Test of chlorine wipes for efficient removal of DNA from forensic genetics laboratories

Sodium hypochlorite is an efficient reagent for removal of unwanted DNA from laboratory surfaces. Here, we tested two different chlorine wipes and compared their performance to a 0.9–1.8% hypochlorite solution. WipeClean Chlorine Disinfection wipes contain > 0.1 g sodium hypochlorite/kg, whereas WetWipe Chlorine Desinfection wipes contain > 1000 ppm active chlorine. Clean surfaces were contaminated with 10 µL 0.5 ng/µL of massively parallel sequencing libraries. The DNA was dried and left for 45 min before any treatment. The surfaces were cleaned using either 1) a 0.9–1.8% hypochlorite solution and clean wipes, 2) a WipeClean wipe, 3) a WetWipe, or 4) the surface was not cleaned. All experiments were repeated three times. Subsequently, the surfaces were swabbed using cotton swabs. DNA was extracted from the swabs and the DNA concentrations were determined in quadruplicates by real-time PCR. This protocol was repeated after the soft plastic wrapping around the wipes were left open or closed for several weeks. The results showed that the WipeClean wipes efficiently removed DNA for up to four weeks after the box with the wipes were opened, whereas the WetWipe wipes dried faster and gradually lost their cleaning effect.     

Evaluation of different sampling media for their potential use as a combined swab for the collection of both organic and inorganic explosive residues

Commercially available skin cleansing alcohol wipes and conventional swabs were investigated for their use as a universal sampling medium for the simultaneous collection of both organic and inorganic explosive residues. Six compounds with the potential to be encountered in casework [pentaerythritol tetranitrate (PETN), 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), triacetone triperoxide (TATP), ammonium nitrate, and sodium chlorate] were selected as representative target compounds. Quantities of these target compounds were deposited on four different substrates (glass, plastic, aluminium foil and laminate). Two chosen alcohol wipes demonstrated better overall performance in the recovery of both the organic and inorganic representative compounds from each of the test surfaces compared to the results obtained using conventional cotton and polyester swabs, pre-moistened with various solvents, and a direct methanol wash (used as a control). Results obtained using dry cotton swabs indicated that it was not an effective swabbing system for the collection of both organic and inorganic explosive residues on common substrates.     

Bioterrorism: processing contaminated evidence, the effects of formaldehyde gas on the recovery of latent fingermarks

In the present age of heightened emphasis on counter terrorism, law enforcement and forensic science are constantly evolving and adapting to the motivations and capabilities of terrorist groups and individuals. The use of biological agents on a population, such as anthrax spores, presents unique challenges to the forensic investigator, and the processing of contaminated evidence. In this research, a number of porous and nonporous items were contaminated with viable anthrax spores and marked with latent fingermarks. The test samples were then subjected to a standard formulation of formaldehyde gas. Latent fingermarks were then recovered post decontamination using a range of methods. Standard fumigation, while effective at destroying viable spores, contributed to the degradation of amino acids leading to loss of ridge detail. A new protocol for formaldehyde gas decontamination was developed which allows for the destruction of viable spores and the successful recovery of latent marks, all within a rapid response time of less than 1 h.     

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.     

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.     

Comparison of quantitative PCR and culture-based methods for evaluating dispersal of Bacillus thuringiensis endospores at a bioterrorism hoax crime scene

Since the anthrax mail attacks of 2001, law enforcement agencies have processed thousands of suspicious mail incidents globally, many of which are hoax bioterrorism threats. Bio-insecticide preparations containing Bacillus thuringiensis (Bt) spores have been involved in several such threats in Australia, leading to the requirement for rapid and sensitive detection techniques for this organism, a close relative of Bacillus anthracis. Here we describe the development of a quantitative PCR (qPCR) method for the detection of Bt crystal toxin gene cry1, and evaluation of the method's effectiveness during a hoax bioterrorism event in 2009. When combined with moist wipe sampling, the cry1 qPCR was a rapid, reliable, and sensitive diagnostic tool for detecting and quantifying Bt contamination, and mapping endospore dispersal within a mail sorting facility. Results from the cry1 qPCR were validated by viable counts of the same samples on Bacillus-selective agar (PEMBA), which revealed a similar pattern of contamination. Extensive and persistent contamination of the facility was detected, both within the affected mailroom, and extending into office areas up to 30m distant from the source event, emphasising the need for improved containment procedures for suspicious mail items, both during and post-event. The cry1 qPCR enables detection of both viable and non-viable Bt spores and cells, which is important for historical crime scenes or scenes subjected to decontamination. This work provides a new rapid method to add to the forensics toolbox for crime scenes suspected to be contaminated with biological agents.     

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.     

