A systematic approach to the analysis of illicit drugs for DNA with an overview of the problems encountered.

Due to the restricted nature of illicit drugs, it is difficult to conduct research surrounding the analysis of this drug material for any potential DNA in sufficient quantities acceptable for high numbers of replicates. Therefore, the current research available in peer reviewed journals thus far regarding analysing illicit drugs for DNA has been performed under varying experimental conditions, often using surrogate chemicals in place of illicit drugs. The data presented within this study originated from the analysis of genuine illicit drugs prepared both in controlled environments and those seized at the Australian border (and therefore from an uncontrolled environment) to determine if DNA can be obtained from this type of material. This study has been separated into three main parts (total n=114 samples): firstly, methamphetamine synthesised within a controlled environment was spiked with both saliva and trace DNA to determine the yield following DNA extraction; secondly, methamphetamine also synthesised in a controlled environment but on a larger scale was tested for the amount of DNA added incidentally throughout the synthesis, including the additional steps of recrystallising, homogenising and "cutting" the drug material to simulate preparation for distribution; and thirdly, the detection of human DNA within samples of cocaine and heroin seized at the Australian border. The DNA Fast Flow Microcon Device was utilised to concentrate all replicates from the same source into one combined extract to improve the DNA profiles for the samples where no DNA spiking occurred. Full STR profiles were successfully obtained from drug samples spiked with both saliva and trace DNA. Methamphetamine was present in the final DNA extracts and caused incompatibilities with the quantification of DNA using Qubit. The yields of DNA from drugs not spiked with DNA sources were much lower, resulting in 36 % of samples yielding alleles where all others did not. These results were not unexpected given these were realistic drug samples where the history of the drug material was unknown. This is the first study to obtain DNA profiles from genuine illicit drug material in both controlled and uncontrolled environments and indicates that the analysis of illicit drugs for DNA is an avenue worth pursuing to provide information which can in turn assist with disrupting the supply of these drugs. Given that DNA profiling is carried out worldwide using essentially the same systems as described within this study, the potential for impact is on a national and international scale.

Shedding more light on shedders.

We report on testing 100 individuals for their shedder status with the aim of demonstrating whether the process of cell staining is reproducible when testing a large number of people. A previous report using the same method was based on 11 donors and indicated that there may be a continuum of shedder types within this small sample set. In this report we also expand the time points post-handwashing to 0, 15, 30, 60, and 180 min. Triplicate samples were collected from both the right and left thumbs. Samples were collected by donors placing a thumb on a clean glass slide and then adding a DNA binding dye. The number of cells were recorded within three separate square millimetre areas (cells/mm2) at 220x magnification. The experiments were conducted in triplicate on three different days, giving a total of 72 thumbprints per individual. Finally, there were 3438 observed frames in the entire dataset. Of the 100 donors, 98 gave consistent and reproducible cell number deposition. There was no difference between the cells deposited by the left and right thumbs in 13 of 15 tested. Males tended to deposit more cells than females. If applying arbitrary boundary to a cell count to definitively determine shedder status, then many of the donors fell within two categories. This study based on 100 individuals strongly suggests that shedder status is a continuum phenomenon.

Comparison of three DNA extraction methods tested on illicit drug-related powders.

The detection of human DNA on and within illicit drug preparations is novel and a focus of current research. Previous studies have indicated that certain drug-related powders present in illicit drug preparations can interfere with downstream DNA analysis when directly added to the PCR. Therefore, it is important to determine if these drug-related powders are effectively removed during the DNA extraction or whether traces of powder remain to interfere with DNA processing. Three extraction methods were selected to assess their efficiency at removing drug-related powders for downstream processes using DNA from both saliva and touch depositions. This is the first study to compare efficiencies of DNA extraction methods from drug-related powders. The extraction methods compared were the DNA IQ™ System, the QIAamp® DNA Investigator Kit and the combination of a simple lysis step followed by use of the Microcon® DNA Fast Flow device. Saliva was added to dimethylsulfone (DMS), nitrostyrene and PROSOLV® tablet mixture to determine the effect of powder type (based on solubility). Saliva was also added to 0, 50, 200 and 400 mg of DMS to determine the effect of an increase in DMS quantity. Trace DNA was deposited onto DMS using a worn glove approach. These samples were re-tested six months post-DNA deposition and profiled for further comparisons. Ten replicates were conducted for each condition with five replicates of saliva positive controls per method (n = 255 samples). A subset of samples was chemically analysed to determine if DMS was present in the final DNA eluant. The readily soluble DMS did not interfere with any of the extraction methods at lower amounts, however increasing the DMS to 400 mg reduced the relative DNA yields using the Microcon® and Investigator methods. The tablet mixture reduced the relative DNA yield of all three methods, however the nitrostyrene (which was relatively insoluble) only reduced the relative DNA yield of the DNA IQ™. The Investigator method performed the best with the trace samples, followed by the Microcon® method and then the DNA IQ™. DMS was detected in all extracts chemically analysed from the DNA IQ™ and Microcon®, whereas only one sample tested from the Investigator kit contained DMS in the extract and was in a relatively low amount compared to the other samples. Not one kit outperformed the others in all comparisons, however the Investigator kit was the most efficient overall at optimising the DNA yield whilst also removing the powders more effectively.

