Internal Validation of RapidHIT®ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†

A new rapid DNA solution, the RapidHIT^®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT^®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.     

Optimized Development of Sebaceous Fingermarks on Nonporous Substrates with Conformal Columnar Thin Films

A form of physical vapor deposition, called the conformal‐evaporated‐film‐by‐rotation (CEFR) method, was optimized for the conformal deposition of columnar thin films (CTFs) on sebaceous fingermarks. Relying on the surface topology of the fingermark, the CTF development technique is different from traditional development techniques. After the optimization of the development conditions, the CTF development technique was found to be superior to traditional development methods on several nonporous substrates: the smooth side of Scotch^® Multitask, Gorilla^®, and Scotch^® Duct tapes; clear and black soft plastics; stained and sealed walnut and cherry woods; partial bloody fingermarks on stainless steel; and discharged cartridge casings. It was equally as good as other development techniques on other substrates, but worse on a few. The optimization study is expected to assist in designing a mobile CEFR apparatus capable of on‐scene development of fingermarks.     

Optical enhancement of fingerprint deposits on brass using digital color mapping

Abstract: The reflection of visible light from α‐phase brass subject to surface oxidation in air at elevated temperatures is investigated. X‐ray photoelectron and auger electron spectroscopy confirm that covered areas of brass (not exposed to air) display dezincification but an absence of significant surface oxidation, confirming a differential oxidation mechanism. Visualization of differential oxidation is shown to be enhanced by selective digital mapping of colors reflected from the surface of the brass using Adobe^® Photoshop^®. Enhancement is optimal when the brass is heated to ～250°C with areas of oxidation having a mirror‐like appearance. The use of this enhancement method to produce a faithful image of fingerprint ridge characteristics is demonstrated on brass shell casings where fingerprints were deposited prefiring.     

A Comparison of Chemical Enhancements for the Detection of Latent Blood,,

Luminol, Bluestar^®, and Hemascein^® were tested to compare detection sensitivities to latent blood. Untreated, EDTA‐treated human blood, and a catalytically similar blood substitute were diluted (neat to 1:1,000,000) and pipetted onto a variety of substrates. Luminol and Bluestar^® performed similarly on all surfaces and fabrics. Hemascein^® yielded poor results on wood surfaces, but performed well in the detection of latent blood on fabrics. Results from untreated, EDTA‐treated, and synthetic blood results indicate that EDTA‐treated blood is similar or slightly less sensitive than untreated blood at all dilutions and on all substrates, and the synthetic blood is less sensitive than real blood, but consistent in detection threshold and thus is useful as a training aid. Additionally, some foods and household chemicals that have previously been shown to cross‐react were tested with Bluestar^®, Hemascein^®, and luminol. Hemascein^® cross‐reacted with many substances, while both luminol reagents were more discriminating.     

Performance Evaluation of a Registered Ballistic Database Using the Evofinder® System

Firearms for police in China are registered along with their fired bullets and cartridge cases. A Registered Ballistic Database (RBD) of 1000 Norinco QSZ‐92 pistols with registered ammunition was established and was evaluated through the Evofinder® system. In this research, 1000 bullets and 1000 cartridge cases were randomly selected and correlated against an RBD of 2996 bullets and 2999 cartridge cases. Examiners found that successful identifications all ranked 1st, supported with land (100%), groove (97%) engraved areas, and primary marks (85.6%) for bullets, and firing pin impressions (99.8%), and breech face marks (99.9%) for cartridge cases. Two known matches (KM) for the same pistol rank in the top two (100%). The distribution of similarity scores varies from marks; however, the Evofinder® system could still effectively distinguish known matches from known nonmatches (KNM) for either bullets or cartridge cases. This study demonstrates the efficiency of the RBD.     

An Accelerated Analytical Process for the Development of STR Profiles for Casework Samples

Significant efforts are being devoted to the development of methods enabling rapid generation of short tandem repeat (STR) profiles in order to reduce turnaround times for the delivery of human identification results from biological evidence. Some of the proposed solutions are still costly and low throughput. This study describes the optimization of an analytical process enabling the generation of complete STR profiles (single‐source or mixed profiles) for human identification in approximately 5 h. This accelerated process uses currently available reagents and standard laboratory equipment. It includes a 30‐min lysis step, a 27‐min DNA extraction using the Promega Maxwell^®16 System, DNA quantification in <1 h using the Qiagen Investigator^® Quantiplex HYres kit, fast amplification (<26 min) of the loci included in AmpF?STR^® Identifiler^®, and analysis of the profiles on the 3500‐series Genetic Analyzer. This combination of fast individual steps produces high‐quality profiling results and offers a cost‐effective alternative approach to rapid DNA analysis.     

