Forensic Laboratories Research Articles

Evaluation of in-house drug evidence testing by King County Medical Examiner's Office in "real-time" fatal overdose surveillance.

With the escalating overdose epidemic, many surveillance efforts have appeared. In 2018, King County Medical Examiner's Office (KCMEO) initiated a fatal overdose surveillance project aimed at expediting death certification and disseminating timely information. In this project, KCMEO investigators collected items of evidence of drug use from overdose death scenes, which were tested by five in-house methods, four using handheld devices: TruNarc Raman spectrometer, with and without the manufacture's H-Kit, Rigaku ResQ Raman spectrometer, and MX908 mass spectrometer. The fifth in-house method used fentanyl-specific urine test strips. Results from in-house testing were compared with results from Washington State Patrol (WSP) Materials Analysis Laboratory. From 2019 to 2022, there were 4244 evidence items of drugs and paraphernalia collected from 1777 deaths scenes. A total of 7526 in-house tests were performed on collected specimens, and 2153 tests were performed by the WSP laboratory using standard analytical methods. The WSP results served as reference standards to calculate performance metrics of the in-house methods. Sensitivities, specificities, and predictive values ranged from good to poor depending on the method, drug, and evidence type. Certain drugs were often associated with specific evidence types. Acetaminophen was frequently found in combination with fentanyl. Fentanyl test strips gave good scores for detecting fentanyl; otherwise, in-house methods using handheld devices had poor performance scores with novel drugs and drugs diluted in mixtures. The results showed that in-house testing of drug evidence has value for medical examiner overdose surveillance, but it is resource intensive, and success depends on collaboration with forensic laboratories.

Cross-reactivity of 24 cannabinoids and metabolites in blood using the Immunalysis Cannabinoids Direct enzyme-linked immunosorbent assay.

With wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥20 ng/mL, with increasing alkyl side-chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

Optimizing analytical precision in the identification of synthetic cathinones and isomers: a comparative assessment of diverse GC-MS operating parameters.

Analyzing new psychoactive substances (NPSs) in forensic laboratories present a formidable challenge globally. Within illicit drug analysis, gas chromatography-mass spectrometry (GC-MS) emerges as a robust analytical tool. This study endeavors to assess and compare peak resolution in the analysis of illicit drugs, specifically focusing on 21 synthetic cathinones, encompassing 9 cathinone isomers. Varied GC-MS operating conditions, including distinct GC-MS columns and thermal gradients, were systematically employed for the simultaneous analysis of these synthetic cathinones. The study utilized HP-1 nonpolar and HP-5MS low-bleed columns to achieve optimal analyte resolution through modulation of GC-MS oven conditions. Mass spectra were meticulously recorded within a mass-to-charge (m/z) range spanning from 40 to 500 in full scan mode. The data showed that the cathinone isomers slightly differed in retention times and mass spectra. The GC oven conditions affected the peak resolution for chromatographic separation even with the same column. The peak resolution improved using a slower thermal gradient heat speed with a prolonged analysis time. Conclusively, the interplay of GC columns and thermal gradients emerged as pivotal factors impacting peak resolution in the analysis of illicit drugs. These empirical insights contribute to a nuanced understanding of peak resolution dynamics and facilitate the identification of synthetic cathinones, including their isomers, in seized materials through the judicious application of GC-MS methodologies.

DNA Extraction Methods

KATIE LONG and MARK FLOOD, Forensic Science Program, Fairmont State University, Fairmont, WV 26554. Evaluation of DNA Extraction Methods. Three forms of DNA extraction types include solution-based, column-based, and magnetic bead-based methodologies, with the latter being the accepted methodology used in forensic laboratories. Magnetic bead-based DNA extraction uses the negative charge of DNA and the positive charge of the magnetic beads to isolate the DNA strands and produce a purified sample. However, solution and column-based extraction methods use the same techniques of washing to purify DNA and typically have comparable DNA yields. Cheek swabs were obtained from ten individuals and the three extraction methods were performed to determine the quantitation of double-stranded DNA and the quality of the gel electrophoresis profile. Preliminary results indicate that solution-based extraction produces a slightly higher yield of double-stranded DNA having a 3.92% difference between the average quantitation of 26.29 ng/µL from solution-based and 25.28 ng/µL from magnetic bead-based methods. However, it is expected that the magnetic bead-based extraction will produce higher quality gel electrophoresis results. Overall, the methods examined produced similar DNA extraction efficiencies. This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant #80NSSC20M0055 and the STaR Sure Grant.

