Use of laboratory data for illicit drug use surveillance and identification of socioeconomic risk factors.

BACKGROUND: Drug overdose is the leading cause of death among people 25-44 years of age in the United States. Existing drug surveillance methods are important for prevention and directing treatment, but are limited by delayed reporting and lack of geographic granularity. METHODS: Laboratory urine drug screen and complete metabolic panel data from patients presenting to the emergency department was used to observe long-term and short-term temporal and geospatial changes at the zip code-level in St. Louis. Multivariate linear regression was performed to investigate associations between zip code-level socioeconomic factors and drug screening positivity rates. RESULTS: An increase in the fentanyl positive drug screens was seen during the initial COVID-19 shutdown period in the spring of 2020. A decrease in cocaine positivity was seen in the fall and winter of 2020, with a return to baseline coinciding with the second major COVID-19 shutdown in the summer of 2021. These changes appeared to be independent of changes in emergency department utilization as measured by complete metabolic panels ordered. Significant short-term changes in fentanyl and cocaine positivity rates between specific time periods were able to be localized to individual zip codes. Zip code-level multivariate analysis demonstrated independent associations between socioeconomic/demographic factors and fentanyl/cocaine positivity rates as determined by laboratory drug screening data. CONCLUSIONS: Analyzing clinical laboratory drug screening data can enable a more temporally and geographically granular view of population-level drug use surveillance. Additionally, laboratory data can be utilized to find population-level socioeconomic associations with illicit drug use, presenting a potential avenue for the use of this data to guide public health and healthcare policy decisions.

A map of neurofilament light chain species in brain and cerebrospinal fluid and alterations in Alzheimer's disease.

Neurofilament light is a well-established marker of both acute and chronic neuronal damage and is increased in multiple neurodegenerative diseases. However, the protein is not well characterized in brain tissue or body fluids, and it is unknown what neurofilament light species are detected by commercial assays and whether additional species exist. We developed an immunoprecipitation-mass spectrometry assay using custom antibodies targeting various neurofilament light domains, including antibodies targeting Coil 1A/1B of the rod domain (HJ30.13), Coil 2B of the rod domain (HJ30.4) and the tail region (HJ30.11). We utilized our assay to characterize neurofilament light in brain tissue and CSF of individuals with Alzheimer's disease dementia and healthy controls. We then validated a quantitative version of our assay and measured neurofilament light concentrations using both our quantitative immunoprecipitation-mass spectrometry assay and the commercially available immunoassay from Uman diagnostics in individuals with and without Alzheimer's disease dementia. Our validation cohort included CSF samples from 30 symptomatic amyloid-positive participants, 16 asymptomatic amyloid-positive participants, 10 symptomatic amyloid-negative participants and 25 amyloid-negative controls. We identified at least three major neurofilament light species in CSF, including N-terminal and C-terminal truncations, and a C-terminal fragment containing the tail domain. No full-length neurofilament light was identified in CSF. This contrasts with brain tissue, which contained mostly full-length neurofilament and a C-terminal tail domain fragment. We observed an increase in neurofilament light concentrations in individuals with Alzheimer's disease compared with healthy controls, with larger differences for some neurofilament light species than for others. The largest differences were observed for neurofilament light fragments including NfL165 (in Coil 1B), NfL324 (in Coil 2B) and NfL530 (in the C-terminal tail domain). The Uman immunoassay correlated most with NfL324. This study provides a comprehensive evaluation of neurofilament light in brain and CSF and enables future investigations of neurofilament light biology and utility as a biomarker.

Biomarkers and Clinical Laboratory Detection of Acute and Chronic Ethanol Use.

BACKGROUND: Ethanol use can lead to many health and socio-economic problems. Early identification of risky drinking behaviors helps provide timely clinical and social interventions. Laboratory testing of biomarkers of ethanol use supports the timely identification of individuals with risky drinking behaviors. This review provides an overview of the utility and limitations of ethanol biomarkers in the clinical laboratory. CONTENT: Direct assessment of ethanol in tissues and body fluids has limited utility due to the pharmacokinetics of ethanol. Therefore, the evaluation of ethanol use relies on nonvolatile metabolites of ethanol (direct biomarkers) and measurement of the physiological response to the toxic metabolites of ethanol (indirect biomarkers). Ethanol biomarkers help monitor both chronic and acute ethanol use. The points discussed here include the clinical utility of ethanol biomarkers, testing modalities used for laboratory assessment, the specimens of choice, limitations, and clinical interpretation of results. Finally, we discuss the ethical principles that should guide physicians and laboratorians when using these tests to evaluate alcohol use. SUMMARY: Indirect biomarkers such as carbohydrate-deficient transferrin, mean corpuscular volume, and liver enzymes activities may suggest heavy ethanol use. They lack sensitivity and specificity for timely detection of risky drinking behavior and have limited utility for acute ethanol use. Direct biomarkers such as ethyl glucuronide, ethyl sulfate, and phosphatidylethanol are considered sensitive and specific for detecting acute and chronic ethanol use. However, laboratory assessment and result interpretation lack standardization, limiting clinical utility. Ethical principles including respect for persons, beneficence, and justice should guide testing.

