J Forensic Sci, 2015 doi: 10.1111/1556-4029.12704 Available online at: onlinelibrary.wiley.com
TECHNICAL NOTE CRIMINALISICS
Shakhawan K. Mawlood,1 M.S.; Majid Alrowaithi,1 Ph.D.; and Nigel Watson,2 Ph.D.
Advantage of ForensiX Swabs in Retrieving and Preserving Biological Fluids*
ABSTRACT: This study compares two novel swabs (forensiX) with a standard cotton swab (EUROTUBE) for the collection of saliva stains
on glass slide for STR analysis. ForensiX collection tubes are a standard cotton swab in an “active drying” tube, where swab sample is soon dried by its innovative tube surface of the wall. The other is forensiX Nylon Flocked Swab. The study is two phases: The first “phase” assesses swab types regarding to retrieve ability of saliva. The second “phase” compares the drying ability of each swab to assess how crime samples would fare when left in storage. The main result showed that “active drying” is effective to store swabbed sample. The forensiX swabs generally are effective for higher (twofold to fourfold) DNA yield compared to delta lab swab (around 750 pg and 250 pg from 0.5 lL of saliva), respectively. These findings demonstrate the importance of drying performance in the preservation of DNA and swab selection.
KEYWORDS: forensic science, DNA extraction, swab types, active drying, retrieve ability, preservation
Human identification by DNA testing is now a highly developed technique. A limiting step in the process is the retrieval of the DNA from the biological stains which are found at crime scenes. There is more than one method for sample collection, but a common approach to recovering dried stains is the double swab technique (1,2).In this technique, a wet swab is used to rehydrate and lift cells, before a second, dry, swab is applied to further recover any remaining cells (3). For DNA collection purposes, various swabs are commercially available, each of which contains different characteristics and comes at a different price. A previous study which compared the retrieval ability of nylon with cotton swabs showed that the retrieval ability of these swabs was different depending on the extraction method (4). Recently a new type of “swab” with an active drying property was manufactured. In this type of swabs, the active drying takes the form of a special internal lining of the swab case that actively absorbs moisture from around the swab when it is returned to the swab case or sheath. The manufacturers claim that this feature improves the rate of drying in comparison with air-drying or air-freezing (i.e., passive drying) to limit DNA samples’ degradation prior to their arrival at the laboratory (5–8). In addition, the drying occurs within the swab independently of outside conditions and this consequently minimizes the opportunity for contamination. 1 Centre for Forensic Science, Department of Pure and Applied Chemistry (WestCHEM), University of Strathclyde, Royal College R 6.20, 204 George Street, G1 1XW Glasgow, UK. 2 Centre for Forensic Science, Department of Pure and Applied Chemistry, University of Strathclyde, Royal College 204 George Street, Glasgow G1 1XW, UK. *Presented in part at the International Symposium on Human Identification 24 (ISHI24), October 7-10, 2013, in Atlanta, GA. Funded by Kurdistan Forensic Laboratory as a scholarship support. Received 11 Dec. 2013; and in revised form 13 April 2014; accepted 27 April 2014.
© 2015 American Academy of Forensic Sciences
This study was designed to determine whether or not swabs which have been described by the manufacturers as “actively drying,” that is, where the swab tube contains a propriety drying system, are in fact more efficient in the collection and release of dried biological fluids than commercially available sample tools. Three types of swabs from two different manufacturers were tested in this experiment (the manufacturers were delta lab and Prionics). The cotton swabs used for comparison were the EUROTUBEâ Collection Swab from (Deltalab, Spain), and the forensiX Evidence Collection Tube (Prionics AG, SchlierenZurich, Switzerland)., which contain an active desiccant system and either a cotton swab (forensiX Buccal Swab tube) or a nylon-flocked swab (forensiX Nylonâ Flocked Swab tube) as shown in (Fig. 1). The forensiX swabs consist of an active drying tube and a cotton swab. All of these were commercially available at the time of testing. The swabs differ from each other primarily in terms of the swab head, size, and material type (Table. 1). This study consisted of two phases (of 120 samples each) with saliva used in both phases. The first phase tested the collection of a different volume of saliva (0.5–10 lL) to compare the efficiency of each type of swab in instances, where measured quantities of saliva, collected from the same person at the same time, were used. The purpose of this phase was to investigate whether or not there was a difference in the volume of biological material that each type of swab could retrieve. However, this does not represent the circumstances typically encountered at a crime scene, where the amount of saliva contributing to a stain is unknown and where there may not be the opportunity to treat the swab or to let it dry before returning it to its tube. Accordingly, the second phase of this study examined the extraction and subsequent storage of saliva for different periods of time, with regard to DNA extractions made after 0 h, 4 h, 1 day, and 40 days of preservation. The aim of this phase was to find out whether the drying performance had a significant influence on 1
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was applied on all slides (each of the 40 slides was swabbed using one of the three tested swab types). The swabs were then placed back into their packaging. Then, the swab type was classified into four groups marked as 0 h, 4 h, 1 day, and 40 days (in total, each group contained ten of each swab type). The first group marked as 0 h was immediately extracted. The remaining three sets of swabs were stored at room temperature and extracted when they reach the desirable time as designated in the second phase (i.e., 4 h, 1 day, and 40 days). Generally, swab heads were removed from the swab shaft, while the cotton swabs of EUROTUBE were cut by cleaned scissors. Then, the swabs were placed into sterile (2 mL) micro centrifuge tubes, ready for extraction. Extraction and Quantitation FIG. 1––The used three tested swab types. (A) EUROTUBEâ Collection Swab (delta lab, Spain). (B) forensiX Buccal Swab tube. (C) forensiX Nylonâ Flocked Swab tube (Prionics AG, Schlieren-Zurich, Switzerland). Figure of (Swab B) illustrated from Prionics.
