Evaluation of local hemoglobinopathy prevalence and promotion of accurate hemoglobin A1c testing using historical data retrieval.

OBJECTIVES: Heterozygous hemoglobin variants are known to cause method- and variant-specific interference with hemoglobin A1c (HbA1c) quantitation. Less attention has been paid to the role of other hemoglobin variants in confounding HbA1c testing. Here we evaluated the frequency with which enzymatic (ENZ) and immunoassay (IA) HbA1c quantitation methods, i.e., those unable to routinely detect the presence of hemoglobin variants, were used within our healthcare system for HbA1c analysis in patients with elevated fetal hemoglobin as well as compound heterozygous and homozygous variants. DESIGN & METHODS: This analysis was enabled by automated review of HbA1c result history, implemented to promote detection of variants prior to HbA1c result reporting. RESULTS: During a 54-week period, 319,290 HbA1c analyses were performed. We observed 110 unique patient cases (0.03% problem identification rate) in which HbA1c testing was ordered in the presence of either a homozygous or compound heterozygous hemoglobin variant or elevated hemoglobin F beyond the tolerance of the method. Among the 110 cases identified, 55 (50%) showed a compound heterozygous or homozygous hemoglobin variant while 55 (50%) showed elevated hemoglobin F. Of those cases involving a compound heterozygous or homozygous variant, 8/55 (15%) involved patients who had one or more ENZ or IA HbA1c results reported previously within our system. Of the 55 total compound heterozygous or homozygous variants identified, 37 (67%) were hemoglobin E, 10 (18%) hemoglobin S/C, 4 (7%) hemoglobin S, 2 (4%) hemoglobin C, 1 (2%) hemoglobin Camden, and 1 (2%) unidentified variant. CONCLUSIONS: Exclusive use of methods unable to routinely detect the presence of hemoglobin variants may lead to reporting of HbA1c results that are not clinically meaningful.

Diagnostic performance of a rapid, novel, whole blood, point of care high-sensitivity cardiac troponin I assay for myocardial infarction.

BACKGROUND: We evaluated the diagnostic performance of a whole blood, point of care (POC) high-sensitivity cardiac troponin I (hs-cTnI) assay for myocardial infarction (MI) compared to central laboratory assays. METHODS: Consecutive patients presenting to the emergency department with symptoms of ischemia were studied. Serial hs-cTnI testing was based on clinical indication at presentation. Parallel measurements were made using fresh whole blood on Siemens Atellica VTLi POC assay, EDTA plasma on Abbott ARCHITECT i2000 used in practice, and heparin plasma on Siemens Atellica. MI was determined according to the Fourth Universal Definition of MI using 99th percentiles. Sensitivities and negative predictive values (NPV) were calculated using 99th percentile URLs. RESULTS: 1089 Patients, 418 females and 671 males, were enrolled. There were 91 (8.4%) MIs. At baseline (0 h), POC hs-cTnI assay had a sensitivity of 65.7% (95% CI 47.8-80.9) for females and 67.9% (54.0-79.7) for males and NPV of 96.4% (93.9-98.1) for females and 96.7% (94.9-98.0) for males. At 2 h, sensitivity improved to 82.9% (66.4-93.4) for females and 80.4% (67.6-89.8) for males, while NPV improved to 98.2% (96.1-99.3) and 97.9% (96.3-99.0), respectively. For central laboratory assays, comparable diagnostics were observed at 2 h: females - sensitivity 94.3% (80.8-99.3) for ARCHITECT and 79.4% (62.1-91.3) for Atellica, and NPV 99.3% (97.6-99.9) and 98.0% (95.8-99.2), respectively; males - sensitivity 87.5% (75.9-94.8) for ARCHITECT and 80.4% (67.6-89.8) for Atellica, NPVs of 98.7% (97.3-99.5) and 97.9% (96.3-99.0), respectively. CONCLUSIONS: The POC, whole blood Atellica VTLi hs-cTnI assay demonstrated comparable diagnostic accuracy for MI to central laboratory assays using 99th percentiles.

Urine Drug Screening in the Era of Designer Benzodiazepines: Comparison of Three Immunoassay Platforms, LC-QTOF-MS and LC-MS-MS.