A Comparison of Solvent Extract Cleanup Procedures in the Analysis of Organic Explosives

The use of an organic solvent to extract explosive residues from hand swabs and postblast debris inevitably leads to the coextraction of unwanted materials, usually in far greater quantities than any explosive residue. In this study, the extraction efficiency of a number of solvent cleanup procedures including solid‐phase extraction (SPE), adsorbent resins such as Chromosorb‐104, and traditional materials such as silica and Florisil was calculated using a quantitative liquid chromatography–ultraviolet (LC‐UV) detection procedure. The Oasis^® HLB cartridge outperformed other cleanup procedures, with analyte recoveries approaching 95%, while the Amberlite XAD‐7 procedure returned the lowest overall recoveries. The matrix rejection ability of each method was then determined using a simulated highly contaminated matrix, with the adsorbent resins showing a higher degree of matrix rejection, which is seen as a reduction in background noise in the UV chromatogram using 210 nm detection.     

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.     

To destroy snail mail: Is this the sole solution for anthrax contaminated letters?

Suspicious packages, strange addresses on envelopes and/or the presence of particular powders: these are the most popular aspects of letters containing Bacillus anthracis. Since the World Trade Center tragedy, alarmism about chemical or biological attacks is always in force. The Italian Ministry of Foreign Affairs introduced new procedures to be followed in case suspected anthrax letters are identified (Ministry of Health PROT. 400.3/120.33/4786 of 23/10/2001). Scientists have to collect samples from surfaces and infectious waste have to be placed in autoclavable bags for decontamination. After the sterilization, mails and packages are burnt thus eliminating every biological trace present on their surface. As a matter of fact, after sterilization, DNA is still present and can be analyzed for forensic purposes: for this reason, here we report on the importance of preserving sterilized substrates. We recreated false infected mails with biological traces on their surfaces, sterilized them and, subsequently, we took samples of biological stains and processed them for DNA quantification and typing. We recreate different time conditions consistent with those of the postal service too. Real-Time PCR and DNA typing showed that, even if sterilization destroys the bacillus, human genomic traces still persist and we obtained both complete and partial profiles of samples’ donors. To conclude, the problem of anthrax contaminated letter call for peculiar and standardized procedures; nonetheless, we show that burning evidences after the sterilization process does not appear to be the best solution since there is a loss of biological material which could be decisive for forensic purposes.     

Responding to chemical,biological, or nuclear terrorism: the indirect and long-term health effects may present the greatest challenge

The possibility of terrorists employing chemical, biological, or nuclear/ radiological (CBN) materials has been a concern since 1995 when sarin gas was dispersed in a Tokyo subway. Contingency planning almost exclusively involved detection. containment, and emergency health care for mass casualties. However, it is clear that even small-scale CBN incidents--like the recent spread of anthrax spores through the mail--can cause widespread confusion, fear, and psychological stress that have lasting effects on the health of affected communities and on a nation's sense of well-being. More emphasis therefore needs to be placed on indirect effects and on the medical, social, economic, and legal consequences that follow months to years afterward. To respond effectively to CBN attacks, a comprehensive strategy needs to be developed that includes not only emergency response, but also long-term health care, risk communication, research, and economic assistance. Organizing an effective response challenges government institutions because the issues involved--eligibility for health care, the effects of low-level exposure to toxic agents. stress-related illnesses, unlicensed therapeutics. financial compensation--are complex and controversial.     

Firearms discharge residue sample collection techniques.

Critical comparisons of Ba and Sb in firearms discharge residue were made on samples collected by three independent collection technqiues. Collection materials studied were transparent adhesive tape, (Scotch Brand), a solution of cellulose acetate in acetone ("Film Lift"), and plastic-shafted cotton swabs wetted with dilute nitric acid. Flameless atomic absorption analyses were performed with a Jarrell-Ash Model 810 instrument equipped with a tantalum strip atomizer. Tape and cotton swabs gave comparable positive indications of residue, with frequencies of 90 and 80%, respectively. The plastic Film Lift gave fewer positives, with a frequency of 50%. With the transparent tape lift, gunshot residue particles are discernible, making nondestructive microscopic identification possible prior to destructive elemental analysis.     

Extraction and analysis of human nuclear and mitochondrial DNA from electron beam irradiated envelopes

The United States Postal Service is considering methods such as electron beam irradiation to neutralize biological agents sent through the mail. While this is proven to reduce/eliminate pathogenic organisms, it may also degrade human genomic DNA and therefore hinder the ability to garner forensically informative genetic profiles. To determine the effects of electron beam irradiation on DNA typing, 16 white, standard letter-sized envelopes were licked. Half of the envelopes served as nonirradiated controls while the other half underwent irradiation at dosages sufficient to kill anthrax spores (29.3 and 51.6 kGy). Total cellular DNA was extracted from all envelopes; nuclear short tandem repeat loci, as well as the hypervariable region I from mitochondrial DNA, were amplified by means of the polymerase chain reaction. Short tandem repeat profiles and mitochondrial DNA sequence haplotypes were acquired on an ABI Prism 310 Genetic Analyzer platform. Analysis of data from irradiated samples revealed evidence of DNA degradation; however, the ability to construct full genetic profiles from both nuclear and mitochondrial DNA remained largely unaffected. The use of the polymerase chain reaction, coupled with florescent fragment analysis and mitochondrial DNA sequencing, should be considered to profile biological material from evidence enduring irradiation to inactivate infectious agents.     

Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin.     

