Snozek, Christine L.H.

Abstract Toxicology testing for drugs associated with scenarios such as recreational use or substance use disorder can be performed in support of the emergency department (ED) for specific patient populations such as pediatrics and trauma. These compounds were historically referred to as drugs of abuse (DOA); although the word “abuse” is recognized as potentially stigmatizing, no replacement terminology for DOA has emerged in current guidelines. This document refers to these compounds as drugs or substances of misuse, and acknowledges the need for less-stigmatizing language that more fully encompasses the range of uses for these drugs. This literature-driven, consensus guidance document provides recommendations primarily targeted to US hospital-based laboratories performing urine drug testing (UDT) in support of the ED. Indications for ordering UDT and related testing in both pediatric and adult populations are summarized. Further, recommendations are made for testing that should be available at all facilities with rapid turnaround, and how to perform and report testing. The advantages and disadvantages of immunoassays and mass spectrometry, as well as common challenges, are reviewed. Indications for mass-spectrometry assays and more extensive testing (e.g., novel psychoactive substances) are also provided. Future directions for improvements in laboratory technology to improve the utility of this testing are outlined. All laboratories should collaborate with ED leadership, medical toxicologists and poison control centers to optimize and update test menus to reflect local drug use patterns, ensure test methodologies and results meet clinical needs, and educate clinical staff regarding assay limitations and accurate test interpretation.

Abstract Background Hemoglobin A1c is used to assess glycemic control to both diagnose and monitor diabetes mellitus. Several different methodologies are available including high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and immunoassay (IA). These methods are standardized and traceable to the Diabetes Control and Complications Trial (DCCT). The aim of this study was to evaluate the difference in HbA1c results between methods and percent of reportable results in variant samples. Methods The HPLC (Bio-Rad Laboratories, Inc; D-100), the CE(Sebia Inc; Capillarys 3), and IA(Roche Diagnostics Inc; Tina-quant Generation 3) were compared using two sets of residual patient samples left after physician ordered testing was complete: 1) samples with an absence of abnormal hemoglobin peaks (i.e. non-variant) detected by HPLC; 2) samples with abnormal hemoglobin peaks (i.e. variant) characterized by hemoglobin electrophoresis performed clinically or lacking an A1c peak by HPLC/CE. The samples were tested within manufacturers stability claims. 317 non-variant samples were compared between the HPLC and CE methods. The absolute difference was calculated for each method using this equation (HbA1c%avg – HbA1c%X) where HbA1c%avg is the average of the methods that produced reportable results and X is the result from each method evaluated. Differences less than or equal to +/-0.3% were considered acceptable. 28 of the 317 non-variant and 47 variant samples were tested by all 3 methodologies. The 47 variant samples grouped into 3 cohorts: common heterozygous variants (HbC, HbD, HbE, HbS and HbF, n=10), uncommon heterozygous variants (n=29) and homozygous variants lacking HbA (n=8). The absolute differences in variant sample results were calculated for samples that did not surpass manufacturer’s claims for producing an accurate result. The frequency of producing a numeric result in variant samples was calculated for each method. Results The HPLC/CE non-variant sample comparison (N=317) had results spanning the measuring range (3.8% - 14.0%) with average(range) of absolute difference=0.1(-0.4to0.6%) with 293/317(92.4%) samples within +/-0.3%. The HPLC/CE/IA non-variant sample comparison (N=28) had a result range=4.0%-9.4%, the average(range) of difference=0.1(-0.2to0.5%) with 25/28 (89.2%) of samples within +/-0.3%. Common heterozygous variant samples gave numeric results with no disqualifying flags in 9 of 10(HPLC), 8 of 10(CE), and 9 of 10(IA) samples tested. The average(range) difference from average=0.0(-0.3,0.5%) for HPLC, 0.0(-0.2,0.3%) for CE, and -0.1(-0.6,0.2%) for IA. Uncommon variant samples gave results in 4 of 29 (HPLC), 20 of 29 (CE), and 26 of 29 (IA) of samples tested. The average(range) difference from average=-0.1(-0.3,0.1%) for HPLC, 0.0(-1.0,1.1%) for CE, and 0.0(-1.1,1.0%) for IA. The homozygous variant (no HbA) samples gave results in 0 of 8 (HPLC), 0 of 8 (CE), and 4 of 8 (IA) of those tested. Conclusion All three methods meet their claims for accurately measuring hemoglobin A1c from non-variant and common hemoglobin variants. The advantage of HPLC and CE is the ability to identify hemoglobin variants that may impact HbA1c quantitation. The IA method provides the most numeric results. IA gave results even when HbA was absent. The lab should understand the capabilities and limitations of their HbA1c method on the population served.