This study investigated the presence of designer benzodiazepines in 35 urine specimens obtained from emergency department patients undergoing urine drug screening. All specimens showed apparent false-positive benzodiazepine screening results (i.e., confirmatory testing using a 19-component liquid chromatography-tandem mass spectrometry (LC-MS-MS) panel showed no prescribed benzodiazepines at detectable levels). The primary aims were to identify the possible presence of designer benzodiazepines, characterize the reactivity of commercially available screening immunoassays with designer benzodiazepines and evaluate the risk of inappropriately ruling out designer benzodiazepine use when utilizing common urine drug screening and confirmatory tests. Specimens were obtained from emergency departments of a single US Health system. Following clinically ordered drug screening using Abbott ARCHITECT c assays and laboratory-developed LC-MS-MS confirmatory testing, additional characterization was performed for investigative purposes. Specifically, urine specimens were screened using two additional assays (Roche cobas c502 and Siemens Dimension Vista) and LC-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to identify presumptively positive species, including benzodiazepines and non-benzodiazepines. Finally, targeted, qualitative LC-MS-MS was performed to confirm the presence of 12 designer benzodiazepines. Following benzodiazepine detection using the Abbott ARCHITECT, benzodiazepines were subsequently detected in 28/35 and 35/35 urine specimens using Siemens and Roche assays, respectively. LC-QTOF-MS showed the presumptive presence of at least one non-Food and Drug Administration (FDA)-approved benzodiazepine in 30/35 specimens: flubromazolam (12/35), flualprazolam (11/35), flubromazepam (2/35), clonazolam (4/35), etizolam (9/35), metizolam (5/35), nitrazepam (1/35) and pyrazolam (1/35). Two or three designer benzodiazepines were detected concurrently in 13/35 specimens. Qualitative LC-MS-MS confirmed the presence of at least one designer benzodiazepine or metabolite in 23/35 specimens, with three specimens unavailable for confirmatory testing. Urine benzodiazepine screening assays from three manufacturers were cross-reactive with multiple non-US FDA-approved benzodiazepines. Clinical and forensic toxicology laboratories using traditionally designed LC-MS-MS panels may fail to confirm the presence of non-US FDA-approved benzodiazepines detected by screening assays, risking inappropriate interpretation of screening results as false positives.

Retinoic Acid Signaling Modulates Recipient Gut Barrier Integrity and Microbiota After Allogeneic Hematopoietic Stem Cell Transplantation in Mice.

Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). An impaired intestinal epithelial barrier is an important component of GVHD pathogenesis. However, contributing host factors that modulate mucosal barrier integrity during GVHD are poorly defined. We hypothesized that vitamin A and retinoic acid (RA) exert positive impacts on maintaining intestinal barrier function after HSCT, thus preventing or dampening GVHD severity. Unexpectedly, we found that exogenous RA increased intestinal permeability of recipient mice after allogeneic HSCT. Serum bacterial endotoxin levels were significantly higher in GVHD mice fed a vitamin A-high (VAH) diet compared to those fed a vitamin A-normal (VAN) diet, indicating a more compromised intestinal barrier function. Furthermore, VAH mice showed more severe lung GVHD with increased donor T cell infiltration in this tissue and died significantly faster than VAN recipients. 16S rRNA sequencing of fecal samples revealed significant differences in the diversity and composition of gut microbiota between VAN and VAH transplant recipients. Collectively, we show that retinoic acid signaling may negatively impact intestinal barrier function during GVHD. Mild vitamin A supplementation is associated with increased lung GVHD and more profound gut dysbiosis. Micronutrients such as vitamin A could modulate complications of allogeneic HSCT, which may be mediated by shaping gut microbiota.

Independent and combined effects of biotin and hemolysis on high-sensitivity cardiac troponin assays.