Forensic Evidence Management Using Blockchain Technology

In the realm of forensic investigations, the management of evidentiary artifacts is a critical aspect that influences the integrity and admissibility of evidence in legal proceedings. The trustworthiness of evidence can be jeopardized by traditional evidence management systems’ frequent problems with data manipulation, unauthorized access, and lack of openness. Blockchain technology has surfaced as a viable approach to tackle these issues by offering a decentralized, transparent, and immutable foundation for data management in recent times. This paper explores the application of blockchain technology in forensic evidence management, highlighting its potential to enhance the security, integrity, and traceability of evidentiary artifacts throughout their lifecycle. By leveraging blockchain's inherent features such as cryptographic hashing, consensus mechanisms, and smart contracts, forensic practitioners can establish a tamper-proof chain of custody, ensure data integrity, and streamline the evidence management process. Moreover, blockchain- based evidence management systems offer benefits such as enhanced transparency, reduced reliance on centralized authorities, and improved collaboration among stakeholders. The paper discusses various use cases and implementations of blockchain technology in forensic evidence management, ranging from digital chain of custody records to decentralized forensic laboratories. Through a comprehensive analysis of the potential benefits and challenges, this paper aims to provide insights into the transformative impact of blockchain technology on forensic evidence management practices, paving the way for more secure, efficient, and trustworthy forensic investigations in the digital age.

Local anesthetic marcaine-bupivacaine hydrochloride and its metabolites in Forensic Samples: A case study

The local anaesthetic bupivacaine and its metabolites such as Desbutyl bupivacaine, 3-hydroxybupivacaine, and 4-hydroxybupivacaine were extracted and found in the forensic sample from postmortem samples such as cerebrospinal fluid (CSF) and postmortem blood. To separate and detect the aforesaid substances in biological matrices, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and ATR-FTIR methods were used. When a couple of husband and wife who were riding on the side of road by a motor cycle which was abruptly collided by an unknown motor cycle. The couple were injured, especially in the left leg of the lady was severe pain due to bone fracture. Both were immediately admitted in nearby a Government Hospital for treatment. Anaesthetic was injected to the lady before starting the procedure of surgery. Within a few minutes she suffered pulmonary consequence and the mortality immediately occurred. Due to a therapeutic amount of bupivacaine was given, postmortem samples CSF and blood were received at forensic laboratory and extracted using a liquid-liquid extraction method (LLE). Certain chromatographic techniques and ATR-FTIR method were used to separate and detect bupivacaine and its metabolites in a forensic sample.

Interlaboratory validation and one-year retrospective review of hemp and marijuana decision-point assays