TABLE 1––Summary of the swab types and structure.
Swab Type â
Manufacture and Country
Cat#
A
EUROTUBE Collection Swab
(Delta lab, Spain)
30252
B
ForensiX Buccal Swab tube
9012030
C
ForensiX Nylonâ Flocked Swab tube
(Prionics AG, Schlieren-Zurich, Switzerland) (Prionics AG, Schlieren-Zurich, Switzerland)
9022045
Made of Wood shaft and cotton head Wood shaft and cotton head Plastic shaft and nylon head
the DNA yield for each of the swab types for different time periods. Saliva sample was taken from one donor and it was thoroughly mixed to ensure homogeneity of the samples. Saliva from this batch was dispensed on all the swabs used in the test. The three different swab types were employed and the DNA recovery of each was compared.
Material and Method Slide Preparation and Swabbing In the first phase, saliva was collected in a clean tube from one volunteer on a single occasion. The saliva was then mixed thoroughly for 30 sec to form a homogenous solution, with a brief spin down to remove debris. Four varied quantities of saliva (of 0.5, 2.5, 5, and 10 lL) were immediately pipetted onto sterilized, labeled, microscope slides in order to prepare four sets of glass slides (30 for each set) of the three types of swab to be tested. The slides were left to dry for 30 min at room temperature inside a sterile laminar flow hood. Finally, the dried saliva on each slide was swabbed using a double swab technique (which mimics the technique of stain collection in real case scenarios at a crime scene) and extracted directly. A separate sterile clean glass slide was used with each set as a negative control. In the second phase of the experiment, 1 lL of saliva was aliquoted onto 120 sterilized, labeled, microscope slides. After 30 min of drying at room temperature, a double swab technique
The total DNA was isolated using the QIAamp DNA Investigator Kit (QIAGEN, Crawley, UK). The elution value was set as 50 lL of ATE buffer (QIAGEN, Crawley, UK). After extraction, each of the sets of swabs from both phases was quantified using the Qiagen Investigator Quantiplex kit (Cat. # 387016) with a Stratagene Mx3005P thermo cycler according to the manufacturer’s instructions. The quantities of DNA were compared to one another and also to the negative control extracted using the same extraction method. If any of the swabs did not work with this protocol, this was noted. DNA Amplification and Profiling Some samples from each of the sets were amplified using a 2720 thermal cycler (Life Technologies, UK) with the Investigator Decaplex SE kit (Cat. # 381025) from (QIAGEN). After running these samples on the 3130 Genetic Analyzer (Applied Biosystem, Life Technologies, Paisley, UK), the expected DNA profile was produced. There was no evidence of contamination. Statistical Analysis Microsoft Excel and Minitabâ 16 software were used to conduct the statistical analysis for this experiment, using a one-way ANOVA test for significance between the results from the three different types of swabs. Results In Phase One, the results of DNA recovery among the three swab types (EUROTUBE collection swab (delta lab), buccal swab tube, and nylon-flocked swab tube (forensiX)) demonstrated that the nylon-flocked swab tube was the most effective performer in terms of sample recovery of the three swabs. Regarding the saliva stains with volumes of 2.5, 5, and 10 lL, the three tested swabs showed different DNA recovering abilities. Generally, forensiX Evidence Collection tubes generated higher results than the EUROTUBE collection swab, and the average recovered DNA with the nylon-flocked swab was two and three times higher than the buccal swab tube and EUROTUBE collection swab, respectively (Fig. 2). One of the most interesting results related to the saliva stain with the smallest volume (0.5 lL), where 750 pg of DNA per microliter was recovered with the nylon-flocked swab. In comparison, only one-third of this result was retrieved with the EUROTUBE swab (Fig. 3).