OBJECTIVES: This study compared the independent and combined effects of hemolysis and biotin on cardiac troponin measurements across nine high-sensitivity cardiac troponin (hs-cTn) assays. METHODS: Parallel cTn measurements were made in pooled lithium heparin plasma spiked with hemolysate and/or biotin using nine hs-cTn assays: Abbott Alinity, Abbott ARCHITECT i2000, Beckman Access 2, Ortho VITROS XT 7600, Siemens Atellica, Siemens Centaur, Siemens Dimension EXL cTnI, and two Roche Cobas e 411 Elecsys Troponin T-hs cTnT assays (outside US versions, with and without increased biotin tolerance). Absolute and percent cTn recovery relative to two baseline concentrations were determined in spiked samples and compared to manufacturer's claims. RESULTS: All assays except the Ortho VITROS XT 7600 showed hemolysis and biotin interference thresholds equivalent to or greater than manufacturer's claims. While imprecision confounded analysis of Ortho VITROS XT 7600 data, evidence of biotin interference was lacking. Increasing biotin concentration led to decreasing cTn recovery in three assays, specifically both Roche Cobas e 411 Elecsys Troponin T-hs assays and the Siemens Dimension EXL. While one of the Roche assays was the most susceptible to biotin among the nine studied, a new version showed reduced biotin interference by approximately 100-fold compared to its predecessor. Increasing hemolysis also generally led to decreasing cTn recovery for susceptible assays, specifically the Beckman Access 2, Ortho VITROS XT 7600, and both Roche Cobas e 411 Elecsys assays. Equivalent biotin and hemolysis interference thresholds were observed at the two cTn concentrations considered for all but two assays (Beckman Access 2 and Ortho VITROS XT 7600). When biotin and hemolysis were present in combination, biotin interference thresholds decreased with increasing hemolysis for two susceptible assays (Roche Cobas e 411 Elecsys and Siemens Dimension EXL). CONCLUSIONS: Both Roche Cobas e 411 Elecsys as well as Ortho VITROS XT assays were susceptible to interference from in vitro hemolysis at levels routinely encountered in clinical laboratory samples (0-3 g/L free hemoglobin), leading to falsely low cTn recovery up to 3 ng/L or 13%. While most assays are not susceptible to biotin at levels expected with over-the-counter supplementation, severely reduced cTn recovery is possible at biotin levels of 10-2000 ng/mL (41-8,180 nmol/L) for some assays. Due to potential additive effects, analytical interferences should not be considered in isolation.

Prevalence of Detectable Biotin in Five US Emergency Department Patient Cohorts.

BACKGROUND: The objective of this study was to estimate the prevalence of biotin supplementation in United States emergency department patients using a multi-site, geographically distributed sampling model. METHODS: Biotin was measured using an Abbott ARCHITECT Biotin research use only assay in 7118 emergency department patient serum or plasma samples from five US medical centers. Samples with biotin ≥10 ng/mL underwent additional LC-MS/MS confirmatory testing for biotin and its primary metabolites. The overall and site-specific prevalence of detectable biotin was determined using the screening assay while biotin speciation (i.e., prevalence of detectable metabolites) was determined using LC-MS/MS. RESULTS: Of 7118 samples screened, 291 (4.1%) had biotin ≥10 ng/mL and were considered positive. Across five medical centers, the fraction of positive samples ranged from 2.0% to 5.4%. The maximum biotin concentration observed was 355 ng/mL. Of the 285 positive screens that underwent additional LC-MS/MS testing, 89 (31%) showed detectable biotin, bisnorbiotin, and/or biotin sulfoxide. Biotin, bisnorbiotin, and biotinsulfoxide were detected in 82/89 (92.1%), 61/89 (68.5%), and 18/89 (20.2%) samples, respectively; biotin was detected in the absence of either metabolite in 18/89 (20.2%) samples. CONCLUSIONS: Using a screening assay, 4.1% of emergency department patient samples were found to be potentially susceptible to interference from biotin. Confirmatory testing showed detectable biotin and/or biotin metabolites in 31% of positive screens (1.3% overall). The prevalence of biotin ≥10 ng/mL varied 2-3-fold across US emergency department patient cohorts. Biotin metabolites were observed in 80% of samples confirmed to have detectable biotin species by LC-MS/MS, suggesting that rigorous assessments of assay susceptibility to biotin interference, often performed using in vitro studies, should consider the potential role of biotin metabolites present in vivo.

Critical Analysis of Laboratory Testing Methodologies When Interpreting Conflicting Results at Autopsy.

ABSTRACT: Toxicological analysis is an important diagnostic component of a postmortem examination and may involve both antemortem and postmortem specimens. Here, we present a case in which an antemortem specimen, when reanalyzed in the forensic toxicology laboratory, resulted in values that contradicted the reported values from the medical record and required further investigation. This case involves a 51-year-old man decedent with a medical history of chronic alcohol abuse. His antemortem urine drug screen, performed upon admission to an emergency department, was negative. His serum blood alcohol level at presentation was reported as 0.960 g/dL and, repeated 4 hours later, was 0.500 g/dL with a comment indicating that there was significant lipemia interfering with the results. At autopsy, the antemortem blood sample collected from the hospital, postmortem blood, and vitreous humor samples were analyzed and all 3 samples were found to be negative for ethanol. The hospital laboratory used an enzymatic assay for ethanol detection, which is known to be impacted by lipemia, and the forensic laboratory used head-space gas chromatography, which is not impacted by lipemia. This highlights the need to critically analyze laboratory testing methodologies when interpreting conflicting results at autopsy.