The effect of electron beam irradiation on forensic evidence. 1. Latent print recovery on porous and non-porous surfaces

The recent use of the postal system as a means of delivering anthrax spores via several contaminated envelopes has led to the selective irradiation of mail. These as yet unsolved attacks and the U.S. Postal Service's decision to irradiate certain types of mail has led to some unexpected complications. The high doses of radiation required to destroy biological agents like anthrax are sufficient to induce damage to other materials present in the envelope. There have been reports of damage to many different items that have been subjected to irradiation, including paper, precious gems, plastic, computer discs, and electronics. However, few studies have examined the effect of such treatments on items of forensic interest. In this paper, the authors focused on the impact of the irradiation process on the ability to visualize latent prints. This experiment involved using several donors, substrates (both porous and non-porous), and visualization reagents. The results indicate that the irradiation process can have a detrimental effect on the success of certain visualization reagents.     

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.     

A Study of Background Levels of Antimony,Barium, and Lead on Vehicle Surface Samples by Graphite Furnace Atomic Absorption

Law enforcement agencies routinely sample for gunshot residue (GSR) by bulk techniques and often submit swabs taken from other surfaces besides the hands of the suspect shooter. This study aims to establish the prevalence of antimony, barium, and lead on normally handled automobile surfaces by graphite furnace atomic absorption analysis. No positives were determined on 50 sampled automobile surfaces above cutoff (positive) from background levels. Transfers of GSR particles from shooter hands to automobile surfaces were found to potentially allow for positive GSR determinations, but such transfers seem to be dependent on the shooting conditions and length of GSR exposure. We determined that our bulk analysis method yields an overall 87.94 ± 5.52% extraction efficiency from cotton swabs, while the LOQ determinations strengthening the fact that bulk analysis methods are valid and valuable tools for GSR investigations.     

Differentiation of epithelial cell types by cell diameter

Genital swabs play an important role in cases of alleged sexual assault. The aim of our study was to see if epithelial cells from the vagina, glans penis, or mouth could be distinguished on the basis of size. Vaginal swabs were taken from 12 women in different phases of their menstrual cycles; penile swabs were taken from 5 men, and mouth swabs were taken from 6 men and 6 women. For each swab, a sample was smeared across a microscope slide and allowed to dry. The dried epithelial samples were then viewed without any further processing with a "SteReoLumar.V12" stereo microscope. The microscope slide surfaces were divided into grids and all single epithelial cells whose contours could be clearly distinguished were photographed. The maximum diameter for each photographed cell was digitally determined using the Axiovision software. In total, 995 vaginal epithelial cells, 211 penile epithelial cells, 329 male oral epithelial cells, and 525 female oral epithelial cells were measured. Menstrual cycle phase did not affect vaginal epithelial cell diameter. The mean vaginal epithelial cell diameter was 63.95 microm (min. = 28.08 microm, max. = 108.06 microm, s = 11.50 microm). The mean penile epithelial cell diameter was 39.24 microm (min. = 28.38 microm, max. = 51.02 microm, s = 4.84 microm). The diameter of oral epithelial cells hardly differed for both sexes, although the female cells were, on the whole, slightly larger. On the basis of these results, it is not possible to conclude that epithelial cells of less than a certain diameter found in the assessment of a vaginal swab must be of penile origin. It is also not possible to usefully distinguish vaginal epithelial cells from male or female oral epithelial cells on the basis of the diameter. However, finding epithelial cells with a diameter distinctly greater than 50 microm in a penile swab sample suggests the presence of vaginal or oral epithelial cells. Epithelial cells examined with the presented method can be used without restrictions for further examinations, such as single-cell DNA analysis after single-cell picking with the micromanipulator developed by Aura Optik (Jena).     

Surface-sampling and analysis of TATP by swabbing and gas chromatography/mass spectrometry

The method of sample recovery for trace detection and identification of explosives plays a critical role in several criminal investigations. After bombing, there can be difficulties in sending big objects to a laboratory for analysis. Traces can also be searched for on large surfaces, on hands of suspects or on surfaces where the explosive was placed during preparatory phases (e.g. places where an IED was assembled, vehicles used for transportation, etc.).In this work, triacetone triperoxide (TATP) was synthesized from commercial precursors following reported methods. Several portions of about 6 mg of TATP were then spread on different surfaces (e.g. floors, tables, etc.) or used in handling tests. Three different swabbing systems were used: a commercial swab, pre-wetted with propan-2-ol (isopropanol) and water (7:3), dry paper swabs, and cotton swabs wetted with propan-2-ol. Paper and commercial swabs were also used to sample a metal plate, where a small charge of about 4 g of TATP was detonated. Swabs were sealed in small glass jars with screw caps and Parafilm^® M and sent to the laboratory for analysis. Swabs were extracted and analysed several weeks later by gas chromatography/mass spectrometry. All the three systems gave positive results, but wetted swabs collected higher amounts of TATP. The developed procedure showed its suitability for use in real cases, allowing TATP detection in several simulations, including a situation in which people wash their hands after handling the explosive.     

Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide

This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption.