Abbott ARCHITECT iPhenytoin assay versus similar assays for measuring free phenytoin concentrations.

OBJECTIVE: To measure free phenytoin (FP) concentrations in filtered specimens using the Abbott ARCHITECT iPhenytoin assay and to compare results from this method with results from the Abbott TDx/FLx assays. METHODS: We verified accuracy, analytic measurement range, and precision for FP measurements. For correlation and therapeutic interval studies, we used filtered calibrators, controls, proficiency-testing materials, and surplus clinical samples. After implementation, we determined proficiency testing results. RESULTS: The analytic measurement range was 2.0 to 25.0 micromol/L. Quality control materials (6.1, 12.6, and 20.1 micromol/L) provided mean (SD) recoveries of 96.1 (5.0%), 99.2 (5.0%), and 99.3 (5.7%), respectively, and coefficients of variation of 5.2%, 5.0%, and 5.8%, respectively. Clinical specimens produced mean (SD) FP recovery levels of 103.7 (10.6%) (bias, 0.1 [0.3] micromol/L). Altering the FP therapeutic range (4.0-8.0 micromol/L) was unnecessary. Proficiency testing yielded consistently acceptable results. CONCLUSION: Our accuracy, precision, and correlation results were similar for the TDx/FLx and ARCHITECT assays, which demonstrates that the ARCHITECT iPhenytoin assay is acceptable for clinical FP measurements.

Persistence and Protective Potential of SARS-CoV-2 Antibody Levels After COVID-19 Vaccination in a West Virginia Nursing Home Cohort.

Importance: West Virginia prioritized SARS-CoV-2 vaccine delivery to nursing home facilities because of increased risk of severe illness in elderly populations. However, the persistence and protective role of antibody levels remain unclear. Objective: To examine the persistence of humoral immunity after COVID-19 vaccination and the association of SARS-CoV-2 antibody levels and subsequent infection among nursing home residents and staff. Design, Setting, and Participants: In this cross-sectional study, blood samples were procured between September 13 and November 30, 2021, from vaccinated residents and staff at participating nursing home facilities in the state of West Virginia for measurement of SARS-CoV-2 antibody (anti-receptor binding domain [RBD] IgG). SARS-CoV-2 infection and vaccination history were documented during specimen collection and through query of the state SARS-CoV-2 surveillance system through January 16, 2022. Exposure: SARS-CoV-2 vaccination (with BNT162b2, messenger RNA-1273, or Ad26.COV2.S). Main Outcomes and Measures: Anti-RBD IgG levels were assessed using multivariate analysis to examine associations between time since vaccination or infection, age, sex, booster doses, and vaccine type. Antibody levels from participants who became infected after specimen collection were compared with those without infection to correlate antibody levels with subsequent infection. Results: Among 2139 SARS-CoV-2 vaccinated residents and staff from participating West Virginia nursing facilities (median [range] age, 67 [18-103] years; 1660 [78%] female; 2045 [96%] White), anti-RBD IgG antibody levels decreased with time after vaccination or infection (mean [SE] estimated coefficient, -0.025 [0.0015]; P < .001). Multivariate regression modeling of participants with (n = 608) and without (n = 1223) a known history of SARS-CoV-2 infection demonstrated significantly higher mean (SE) antibody indexes with a third (booster) vaccination (with infection: 11.250 [1.2260]; P < .001; without infection: 8.056 [0.5333]; P < .001). Antibody levels (calculated by dividing the sample signal by the mean calibrator signal) were significantly lower among participants who later experienced breakthrough infection during the Delta surge (median, 2.3; 95% CI, 1.8-2.9) compared with those without breakthrough infection (median, 5.8; 95% CI, 5.5-6.1) (P = .002); however, no difference in absorbance indexes was observed in participants with breakthrough infections occurring after specimen collection (median, 5.9; 95% CI, 3.7-11.1) compared with those without breakthrough infection during the Omicron surge (median, 5.8; 95% CI, 5.6-6.2) (P = .70). Conclusions and Relevance: In this cross-sectional study, anti-RBD IgG levels decreased after vaccination or infection. Higher antibody responses were found in individuals who received a third (booster) vaccination. Although lower antibody levels were associated with breakthrough infection during the Delta surge, no significant association was found between antibody level and infection observed during the Omicron surge. The findings of this cross-sectional study suggest that among nursing home residents, COVID-19 vaccine boosters are important and updated vaccines effective against emerging SARS-CoV-2 variants are needed.

Fentanyl Quality Assurance Project Prompted Change in Clinical Workflow and Test Configurations.

BACKGROUND: Deaths attributable to fentanyl (FEN, a synthetic opioid) are high in Appalachia and highest in West Virginia. The goal of the study was to determine FEN prevalence among specimens submitted for definitive opioid testing and monitor responses to provider notifications of unexpected FEN findings during Q1 2020. METHODS: All definitive opioid test data were reviewed daily for FEN signatures in Q1 2020. Unexpected FEN results were communicated to providers and monitored for 10 days to record actions taken. Prevalence data were categorized. Behavioral Medicine (BMED) leaders analyzed January data and implemented FEN screening in the clinic. BMED Q1 clinic visits and order volumes for drug screens were reviewed after Q1. RESULTS: FEN positivity was 11% in Q1; >60% of findings were unexpected. Actions were taken for 54% of notifications in January but only 18% in March. Notifications required 70 hours of combined laboratory effort each month. BMED providers ordered 44% of definitive opioid tests and 69% of definitive FEN tests. Data prompted the addition of FEN to routine drug screen panels in the laboratory, and a 10% random FEN screening rate in the BMED opioid use disorder clinics (COAT). CONCLUSIONS: Prevalence of FEN positivity was higher than initially expected, even for this region in Appalachia. Expanded presence of FEN screening should assist BMED providers with clinical efforts and help identify patients in need of intervention/therapy.

Evidence for injurious effect of cocaethylene in human microvascular endothelial cells.

BACKGROUND: Cocaethylene (CE) is a conjugate of cocaine and ethanol that may contribute to the pathogenesis of systemic vascular diseases. This study was conducted to investigate the effect of CE on human microvascular endothelial cells (HMEC-1) in culture. METHODS: Proliferating and confluent monolayers of HMEC-1 were used for assessing growth kinetics, viability, cytotoxicity, and morphologic/barrier alterations after CE treatment (0-1 mmol/l) for up to 7 days. The Trypan blue exclusion, lactate dehydrogenase (LDH) release assay, manual cell counts, and silver nitrate staining technique were used. RESULTS: The doubling times of 30.0 and 31.4 h for the 0.5 and 1.0 mmol/l CE-treated HMEC-1, respectively, were significantly longer than the 28.6 h for the control group (p < 0.05). The viabilities of 90.4 +/- 3.8% (control) and 93.1 +/- 1.9% (CE-treated) from the Trypan blue exclusion-staining experiments indicated non-lethality of CE. LDH activities of 173 +/- 33 U/l (control) and 157 +/- 43 U/l (CE-treated) confirmed the absence of CE cytotoxicity. Silver staining results indicated increased monolayer permeability as demonstrated by the formation of intercellular gaps after 1 h of exposure. CONCLUSIONS: HMEC-1 exposure to CE induced cellular injury that could affect the permeability of small blood vessels. These cellular changes could in part be the pivotal point for studies to explain the edema and inflammation in surrounding tissues of individuals exposed to CE.

Workflow analysis comparing manual and automated specimen processing for mass spectrometry-based vitamin D testing.

OBJECTIVE: To quantify the benefits of automating specimen extraction in terms of specimen-preparation times and labor usage. METHODS: We used workflow modeling and time-motion studies to compare manual and automated solid-phase extraction methods to prepare specimens for a mass spectrometry-based vitamin D assay. We processed 20 batches, that included 5 to 90 specimens each, with both methods in parallel and randomly over a 4-week period. Technologist discomfort/fatigue was subjectively measured. RESULTS: Batch preparation time, per-specimen processing time, and labor requirements were significantly lower for all batch sizes on the Tecan Freedom EVO 150 robotic liquid-handling system (EVO). Technologist fatigue was significant when batch sizes reached 60 specimens. Cycle times were more uniform on the EVO. Automation provided as many as 85 minutes of useable technologist idle time for the 90-specimen batch. CONCLUSIONS: Automated specimen preparation should be considered when batch sizes reach 35 to 40 specimens per day.

Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-kappaB DNA-binding activity.

BACKGROUND: Cocaethylene (CE) is known to increase the permeability of human microvascular endothelial cell monolayers. The molecular mechanism underlying this increase may involve calcium-modulated signaling pathways such as the p38 mitogen-activated protein kinase (p38 MAPK) and the nuclear factor-kappaB (NF-kappaB) family of transcription factors. The hypothesis of this study was that CE-mediated endothelial permeability change may be mediated by the p38 MAPK and consequently NF-kappaB dimers. METHODS: We used sandwich ELISA to detect phosphorylated p38 MAPK in the cell line human microvascular endothelial cell 1 (HMEC-1) after treatment with 1 mmol/L CE. We used electrophoretic mobility shift assay to detect changes in NF-kappaB dimers present in HMEC-1 and their DNA-binding activity after treatment with CE. Lipopolysaccharide (LPS) from Salmonella typhosa was used as a positive control for all experiments. RESULTS: Treatment with CE and LPS had similar effects on HMEC-1 p38 MAPK phosphorylation and NF-kappaB DNA-binding activity. Both treatments increased the phosphorylation of p38 MAPK, consistent with activation of proinflammatory cell signaling. Treatment of HMEC-1 with CE decreased DNA binding of both the RelA/p50 and p50/p50 dimers of the NF-kappaB transcription factor family, whereas treatment with LPS decreased and then increased the DNA binding of these dimers. CONCLUSION: In addition to increasing HMEC-1 monolayer permeability, CE also alters transcription factor and kinase activity related to inflammation. Thus, CE causes endothelial activation that can elicit a prolonged and organized cellular response, rather than being directly toxic to endothelial cells.