Microwave-assisted extraction and differential scanning calorimetry in the chemical identification of sliming agents apprehended in the south region of Brazil

Over the past decades, consume of slimming agents considerably increased in several countries, including Brazil, due to weight-loss and stimulant properties. Since these drugs are controlled to prevent illicit and indiscriminate use, there is a parallel illegal market that uses the Internet and irregular pharmacies in order to distribute these formulations. Slimming agents produced by these illegal sources are known for being manufactured with little or none quality control resulting in uncertain and unknown formulations. For forensic purposes, apprehended pharmaceuticals have to undergo a process of chemical identification that can be difficult due to its complex matrix. In this sense, application of assisted energies in the extraction step such as microwave irradiation can be a promising method to increase the recuperation of the target molecules of the sample. Therefore, the aim of this research was to identify four slimming agents apprehended in Brazil by means of visual inspection, Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry and Gas Chromatography – Mass Spectrometry. Moreover, the efficiency of solid-liquid extraction and microwave-assisted extraction was compared. It should be noted that our work was one of the few to use Differential Scanning Calorimetry and the application of microwave irradiation in the analysis of apprehended materials. Results showed that the majority of the samples was counterfeit being composed of one or several adulterants or contaminants. Initially, visual inspection resourcefully screened the slimming agents for possible signs of falsification, however it failed to detect fraudulent products that were very similar to veridical medicines. Sequentially, Fourier Transform Infrared Spectroscopy detected functional groups present in the samples while the presence or absence of the alleged active ingredients were successfully measured with Differential Scanning Calorimetry and, thus, providing a full chemical screening of the apprehended materials. Gas Chromatography- Mass Spectrometry confirmed the presence of adulterants such as caffeine, fluoxetine and phenolphthalein as well as contaminants such as sulfurol in the falsified samples. Finally, comparison of extraction procedures indicated that microwave-assisted extraction increased the recovery of compounds detected in chromatographic analysis to a greater extent than solid-liquid extraction.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Determination of DNA yield rates in six different skeletal elements in ancient bones

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18^th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.     

How to avoid driving DNA caseworkers crazy: CaseSolver,an expert system to investigate complex crime scenes

DNA analyses can be used for both investigative (crime scene-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, low template LT-DNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches of national DNA databases is problematic because it is realistically restricted to clear major contributors and the occurrence of masking and drop-out means that there will not be a definitive DNA profile to perform the search with.CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio (LR) based on a qualitative model (forensim) c) LR based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the LRs, which can be exported as a report. The software can also identify contributors from small or large databases (e.g., staff database or 1 mill. individuals). In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we describe recent improvements made to the software in version v1.5.0, made in response to user requirements during intensive casework usage.     

Development of a new 17 Y-STRs system using fluorescent-labelled universal primers and its application in Shanxi population in China

Y-chromosomal short tandem repeats (Y-STRs) polymorphisms are useful in forensic identification, population genetics and constructing of human structures. Increasing the number of Y-STRs and their polymorphism will drastically narrow down the matching number of genealogy populations or pedigrees when searching against a forensic DNA databank. In this study, we develop a system containing 17 complementary Y-STRs that are compatible and reinforce the current commercially available Y-STR kits. This system was constructed by multiplex PCR with expected size of 126bp-400bp using home-made universal primers labeled by different fluorescence (DYS715, DYS709, DYS716, DYS713, DYS607, DYS718, DYS723, DYS708, DYS714, DYS712, DYS717, DYS721, DYS605, DYS719, DYS726, DYS598 and DYS722). The genetic data were obtained from 394 individuals in Shanxi province, China. The Y-STR system has 131 haplotypes and high discrimination power is 1. In conclusion, our study provides a robust, sensitive and cost-effective genotyping method for human identification, which is beneficial for narrowing the searching scope when applying to the genealogy searching with Y-STR DNA databank.     

The best possible result from the minimum available

In accordance with the Italian DNA legislation (DPR 7 April 2016, n. 87) a number of markers lower than seven are not considered usable for inclusion in the Italian forensic DNA database. For this reason, if the forensic DNA analysis performed in our laboratory do not provide acceptable results for a number greater than or equal to seven, the profile is not indicated in the final report. Thus, having indications about the possible success of an analysis before executing it, is a crucial point in the validation process of the accreditated method used in our laboratory.To achieve this goal, the quantification of extracts before typing plays a fundamental role. Especially when touched objects need to be examined tens or hundreds of nanograms may be present, but also very few or no cell can be present on the object. As such, quantification of every sample can ensure the maximum efficiency and prevent repeat analyses, over-amplified samples or completely useless examination.Quantifiler® Trio DNA quantification kit was validated in our laboratory according the guidelines approved by the ENFSI and always used before STR amplification of forensic casework DNA samples. Our attention has focused in particular on the definition of a minimum threshold at which it is useless to carry out DNA typing defining correlation of the negative results of the quantification by the absence of genetic profiles, as a result of DNA typing. Moreover, the validation of the Savant™ SPD131DDA SpeedVac™ Concentrator to get the maximum possible yield from DNA extracts was also investigated.     

How many DNA analyses are performed on adult sexual assault victims in Milan (Italy): A ten-year review

The aim of this study is to evaluate the actual use of serological/DNA analyses in the investigations carried out on adult sexual violence victims in Italy during the years 2006–2015.The victims were assisted in the largest Italian rape center, in Milan (Soccorso Violenza Sessuale e Domestica – SVSeD - Service for Sexual and Domestic Violence).The total number of sexual violence victims examined during the years 2006–2015 (adults and minors) was 3521, in 1697 of cases, biological evidence had been collected, while the number of adult victims (>18 y.o.) examined was 2300, in 1211 of cases biological evidence had been collected.Biological evidence was collected from the victims’ bodies using two swabs in five anogenital areas (labia maiora, labia minora, perineum, perianal and anal/rectum regions) and two swabs in all other skin areas suggested by the victims as areas of possible contact (double swab technique). Clothes were also collected on a case by case basis for the search of biological stains. Despite the proper collection, handling and chain of custody for all the swabs/items collected, serological/DNA analyses were requested in 86 cases out of 1211 only (710%). This percentage dropped to 190% when considering adolescent victims (13–19 y.o.).The reason why Italian Magistrates make little use of the powerful tool of DNA analyses in sexual assault cases, still remains unclear. Legal and procedural aspects are therefore also discussed.     

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.