After the passage of the Agriculture Improvement Act of 2018 (Farm Bill) that defined hemp as Cannabis sativa L. if it contained ≤0.3 % Δ9-THC on a dry weight basis, forensic laboratories were faced with the challenge of distinguishing marijuana from hemp in seized drug samples. This study reports the results of an interlaboratory validation of a previously developed qualitative decision-point assay for the differentiation of illegal marijuana from legal hemp, utilizing gas chromatography-mass spectrometry (GC–MS) with a 1 % Δ9-THC threshold. The method was validated in terms of selectivity, linearity, carryover, precision, accuracy, extract stability, dilution integrity, and measurement uncertainty. Other elements such as decarboxylation rate and potential for CBD interference were also evaluated. Specificity and positive predictive value were both 100 % using known cannabis reference materials. A total of 8 false negative results were observed among the 280 analyses, resulting in an overall assay sensitivity of 94 % and a negative predictive value of 95 %. The small number of false negative results near the 1 % threshold were attributed to decarboxylation inefficiency in some laboratories. While qualitative in nature, the measurement uncertainty of the assay at the 0.3 % Δ9-THC cutoff (legal threshold) using a 95.45 % confidence interval (k = 2) ranged from 12.2 % to 21.8 % among laboratories (equivalent to 0.3 % ± 0.04 % to 0.07 % Δ9-THC by weight). A one-year retrospective study across three agencies and fifteen laboratories was undertaken. Among the three agencies participating in the original study, 93–96 % of all suspected cannabis seizures yielded results above the administrative threshold of 1 % Δ9-THC (n = 3,288). The interlaboratory method development highlighted differences in performance between sites, ultimately improving the overall robustness of the method. Observations highlight the need for site-specific validation and vigilance. While multi-agency collaborations and interlaboratory validations are valuable, they do not replace the necessity for full, independent, and rigorous validation.

Species and origin determinations of an ivory chess set: An application of the ivory workflow implemented by California’s Wildlife Forensic Laboratory

Despite the prevalence of elephant poaching and ivory trafficking, domestic and international ivory markets around the world are slowly closing due to increased education and enforcement efforts. This includes California’s ivory market in 2016 after the passage of Assembly Bill 96 (AB 96), which prohibits the purchase, sale, offer for sale, possession, or importation with intent to sell, of ivory from elephant, mammoth, and mastodon along with other non-proboscidean species. To assist with enforcement efforts, the California Department of Fish and Wildlife’s Wildlife Forensic Laboratory (CDFW-WFL) has created and implemented a scientific workflow to taxonomically identify, geographically assign, and age California’s seized ivory. Here we discuss the application of this scientific workflow to a 32-piece ivory chess set, which was purchased in 1969 and donated to and examined by the WFL in 2021. Genetic data revealed 11 unique haplotypes and 19 unique genotypes, suggesting a diverse set of African elephants from numerous locations in Africa were used to assemble the chess set. Stable isotope data corroborated these findings and radiocarbon dating suggested the ivory used to carve these chess pieces grew approximately 6 years prior to the chess set being purchased. Our results indicate that the use of a variety of scientific techniques provides a wide scope of information; furthermore, taxonomically identifying, geographically assigning, and aging the ivory chess set demonstrates to law enforcement officers how our ivory workflow can assist them in coordinating efforts locally, nationally, and internationally to help stop the illegal importation of ivory into California.

Screening for Forensically Relevant Drugs Using Data-Independent High-Resolution Mass Spectrometry.

Forensic and clinical laboratories are expected to provide a rapid screening of samples for a wide range of analytes; however, the ever-changing landscape of illicit substances makes analysis complicated. There is a great need for untargeted methods that can aid these laboratories in broad-scope drug screening. Liquid chromatography hyphenated with high-resolution mass spectrometry (LC-HRMS) has become a popular technique for untargeted screening and presumptive identification of drugs of abuse due to its superior sensitivity and detection capabilities in complex matrices. An untargeted extraction and data acquisition method was evaluated for the broad screening of high-priority drugs of abuse in whole blood. A total of 35 forensically relevant target analytes were identified and extracted at biologically relevant low and high (10× low) concentrations from whole blood using supported liquid extraction. Data-independent acquisition was accomplished using ultraperformance liquid chromatography and a quadrupole time-of-flight mass spectrometry. Results were acceptable for screening assays, with limits of detection at or below the recommended low-concentration cutoffs for most analytes. Analyte ionization varied from 30.1 to 267.6% (average: 110.5%) at low concentrations and from 8.6 to 383.5% (average: 93.6%) at high concentrations. Extraction recovery ranged from 8.5 to 330.5% (average: 105.3%) at low concentrations and from 9.4 to 127.5% (average: 82.7%) at high concentrations. This variability was also captured as precision, ranging from 4.7 to 135.2% (average: 36.5%) at low concentrations and from 0.9 to 59.0% (average: 21.7%) at high concentrations. The method described in this work is efficient and effective for qualitative forensic toxicology screening, as demonstrated by analysis of 166 authentic suspected impaired driver and postmortem specimens. That said, it is critical that laboratories establishing untargeted LC-HRMS screening assays be aware of the strengths and limitations across diverse drug categories and chemical structures.