MAWLOOD ET AL.
FIG. 2––The retrieval ability of swab types. Different saliva stain (2.5, 5, 10 lL) volumes using different swab types (the EUROTUBE collection swab, the forensiX buccal swab tube, and the forensiX nylon-flocked swab).
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ADVANTAGE OF FORENSIX SWABS
3
FIG. 4––Swab preservation ability. Bar chart of retrieval ability of collected saliva stains preserved for different periods of time (0 h, 4 h, 1 day, and 40 days).
In addition, after storage for 40 days at room temperature (samples 91–120), both forensiX swabs (the nylon-flocked and the buccal swab tube) recovered more DNA. This amount was more than four times that recovered by the EUROTUBE collection swab. In contrast, with a preserved saliva stain with EUROTUBE collection swab after the same period (40 days), only a few picograms of DNA was recovered, and in some samples, no DNA was recovered at all. Comparison of the results under different storage conditions using a one-way ANOVA test revealed a highly significant difference among the swabs (p ˂ 0.0001). The pairwise comparison study showed that the DNA yield of the three types of swabs significantly differed from one another. FIG. 3––The retrieval ability of 0.5 lL saliva. Using different swab types (the EUROTUBE collection swab, the forensiX buccal swab tube, and the forensiX nylon-flocked swab).
The results of a one-way ANOVA test showed that the DNA yield of the three types of swabs was significantly different (F (2,117) P-value ˂0.001). In addition, a post hoc pairwise comparison was performed to identify which swab type’s DNA yield differed from the others. The results showed a significant difference between the yield of the forensiX nylon-flocked tube and that of either the forensiX buccal swab tube or of the EUROTUBE collection swab (F(1,78) p-value ˂0.001 and F(1,78) p-value ˂0.005, respectively). In addition, the yield of the forensiX buccal swab tube was not significantly different from the EUROTUBE collection swab (F(1,78) p-value ˂0.001). Figure 4 shows the results of Phase Two of the experiment, concerning the effectiveness of preservation for different time periods (0 h, 4 h, 1 day, and 40 days) across the three swabs. As the figure illustrates, the amount of recovered saliva was nearly the same among the swab types in the first set of swabs which were extracted immediately (samples 1–30). The differences in the DNA yield become more prominent between the different swab types when the preservation time is increased. The amount after 4 h (samples 31–60) and 1 day (samples 61–90) was around 0.30 ng/lL and 0.35 ng/lL for the EUROTUBE collection swab and buccal swab tube, respectively. For the nylon-flocked swab tubes, the average amount of recovered DNA was above 0.6 ng/lL for the samples which had been stored for 4 h and for 1 day.
Discussion This study was designed to compare the forensiX evidence collection swab tubes to the EUROTUBEâ Collection swab through simulating the collection of saliva stains as would be encountered at a crime scene. Obtaining the DNA profiles of a possible contributor is usually a long process, and collection of the samples with a swab is the first step. To reduce DNA degradation of the collected biological fluids, the samples may be stored for days, weeks or even more in the laboratory before the DNA extraction proceeds. Biological degradation is therefore highly likely in some cases, and this is most intricate for a small DNA sample, such as a trace DNA sample. In the first phase of the study, generally, it was found when using different volumes of saliva that the forensiX nylon-flocked swab tubes retrieved a larger quantity of DNA than other cotton collection swabs. This was particularly apparent with the smallest tested volume, that is, 0.5 lL saliva. In contrast, collection via EUROTUBE swabs recovered only a few picograms of DNA. Results were close to the negative controls in some samples. Several factors affect the efficiency of DNA retrieval among the swabs, these factors may result in the observed difference in DNA yield. One such factor that should be taken into consideration is the saliva recovery capability of each of the three tested swabs. Another significant factor is the efficiency of the swab to release the evidence which has been collected. The swab may be effective at saliva collection, but not efficient at release during the extraction process, in which case the DNA yield will be reduced.