Abstract Background Alcohol biomarkers including ethyl glucuronide (EtG) and phosphatidylethanol (PEth) are ordered frequently in clinical and forensic settings including solid organ transplantation. PEth provides a long detection window but can be insensitive to light drinking. In contrast, EtG and ethyl sulfate (EtS) can be elevated after light alcohol consumption and might complement PEth testing. Methods Urine EtG/EtS and whole blood PEth results were evaluated from all clinically-ordered testing between 2014 and 2024. PEth and EtG/EtS confirmation were performed by liquid chromatography tandem mass spectrometry at two reference laboratories, using cutoffs: Lab A, PEth 20 ng/ml, EtG and EtS 500 and 250 ng/ml; Lab B, PEth 10 ng/ml, EtG and EtS 250 and 100 ng/ml. Only Lab B performed EtG screening by immunoassay, using a 500 ng/ml cutoff. Results Phosphatidylethanol was positive in 1269 (15.6%) of 8131 samples, compared to 769 (6.7%) confirmed EtG/EtS positives from 11 555 samples. EtG screening (n = 9668) was positive in 743 (7.7%) samples, of which 30 (4.0%) confirmed negative (false positives); the screen was indeterminate in 267 (2.8%) samples, 66 of which confirmed positive and 172 negative. Of 3132 paired PEth and EtG samples, 2887 (92.2%) were concordant, 224 (7.2%) were PEth-positive, and 21 (0.7%) were EtG-positive. PEth was significantly more sensitive in paired samples (P < .001), even after accounting for potential confounders such as transfusion. Limiting testing to PEth would have correctly identified alcohol consumption in 331 of 373 (88.7%) instances versus EtG/EtS in 149 (39.9%), and reduced charges by >$720 000 USD. Discussion Phosphatidylethanol outperformed EtG/EtS in detecting alcohol consumption in a predominantly abstinent transplant population. Compared to PEth, EtG/EtS had lower overall positivity and poorer sensitivity in paired samples; additionally, EtG screening demonstrated false positives and indeterminate results. EtG testing provided little added value beyond PEth in this population, and did not warrant the increased cost of performing both tests.

Abstract Single‐molecule immunoassay is a reliable technique for the detection and quantification of low‐abundance blood biomarkers, which are essential for early disease diagnosis and biomedical research. However, current single‐molecule methods predominantly rely on endpoint detection and necessitate signal amplification via labeling, which brings a variety of unwanted effects, like matrix effect and autofluorescence interference. This study introduces a real‐time mass imaging‐based label‐free single‐molecule immunoassay (LFSMiA). Featuring plasmonic scattering microscopy‐based mass imaging, a 2‐step sandwich assay format enables background reduction, minimization of matrix effect by dynamic tracking of single binding events, and fully leveraging real‐time data for improved measurement precision through a Bayesian Gaussian process model, the LFSMiA enables ultra‐sensitive and direct protein detection at the single‐molecule level in neat blood sample matrices. LFSMiA measurement is demonstrated for interleukin‐6 and prostate‐specific antigen in buffer, undiluted serum, and whole blood with sub‐femtomolar detection limits and eight logs of dynamic ranges. Moreover, comparable performance is achieved with an inexpensive miniaturized setup. To show its translational potential to clinical settings and point‐of‐care diagnostics, N‐terminal pro‐B‐type natriuretic peptide is examined in patient whole blood samples using the LFSMiA and results in a strong linear correlation (r > 0.99) with standard clinical lab results.

