridge case and firearm of 35 % (12 of 34) and 71 % (5 of 7), respectively. The second batch was collected days to weeks after the shooting incidents, during which it had rained at least once. Additionally, the majority of the cartridge cases from the second batch (10 of 13) showed signs of damage, possibly from having been run over by at least one car. These cartridge cases did not produce DNA profiles suitable for comparison purposes. The only confirmed incident of contamination for all FCCs collected with the cartridge case collector occurred in Batch 2 when the FCCs had been damaged causing the opening to be significantly reduced. Although damaged, the collecting officer still attempted to use the collector by forcing the FCC onto the
tripeptide GGH to the swabbing solution decreases the DNA Degradation Index when compared to swabbing with water alone (Fig. 4B and Supplemental Data, S1A). In Experiments 2 and 3, the rinse-and-swab method with BTmix reproducibly demonstrated increases in total DNA recovered (approximately threefold; Fig. 5 and Supplemental Data, S1B) and average peak height per locus (over fourfold; Fig. 6 and Supplemental Data, S1D) compared to the traditional double swab method using water as the swabbing solution on FCCs handled and fired in the laboratory. Inhibition may still play a part in affecting the successful analysis of these samples, however, the actual electropherograms and quantification IPC data did not suggest inhibition was a major factor. While mock samples prepared in the laboratory provide valuable data, analyzing real-world samples is the true test of a new method. When the confirmed contamination samples in Batch 2 are removed the
Table 1 Summary of results for crime scene FCCs analyzed individually and in pairs. One cartridge case in Batch 5 was analyzed individually. *: Two DNA samples generated and the subsequent DNA profiles were removed from Batch 2 due to contamination during collection. Analyzed Individually (Group 1)
# of Cartridge Cases # of DNA Samples Generated # of Profiles Suitable % Success / Sample
Analyzed in Pairs (Group 2)
Batch 1
Batch 2
Batch 3
Total
Batch 4
Batch 5
Batch 6
Total
14 14 4 29 %
13 11* 0* 0%
20 20 8 40 %
47 45 12 27 %
20 9 1 11 %
9 5 0 0%
14 6 2 33 %
43 20 3 15 %
6
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Table 2 Summary of results per firearm when the FCCs were analyzed individually and in pairs. *: One firearm was removed from Batch 2 due to contamination during collection. Analyzed Individually (Group 1)
# of Firearms Successful Analysis % Success / Firearm
Analyzed in Pairs (Group 2)
Batch 1
Batch 2
Batch 3
Total
Batch 4
Batch 5
Batch 6
Total
4 3 75 %
2* 0 0%
3 2 67 %
9 5 56 %
2 1 50 %
1 0 0%
2 2 100 %
5 3 60 %
FCCs (18 cartridge cases total) were covered in dried mud and produced a single DNA profile suitable for comparison. A third set of five FCCs produced only one DNA extract with a detectable amount of DNA, but did not result in a DNA profile suitable for comparison purposes. The final two sets of FCCs, however, both produced at least one DNA profile suitable for comparison and resulted in quantifiable DNA in four of the seven DNA extracts. Both this study and Montpetit et al. [3] studies found that mixed DNA profiles greatly outnumber single source profiles at a ratio of at least 2 to 1 (Table 3). The cause of the mixtures can only be speculated, but some possible sources may be a shared ammunition magazine, the chamber of the firearm, secondary transfer from the handler, victim blood, or shared ammunition stored loose in a communal bag. These results could be used as an argument to analyze the FCCs individually to prevent the creation of uninterpretable mixtures where a DNA profile suitable for comparison might have existed. Additionally, total DNA quantities obtained from FCCs in this study are consistent with the results of Montpetit et al. [3]. This study demonstrated that 40 % of the FCCs from casework analyzed individually with the rinse-and-swab method using BTmix yielded at least 50 pg of total DNA that is similar to the 33 % described by Montpetit et al. During the course of this study, in addition to the collaboration with KCPD and KCPCL, FCCs from two other investigations involving a total of three firearms (a total of 30 FCCs) were analyzed using the rinse-andswab method with BTmix. These FCCs were submitted to the ATF Laboratory individually wrapped tightly in paper towels and placed in a small cardboard box several months earlier. In all three instances, at least one DNA profile suitable for comparison was developed from each group of FCCs assumed to have originated from the same firearm. One of these profiles was consistent with the person of interest and another resulted in a CODIS hit. This demonstrates that even with non-ideal circumstances, probative DNA profiles can be obtained from FCCs using the rinse-and-swab method. As with analysis of all evidence, the probative value and order of the examinations must be weighed against the timing and potential loss of valuable information. Efforts are currently underway to increase the percentage of recovered firearms and FCCs entered into the NIBIN database within 48 h to maximize their value for producing investigative leads. The process of entering FCCs into NIBIN, which includes cleaning the cartridge cases, does not allow for downstream DNA analysis. As part of this study, we wanted to determine how the rinse-and-swab method would affect the timing for entry of FCCs into NIBIN. DNA collection from individual cartridge cases took approximately five minutes each. Therefore, the DNA collection from twelve cartridge cases could be completed within an hour. For most of the samples