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The manufacturer claims that the forensiX nylon-flocked swabs improve the yield in two ways (because they are made of nylon instead of cotton). First, sample absorption into the swab is improved because the sample is more quickly absorbed by capillary action. Secondly, sample retention in the swab is reduced, as the material is stored in close proximity to the surface of the swab rather than being internalized, thereby improving the release of the sample material when it is needed (3,9). This hypothesis may explain the differences between the forensiX product itself (nylonflocked and buccal swab tubes) on the one hand, and EUROTUBE collection swab on the other. In addition, the shaft part of the nylon-flocked swab is also made of nylon. This could be another reason for its higher level of efficiency. The shaft part of the other two swab types is made of wood, which may absorb some moisture and biological material (10). In the second phase of the experiment, the results illustrated the advantage of using the forensiX swab tube rather than the EUROTUBE collection swabs to preserve samples for long periods of time. Interestingly, after a significant period of time (of up to 40 days), it was still possible to retrieve nearly the same amount of DNA which had been immediately retrieved by direct extraction (at 0 h). The forensiX collection tool consists of two parts: a swab and a plastic tube. The inner surface area of the plastic tube contains a “SafeDry” desiccant which is composed of a molecular sieve drying agent in a polymer matrix. This system allows the sample swab to be actively dried by absorbing the moisture and the humidity from the air space around the sample. Therefore, this rapid active drying method improves the retrieval results because it at least partially prevents the bacteria and fungi existing in collected saliva from degrading the DNA. Furthermore, the tube isolates the saliva on the swab head from exposure to the influences of the external environment. Recently, Garvi et al. (10) demonstrated that an active drying method is better than other methods which some laboratories were following to store collected biological evidence, for example, passive drying, cooling, and freezing (10). On the other hand, the amount of DNA retrieved with the EUROTUBE collection swab decreased dramatically with an increased time of storage. This was most apparent among the samples that were stored for 40 days, where some of the samples showed a nonquantifiably low level of DNA. The improved drying performance of the forensiX nylonflocked swab tubes may be due not only to the active drying system which they feature, but also to the nature of the swab head, which consists of a brush-like head, from which a solution containing the biological material from a stain might be more readily eluted. It is possible that it is a combination of these factors that contributes to the improved performance of these swabs. Another advantage of these swabs is that the design of the swab shaft of the forensiX evidence collection tool has a head which can be readily cut off for deposition in a tube for extraction and that it also includes the tube identification number on both the tube sheath and the swab itself. It is therefore probable that although the forensiX swab tubes cost more than commercially collection swabs, the performance ability advantage of the forensiX swab tubes will counterbalance the higher price.
Conclusions In conclusion, the present study has demonstrated improved yields and the preservation of the DNA by the forensiX swab tools (both Nylon and Cotton) when compared with the EUROTUBE collection swabs. This finding indicates the importance of using such a tool, particularly with regard to the small saliva stains which may be stored for long period of time, that are common from crime scenes. Additional investigation should be conducted with different drying systems, concentrations, longer periods of storage, use of the swabs on other substrates, and, finally, studies which can draw comparisons with additional types of swab, for example, Copan 4N6FLOQSwabsTM (Copan Italia, Brescia, Italy) as recently studied by Dadhania et al. (5). Acknowledgment The authors would like to thank (Lardi Elges) from Prionics, who provided the swabs and the volunteer for donation the sample. References 1. Sweet D, Lorente J, Valenzuela A, Lorente M, Villanueva E. PCR-based DNA typing of saliva stains recovered from human skin. J Forensic Sci 1997;42(3):447. 2. Sweet D, Lorente M, Lorente JA, Valenzuela A, Villanueva E. An improved method to recover saliva from human skin: the double swab technique. J Forensic Sci 1997;42:320–2. 3. Pang B, Cheung B. Double swab technique for collecting touched evidence. Leg Med 2007;9(4):181–4. 4. Brownlow RJ, Dagnall KE, Ames CE. A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods. J Forensic Sci 2012;57:713–7. 5. Dadhania A, Nelson M, Caves G, Santiago R, Podini D. Evaluation of Copan 4N6FLOQSwabsTM used for crime scene evidence collection. Forensic Sci Int-Gen Sup Series 2013;4(1):e336–7. 6. Hofreiter M, Serre D, Poinar HN, Kuch M, P€a€abo S. Ancient DNA. Nat Rev Genet 2001;2(5):353–9. 7. Lindahl T. Instability and decay of the primary structure of DNA. Nature 1993;362(6422):709–15. 8. Overballe-Petersen S, Orlando L, Willerslev E. Next-generation sequencing offers new insights into DNA degradation. Trends Biotechnol 2012;30(7):364–8. 9. Dalmaso G, Bini M, Paroni R, Ferrari M. Qualification of high-recovery, flocked swabs as compared to traditional rayon swabs for microbiological environmental monitoring of surfaces. PDA J Pharm Sci Technol 2008;62(3):191–9. 10. Garvin AM, Holzinger R, Berner F, Krebs W, Hostettler B, Lardi E, et al. The forensiX evidence collection tube and its impact on DNA preservation and recovery. Biomed Res Int 2013; vol. 2013: Article ID 105797, 7 pages, 2013. doi:10.1155/2013/105797. Additional information and reprint requests: Shakhawan K. Mawlood, M.S. Centre for Forensic Science Department of Pure and Applied Chemistry (WestCHEM) University of Strathclyde Royal College R 6.20, 204 George Street Glasgow G1 1XW, Scotland, UK E-mail: [emailprotected]