Comparison of the Clinical Performances of the Abbott Alinity IgG, Abbott Architect IgM, and Roche Elecsys Total SARS-CoV-2 Antibody Assays.

Critical evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic assays is needed to guide clinical decision-making and ensure that these assays provide optimal benefit to patients and the public. Here, three commercially available assays with widespread distribution capabilities are compared. A total of 667 specimens, 103 from patients with confirmed SARS-CoV-2 infections and 564 collected prior to the emergence of SARS-CoV-2, were analyzed in parallel using the Roche Elecsys SARS-CoV-2 total antibody and Abbott Alinity SARS-CoV-2 IgG assays; a subset of 55 samples from patients with confirmed SARS-CoV-2 infections was additionally evaluated using the Abbott Architect SARS-CoV-2 IgM assay. Qualitative agreement between the Abbott IgG and Roche total antibody assays was 98.7% (658/667), with Cohen's kappa value of 0.919 (95% confidence interval [CI], 0.867 to 0.972). Qualitative agreements with the Abbott IgM assay were 92.7% (51/55, Abbott IgG) and 85.5% (47/55, Roche total antibody). Diagnostic specificities determined using pre-COVID-19 samples for the Abbott IgG and Roche total antibody assays were 99.65% (95% CI, 98.72 to 99.90%) and 100.00% (95% CI, 99.32 to 100.00%), respectively, spanning claims made by each manufacturer. Diagnostic sensitivities increased for all three assays with increasing time since the onset of symptoms. Among 51 patients with confirmed SARS-CoV-2 infections, 23 (45.1%), 24 (47.1%), and 22 (43.1%) were reactive by the Abbott IgG, Roche total antibody, and Abbott IgM assays, respectively, with sampling times 0 to 56 days post-positive PCR (median/mean, 2/6.2 days). Combining IgG and IgM screening identified 4/55 additional samples with detectable antibodies that would not have been observed using the assays independently. Notably, one immunocompromised patient with confirmed SARS-CoV-2 infection showed no detectable antibodies using any of the three assays 43 days after onset of symptoms.

Prospective Evaluation of Late-Night Salivary Cortisol and Cortisone by EIA and LC-MS/MS in Suspected Cushing Syndrome.

CONTEXT: Late-night salivary cortisol (LNSC) measured by enzyme immunoassay (EIA-F) is a first-line screening test for Cushing syndrome (CS) with a reported sensitivity and specificity of >90%. However, liquid chromatography-tandem mass spectrometry, validated to measure salivary cortisol (LCMS-F) and cortisone (LCMS-E), has been proposed to be superior diagnostically. OBJECTIVE SETTING AND MAIN OUTCOME MEASURES: Prospectively evaluate the diagnostic performance of EIA-F, LCMS-F, and LCMS-E in 1453 consecutive late-night saliva samples from 705 patients with suspected CS. DESIGN: Patients grouped by the presence or absence of at least one elevated salivary steroid result and then subdivided by diagnosis. RESULTS: We identified 283 patients with at least one elevated salivary result; 45 had an established diagnosis of neoplastic hypercortisolism (CS) for which EIA-F had a very high sensitivity (97.5%). LCMS-F and LCMS-E had lower sensitivity but higher specificity than EIA-F. EIA-F had poor sensitivity (31.3%) for adrenocorticotropic hormone (ACTH)-independent CS (5 patients with at least 1 and 11 without any elevated salivary result). In patients with Cushing disease (CD), most nonelevated LCMS-F results were in patients with persistent/recurrent CD; their EIA-F levels were lower than in patients with newly diagnosed CD. CONCLUSIONS: Since the majority of patients with ≥1 elevated late-night salivary cortisol or cortisone result did not have CS, a single elevated level has poor specificity and positive predictive value. LNSC measured by EIA is a sensitive test for ACTH-dependent Cushing syndrome but not for ACTH-independent CS. We suggest that neither LCMS-F nor LCMS-E improves the sensitivity of late-night EIA-F for CS.