Fentanyl transdermal patches: Extraction and evaluation of a novel disposal method using NarcX®

Currently, there is no known commercially available product for disposing of used fentanyl transdermal patches. To eliminate the potential for harm and abuse, a proper disposal method is needed–one that neutralizes the dangerous amount of residual fentanyl that remains after therapeutic use of the fentanyl patch. The patent-pending liquid solution of activated carbon, known as NarcX^®, was investigated as a potential fentanyl adsorbing agent. In order to determine the amount of fentanyl remaining after a patch is treated with NarcX^®, here, we utilized hexanes to first dissolve the patch adhesive and then followed with liquid-liquid extraction with methanol to recover the fentanyl. Using liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS), the extracts obtained with this method yielded between 85% and 117% recovery of fentanyl from new and unused patches. Further optimization of this method allowed for a quantitative evaluation of NarcX^®-treated fentanyl patches. 100 µg/h Apotex brand fentanyl patches were exposed to NarcX^® for 1, 24, 48, and 72 h. NarcX^® was shown to adsorb fentanyl from the patches with varying degrees of success, demonstrating an average of 66.98 ± 0.75% fentanyl adsorption after 72 h. These findings suggest that more work is needed to successfully neutralize the fentanyl patches in their entirety using NarcX^®; however, this work does demonstrate proof of concept.     

Can the RUVIS Reflected UV Imaging System Visualize Fingerprint Corrosion on Brass Cartridge Casings Postfiring?

Comparisons are made between the visualization of fingerprint corrosion ridge detail on fired brass cartridge casings, where fingerprint sweat was deposited prefiring, using both ultraviolet (UV) and visible (natural daylight) light sources. A reflected ultraviolet imaging system (RUVIS), normally used for visualizing latent fingerprint sweat deposits, is compared with optical interference and digital color mapping of visible light, the latter using apparatus constructed to easily enable selection of the optimum viewing angle. Results show that reflected UV, with a monochromatic UV source of 254 nm, was unable to visualize fingerprint ridge detail on any of 12 casings analyzed, whereas optical interference and digital color mapping using natural daylight yielded ridge detail on three casings. Reasons for the lack of success with RUVIS are discussed in terms of the variation in thickness of the thin film of metal oxide corrosion and absorption wavelengths for the corrosion products of brass.     

Lead isotope measurement of primer gunshot residues and likelihood ratio predictions for forensic cartridge discrimination and individualization in China

Gunshot residues (GSR), cartridge projectiles, and casings are frequently encountered evidence in gun-related forensic investigations. However, in circumstances where the investigation of striation marks is impossible, such as unrecovered or deformed projectiles and cartridge casings, GSR deposited on the hands or clothes of the shooter and victim-related items can provide information to establish a link between the suspect, the firearms used, and the victim. Since the formula of primers used by all cartridge manufacturers in China is identical, links based on the conventional morphological and compositional analysis of GSR are difficult to establish. However, the abundance of lead isotopes in primer components of lead styphnate varies significantly, and a fundamental understanding of these differences may facilitate the validation of primer (p)GSR evidence in forensic investigations. Here, 44 pGSR samples were characterized by Pb isotope ratios of ²⁰⁶Pb/²⁰⁴Pb, ²⁰⁷Pb/²⁰⁴Pb, and ²⁰⁸Pb/²⁰⁴Pb using laser ablation multicollector inductively coupled plasma mass spectrometry. There was no obvious mass fractionation of the lead isotope ratios of the primers from individual cartridges analyzed before and after the shooting process, thereby establishing a basis for the comparison of pGSR and unfired cartridges. Evaluation of the results using univariate likelihood ratio (LR) computations revealed low rates of misleading evidence (<0.53%) The results demonstrated that lead isotope ratio analysis of pGSR and LR predictions can provide a practicable method for forensic cartridge discrimination and individualization.     

