Determination of Both Fetus’ and Mother's Blood Type from an Autopsy Case Immersed in Formalin for Over 50 Years

A female fetus which had been immersed in formalin for more than 50 years was found in Japan. Because no liquid blood could be obtained, we tried to use immunohistochemistry (IHC) methods to tissue samples obtained at autopsy to identify both the fetal and mother's blood type. We detected B antigens on endothelial cells in paraffin sections of the fetal organs. Furthermore, we observed both anti‐A‐ and anti‐B‐positive red blood cells in the intervillous space, which is indicative of the mother's blood type. To our knowledge, this is the first case report on determining the blood type of both the fetus and the mother from tissue immersed in formalin for such a long time. The results suggest that IHC is valuable for the determination of ABO blood type in circumstances of long postmortem duration and unfavorable storage conditions.     

Linking ammonium nitrate-aluminum (AN-AL) post-blast residues to pre-blast explosive materials using isotope ratio and trace elemental analysis for source attribution

Forensic science practitioners are often called upon to attribute crimes using trace evidence, such as explosive remnants, with the ultimate goal of associating a crime with a suspect or suspects in order to prevent further attacks. The explosive charge is an attractive component for attribution in crimes involving explosives as there are limited pathways for acquisition. However, there is currently no capability to link an explosive charge to its source via post-blast trace residues using isotope ratios or trace elements. Here, we sought to determine if pre-blast attribution signatures are preserved after detonation and can be subsequently recovered and detected. A field study was conducted to recover samples of post-blast explosives from controlled detonations of ammonium nitrate-aluminum (AN-Al), which were then analyzed via isotope ratio mass spectrometry (IRMS) and inductively coupled plasma-mass spectrometry (ICP-MS) for quantitation and profiling of isotopes ratio and trace element signatures, respectively. Oxygen and nitrogen isotope ratios from AN-Al yielded some of the most promising results with considerable overlap within one standard deviation of the reference between the spreads of pre- and post-blast data. Trace element results from AN-Al support the findings in the isotope ratio data, with 26 elements detected in both pre- and post-blast samples, and several elements including B, Cd, Cr, Ni, Sn, V, and Zn showing considerable overlap. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute signatures from post-blast debris to signatures in pre-blast explosive materials for use in future investigations.     

An interlaboratory study to evaluate the utility of gas chromatography-mass spectrometry and gas chromatography-infrared spectroscopy spectral libraries in the forensic analysis of fentanyl-related substances

Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.     

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49–246 ng/mg for caffeine abusers, and 0.25–363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.     

Evaluation of portable gas chromatography–mass spectrometry (GC–MS) for the analysis of fentanyl,fentanyl analogs,and other synthetic opioids

Potent synthetic opioids including fentanyl and its analogs are frequently encountered in the field and require detection and identification by first responders to maintain the safety of drug abusers, first responders, health-care providers, and the public at large. Due to the low concentration at which these substances may be encountered and the complicating matrices within which they may be dispersed, the use of portable gas chromatography–mass spectrometry (GC–MS) for their identification in the field offers great potential value. This research established that portable GC–MS is a useful method for the detection and identification of a large number of synthetic opioids, especially fentanyl and its analogs. In this study, 250 synthetic opioids and related substances including 210 fentanyl analogs were analyzed using portable GC–MS. It was concluded that 225 of the 250 (90.0%) opioids analyzed were successfully detected onboard at the time of analysis and identified as either the substance (55.2%) or an analog (34.8%). These outcomes have equivalent benefit for the field analysis of illicit drugs due to both initiating the same subsequent actions by first responders.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Ethanol stability in unpreserved refrigerated antemortem blood

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8 mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003–0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.     

Forensic examination of security features of the CDC-issued COVID-19 vaccination card and suggested improvements to minimize counterfeiting

During the COVID-19 pandemic, the United States released the CDC-issued vaccination card to document the status of immunizations. It eventually was used as a verification system to allow patrons to safely access public venues. This is/was apparently a simple printed piece of paper with minimal security features which made it an easy target for counterfeiters with little expertise required. Those unvaccinated who wished to enter areas that required proof of the COVID-19 vaccine were soon finding alternative illegal methods to do so which led to public health concerns. This research was divided into two parts. Part A aimed to forensically identify the security features with a video spectral comparator and a stereomicroscope. Ten “known” blank cards were collected directly from dispensaries that administered the vaccine and 68 “unknowns” from individuals who received the COVID-19 vaccine. Part B aimed to evaluate the authentication systems employed by countries outside the United States to propose a model on how the vaccination card could be improved and what security features should be included for future instances requiring proof of immunization. The results revealed the CDC document was produced with minimal security features making the document prone to counterfeiting. Furthermore, based on the information collected from other countries' vaccination certificates, it is recommended to develop a dual authentication system, with digital and printable aspects, that contains a QR code linked to a database. Other security features may include a non-optical brightener substrate, security fibers, a watermarked logo, offset printing, and a geographical color-coded system.     

New materials used for the synthesis of 2-chlorophenyl cyclopentyl ketone seized from an illicit ketamine manufacturing unit

Ketamine deemed as a psychoactive substance has gained popularity for recreational use owing to its hallucinogenic and dissociative effects. Understanding the synthetic processes of ketamine can provide essential clues for law enforcement officers against illicit ketamine manufacturing. In this case report, a chemical company was being monitored by law enforcement officers due to its importation of precursors and materials that could be used for the synthesis of illicit drugs. After materials and products seized from this chemical company were employed for analyses using gas chromatography–mass spectrometry, liquid chromatography–high-resolution mass spectrometry, and nuclear magnetic resonance analyses, ketamine, hydroxylamine, 2-chlorophenyl cyclopentyl ketone, and cyclopentanone p-toluenesulfonylhydrazone were identified. In addition, a novel process for the synthesis of ketamine precursor 2-chlorophenyl cyclopentyl ketone from cyclopentanone p-toluenesulfonylhydrazone and 2-chlorobenzaldehyde was validated. This is the first report to uncover this novel process for the synthesis of 2-chlorophenyl cyclopentyl ketone and can be used to increase awareness among law enforcement officers and forensic practitioners about these novel starting materials for the synthesis of ketamine.     

Visualization of latent fingerprints using fluorescence lifetime imaging on paper emitting strong fluorescence

Latent fingerprints were successfully visualized using fluorescence lifetime imaging (FLIM) on paper which emits strong fluorescence with a lifetime close to that of fingerprints and thus from which it is difficult for time-resolved spectroscopy to visualize fingerprints. Latent fingerprint samples on paper were excited using a 450 nm or 532 nm nanosecond pulsed-laser, and time-resolved fluorescence images were obtained at a delay time of 6–16 ns in intervals of 1 ns, to the excitation pulse. The excitation beam was expanded using a lens, and the fluorescence from the fingerprints was captured using an intensified CCD camera. Because of the large fluorescence intensity of the background paper of approximately two to four orders of magnitude larger than that of the fingerprint, the fingerprint was not visualized on each fluorescence image by time-resolved spectroscopy. However, the fingerprint was visualized in a FLIM image constructed using a series of the fluorescence images for the case with the fluorescence intensity of the background paper being four orders of magnitude larger than that of the fingerprint. The difference in fluorescence lifetime in the FLIM image of the visualized fingerprint and background paper was in the order of 0.1 ns, which was an order of magnitude smaller than the inherent fluorescence lifetime of a few nanoseconds for the fingerprints and paper. It was demonstrated that, at a background fluorescence intensity with a certain order of magnitude larger than that of fingerprints, FLIM has the potential to visualize latent fingerprints which cannot be visualized by time-resolved spectroscopy.