Persistence of touch DNA on commonly encountered substrates in different storage conditions.

The persistence of touch DNA deposited after realistic handling of items typically encountered in forensic investigations has been the subject of few studies. Understanding the long-term persistence of touch DNA on different substrates in varying conditions can be central to the effective triage of samples for further processing. As the time between an alleged incident and collection of evidence may vary from a few days to years after an alleged event, this study assessed three different common substrates for the persistence of touch DNA over a time span up to 9 months. These substrates included fabric, steel, and rubber, each of which were handled in a way to imitate what may happen during a criminal act. The three substrates were exposed to two different environments for up to 9 months: inside a dark cupboard with no traffic to act as a control and an outside semi-exposed environment. Ten replicates from each of the 3 substrates were tested at 5 time points to create 300 samples. All samples were processed using a standard operating workflow to provide genotype data after exposure to different environments. It was found that the fabric samples produced informative STR profiles (defined here as 12 or more alleles) up to the 9 month timepoint for either environment. The rubber and steel substrates for the inside condition produced informative STR profiles up to the 9 month timepoint, but only generated informative STR profiles for the outside condition up to 3 and 6 months, respectively. These data add to our understanding of the external factors that affect DNA persistence.

Improvements, factors, and influences on DNA recovery from firearms.

Touch DNA recovery from firearms can be central to many criminal investigations, yet the generation of DNA profiles from these items remains poor. Currently in Australia, published casework data highlights extremely poor DNA success from samples recovered from firearms. Only between 5% and 25% of samples result in useful DNA data and therefore increasing the success of DNA recovered from firearms is highly important but has not yet been explored in-depth. This study focused on increasing the recovery of DNA from ten firearm components that were held for 15 s. Multiple recovery methods were used, and the resulting genetic data compared. DNA evidence may be deliberately removed from firearms after discharge to hamper forensic investigations, therefore this study examined the effect of wiping down the components or handling them with gloves. A standard double swab and rinse swab recovery method resulted in an average of 73% cellular recovery. A cumulative swab process had the highest average recovery at 86%, although it was found that increasing the DNA yield led to an increase in mixture complexity. Wiping over the components was observed to remove on average 69% of cellular material, compared with 33% when handed with gloves. However, the size and texture of the components affected the efficiency of cellular material removal. The results from this study allow for prioritisation of areas to sample on firearms, as well as suggesting techniques that can be applied for the optimum process of cellular recovery and subsequent generation of STR DNA data.

Microbial degradation products of lurasidone and their significance in postmortem toxicology.

Recent research reported that lurasidone degrades in unpreserved ante-mortem human whole blood inoculated with microorganisms known to dominate postmortem blood specimens. In vitro degradation occurred at a similar rate to risperidone, known to degrade in authentic postmortem specimens until below analytical detection limits. To identify the lurasidone degradation products formed, an Agilent 6520 liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode was used. Numerous degradation products not previously reported in prior in vitro or in vivo pharmacokinetic studies or forced degradation studies were detected. Accurate mass data, mass fragmentation data, acetylation experiments, and a proposed mechanism of degradation analogous to risperidone supports initial identification of the major degradation product as N-debenzisothiazole-lurasidone (calculated m/z [M + H]+ = 360.2646). A standard was unavailable to conclusively confirm this identification. Retrospective data analysis of postmortem cases involving lurasidone identified the presence of the major degradation product in four of six cases where lurasidone was also detected. This finding is significant for toxicology laboratories screening for this drug in postmortem casework. The major postmortem lurasidone degradation product has consequently been added to the LC-QTOF-MS drug screen at Forensic Science SA (FSSA) to indicate postmortem lurasidone degradation in authentic postmortem blood specimens and as a marker of lurasidone administration in the event lurasidone is degraded to concentrations below detection limits.