Performance Evaluation of an Automated Fentanyl Immunoassay.

BACKGROUND: High-throughput fentanyl immunoassays have recently emerged for clinical use, but early reports have demonstrated relatively high false-positive rates. The purpose of this study was to compare 2 immunoassays, the ARK and ARK II fentanyl immunoassays, and to demonstrate the clinical impact of implementing the ARK II assay. METHODS: The ARK and ARK II fentanyl assays were performed on a Roche c 502 chemistry analyzer. Positive and negative percentage agreement was assessed for each assay with 112 residual patient specimens relative to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cross-reactivity was assessed with the primary metabolite, norfentanyl, and analogs acetylfentanyl, acrylfentanyl, and furanylfentanyl. The proportion of specimens that did not confirm was assessed retrospectively from the laboratory information system. RESULTS: The concordance of the ARK assay was 75% (kappa 0.46, 95%CI 0.28-0.63) and the ARK II was 93% (kappa 0.86, 95%CI 0.76-0.95) with LC-MS/MS. 30 ng/mL of norfentanyl was required for a positive result by ARK and 15 ng/mL by ARK II. Similar cross-reactivity was observed when fentanyl and norfentanyl were both present in the specimen and with fentanyl analogs. After implementing the ARK II assay, the proportion of specimens that did not confirm by LC-MS/MS decreased from 11.7% per month to 2.0% per month. CONCLUSIONS: The ARK II fentanyl immunoassay has improved concordance relative to the original ARK fentanyl immunoassay using LC-MS/MS as the comparator method. Improved analyte specificity resulted in a reduced proportion of clinical samples that do not confirm.

Detecting Fentanyl Analogs in Urine Using Precursor Ion Scan Mode.

The opioid crisis has led many providers to inquire about the capabilities of urine drug testing to detect contemporary compounds such as fentanyl and fentanyl analogs. However, current methods for clinical urine drug testing, including immunoassays and targeted liquid chromatography tandem mass spectrometry, are not designed to broadly screen for the variety of fentanyl analogs that may be encountered. In this proof-of-principle study we developed a precursor ion scan method to enable semi-targeted data acquisition for structurally related fentanyl analogs. Based on the knowledge that many analogs fragment to m/z=188 and m/z=105, data was acquired on all precursor ions 250-400 Da that fragmented to these product ions. Using a tandem mass spectrometer we analyzed 102 residual urine specimens, in which we identified fentanyl, acetylfentanyl and acrylfentanyl. In 30 contrived urine samples, the precursor ion scan was also able to identify furanylfentanyl, butryrlfentanyl, 4-fluroisobutrylfentanyl, and despropionylfentanyl with accuracy ranging from 83-100%.

Comparison of Two Commercially Available Fentanyl Screening Immunoassays for Clinical Use.

BACKGROUND: Fentanyl is a synthetic opioid associated with illicit drug use and overdose deaths. The SEFRIA Immunalysis (IAL) and ARK fentanyl assays are both FDA-cleared, open channel immunoassays for fentanyl detection in urine. However, limited data are available in the literature comparing these assays. The objective of this study was to perform a direct comparison of these two fentanyl immunoassays. METHODS: IAL and ARK fentanyl immunoassays were performed on a Roche Cobas e602 automated chemistry analyzer. Repeatability and total imprecision were compared by diluting fentanyl into urine at concentrations above, below, and at the manufacturers' cutoffs of 1.0 ng/mL. Cross-reactivity was assessed for norfentanyl and the fentanyl analogs acetylfentanyl, acrylfentanyl, and furanylfentanyl. Concordance was assessed in 90 patient samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard. RESULTS: Repeatability varied from 11.4%-17.8% on the IAL assay and 2.8%-5.5% on the ARK assay. Total imprecision was 18.9%-40.7% on the IAL assay and 2.9%-6.4% on the ARK assay. Both assays cross-reacted with acetylfentanyl (∼100%), acrylfentanyl (∼100%), and furanylfentanyl (∼20%), but only the ARK assay cross-reacted with norfentanyl (∼3%). An admixture of 0.5 ng/mL fentanyl and 6 ng/mL norfentanyl produced a positive result on the ARK assay. Total concordance between IAL and ARK for 90 tested patient samples was 93% (kappa = 0.85). Relative to LC-MS/MS, the IAL assay had a concordance of 90% (kappa = 0.79) and the ARK assay had a concordance of 94% (kappa = 0.88). Including norfentanyl in the LC-MS/MS confirmation increased the concordance of the ARK to 96% (kappa = 0.90). CONCLUSIONS: The ARK assay recognized the metabolite norfentanyl, demonstrated superior precision, and had better concordance with LC-MS/MS compared to the IAL assay.