Embutramide,a Component of Tanax®(T‐61) as a New Drug of Abuse?

Tanax^®(T‐61) is a euthanasia solution commonly used in veterinary medicine in Europe. It consists of three active components: embutramide, mebezonium iodide, and tetracaine hydrochloride. Human consumption of Tanax^®(T‐61) is usually associated with suicide attempts. In our 15‐year‐long practice, embutramide was detected only three times but within a short period. First, it was found in the urine of a 42‐year‐old veterinarian, and the other two observations were made in a 16‐year‐old young man. Urine samples were analyzed using Shimadzu Prominence TOX.I.S.II. HPLC–DAD system with online SPE extraction system. Both of the two patients denied any intention to die. These cases show that this veterinary drug may also be considered as potential drugs of abuse.     

Discriminating Hodgdon Pyrodex® and Triple Seven® Using Gas Chromatography–Mass Spectrometry

Abstract: Pyrodex ^® and Triple Seven ^® are black powder substitutes that often find use as fillers in improvised explosive devices, such as pipe bombs. These propellants have essentially the same overall appearance and oxidizers, but different fuels. For example, Pyrodex ^® contains sulfur, sodium benzoate, and dicyandiamide (DCDA), whereas Triple Seven ^® lacks sulfur but also contains 3‐nitrobenzoic acid. In this method, intact particles and postblast solid residues were reacted with bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane in acetonitrile for 30 min at 60°C. The resultant trimethylsilyl derivatives of the organic fuels were then analyzed by gas chromatography–mass spectrometry. Each derivative was clearly resolved from other components, and high‐quality mass spectra were obtained. In addition, characteristic fragments resulting from loss of a methyl radical from the molecular ion (m/z 163 for sulfur, m/z 171 for DCDA, m/z 179 for benzoic acid, and m/z 224 for nitrobenzoic acid) were able to be monitored.     

An investigation into the use of a portable cyanoacrylate fuming system (SUPERfume®) and aluminum powder for the development of latent fingermarks

Abstract: Few techniques offer “in situ” methods of friction ridge skin mark development. “In situ” development reduces mark transportation, degradation, and often cost. The effectiveness of cyanoacrylate fuming using the SUPERfume^® and dusting with aluminum powder for latent fingermark development on several nonporous surfaces, stored in various temperature environments for time periods up to 52 weeks, was investigated. Five thousand and four hundred latent fingermarks were deposited under controlled conditions and graded. The results suggested that cyanoacrylate fuming (SUPERfume^®, Foster and Freeman, U.K.) was more effective at developing latent fingermarks on textured and smooth plastic surfaces and for marks stored in temperatures of 37°C, whereas aluminum powder was more effective on glass, enameled metal paint, and varnished wood, and for storage temperatures below 20°C. There were no significant benefits to using either technique for marks older than 24 h, but it was possible to develop fingermarks following 52 weeks of storage using both techniques.     

Recovery of Latent Fingerprints and DNA on Human Skin*

Abstract: The project “Latent Fingerprints and DNA on Human Skin” was the first systematic research in Europe dealing with detection of fingerprints and DNA left by offenders on the skin of corpses. One thousand samples gave results that allow general statements on the materials and methods used. The tests were carried out according to a uniform trial structure. Fingerprints were deposited by natural donors on corpses. The latent fingerprints were treated with magnetic powder or black fingerprint powder. Afterward, they were lifted with silicone casting material (Isomark^®) or gelatine foil. All lifts were swabbed to recover DNA. It was possible to visualize comparable and identifiable fingerprints on the skin of corpses (16%). In the same categories, magnetic powder (18.4%) yielded better results than black fingerprint powder (13.6%). The number of comparable and identifiable fingerprints decreased on the lifts (12.7%). Isomark^® (14.9%) was the better lifting material in comparison with gelatine foil (10.1%). In one‐third of the samples, DNA could be extracted from the powdered and lifted latents. Black fingerprint powder delivered the better result with a rate of 2.2% for full DNA profiles and profiles useful for exclusion in comparison with 1.8% for the magnetic powder traces. Isomark^® (3.1%) yielded better results than gelatine foil (0.6%).     

