Contribution to the Development of Guidelines in the Analysis of Biological Evidence in Sexual Assault Investigations

This investigation intends to study materials and techniques used for biological evidence collection in sexual assault cases and is divided into two stages: in stage one, methods for biological evidence collection (the single swab (including three variants) and the “double swab technique”) were compared; in stage two, swabs’ component material was compared. The sampling was composed of 42 heterosexual couples who provided mock samples. The collection methods in which the whole swab is covered by evidence presented significantly better outcomes (p < 0.001), such as the “double swab technique.” Additionally, nylon swabs proved to present significantly better features regarding the capacity of sample elution, providing significantly higher amounts of DNA (p ≤ 0.034). This study provides guidelines for better collection of biological evidence regarding the collection method using a swab and the proper swab material to utilize.     

Advantage of ForensiX Swabs in Retrieving and Preserving Biological Fluids

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 μL of saliva), respectively. These findings demonstrate the importance of drying performance in the preservation of DNA and swab selection.     

Nondestructive Biological Evidence Collection with Alternative Swabs and Adhesive Lifters

In forensic science, biological material is typically collected from evidence via wet/dry double swabbing with cotton swabs, which is effective but can visibly damage an item's surface. When an item's appearance must be maintained, dry swabbing and tape‐lifting may be employed as collection techniques that are visually nondestructive to substrates' surfaces. This study examined the efficacy of alternative swab matrices and adhesive lifters when collecting blood and fingerprints from glass, painted drywall, 100% cotton, and copy paper. Data were evaluated by determining the percent profile and quality score for each STR profile generated. Hydraflock^® swabs, BVDA Gellifters^®, and Scenesafe FAST? tape performed as well as or better than cotton swabs when collecting fingerprints from painted drywall and 100% cotton. Collection success was also dependent on the type of biological material sampled and the substrate on which it was deposited. These results demonstrated that alternative swabs and adhesive lifters can be effective for nondestructive DNA collection from various substrates.     

The Extraction and Recovery Efficiency of Pure DNA for Different Types of Swabs

The extraction and recovery efficiency of swabs used to collect evidence at crime scenes is relatively low (typically <50%) for bacterial spores and body fluids. Cell‐free deoxyribonucleic acid (DNA) is an interesting alternative compared to whole cells as a source for forensic analysis, but extraction and recovery from swabs has not been tested before using pure DNA. In this study cotton, foam, nylon flocked, polyester and rayon swabs are investigated in order to collect pure DNA isolated from saliva samples. The morphology and absorption capacity of swabs is studied. Extraction and recovery efficiencies are determined and compared to the maximum theoretical efficiency. The results indicate that a substantial part of DNA is not extracted from the swab and some types of swab seem to bind effectively with DNA. The efficiency of the different types of swab never exceeds 50%. The nylon flocked 4N6FLOQSwab used for buccal sampling performs the best.     

A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods

The Metropolitan Police Service currently uses cotton swabs to retrieve DNA for forensic profiling. Recently, a new nylon flocked swab type has become available from Copan (MicroRheologics, Brescia, Italy) that it is claimed, offers increased sample recovery and release yields. If true, the flocked swab may have important applications in DNA evidence retrieval. This study examines the DNA retrieval capability of cotton and nylon flocked swabs when extracted using three common extraction platforms (QIAcube, BioRobot EZ1 and manually processed QIAamp DNA investigator kit). Results indicate that both swab types are capable of recovering high percentages of DNA (>50%); however, the extraction platform selected was shown to have a significant effect upon DNA retrieval. Across all experiments, the cotton swab combined with the spin-column extractions was shown to be most effective, with the nylon swab and BioRobot EZ1 combination being the least effective. These findings illustrate the importance of extraction method selection.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

A Comparison of Common Swabbing Materials for the Recovery of Organic and Inorganic Explosive Residues

The efficiency of solvent based extraction methods used to remove explosive residues from four different swab types was investigated. Known amounts of organic and inorganic residues were spiked onto a swab surface with acetonitrile or ethanol:water combined with ultrasonication or physical manipulation used to extract the residues from each swab. The efficiency of each procedure was then calculated using liquid chromatography‐ultraviolet detection for organic residues and ion chromatography for inorganic residues. Results indicated that acetonitrile combined with physical agitation proved to be the most efficient method; returning analyte recoveries c. 95% for both alcohol based swabs and cotton balls. Inorganic residues were efficiently extracted using ethanol:water, while the use of acetonitrile followed by water significantly reduced the recovery of inorganic residues. Swab storage conditions were then investigated with results indicating decreased storage temperatures are required to retain the more volatile explosives.     