An inter-laboratory comparison of probabilistic genotyping parameters and evaluation of performance on DNA mixtures from different laboratories

Probabilistic genotyping (PG) is becoming the preferred standard for evidence interpretation, amongst forensic DNA laboratories, especially those in the United States. Various groups have expressed concern about reliability of PG systems, especially for mixtures beyond two contributors. Studies involving interlaboratory testing of known mixtures have been identified as ways to evaluate the reliability of PG systems. Reliability means different things in different contexts. However, it suffices here to think about it as a mixture of precision and accuracy. We might also consider whether a system is prone to producing misleading results – for example large likelihood ratios (LRs) when the POI is truly not a contributor, or small LRs when the POI is a truly a contributor. In this paper we show that the PG system STRmix™ is relatively unaffected by differences in parameter settings. That is, a DNA mixture that is analyzed in different laboratories using STRmix™ will result in different LRs, but less than 0.05% of these LRs would result in a different, or misleading conclusion as long as the LR is greater than 50. For the purposes of this study, we define LRs assigned using different parameters for the same mixtures as similar if the LR of the true POI is greater than the LRs generated for 99.9% of the general population. These findings are based on an interlaboratory study involving eight laboratories that provided twenty known DNA mixtures of two to four contributors and their individual laboratory STRmix™ parameters. The eight sets of laboratory parameters included differences in STR kits and PCR cycles as well as the peak, stutter, and locus specific amplification efficiency variances.

InDEL instability in two different tumoral tissues and its forensic significance.

There may be cases where malignant tumor samples can be used for forensic DNA profiling studies. STRs are the first systems preferred in forensic science laboratories for identification purposes. However, genetic instability in tumoral tissues causes STR polymorphism to change, leading to erroneous results. On the other hand, insertion/deletion polymorphism (InDels) are used as genetic markers in forensic science, as they have features that make both STR and SNPs preferable. Although previous studies approved that STR instability is observed in many different tumors, there are only a few studies that have displayed the instability of InDels in tumoral tissues before. In this study, it was aimed to determine whether instability is observed in formalin-fixed paraffin-embedded breast and thyroid tumoral tissues at 36plex InDel Panel. A total of 47 cases, 26 of which were diagnosed as breast cancer and 21 as thyroid cancer, were included in the study. In 21 of 26 (80.76%) breast cancers mutational changes were observed, however only 6 of 21 (28.57%) thyroid carcinoma cases displayed instability.Moreover, in these six cases, mutations were detected at only 1 or 2 loci. The most common change in both tissues was loss of heterozygosity. These findings suggest that paraffin embedded tissues of thyroid tumor can be used in cases of forensic genetic identification, however paraffin embedded breast cancer tissues should be examined with care. In conclusion, low InDel mutation rates compared to STR instability, make InDel analysis from paraffin blocks suitable for forensic genetic identification. However, researchers should keep in mind that there may be differences between the profiles of the tumoral tissues taken as reference and the actual case. In addition, by incorporating additional markers such as SNPs and microhaplotypes with low mutation rates into the study alongside Indels, researchers can significantly enhance the discrimination power in identification processes.