Abstract Objective: To retrospectively observe procalcitonin (PCT) and antibiotic ordering practices in patients hospitalized with community-acquired pneumonia (CAP). Design: Retrospective, exact matched, multicenter cohort study from October 1, 2018 – March 31, 2023. Setting: All hospitals across the Mayo Clinic Enterprise. Participants: Adult patients with CAP, identified using pneumonia diagnosis codes and receipt of systemic antibiotics with an indication of “respiratory tract infection” within 48 hours of hospitalization. Methods: PCT testing within the first 7 days of hospitalization was compared to non-PCT care (nPCT). The primary outcomes were treatment duration, antibiotic days of therapy (DOT), and length of stay (LOS). Results: 15364 patients met inclusion criteria. PCT testing occurred in 42.4% (6515/15364) of encounters, totaling 8214 PCT results. 12880 unique patient encounters were matched 1:1, 6440 in each group. Treatment duration was longer in the PCT group compared to the nPCT group (5.1 vs 4.6 days, respectively, P < 0.001). Patients in the PCT group also received more DOT (8.6 vs 7.6 DOT, P < 0.001) and had a longer LOS (6.8 vs 5.9 days, P < 0.001), respectively. There was no difference in 30-day all-cause mortality or C. difficile infection between groups. In a sensitivity analysis of nPCT patients compared to those with a peak value <0.25 ng/mL (i.e. normal result) there was no difference in treatment duration (4.6 days nPCT vs 4.7 days normal PCT, P = 0.104) or LOS (5.9 days nPCT vs 6.0 days normal PCT, P = 0.134). Conclusion: PCT testing in patients hospitalized with CAP was not associated with reduced antimicrobial utilization, LOS, or 30-day all-cause mortality.

BACKGROUND While most hypertriglyceridemia is asymptomatic, hypertriglyceridemia-associated acute pancreatitis (HTG-AP) can be more severe than AP of other etiologies. The reasons underlying this are unclear. We thus examined whether lipolytic generation of nonesterified fatty acids (NEFAs) from circulating triglycerides (TGs) could worsen clinical outcomes. METHODS Admission serum TGs, NEFA composition, and concentrations were analyzed prospectively for 269 patients with AP. These parameters, demographics, and clinical outcomes were compared between HTG-AP (TGs >500 mg/dL; American Heart Association [AHA] 2018 guidelines) and AP of other etiologies. Serum NEFAs were correlated with serum TG fatty acids (TGFAs) alone and with the product of TGFA serum lipase (NEFAs – TGFAs × lipase). Studies in mice and rats were conducted to understand the role of HTG lipolysis in organ failure and to interpret the NEFA-TGFA correlations. RESULTS Patients with HTG-AP had higher serum NEFA and TG levels and more severe AP (19% vs. 7%; P < 0.03) than did individuals with AP of other etiologies. Correlations of long-chain unsaturated NEFAs with corresponding TGFAs increased with TG concentrations up to 500 mg/dL and declined thereafter. However, NEFA – TGFA × lipase correlations became stronger with TGs above 500 mg/dL. AP and intravenous lipase infusion in rodents caused lipolysis of circulating TGs to NEFAs. This led to multisystem organ failure, which was prevented by pancreatic TG lipase deletion or lipase inhibition. CONCLUSIONS HTG-AP is made severe by the NEFAs generated from intravascular lipolysis of circulating TGs. Strategies that prevent TG lipolysis may be effective in improving clinical outcomes for patients with HTG-AP. FUNDING National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, NIH) (RO1DK092460 and R01DK119646); Department of Defense (PR191945 under W81XWH-20-1-0400); National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH (R01AA031257).