In vitro degradation of ziprasidone in human whole blood.

A systematic study was performed into the degradation of ziprasidone in simulated postmortem blood. Fifteen potential degradation products not previously reported in the literature were observed. Four resulted from degradation in human blood, whereas the remaining products resulted from reaction with solvents: four from alkaline degradation, four from reaction with acetaldehyde, and three from reaction with acetone. To identify possible degradation products, a liquid chromatograph-diode array detector (LC-DAD) and liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS) operating in auto-MS/MS mode were used. It was indicated from red-shifted UV-Vis spectra, accurate mass data, mass fragmentation data, and a deuteration experiment that the site of ziprasidone degradation, in the in vitro blood experiments, was the methylene carbon of the oxindole moiety. The major in vitro blood degradation products were proposed to be E/Z isomers of 3-ethylidene-ziprasidone. Further, another in vitro degradation product in microbially inoculated blood specimens was proposed to be 3-ethyl-ziprasidone. 3-Ethylidene-ziprasidone was hypothesized to form from the reaction of ziprasidone with acetaldehyde derived from the ethanol used to spike ziprasidone into the in vitro blood experiments. Data from two postmortem investigations were available for retrospective reanalysis. Attempts were made to detect degradation products of ziprasidone, but none were found.

Recovery of integrated and surface trace DNA from illicit drug tablets.

In many parts of the world, tablets are a commonly encountered form of illicit drug preparation. Whilst previous research has investigated the feasibility of detecting trace DNA on illicit drug capsules, this has not been performed for tablets. Tablets have a unique substrate surface and therefore the amount of DNA transferring to them and persisting on them may be different to capsules; there may also be differences in the collection efficiency and the outcome of downstream DNA processing and analysis steps. The ability to profile the DNA from individuals who handled tablets during their preparation and distribution would add another level of discrimination between various drug seizures or corroborate chemical profiling outcomes which may link various seizures to a common origin. DNA from two different individuals (male and female) was added to the tablets in two stages. Firstly, tablet powder was spiked with DNA from one individual to mimic the situation where DNA traces are incorporated during the drug synthesis or final drying stages. The powder was then pressed into tablets in a clean environment without intentional addition of DNA. Subsequently, a second individual counted out the tablets into bags of ten to mimic the preparation for distribution at a user level. The exterior of the tablet was swabbed and then the entire tablet and the swab were put through separate DNA extractions, yielding two DNA extracts for each tablet. Swabs of the exterior tablet surface yielded single source DNA profiles that identified the tablet handler in 100 % of samples. The tablet extract yielded the donor of the DNA intentionally added within the drug powder in 80% of samples with varying levels of support, however contributions of the exterior handler were detected in 60 % of samples. The identification of individuals potentially involved in the synthesis of the drugs compared to the distribution of the tablets will provide invaluable strategic intelligence related to illicit drug investigations and to law enforcement agencies.

DNA on drugs (part 2): An extended study into the transfer and persistence of DNA onto illicit drug capsules using realistic scenarios.