Evaluation of the PowerPlex Fusion System in a sample from East Timor

East Timor is a country located in Southeast Asia. In this study, we determined allele frequencies and forensic parameters for 24 STR autosomal loci included in the PowerPlex^® Fusion System. Autosomal STR data was collected from saliva samples of 100 individuals from East Timor. The amplification of the 24 autosomal STRs was performed using PowerPlex^® Fusion System (Promega Corporation) and the amplified products were analysed on 3500 Genetic Analyser using GeneMapper^® ID-X 1.2 Software (Applied Biosystems). All the analysed loci meet Hardy–Weinberg equilibrium after Bonferroni correction. In our samples, we found “off-ladder” alleles (D2S441, Penta E and FGA locus) confirmed by reamplification, amplification with others kits and sequenced when justified.     

Manual and Automated Cardiopulmonary Resuscitation (CPR): A Comparison of Associated Injury Patterns,

The purpose of this study was to identify and compare patterns of trauma associated with AutoPulse^® CPR and manual CPR. Finalized autopsy records from 175 decedents brought to the Harris County Institute of Forensic Sciences were reviewed, 87 received manual‐only CPR, and 88 received AutoPulse^® CPR (in combination with manual CPR as per standard protocol). The characteristic pattern observed in manual‐only CPR use included a high frequency of anterior rib fractures, sternal fractures, and midline chest abrasions along the sternum. The characteristic pattern observed in AutoPulse^® CPR use included a high frequency of posterior rib fractures, skin abrasions located along the anterolateral chest and shoulder, vertebral fractures, and a few cases of visceral injuries including liver lacerations, splenic lacerations, and hemoperitoneum. Knowledge of the AutoPulse^® CPR injury pattern can help forensic pathologists differentiate therapeutic from inflicted injuries and therefore avoid an erroneous assessment of cause and manner of death.     

Powdered Activated Carbon: An Alternative Approach to Genomic DNA Purification

Forensic evidence samples are routinely found as stains on various substrates, which may contain substances known to inhibit polymerase chain reaction (PCR). The goal of this study was to evaluate post‐Chelex^®100 purification using powdered activated carbon (PAC). Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the DNA recovery and inhibitor removal using PAC with those of the Amicon^®Ultra 100K. For extracted bloodstains on soil and wood substrates, PAC and Amicon^®Ultra 100K generated similar DNA yield and quality. Moreover, the two methods significantly decreased the concentration of humic substances and tannins compared to nonpurified extracts (p < 0.001). In instances where extracts contained indigo dye (bloodstains on denim), Amicon^®Ultra 100K performed better than PAC due to improved amplifiability. Efficient adsorption of humic substances and tannins, which are common inhibitors, indicates PAC's potential application in the purification of high‐template DNA extracts.     

An Investigation of PCR Inhibition Using Plexor®‐Based Quantitative PCR and Short Tandem Repeat Amplification

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor^® real‐time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and C_T takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex^® 16 HS System. The overall results demonstrate that real‐time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.     

Low-cost direct PCR for aged and processed wildlife sample analysis

Direct PCR has been used successfully in wildlife forensic DNA analysis from several types of biological samples using specialized, commercial direct PCR kits. This is attributed to the proprietary chemicals provided in the kits such as pre-PCR buffer and modified DNA polymerases. These reagents can be expensive, thereby limiting their widespread adoption in developing countries, where wildlife crimes are often encountered. We report on a study to evaluate the possibility of using low-cost direct PCR assay for degraded and processed wildlife sample analysis. Phire^® and Q5^® polymerases were used, due to their relatively low cost, for direct amplification of six aged and processed sample types (dried skin, 30-year old hair, muscle tissue, bone, trace blood mixed in vodka, and dried soft antler). The result indicated that Phire^® Hot Start II DNA polymerase and Q5^® DNA polymerase performed similarly to commercially available direct PCR kit. The low-cost amplification could efficiently identify species origin from all aged and processed samples. We observed a rate of more than 80% amplification success and high PCR product concentrations sufficient for further sequencing. The assay proved to be cost effective and robust; thus, we expect it to be adopted by wildlife forensic laboratories in developing countries.     

Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance

Abstract: Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit is an improved version of the AmpF?STR^® Identifiler^® PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex‐determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit for human identity and parentage testing.