Quantitation of propofol metabolites by LC–MS/MS demonstrating long detection window for urine drug monitoring

IntroductionChromatographic methods for analysis of propofol and its metabolites have been widely used in pharmacokinetic studies of propofol distribution, metabolism, and clearance. Application of chromatographic methods is also needed in clinical and forensic laboratories for detecting and monitoring propofol misuse. ObjectiveWe report a method for sensitive analysis of propofol, propofol 1-glucuronide (PG), 4-hydroxypropofol 1-glucuronide (1-QG), 4-hydroxypropofol 4-glucuronide (4-QG) and 4-hydroxypropofol 4-sulfate (4-QS) in urine by LC–MS/MS analysis. The method employs a simple dilute-and-analyze sample preparation with stable isotope internal standardization. ResultsValidation studies demonstrate a linear calibration model (100–10,000 ng/mL), with dilution integrity verified for the extended range of concentrations experienced in propofol use. Criteria-based validation was achieved, including an average coefficient of variation of 6.5 % and a percent bias of −4.2 ng/mL. The method was evaluated in 12 surgical patients, with monitoring periods lasting up to 30 days following intravenous propofol administrations of 100–3000 mg on the day of surgery. While the concentration ratio of PG to 4-hydroxy propofol metabolite decreased significantly in the days following surgery, PG maintained the highest concentration in all specimens. Both PG and 1-QG were detectable throughout the monitoring periods, including in a patient monitored for 30 days. Lower concentrations were determined for 4-QG and 4-QS, with evidence of detection up to 20 days. Propofol was not detectable in any urine specimens, thereby proving ineffective for identifying drug use. ConclusionThe validated method for quantifying propofol metabolites demonstrates its applicability for the sensitive detection of propofol misuse over a long window of drug-use detection.

Analysis of Cannabis and its Products in the Context Forensic Science

Legalization of low-tetrahydrocannabinol-high-cannabidiol cannabis plants is gaining momentum due to growing demand for these products. In some countries, the testing of cannabis and products containing it is a legal requirement: cannabis is identified, the amount of tetrahydrocannabinol or cannabidiol and their precursor acids is assessed. Current research requires more sensitive and reliable analytical methods for accurate identification and quantification of cannabis components. This article purpose is to review the latest advances in the scientific literature on the isolation and analysis of cannabis and cannabis-containing products in the context of forensic science. Research methods included analysis of information on this topic using search engines, including Google, Google Scholar, Web of Science, PubMed, and ScienceDirect. Search requests took into account scientific, analytical and statistical reports in the field of forensic research. Since the forensic expert must be well-versed in modern trends in the analysis of prohibited substances and use the latest data from the analytical and forensic literature in his work, such information will help them choose research methods, taking into account the actual resources and equipment of forensic laboratories. According to the article purpose, advantages and disadvantages of existing methods of extraction and analysis of objects containing cannabis for identification, quantification, profiling and age estimation of cannabis are considered.

Analysis of markers for forensic plant species identification

While plant species identification in forensics can be useful in cases involving poisonous, psychoactive, or endangered plant species, it can also become quite challenging, especially, when dealing with processed, decaying, colonized or infected material of plant origin. The Animal Plant and Soil Traces expert working group of the European Network of Forensic Science Institutes in their best practice manual has recommended several markers for plant species identification. Current study is a part of implementation of method in a forensic laboratory and its aim is to evaluate four of the recommended markers (ITS, matK, rbcL, and trnH-psbA) for species identification of forensically important plant species including medicinal, poisonous, psychoactive, and other plants. Such parameters as PCR and sequencing success, sequence length, species resolution rate and species cover in GenBank were analysed. Blind testing was performed to evaluate use of the markers for identification of forensically more complicated samples.According to results, a combination of ITS, matK and trnH-psbA is the best choice for plant species identification. The best results with fresh plant material can be achieved with ITS, trnH-psbA, and matK, while ITS and matK are the best choice when working with low quality plant material. rbcL due to its low species discrimination rate can be used only as an indicative marker.

Why Down-managing Backlog Forensic DNA Case Entries Matters

Forensic laboratories face a backlog of case files, affecting service delivery, causing delays. Forensic laboratories face a backlog of case ϐiles, affecting service delivery and causing delays. The backlog points to underfunding, poor planning, and inadequate support, hindering deoxyribonucleic acid (DNA) analysis. Resolving casework backlogs may initially seem like a straightforward and attainable measure to improve the arrest of offenders and promote justice. Longer turnaround times impede investigative leads, emphasising the need for efficient strategies and a comprehensive approach to address and prevent backlogs in forensic laboratories. No study has been published on the forensic DNA backlogs in South Africa. The article explicitly addresses one aspect of a Doctor of Philosophy study and aims to ascertain the impact of backlogs of forensic DNA case entries. The study article’s research questions included the following: “What cases are considered as backlog?”; “What is the current backlog in forensic DNA case entries in South Africa?” and “What are the main reasons for the backlog of cases involving forensic DNA?” The prompt processing of DNA evidence is vital not only for safeguarding individuals falsely accused of crimes based on circumstantial evidence but also for adding prosecutors and providing justice for crime victims.