Capsules are now the main form of ecstasy rather than tablets in Australia and therefore their examination is of interest to forensic drug chemists in Australia and possibly elsewhere. Recently, we used controlled experimental conditions to show that capsules may be a source of DNA that can be used to identify those involved in production and distribution of illicit drugs. The question remains: in realistic scenarios where there are more unknowns, can we still detect DNA, and determine whose it is, on the exterior of capsules? The concept of comprehensive forensic intelligence and investigations - utilizing both biological and chemical signatures - relating to illicit drug preparations (i.e., the capsules and their contents) may be of great use to law enforcement. Experiments were conducted with both semi-realistic and realistic scenarios where two volunteers were asked to firstly use an encapsulator and mimic the loading of capsules, then Volunteer 1 would count out the capsules that Volunteer 2 prepared, and vice versa. This was to simulate the scenario where one person was involved in the assembly of the capsules which were then separated into smaller bags of 10 capsules by a second person for distribution. Gelatine and vegetable capsules were tested, with 10 replicates used per capsule type, scenario, and volunteer (total n = 80 capsules). Volunteer 2 was included as a contributor to the DNA profiles generated from 100% of samples handled by them within the semi-realistic scenario, whereas the other volunteer could be included as a contributor in 65% of samples. For the realistic scenario, profiles could be generated with the inclusion of both volunteers as profile contributors in 15% of samples and from just one of the volunteers in a further 50% of samples (therefore in total, either both or one of the volunteers were detected in 65% of realistic samples). Surprisingly, it was not necessarily the case that the last person to handle the capsule was the major or only contributor. The potential variability in the DNA quantities that could be deposited onto the capsules of genuine illicit drugs is high and would vary on a case-by-case basis. Nevertheless, this study has indicated that in realistic scenarios where two people are involved in the later stages of illicit drug capsule preparation, that either one or both individuals may be identified, potentially opening new investigative leads for law enforcement agencies as well as offering new information for intelligence-led policing.

A survey of the effects of common illicit drugs on forensic DNA analysis.

Profiling of DNA associated with illicit drug packages and paraphernalia is a common investigative tool. In addition, research is being conducted regarding the analysis of trace DNA present within illicit drugs and on capsules. The application of trace DNA analysis to illicit drugs has the potential to identify individuals involved in their manufacture and distribution. However, the inhibitory effects of illicit drugs and related compounds on downstream DNA analysis has not yet been investigated. If drug-induced polymerase chain reaction (PCR) inhibition occurs, the quality or informativeness of the resultant DNA profile may be impacted. In this study, the effects of a range of drugs, diluents, adulterants, and synthetic precursors on both quantitative PCR (qPCR) data and short tandem repeat (STR) DNA profiling results were examined. Twenty-two compounds representative of drug compounds and adulterants which may be encountered in drug seizures were spiked with 1 ng/µL and 0.05 ng/µL of control DNA and underwent DNA quantification using Quantifiler™ Trio. A subset of 13 compounds, including the majority that indicated potential inhibition in Quantifiler™ Trio, underwent STR profiling with VeriFiler™ Plus to determine if inhibition also occurred at this stage. The effect of diluting the DNA extract on the extent of inhibition of STR profiling was also investigated. Internal PCR controls within the qPCR were not a reliable indicator of inhibition, although suppression of the short and long autosomal fragments was observed in the presence of many compounds, and four compounds gave inconclusive results. STR internal quality controls indicated inhibition in 5 of the 13 compounds, however, profiles were affected by the presence of 11 of the 13 compounds in various ways such as a decreased average relative fluorescence units (RFU), drop out of certain alleles (some based on allele size range of locus) leading to a decreased likelihood ratio (LR), an increase in the proportion of stutter peaks and the presence of split or shoulder peaks. All profiles improved following a dilution of the compound in the PCR and allowing the generation of LR values in excess of 1 × 1025, indicating inhibition occurred rather than DNA degradation. The data obtained show that removal of some of these compounds is required through an effective DNA extraction process for successful downstream trace DNA profiling. Upon successful PCR, the resultant DNA profiles provide the opportunity for opening new investigative avenues for law enforcement agencies.

Microplastics in intertidal water of South Australia and the mussel Mytilus spp.; the contrasting effect of population on concentration.

Microplastics, plastic particles <5 mm in size, are of global concern as human-caused pollutants in marine and fresh waters, and yet little is known of their distribution, behaviour and ecological impact in the intertidal environment of South Australia. This study confirms for the first time, the presence of microplastic in the South Australian intertidal ecosystem by quantifying the abundance of particles in intertidal water and in the keystone species, the blue mussel, Mytilus spp., an important fisheries species, at ten and six locations respectively, along the South Australian coastline. For a remote region known for its pristine environment, microplastic concentration in intertidal water was found to be low to moderate (mean = 8.21 particles l-1 ± 4.91) relative to global levels and microplastic abundance in mussels (mean = 3.58 ± 8.18 particles individual-1) was within the range also reported globally. Microplastic particles were ubiquitous across sites and bioavailable by size in water (mean = 906.36 µm) and in mussel (mean = 983.29 µm) raising concerns for the health of South Australia's unique coastal ecosystems and for the human food chain. Furthermore, a positive correlation was found between human coastal population size and microplastic concentration in intertidal water, irrespective of influences from industry - tourism, fishing and shipping ports. FTIR analysis determined plastic type to include polyamide (PA), polyethylene (PE), polypropylene (PP), acrylic resin, polyethyleneterephthalate (PET) and cellulose, suggesting synthetic and semi-synthetic particles from single-use, short-life cycle products, fabrics, ropes and cordage. Our findings shed light on the urgent need to establish the local sources of microplastic pollution in order to assist the community, industry and government to reduce the impact of microplastic on the fragile marine systems within South Australian intertidal waters and on the organisms associated with the human food chain.