Comparison of operational DNA recovery methods: Swabs versus tapelifts

It is routine among many jurisdictions to recover DNA using tapelifts on porous substrates (e.g. clothing) and swabs on non-porous substrates (e.g. tool handles). Here, we examine this by comparing the efficiency of the NSW jurisdiction’s specific swabbing and tapelift techniques on a range of porous and non-porous substrates. To test DNA recovery efficiency, 30 μl aliquots of 1:50 and 1:100 saliva dilutions were deposited onto the substrates, left to dry overnight, recovered, extracted, quantified and a subset profiled. Tapelifts recovered more DNA and DNA profiles with more detectable alleles than swabs for both saliva dilutions on porous substrates. For non-porous substrates, similar DNA quantities and profiles were generally recovered with both methods for both saliva dilutions. These data underpin current practices to recover DNA using tapelifts for porous substrates and swabs for non-porous substrates. These data also revealed severe degradation of DNA recovered from brass, supporting the on-going need to improve DNA recovery and analysis methods for brass substrates.     

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water-moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent-based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

Evaluation of the use of freezer mill to improve DNA retrieval from dried cotton swabs

This investigation evaluated the use of a freezer mill to improve retrieval of DNA from dried cotton swabs (using 100 μl saliva) compared to uncrushed swabs and whole saliva by measuring DNA yield and profile average peak height. Three treatments were tested; short, medium (as for a bone sample) and extended. The samples subjected to the freezer mill had the powder that remained on the freezer mill components collected (using a water and agitation method). All freezer mill samples returned a lower average DNA yield than either uncrushed swabs or whole saliva. The powder from the crushed swabs comprised an average of 35% of the total DNA yield, whereas the powder from the components comprised 65%. Allele drop-out was observed in samples exposed to extended treatments. Both short and medium treatments provided significantly higher peak heights than uncrushed cotton swabs with equal quantities of DNA (P < 0.05). Using a freezer mill on dried cotton swabs does not increase the DNA yield. This investigation suggests collecting powder from the freezer mill components will increase DNA yield, especially from trace samples.     

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)

The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues, nails, prints after chemical treatments, skin swabs.     

Assessing DNA recovery and profile determination from bloody snow

Successful DNA typing of forensically relevant evidence is reliant on both the quality and quantity of biological material recovered from a crime scene. In geographical areas of the world exposed to cold climates, it is not uncommon for biological evidence to encounter a diversity of challenging surfaces and environments, including snowy surfaces. Currently, there is no standard protocol for recovery of bloodstain evidence in snow and very few publications exploring adequate methods of recovering biological evidence from snowy surfaces. In this study, three common substrates (e.g., cotton swabs, FTA paper, and untreated filter paper) utilized by investigators for evidence recovery were evaluated for their ability to recover human blood (DNA) evidence from snow that would be viable for traditional forensic DNA typing. Each biological sample was extracted and quantified to evaluate the quality and quantity of DNA recovered. All samples yielded sufficient non-degraded DNA to proceed with DNA profiling, where complete DNA profiles were generated from each collection substrate. The experimental findings presented herein demonstrate that the ability to recover viable DNA from human blood collected on surface snow is possible using all three collection methods tested.     

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non-FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y-STR kits. Autosomal and Y-STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.     