Abstract Objectives Urine drug testing (UDT) is a critical tool used in medical, forensic, and occupational settings, but interpreting results can be challenging. We performed a study to assess the ability of health care professionals to interpret UDT results accurately. Methods In total, 911 clinical and laboratory professionals in the United States and Canada responded to a survey with questions gauging expertise in UDT interpretation. Responses were analyzed to identify knowledge gaps. Results Toxicologists and laboratory PhD scientists performed well, with means of 4.82 and 4.63 questions answered correctly (out of 6 possible), respectively. Physicians specializing in pathology, emergency medicine, primary care, and internal medicine, however, displayed concerning knowledge gaps, as did laboratorians with nondoctoral degrees. Experience and training correlated with interpretation accuracy. Identification of simulated compliance as well as understanding opioid exposure, metabolism, and immunoassay cross-reactivity were among the most clinically significant knowledge gaps. More than 30% of survey respondents indicated that they would seek UDT information from the internet or peers rather than clinical or laboratory experts. Conclusions The study highlighted the need for targeted education and better collaboration between clinical and laboratory experts and other health care professionals to ensure that when physicians order UDT, they can accurately interpret results and reduce harm.

AbstractIntroductionPhosphatidylethanol (PEth) is a long‐term marker of alcohol consumption used frequently in clinical scenarios such as liver transplant evaluation. Recent cases have demonstrated that packed red blood cell (pRBC) transfusion creates the potential for artificial elevation or decrease of observed PEth concentrations in recipients. Very little is known about the prevalence or stability of PEth in pRBCs.MethodsApheresis and whole‐blood (WB) donations were tested for PEth using liquid chromatography – tandem mass spectrometry with limit of quantitation 10 ng/mL. Units were stored under routine blood bank conditions to evaluate the stability of PEth and the impact of irradiation.ResultsOver 40% of apheresis and WB donors had PEth ≥10 ng/mL (maximum observed 587 ng/mL). As WB units were processed into component pRBCs, PEth concentrations increased and were higher than donor WB levels (EDTA sample) prior to collection (maximum observed 711 ng/mL). Storage for up to 5 weeks post donation resulted in mean 17.3% decrease in PEth‐positive units; in contrast to a prior report, we observed no PEth formation in units with negative (<10 ng/mL) baseline concentrations. Irradiation of pRBCs did not substantially affect PEth concentrations in either PEth‐positive or PEth‐negative units.DiscussionPEth concentrations in healthy blood donors may potentially confound alcohol use or abstinence assessment in pRBC recipients. Transfusion medicine services and clinical practices such as transplantation and behavioral medicine should recognize this phenomenon and collaborate on testing protocols to appropriately interpret PEth in pRBC recipients.

Context.— Accurate interpretation of drug test results is key to appropriate patient care in numerous settings, including pain management. Despite recommendations that providers should consult laboratory professionals for guidance when necessary, literature demonstrating laboratorian expertise in drug test interpretation is lacking. Objective.— To evaluate participating laboratories’ performance on the case-based, interpretive (“dry”) challenge included with each Drug Monitoring for Pain Management proficiency testing program from 2012–2023. Design.— All challenges (n = 23) required participants to identify if drug test results were consistent or inconsistent with prescribed medications in the case history. Relevant medications, presumptive and confirmatory drug test results, and participant responses were extracted from program summary reports and examined for performance and common themes. Results.— Overall, 91.8% (6821 of 7431) of participant responses correctly identified whether drug testing was consistent with medications. There were 8 challenges with participant scores less than 91.8% (range, 59.8% [49 of 82 responses] to 88.9% [193 of 217 responses]). Common knowledge gaps identified in these challenges included false-positive presumptive (screening) results, minor metabolism of opiates, and recognizing that presence of a nonprescribed drug is inconsistent with prescribed medications. Although some participants repeatedly responded incorrectly, there were no associations between laboratory type, personnel responding, or analytical performance and incorrect responses to interpretative challenges. Conclusions.— Program participants performed well overall, but several concerning educational gaps were identified. Laboratorians have a role in providing interpretative guidance for drug testing and should emphasize ongoing education to ensure competence in the setting of constantly changing prescribed and nonprescribed drug use.