DNA deposited in whole thumbprints: A reproducibility study.

Touch DNA is pivotal in forensic science therefore understanding the mechanisms and variations of deposition and composition of genetic material in touched deposits is essential. Shedder status is still poorly understood, and the consistency and cohesiveness of research is less developed compared to other transfer and persistence considerations. In this study, the inter- and intra-variations between shedder categories and individuals were investigated by use of a nucleic acid binding dye. Ten volunteers deposited 30 thumbprints under two different time points post handwashing: 15 after a period of 15 min post handwashing (defined) and 15 after a period of at least 60 min post handwashing (undefined). Thumbprints were made on glass slides, marked with a grid of 55 squares, then the marks were stained with Diamond Dye and the cells that fluoresced in each square (7500 total) counted to determine the total number of cells. Shedders were less consistent in cellular deposition when thumbprints were made in the defined condition compared to waiting at least 60 min. Heavy shedders consistently generated informative profiles (defined here as 12 or more alleles); this occurred 73% of the time in the defined condition and 87% in the undefined. Intermediate shedders produced informative profiles, which occurred 50% of the time in the defined and 80% in the undefined condition. Light shedders consistently produced uninformative profiles with only 33% being considered uploadable to a DNA database for the defined condition and 27% informative for the undefined condition.

Impurity profiling of methamphetamine synthesized from methyl α-acetylphenylacetate.

The group of P2P precursors including α-phenylacetoacetonitrile (APAAN), α-phenylacetoamide (APAA) and methyl α-acetylphenylacetate (MAPA) has become increasingly popular in Europe and other parts of the world in the last decade. Previous investigations have reported the use of APAAN in the synthesis of amphetamine and methamphetamine and identified a range of characteristic impurities. This research has expanded upon the current literature by investigating the use of MAPA in the synthesis of methamphetamine. In this study methamphetamine was synthesized via three common clandestine methods: the Leuckart synthesis and two reductive amination methods. We report the identification of seven impurities, four of which are methyl ester equivalents of impurities previously reported for the detection of APAAN. These are methyl 2-phenylbut-2-enoate, methyl 2-phenyl-3-hydroxybutanoate, methyl 3-(methylamino)-2-phenylbut-2-enoate and methyl 3-(methylamino)-2-phenylbutanoate. The other impurities identified are ethyl ester compounds formed via transesterification of the methyl ester due to the reaction solvent. This susceptibility for transesterification suggests that identification of the pre-precursor used may not always be straightforward and may be dependent on the reaction conditions employed. Of the impurities reported, methyl 3-(methylamino)-2-phenylbutanoate was deemed to be a potentially reliable impurity for detection of the use of MAPA; however, it is expected that lower levels of characteristic impurities may be detected in methamphetamine synthesized from MAPA than that from APAAN.

An application example of the likelihood ratio approach to the evaluation of organic gunshot residues using a fictional scenario and recently published data.

The analysis of gunshot residues (GSR) can provide important information with regard to the involvement of a person of interest (POI) in a firearm-related incident. Organic gunshot residues (OGSR) have been investigated in order to provide additional and complementary information to the traditional inorganic gunshot residue (IGSR) particles detected by scanning electron microscopy (SEM). Currently, many procedures and analytical methods have been developed to detect OGSR-related compounds collected from the shooter's hands. However, such studies provide no information regarding the inclusion of such results in an activity level evaluation for discharging a firearm. The aim of this article is to assess the feasibility of using the likelihood ratio (LR) approach as a tool to evaluate OGSR results for activity level propositions. The developed model focuses on the assignment of an LR for several compounds detected in OGSR. A simple worst-case simulation was investigated in order to assess the applicability of the LR approach to evaluate OGSR traces. This simulation highlighted the importance of addressing an appropriate pair of activity level propositions when evaluating the results.