The Ethics of Direct-to-Consumer Testing.

Direct to consumer laboratory testing is a rapidly growing industry. However, the idea of consumers ordering their own laboratory tests has raised ethical concerns. Respect for autonomy, beneficence, nonmaleficence, and justice are core principles of biomedical ethics. Although direct to consumer testing would seem to offer autonomy to consumers, autonomy is only maintained if certain criteria are met, including intentionality, understanding, and noncontrol. There is little published evidence to support either beneficence or maleficence of direct to consumer testing. Finally, there are conflicting opinions about the justice of direct to consumer testing and whether it increases or decreases health disparities.

Biomarkers of Alzheimer Disease.

BACKGROUND: Alzheimer disease (AD) was once a clinical diagnosis confirmed by postmortem autopsy. Today, with the development of AD biomarkers, laboratory assays to detect AD pathology are able to complement clinical diagnosis in symptomatic individuals with uncertain diagnosis. A variety of commercially available assays are performed as laboratory-developed tests, and many more are in development for both clinical and research purposes. CONTENT: The role of laboratory medicine in diagnosing and managing AD is expanding; thus, it is important for laboratory professionals and ordering physicians to understand the strengths and limitations of both existing and emerging AD biomarker assays. In this review, we will provide an overview of the diagnosis of AD, discuss existing laboratory assays for AD and their recommended use, and examine the clinical performance of emerging AD biomarkers. SUMMARY: The field of AD biomarker discovery and assay development is rapidly evolving, with recent studies promising to improve both the diagnosis of symptomatic individuals and enrollment and monitoring of asymptomatic individuals in research studies. However, care must be taken to ensure proper use and interpretation of these assays. For clinical purposes, these assays are meant to aid in diagnosis but are not themselves diagnostic. For individuals without symptoms, AD biomarker tests are still only appropriate for research purposes. Additionally, there are analytical challenges that require careful attention, especially for longitudinal use of AD tests.

Ethics for Laboratory Medicine.

BACKGROUND: Laboratory medicine, like other areas of medicine, is obliged to adhere to high ethical standards. There are particular ethical issues that are unique to laboratory medicine and other areas in which ethical issues uniquely impact laboratory practice. Despite this, there is variability in ethics education within the profession. This review provides a foundation for the study of ethics within laboratory medicine. CONTENT: The Belmont Report identifies 3 core principles in biomedical ethics: respect for persons (including autonomy), beneficence (and its corollary nonmalfeasance), and justice. These core principles must be adhered to in laboratory medicine. Informed consent is vital to maintain patient autonomy. However, balancing patient autonomy with the desire for beneficence can sometimes be difficult when patients refuse testing or treatment. The use of leftover or banked samples is fundamental to the ability to do research, create reference intervals, and develop new tests, but it creates problems with consent. Advances in genetic testing have created unique ethical issues regarding privacy, incidental findings, and informed consent. As in other professions, the emergence of highly contagious and deadly infectious diseases poses a difficult ethical dilemma of helping patients while protecting healthcare workers. CONCLUSIONS: Although many clinical laboratorians do not see or treat patients, they must be held accountable to the highest ethical and professional behavior. Recognition and understanding of ethical issues are essential to ethical practice of laboratory medicine.

Multiple neurosteroid and cholesterol binding sites in voltage-dependent anion channel-1 determined by photo-affinity labeling.

Voltage-dependent anion channel-1 (VDAC1) is a mitochondrial porin that is implicated in cellular metabolism and apoptosis, and modulated by numerous small molecules including lipids. VDAC1 binds sterols, including cholesterol and neurosteroids such as allopregnanolone. Biochemical and computational studies suggest that VDAC1 binds multiple cholesterol molecules, but photolabeling studies have identified only a single cholesterol and neurosteroid binding site at E73. To identify all the binding sites of neurosteroids in VDAC1, we apply photo-affinity labeling using two sterol-based photolabeling reagents with complementary photochemistry: 5α-6-AziP which contains an aliphatic diazirine, and KK200 which contains a trifluoromethyl-phenyldiazirine (TPD) group. 5α-6-AziP and KK200 photolabel multiple residues within an E73 pocket confirming the presence of this site and mapping sterol orientation within this pocket. In addition, KK200 photolabels four other sites consistent with the finding that VDAC1 co-purifies with five cholesterol molecules. Both allopregnanolone and cholesterol competitively prevent photolabeling at E73 and three other sites indicating that these are common sterol binding sites shared by both neurosteroids and cholesterol. Binding at the functionally important residue E73 suggests a possible role for sterols in regulating VDAC1 signaling and interaction with partner proteins.

Multiple functional neurosteroid binding sites on GABAA receptors.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1ß3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and ß3 (labeled residue ß3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues ß3-L294 and G308) in the interface between the ß3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and ß3-α1 intersubunit sites are critical for neurosteroid action.