cap post. When used as designed there is no need to handle the FCC and this extra sample manipulation may be the cause of the contamination. This demonstrates the necessity to train end-users in both contamination prevention techniques during evidence collection and the use of this specific device for the collection of FCCs. Batch 2 demonstrates several important points. First, it is critical to collect the FCCs as soon as possible to avoid loss of DNA due to weather or other environmental factors that may occur in the intervening time. Second, the FCCs should be analyzed as soon as possible due to the loss of DNA over time when deposited on metal surfaces [29]. This same effect has been observed when cells were deposited directly on brass cartridge cases (data not shown). Finally, great care must be taken to avoid contaminating any evidence, and even more so when it is expected to contain trace quantities. The variability in the results reflects the nature of trace DNA analysis on any object. For example, a single firearm in this study was associated with nine FCCs, three of which produced quantifiable DNA and only one of those had greater than 100 pg of total DNA. A different firearm was associated with seven FCCs and produced detectable DNA in all the extracts, four of which contained greater than 100 pg total DNA. Many factors can affect the transfer and persistence of trace DNA. Additionally, the caliber of cartridge case, make and model of firearm, length of time between deposition and collection, and the metal composition may impact the results when analyzing FCCs. The vast majority of the FCCs collected from crime scenes in this study were composed of brass (80 brass, 9 nickel-plated, and 1 unknown). Given that brass substrates were previously demonstrated to be a challenge due to the possible degradative and inhibitory effects, it is encouraging that all of the DNA profiles suitable for comparison were obtained from brass FCCs. With respect to caliber, DNA analysis was successful on three of the four calibers observed (9 mm, .40 caliber, and .45 caliber). Only five .357 caliber cartridge cases were examined and no DNA profiles suitable for comparison were obtained. The surface areas for these calibers are not appreciably different and therefore, surface area would not be expected to be a major factor when comparing these results. Surface area may be more of a factor for smaller caliber FCCs such as .22 and .25, but these are less common. These FCC-specific factors warrant future studies. The analysis of the second group of crime scene FCCs (Group 2), which were analyzed in pairs, was less successful. The success rates per DNA sample and per firearm were 15 % (3 of 20) and 60 % (3 of 5), respectively. Due to the relatively low number of FCCs and assumed associated firearms, it was not possible to determine if the difference between Group 1 and Group 2 is statistically significant. As noted above, many factors can affect the success rate. In Group 2, two sets of
Table 3 Summary of single source, two person mixtures, and three person mixtures for DNA profiles determined to be suitable for comparison purposes. *: Two single source profiles were removed from Batch 2 due to contamination during collection. Types of Profiles
Single Source Profiles
Two Person Mix
Three Person Mix
Total Suitable Profiles
# of Each, Sampled Individually # of Each, Sampled in Pairs # of Each, Total % of Suitable Profiles
1* 0 1 7%
8 3 10 67 %
3 0 4 27 %
12 3 15
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collected in this study, DNA collection was performed within 24 h of the evidence being received by the laboratory and the cartridge cases were entered into NIBIN within the next 12 h. Samples in this study were not processed for latent prints, thus the effects of latent print chemical processing of FCCs on the effectiveness of the rinse-and-swab method with BTmix was not examined; however, traditional methods of latent print development on FCCs have typically resulted in low success rates [30,31]. This does not include the relatively new RECOVER Latent Fingerprint Technology process (Foster and Freeman, Sterling, VA) [32] that may be more effective. Future research should be performed to determine if the rinse-and-swab method with BTmix could be performed prior to the RECOVER:LFT process without affecting the ability to visualize latent prints. One of the aims of this research was to develop a method that was easily incorporated into a laboratory’s workflow. In its current form, the rinse-and-swab method with BTmix in conjunction with the modified QIAamp® DNA Micro extraction protocol requires splitting the lysate into two tubes that are combined onto the same silica column. Although viewed as a manageable burden given the increase in success rate, research is currently underway that indicates similar or better results when a reduced rinse volume is used during the DNA collection steps. Reducing the rinse volume will allow for the use of a single lysate tube, simplify the process, and allow for automation without sacrificing DNA recovery. This study, in addition to other studies examining the DNA analysis of FCCs, demonstrates that it is possible to obtain DNA profiles suitable for comparison from FCCs with a relatively high rate of success; therefore, the forensic science community should view FCCs as viable sources of DNA. The main goal of this study was to develop methods to improve various aspects of the collection and DNA analysis of FCCs that can be practically implemented in order to increase success rates. As discussed in the Montpetit review [10], comparing results among studies performed by multiple independent laboratories is difficult due to differing extraction methods, analysis parameters, amplification kit sensitivities, and interpretation thresholds. A strength of this study is that it offers methods that can be easily adapted into most forensic laboratory’s existing workflows, as it uses standard laboratory equipment and only a minor modification to the collection method and swabbing solution. The results demonstrated a high rate of success using the GlobalFiler™ PCR amplification kits without employing enhanced sensitivity techniques. Both the quantity (approximate threefold increase) and the quality (decreased Degradation Index) of the DNA recovered were improved using the rinse-and-swab method with BTmix compared to conventional double swab method with water. Finally, even the best DNA collection techniques will not overcome the loss of DNA at the crime scene or during collection and transport to the forensic laboratory. This study has shown that in order to maximize successful results, that the entire process must be considered. Proper collection and preservation starting at the scene followed by an improved DNA collection method can combine to significantly improve the success rate of DNA analysis from FCCs.
[3] [4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12] [13] [14]
[15]
[16] [17]
[18]
[19]
[20] [21]
[22]
[23]
[24]
[25] [26] [27]
Declaration of Competing Interest
[28]
None.
[29]
Appendix A. Supplementary data [30]
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigen.2020.102238.
[31]
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