Investigations into the stability of 17 psychoactive drugs in a "simulated postmortem blood" model.

In the postmortem environment, some drugs and metabolites may degrade due to microbial activity, even forming degradation products that are not produced in humans. Consequently, underestimation or overestimation of perimortem drug concentrations or even false negatives are possible when analyzing postmortem specimens. Therefore, understanding whether medications may be susceptible to microbial degradation is critical in order to ensure that reliable detection and quantitation of drugs and their degradation products is achieved in toxicology screening methods. In this study, a "simulated postmortem blood" model constructed of antemortem human whole blood inoculated with a broad population of human fecal microorganisms was used to investigate the stability of 17 antidepressant and antipsychotic drugs. Microbial communities present in the experiments were determined to be relevant to postmortem blood microorganisms by 16S rRNA sequencing analyses. After 7 days of exposure to the community at 37°C, drug stability was evaluated using liquid chromatography coupled with diode array detection (LC-DAD) and with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Most of the investigated drugs were found to be stable in inoculated samples and noninoculated controls. However, the 1,2-benzisothiazole antipsychotics, ziprasidone and lurasidone, were found to degrade at a rate comparable with the known labile control, risperidone. In longer experiments (7 to 12 months), where specimens were stored at -20°C, 4°C, and ambient temperature, N-dealkylation degradation products were detected for many of the drugs, with greater formation in specimens stored at -20°C than at 4°C.

Impurity profiling of methamphetamine synthesised from α-phenylacetoacetonitrile (APAAN).

The rise in popularity of 'designer' precursor compounds for the synthesis of amphetamine-type stimulants poses a significant challenge to law enforcement agencies. One such precursor is α-phenylacetoacetonitrile (APAAN). APAAN emerged in Europe in 2010 and quickly became one of the most popular precursors for amphetamine synthesis in that region. Previous literature has identified four APAAN-specific impurities formed in the synthesis of amphetamine; however, there is currently no research on the use of APAAN in the synthesis of methamphetamine, which is more likely to be employed in a non-European market. In this study methamphetamine was synthesised via three common clandestine methods: the Leuckart method and two reductive amination methods. We report the identification of five new impurities and two previously identified impurities characteristic for the use of APAAN in the synthesis of methamphetamine. The newly identified impurities were characterised by MS and NMR and determined to be (E)-3-(methylamino)-2-phenylbut-2-enenitrile, 3-(methylamino)-2-phenylbutanenitrile, 3-methyl-2,4-diphenylpentanedinitrile, 2-phenylbutyronitrile and 3-hydroxy-2-phenylbutanenitrile.

The influences of dusty environments on the STR typing success of post-detonation touch DNA samples.

As the use of improvised explosive devices (IEDs) in a broad spectrum of offences continues, it is vital that research is performed to assess the capabilities of the forensic DNA profiling technology currently available to provide information as to potential perpetrators. This work investigates some of the most important gaps in our understanding surrounding the poor success rates in DNA profiling obtained through the sampling of touch DNA on post-detonation IED samples. It has been previously suggested that the use of Diamond™ Nucleic Acid Dye may fix cells to a surface, therefore reducing the effect of an experimental process to remove or damage those cells. This was found not to be the case for samples undergoing a detonation as there was no difference in the resultant post-detonation profiles between the stained samples, stained prior to detonation, and unstained samples. The comparison of data from previously performed research, within an enclosed explosives chamber, to real-world outdoor detonation events in a rural and dusty environment was investigated. It was found that there was a significant difference between the environments for the aluminium but not for the battery or electrical tape substrates indicating that environment has the potential to influence STR success through the introduction of PCR inhibitors; humic acid within rural natural dust was introduced here. No difference was observed in cell loss due to the detonation between environments and the dirt within the PCR was higher in the 'outdoor' samples. The effect on cellular retention and damage due to the sample's distance from the charge has been thoroughly investigated through incremental 100 mm exposure. Distance from the charge was found to affect every metric analysed; these being the cell loss from samples, the number of alleles amplified in resultant direct PCR profiles, and the total RFU of the subsequent profiles. These data outline the importance of this work allowing results to be assessed and triage decisions be made accordingly. The analysis of wood, PVC pipe, a mobile phone with rubber buttons, a SIM card, and a circuit board showed that none of these samples at 400 mm from the charge caused substrate specific PCR inhibition. On-site collection teams do not need to triage collection based on these sample types as there was no significant difference observed in their ability to return DNA profiling data. Surface area and inhibitor presence are key variables to consider when determining STR processing workflow for post-detonation samples as for samples with larger surface areas within the outdoor environment PCR post-extraction is preferential to direct PCR.