Reverse pseudohyperkalemia and pseudohyponatremia in a patient with B-cell non-Hodgkin lymphoma.

OBJECTIVES: Investigate concomitant and spurious high potassium and low sodium results in heparinized plasma. METHODS: Potassium and sodium values were measured from heparinized plasma and serum in a patient with B-cell non-Hodgkin lymphoma using both an automated chemistry analyzer (indirect ion selective electrode) and blood gas analyzer (direct ion selective electrode). RESULTS: Potassium levels were significantly increased while sodium levels were significantly decreased in heparinized plasma compared to serum on several occasions. CONCLUSIONS: To our knowledge, concomitant reverse pseudohyperkalemia and pseudohyponatremia has not been reported previously. We postulate the discrepancy between plasma and serum sodium (pseudohyponatremia in plasma) may be unique to cases of reverse pseudohyperkalemia with extreme potassium elevations.

Diagnosing and Managing Sepsis by Probing the Host Response to Infection: Advances, Opportunities, and Challenges.

Sepsis is a major source of mortality and morbidity globally. Accurately diagnosing sepsis remains challenging due to the heterogeneous nature of the disease, and delays in diagnosis and intervention contribute to high mortality rates. Measuring the host response to infection enables more rapid diagnosis of sepsis than is possible through direct detection of the causative pathogen, and recent advances in host response diagnostics and prognostics hold promise for improving outcomes. The current review discusses recent advances in the technologies used to probe the host response to infection, particularly those based on transcriptomics. These are discussed in the context of contemporary approaches to diagnosing and prognosing sepsis, and recommendations are made for successful development and validation of host response technologies.

Appropriateness of Cardiac Troponin Testing: Insights from the Use of TROPonin In Acute coronary syndromes (UTROPIA) Study.

OBJECTIVE: Our objective was to examine the appropriateness of cardiac troponin (cTn) testing among patients with cTn increases. METHODS: This is a planned secondary analysis of the Use of TROPonin In Acute coronary syndromes (UTROPIA, NCT02060760) observational cohort study. Appropriateness of cTn testing was adjudicated for emergency department patients with cTn increases >99th percentile and analyzed using both contemporary and high-sensitivity (hs) cTnI assays according to sub-specialty, diagnoses, and symptoms. RESULTS: Appropriateness was determined from 1272 and 1078 adjudication forms completed for 497 and 422 patients with contemporary and hs-cTnI increases, respectively. Appropriateness of cTnI testing across adjudication forms was 71.5% and 72.0% for cTnI and hs-cTnI, respectively. Compared with emergency physicians, cardiologists were less likely to classify cTnI orders as appropriate (cTnI: 79% vs 56%, P < .0001; hs-cTnI: 82% vs 51%, P < .0001). For contemporary cTnI, appropriateness of 95%, 70%, and 39% was observed among adjudication forms completed by cardiologists for type 1 myocardial infarction, type 2 myocardial infarction, and myocardial injury, respectively; compared with 90%, 86%, and 71%, respectively, among emergency physicians. Similar findings were observed using hs-cTnI. Discordance in appropriateness adjudication forms occurred most frequently in cases of myocardial injury (62% both assays) or type 2 myocardial infarction (cTnI 31%; hs-cTnI 23%). CONCLUSIONS: Marked differences exist in the perception of what constitutes appropriate clinical use of cTn testing between cardiologists and emergency physicians, with emergency physicians more likely to see testing as appropriate across a range of clinical scenarios. Discordance derives most often from cases classified as myocardial injury or type 2 myocardial infarction.

Low pO2 Contributes to Potential Error in Oxygen Saturation Calculations Using a Point-of-Care Assay.

OBJECTIVES: The present study addressed the accuracy of calculated oxygen saturation (sO2) using point-of-care (POC) testing compared with measured values on a blood gas analyzer. METHODS: In total, 3,323 sO2 values were measured in 1,180 patients using a CO-oximeter (ABL 800 Flex; Radiometer, Copenhagen, Denmark). Measured parameters were then used to calculate an expected sO2 for the POC method (Abbott i-STAT; Abbott POC, Princeton, NJ). Cases in which calculated sO2 differed from measured sO2 by 10% or more were analyzed. RESULTS: Of the 3,323 comparisons performed, 260 (8%) showed discrepancies (± ≥10%) between measured and calculated sO2 values. Ninety-four of discrepant measurements (245 of 260) occurred when pO2 was less than 50 mm Hg. pH and bicarbonate distributions shifted to lower values in discrepant vs nondiscrepant cases. CONCLUSIONS: Our results suggest that the likelihood of discrepant sO2 is 27% among patients with pO2 less than 50 mm Hg. Direct measurement of sO2 by CO-oximetry is strongly suggested in this clinical scenario.