Development of a novel seized drug screening method utilizing DART-MS and used weigh paper

The conventional analytical scheme for seized drug analysis utilizes quick and cost-effective presumptive testing, such as color tests, followed by more discriminatory techniques, such as gas chromatography-mass spectrometry (GC–MS). Before the seized drug evidence is analyzed, the evidence is weighed using an appropriate weighing matrix and balance. Typically, the weighing matrix is discarded as chemical waste; however, the process of weighing the evidence leads to the deposition of drug residue onto the weighing matrix, which is then amenable to rapid analysis using ambient ionization approaches, such as direct analysis in real time mass spectrometry (DART-MS). This study demonstrates a DART-MS method for the rapid screening of seized drug evidence using filter paper and glassine paper weighing matrices commonly found in forensic laboratories. Validation experiments were performed using filter paper and glassine paper and compared to the results obtained using glass capillaries to assess the effectiveness of each weighing matrix. A total of 40 authentic samples, 20 filter papers and 20 glassine papers, were analyzed and the resulting spectra were searched using the National Institute of Standards and Technology (NIST) DART-MS Forensics Database and Data Interpretation Tool (DIT). When compared to the ground truth GC–MS results, the developed approach had a 90% correct identification rate, with all misidentifications being due to the presence of tablets rather than powder. The developed DART-MS screening method provides the seized drug community with a method to rapidly screen seized drug residue remaining from the weighing process and to improve the overall laboratory efficiency.

Intelligence and the Value of Forensic Science

Recent research has seen a rapid expansion in the reference to front-end forensics as an indication of the untapped value of forensic science. While some of these contributions have centered on development of forensic intelligence from a single area of investigation, others call for a more fundamental change in the relationship between crime laboratories and policing, particularly relating early laboratory analysis with big datasets to provide leads to investigators. We highlight several recently implemented tactical strategies of crime laboratories that contribute to the body of forensic intelligence. Beyond the scientific gains from these tactical applications, the corresponding details on associated efficiencies, costs, time savings, and quality improvements offer insights towards patterns of success for the community of crime laboratories. Further details expand an interpretation of what constitutes success with an eye on the contributions of the crime laboratory towards public health, safety, and protection of the innocent in addition to societal gains from conviction of the guilty. The economic interpretation of the value provided by the forensic laboratory assists in the cost–benefit review of strategic and tactical decisions and supports the justification for laboratory public funding with measures of the return on investment from public support of the forensic laboratory. Examples of the cost savings from crimes avoided include the returns from testing the backlog of unsubmitted sexual assault kits with USD 81 of costs avoided for each USD 1 spent to high-efficiency laboratories achieving gains of USD 646 for each UD 1 spent on de novo case submissions.