Variation in polymer types and abundance of microplastics from two rivers and beaches in Adelaide, South Australia.

Microplastics are a major source of marine pollution and comprise of many recyclable polymers. For this study, we investigated the prevalence of microplastic polymers in an urban and non-urban setting and determined what type of plastic polymers was most common in these areas. This was conducted by extracting sediment and sand samples from 2 rivers and beaches in Adelaide, South Australia. The microplastics were extracted using density separation and were identified using Fourier-transform infrared spectroscopy. We found a significantly higher abundance of microplastics and variety of polymers in the sediment of the Patawalonga creek, compared to the less urbanised environment. Most of the microplastics found in the study were from recyclable products which highlight the lack of recycling practices undertaken by the inhabitants of that area.

DNA on drugs! A preliminary investigation of DNA deposition during the handling of illicit drug capsules.

DNA profiling from capsules and tablets offers a complementary tool to that of chemical profiling when investigating the manufacture and trade in illicit drugs. By sampling the outside of capsules, individuals who may have handled them during production, assembly or distribution may have deposited their DNA and can be identified if matched to a nominated profile or one on a relevant DNA database. The profiles can also be compared to those found on other capsules to potentially link various drug seizures. This study sampled the exterior of capsules after they had been handled in a controlled scenario to determine if informative DNA profiles could be generated from this brief contact. Two individuals of intermediate shedder status washed their hands and waited for 30 min before handling ten gelatine, vegetable, and enteric vegetable capsules each (n = 60). Contact was made for 15 s. Each capsule was swabbed and DNA isolated. The amount of recovered human DNA was quantified and profiled using the Verifiler Plus DNA profiling kit. Profiles were generated from 82% (49/60) of capsules tested with LR values above 1 × 103 for the inclusion of the volunteer as a contributor. Inhibition of the PCR was detected in 24 of the 60 samples, however 16 of these still produced informative profiles when sufficient template DNA was available and only mild inhibition was detected, or by overcoming inhibition by dilution of the DNA extract. This pilot study demonstrates the potential for forensic science laboratories to recover human DNA from the exterior surface of capsules which are commonly used to encase illicit drugs such as MDMA, thus enabling both biological and chemical profiling methods to contribute to the investigation of clandestine drug production and distribution.

How many cells are required for successful DNA profiling?

Through advances in fluorescent nucleic acid dye staining and visualisation, targeted collection of cellular material deposited, for example by touch or within a saliva deposit, is possible. In regard to the potential evidentiary value of the deposit the questions remain: 'How many cells are required to generate an informative DNA profile?'; 'How many visualised corneocytes within a touch deposit compared to typical nucleated cells are required in order to achieve successful DNA profiling?'. Diamond TM Nucleic Acid Dye (DD) staining of cellular material, and subsequent visualisation utilising portable fluorescence microscopy, was performed for touch and saliva samples to target defined numbers of cells for collection, by swab and tapelift, and subsequent processing via direct PCR and PCR post-extraction. The resulting DNA quantification data and alleles generated within subsequent DNA profiles could be correlated to the number of cells initially collected to determine cellular threshold requirements for DNA profile generation for each workflow. Full profiles were consistently generated using direct PCR when the template was ≥40 buccal cells collected by either a swab or tapelift. By contrast ≥800 corneocytes collected by swabbing or ≥4,000 corneocytes collected by a tapelift were required to generate same number of STR alleles from touch samples. When samples were processed through a DNA extraction workflow, ≥80 buccal cells were required to generate full profiles from both swab and tapelift, while touch samples required ≥4,000 corneocytes collected by a swab and >8,000 corneocytes collected by a tapelift. The data presented within this study allow for informative sample triage and workflow decisions to be made to optimise STR amplification based on the presence and visual quantification of stained cellular material.