Understanding research methods,limitations, and applications of drug data collected by the National Forensic Laboratory Information System (NFLIS-Drug)

The National Forensic Laboratory Information System (NFLIS) is a drug surveillance program of the US Drug Enforcement Administration that systematically collects data on drugs that are seized by law enforcement and submitted to and analyzed by the Nation's forensic laboratories (NFLIS-Drug). NFLIS-Drug data are increasingly used in predictive modeling and drug surveillance to examine drug availability patterns. Given the complexity of the data and data collection, there are some common methodological pitfalls that we highlight with the aim of helping researchers avoid these concerns. The analysis done for this Technical Note is based on a review of the scientific literature that includes 428 unique, refereed article citations in 182 distinct journals published between January 1, 2005, and April 30, 2021. Each article was analyzed according to how NFLIS-Drug data were mentioned and whether NFLIS-Drug data were included. A sample of 37 articles was studied in-depth, and data issues were summarized. Using examples from the literature, this Technical Note highlights eight broad concerns that have important implications for the proper applications, interpretations, and limitations of NFLIS-Drug data with suggestions for improving research methods and accurate reporting of forensic drug data. NFLIS-Drug data are timely and provide key information to inform drug use trends across the United States; however, our present analysis shows that NFLIS-Drug data are misunderstood and represented in the literature. In addition to highlighting these issues, DEA has created several resources to assist NFLIS data users and researchers, which are summarized in the discussion.     

Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H₀) that the difference in intensity is equal to zero. If H₀ is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H₀ is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (t_calc) value. For a given comparison, ions with higher t_calc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.     

An unusual blunt force trauma pattern and mechanism to the cranial vault: Investigation of an atypical infant homicide

This case report presents an unusual fracture pattern in the cranium of a four-month-old infant indicative of child abuse. Upon postmortem examination, the infant presented with numerous bilateral linear cranial fractures running perpendicular to the sagittal suture with depressed and curvilinear fractures apparent on the supra-auricular surfaces of the cranium. Histological evidence indicates multiple traumatic events to the cranium. In addition, the stair-step pattern of a parietal fracture may represent multiple contiguous fractures from repeated loading of the head at different times with variation of the focal points of compressive force. Additionally, the left humerus, left radius, and left ulna have healing metaphyseal fractures, and the left ulna also has an antemortem diaphyseal fracture which resulted in the distal metaphysis being rotated 45 degrees medially. Integration of autopsy, anthropological, and neuropathological reports for this case suggest multiple inflicted injury episodes with a repeated atypical mechanism(s) to the cranial vault of the infant. During investigative interviews, the caretaker admitted to squeezing the infant's head and neck on multiple occasions to quiet the child. This reported abusive mechanism is consistent with the pattern of symmetric cranial fractures and soft tissue injuries indicating asphyxiation. This case report provides forensic investigators with a potential trauma mechanism to explore in cases when a similar pattern of cranial trauma is observed and highlights the need for greater research on fracture propagation and fracture healing in the infant cranium.     

Validation of the 4‐aminophenol color test for the differentiation of marijuana‐type and hemp‐type cannabis

The analysis of cannabis plant material submitted to seized‐drug laboratories was significantly affected by the signing of the Agricultural Improvement Act of 2018, which defined hemp and removed it from the definition of marijuana in the Controlled Substances Act. As a result, field law enforcement personnel and forensic laboratories now are in need of implementing new protocols that can distinguish between marijuana‐type and hemp‐type cannabis. Colorimetric tests provide a cost‐effective and efficient manner to presumptively identify materials prior to submission to a laboratory for analysis. This work presents the validation of the 4‐aminophenol (4‐AP) color test and demonstrates its utility for discriminating between marijuana‐type and hemp‐type cannabis (i.e., typification). Validation studies included the testing of numerous cannabinoid reference materials, household herbs, previously characterized cannabis plant samples, and real‐case samples. The 4‐AP test reliably produces a pink result when the level of Δ⁹‐tetrahydrocannabinol (THC) is approximately three times lower than the level of cannabidiol (CBD). A blue result is generated when the level of THC is approximately three times higher than that of CBD. Inconclusive results are observed when the levels of THC and CBD are within a factor of three from each other, demonstrating the limitations of the test under those scenarios.     

Comparison between lung weight and blood strontium in bodies found in seawater

This paper examines the use of lung weight increase as an indicator of seawater drowning compared to the amount of Sr absorbed by the blood. The study population was limited to male victims older than 20 years. Significant differences between cases of drowning and "non-drowning" were detected in terms of the lung-heart weight ratio (L/H) (p<0.001) or lung-body weight ratio (L/B) (p=0.005). However, using lung weight (L), L/H or L/B to distinguish between seawater drownings and saltwater non-drownings some overlap was produced. The factor rendering least overlap was L/B, which also appeared to be non-dependent on the victim's age. Our findings suggest that a value of L/B higher than 19.5 g/kg could be a useful indicator of death by drowning, but that when a lower value is found, additional drowning diagnoses would be needed to establish the manner of death.     

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.     

Quantification of psilocin in human whole blood using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC–MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7–200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification.     

A “concrete” identification in a dismemberment case

Homicide perpetrators can use concrete as a means to conceal their victims. When concrete encasement is encountered in the forensic field, albeit rarely, it is often coupled with postmortem dismemberment. This method of obscuring the evidence presents unique investigative obstacles, specifically related to identification. Various approaches to obtaining fingerprints from decedents encased in concrete have been suggested and implemented over the years. The presented case is that of an initially unidentified 44-year-old male, who was subject to postmortem dismemberment and concrete encasement. Meticulous excavation techniques facilitated preservation of evidence and an anatomical reconstruction of the body. These techniques enabled inspection of the incision sites of the dismembered remains during the postmortem examination. Identifiable jewelry and tattoos were noted at autopsy. Further, the resulting concrete molds could be utilized to obtain fingerprints. These prints were used to ultimately identify the decedent.     

Latex agglutination for bacterial antigens and meningococcus PCR: two useful tools in legal sudden deaths

Bacterial infections are considered to be a major cause of sudden deaths. The recognition of infections caused by Neisseria meningitidis is an essential duty of medicolegal offices due to the risk of secondary cases. Since other microorganisms, such as Haemophilus influenzae and Streptococcus pneumoniae, are also involved in infectious sudden deaths, the identification of the pathogen responsible for death is essential in order to establish a positive diagnosis while also preventing secondary meningococcal cases. However, because of the unreliability of culture methods used for autopsy specimens and the fragile nature of the microorganisms, other techniques were used. In this study, the detection of specific antigens of N. meningitidis (serogroups A, B, C, Y and W135), H. influenzae type b, S. pneumoniae and Group B Streptococcus was undertaken in 40 samples from sudden death cases in legal procedures with a latex agglutination test. In addition, a meningococcus polymerase chain reaction (PCR) assay (ctrA, crgA and siaD genes) was also used as a corroboration method for positive N. meningitidis agglutinations. Eleven cases of sudden death were confirmed to be due to meningococcus while one case was confirmed to have been caused by H. influenzae type b fulminant epiglottitis. Rapid laboratory diagnosis of meningococcal infection allowed contacts management and notification to the health authorities. From the point of view of the authors, forensic diagnosis of unascertained deaths should include latex agglutination and meningococcus PCR when a fulminant infection by N. meningitidis or H. influenzae is suspected as well as in deaths where the cause is unclear.