Chemical analysis of polymers used for 3D printing of firearms

3D-printed firearms cause challenges in criminal investigations and forensic analysis because they are difficult to trace. Indeed, in addition to being "ghost guns", they may not produce all the conventional ballistic traces normally used for firearm identification. However, 3D-printed firearms produce other very specific traces, such as polymer traces which come from the polymers used to print the firearm. To date, only a few studies have focused on the analysis of polymer traces. This study therefore aims to characterize polymer traces from 3D-printed firearms, using non-destructive spectroscopic techniques readily available in most forensic laboratories (i.e., FTIR and Raman) and evaluate the potential for association of polymer specimens or traces with their source. To do so, the study was divided into four parts. First, the population study conducted among 3D printing companies and individuals practicing 3D printing has revealed that PLA and PLA+ are the most widely used polymer types in Quebec, Canada. Second, FTIR and Raman spectroscopic analysis of polymer samples collected during the population study has allowed the development of a reference polymer spectral database. The analysis and interpretation of these spectra revealed that polymer filaments present very low intravariability, but very high intervariability, due in part to the different polymer types and the pigments used to color them. The use of chemometric tools with the spectra showed that these two spectroscopic methods were highly discriminating. Third, test firing of 3D-printed firearms has allowed for the simulation of a scene involving this type of firearm and the collection of polymer traces generated. Fourth, the comparison of chemical signatures between polymer filaments and polymer traces has allowed for the evaluation of the potential for chemical association. This study highlights the added value of chemical analysis of 3D-printed firearms polymer traces in a criminal investigation by demonstrating that polymer filaments, the polymer from which a 3D-printed firearm is made, as well as polymer traces generated during firing, can be linked chemically and provide relevant information.

Strategies to deal with genetic analyzer-specific DNA methylation measurements.

Targeted bisulfite sequencing using single-base extension (SBE)can be used to measure DNA methylation via capillary electrophoresis on genetic analyzers in forensic labs. Several accurate age prediction models have been reported using this method. However, using different genetic analyzers with different software settings can generate different methylation values, leading to significant errors in age prediction. To address this issue, the study proposes and compares four methods as follows: (1) adjusting methylation values using numerous actual body fluid DNA samples, (2) adjusting methylation values using control DNAs with varying methylation ratios, (3) constructing new age prediction models for each genetic analyzer type, and (4) constructing new age prediction models that could be applied to all types of genetic analyzers. To test the methods for adjusting values using actual body fluid DNA samples, previously reported adjusting equations were used for blood/saliva DNA age prediction markers (ELOVL2, FHL2, KLF14, MIR29B2CHG/C1orf132, and TRIM59). New equations were generated for semen DNA age prediction markers (TTC7B, LOC401324/cg12837463, and LOC729960/NOX4) by drawing polynomial regression lines between the results of the three types of genetic analyzers (3130, 3500, and SeqStudio). The same method was applied to obtain adjustment equations using 11 control DNA samples. To develop new age prediction models for each genetic analyzer type, linear regression analysis was conducted using DNA methylation data from 150 blood, 150 saliva, and 62 semen samples. For the genetic analyzer-independent models, control DNAs were used to formulate equations for calibrating the bias of the data from each genetic analyzer, and linear regression analysis was performed using calibrated body fluid DNA data. In the comparison results, the genetic analyzer-specific models showed the highest accuracy. However, genetic analyzer-independent models through bias adjustment also provided accurate age prediction results, suggesting its use as an alternative in situations with multiple constraints.

Use of the Investigator ESSplex SE QS Kit (QIAGEN) at Half PCR Reaction Volumes for the Analysis of Forensic Samples

The Investigator ESSplex SE QS Kit (Qiagen) is a next-generation polymerase chain reaction (PCR) kit that, in 60 min, amplifies 17 Short Tandem Repeat (STR) markers, including the five European Standard Set (ESS) loci (D10S1248, D12S391, D1S1656, D22S1045, D2S441), the SE33 marker, and the locus Amelogenin for sex determination. Two quality sensors (QS1 and QS2) are also co-amplified to check PCR performance. Since forensic laboratories carry out hundreds of DNA typings annually, we verified the kit’s performance using half reaction volumes with the aim of improving the number of samples that may be amplified with a single kit and consequently reducing laboratory costs. In the present study, intended as a technical note rather than internal validation, some control samples (oral swabs) with known DNA profiles and 40 real casework samples were analyzed. We observed that reducing the total reaction volume, while keeping all component ratios unaltered, yields DNA profiles comparable to those obtained using standard reaction volumes and with allele peaks higher than those with regular volumes. Using half volumes for PCR amplification enables the analysis of a larger number of samples compared to the standard protocol, thereby reducing laboratory costs without compromising the quality of the analysis.