Use of a commercially available hydrogel as a novel DNA surface sampling tool

A common requirement in the military, law enforcement, and forensic mission space is the need to collect trace samples from surfaces using a method that not only readily captures the sample but also retains its integrity for downstream identification and characterization. Additionally, collecting samples from three-dimensional objects (e.g., shell casings) is a challenge for which there is currently no validated standardized approach. Recently, hydrogels have been shown to have the potential for surface collection of trace bacterial spores, amino acids, and DNA. To test whether these hydrogels can serve as a viable collection medium for sampling DNA from surfaces, we carried out a series of preliminary tests examining collection efficiency and suitability of hydrogel material to recover samples of diluted, dried human DNA on a smooth polycarbonate surface. The recovery of surface DNA using a commercially available hydrogel was examined, and the efficiency compared to samples collected using a standard foam collection swab. DNA collected using the hydrogel and swab methods was then examined using quantitative polymerase chain reaction (qPCR) and short tandem repeat (STR) analysis to determine whether the collection material was compatible with these downstream processes. The hydrogel material used for this study collected the experimental DNA with comparable efficiency to standard collection swabs. In addition, qPCR and STR analyses demonstrated compatibility with the hydrogel collection and extraction process. These data suggest that hydrogels have the potential to be used as sample collection materials and deserve further characterization to elucidate their utility in collection from irregularly shaped, three-dimensional surfaces/materials.     

The effect of NucleoSpin® Forensic Filters on DNA recovery from trace DNA swabs

Forensic DNA profiling is a globally accepted method for human identification, however, obtaining full DNA profiles from trace DNA can be challenging. The optimal recovery of DNA from trace DNA swabs is therefore crucial. Methods for extracting DNA from swabs often make use of a spin basket combined with a centrifugation step, to enhance the release of cells from the swab prior to DNA extraction. The NucleoSpin® Forensic Filter (Macherey-Nagel, Düren) is a type of spin basket, but it has not been thoroughly assessed on trace DNA samples. This study aimed to assess if the inclusion of the NucleoSpin® Forensic Filter significantly improved DNA recovery and DNA profiling success from cotton and flocked swabs used to collect trace DNA and buccal cells (control). Buccal cells and trace DNA samples were collected from 25 volunteers using each swab type (cotton and flocked) in duplicate. DNA was extracted from the samples using the NucleoSpin® DNA Forensic kit, one set with, and the other set without, NucleoSpin® Forensic Filters. DNA concentration was assessed using real time PCR, and DNA profiling was done using the PowerPlex® ESX 16 system. The inclusion of the NucleoSpin® Forensic Filters significantly improved DNA concentration for buccal cells that were collected using flocked swabs (p = 0.035). However, no significant differences were noted for trace DNA samples for either swab type. There was also no significant difference in DNA profiling success when NucleoSpin® Forensic Filters were used, regardless of swab and sample type. These results may be helpful for laboratories that are considering the NucleoSpin® Forensic Filters in the DNA extraction workflow, particularly for trace DNA samples.     

Technical note: Comparison of forensic swabs for intravaginal sampling

This study compared the currently used swab Prionics ForensiX Evidence Collection Kit with the alternatives Prionics ForensiX Evidence Collection Tube SafeDry and Sarstedt Forensic Swab XL. Volunteers provided intravaginal swabs collected with all swab types at specific time points after unprotected sexual intercourse. Quantifiable DNA, detectability of seminal fluid component (prostate specific antigen, PSA) and spermatozoa were evaluated to find the best-performing swab type.While Sarstedt XL showed significantly higher DNA quantities for sperm cell fractions than ForensiX Kit, the more concise PSA test results clearly favour ForensiX SafeDry. Reassuringly, mostly complete autosomal STR profiles of male components were obtained for sperm cell fractions at all time points and tested swabs. Switching to the higher performing ForensiX SafeDry with improved sampling and processing properties will also benefit victims, medical personnel, and investigators.     

The Use of Adhesive Tape for Recovery of DNA from Crime Scene Items

Abstract: The selection of the appropriate method of collection of biological material from crime scene items can be crucial to obtaining a DNA profile. The three techniques commonly used for sampling items are: cutting, swabbing, and taping. The tape sampling technique offers an advantage, in that it enables the collection of a potentially highly informative source of DNA, shed epithelial cells, from selected areas on crime scene items (the inside fingers of a glove, for instance). Furthermore, surface collection of biological material by taping reduces co‐sampling of known PCR inhibitors such as clothing dyes. The correct choice of tape for crime scene item sampling is important. Not all tapes are suitable for biological trace evidence collection as well as DNA extraction. We report on one tape that met both these criteria. Three different cases are presented which demonstrate the usefulness of adhesive tape sampling of crime items. Finally, the advantages of the tape collection technique are discussed and guidelines for preferred areas of tape sampling on various casework items are presented.