Sex-specific 99th percentiles derived from the AACC Universal Sample Bank for the Roche Gen 5 cTnT assay: Comorbidities and statistical methods influence derivation of reference limits.

OBJECTIVES: Our purpose was to determine a) overall and sex-specific 99th percentile upper reference limits (URL) and b) influences of statistical methods and comorbidities on the URLs. METHODS: Heparin plasma from 838 normal subjects (423 men, 415 women) were obtained from the AACC (Universal Sample Bank). The cobas e602 measured cTnT (Roche Gen 5 assay); limit of detection (LoD), 3ng/L. Hemoglobin A1c (URL 6.5%), NT-proBNP (URL 125ng/L) and eGFR (60mL/min/1.73m2) were measured, along with identification of statin use, to better define normality. 99th percentile URLs were determined by the non-parametric (NP), Harrell-Davis Estimator (HDE) and Robust (R) methods. RESULTS: 355 men and 339 women remained after exclusions. Overall<50% of subjects had measureable concentrations ≥ LoD: 45.6% no exclusion, 43.5% after exclusion; compared to men: 68.1% no exclusion, 65.1% post exclusion; women: 22.7% no exclusion, 20.9% post exclusion. The statistical method used influenced URLs as follows: pre/post exclusion overall, NP 16/16ng/L, HDE 17/17ng/L, R not available; men NP 18/16ng/L, HDE 21/19ng/L, R 16/11ng/L; women NP 13/10ng/L, HDE 14/14ng/L, R not available. CONCLUSIONS: We demonstrated that a) the Gen 5 cTnT assay does not meet the IFCC guideline for high-sensitivity assays, b) surrogate biomarkers significantly lowers the URLs and c) statistical methods used impact URLs. Our data suggest lower sex-specific cTnT 99th percentiles than reported in the FDA approved package insert. We emphasize the importance of detailing the criteria used to include and exclude subjects for defining a healthy population and the statistical method used to calculate 99th percentiles and identify outliers.

Dynamic silver speciation as studied with fluorous-phase ion-selective electrodes: Effect of natural organic matter on the toxicity and speciation of silver.

The widespread application of silver in consumer products and the resulting contamination of natural environments with silver raise questions about the toxicity of Ag(+) in the ecosystem. Natural organic matter, NOM, which is abundant in water supplies, soil, and sediments, can form stable complexes with Ag(+), altering its bioavailability and toxicity. Herein, the extent and kinetics of Ag(+) binding to NOM, matrix effects on Ag(+) binding to NOM, and the effect of NOM on Ag(+) toxicity to Shewanella oneidensis MR-1 (assessed by the BacLight viability assay) were quantitatively studied with fluorous-phase Ag(+) ion-selective electrodes (ISEs). Our findings show fast kinetics of Ag(+) and NOM binding, weak Ag(+) binding for Suwannee River humic acid, fulvic acid, and aquatic NOM, and stronger Ag(+) binding for Pony Lake fulvic acid and Pahokee Peat humic acid. We quantified the effects of matrix components and pH on Ag(+) binding to NOM, showing that the extent of binding greatly depends on the environmental conditions. The effect of NOM on the toxicity of Ag(+) does not correlate with the extent of Ag(+) binding to NOM, and other forms of silver, such as Ag(+) reduced by NOM, are critical for understanding the effect of NOM on Ag(+) toxicity. This work also shows that fluorous-phase Ag(+) ISEs are effective tools for studying Ag(+) binding to NOM because they can be used in a time-resolved manner to monitor the activity of Ag(+) in situ with high selectivity and without the need for extensive sample preparation.

Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes.

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Effects of Humic and Fulvic Acids on Silver Nanoparticle Stability, Dissolution, and Toxicity.

The colloidal stability of silver nanoparticles (AgNPs) in natural aquatic environments influences their transport and environmental persistence, while their dissolution to Ag(+) influences their toxicity to organisms. Here, we characterize the colloidal stability, dissolution behavior, and toxicity of two industrially relevant classes of AgNPs (i.e., AgNPs stabilized by citrate or polyvinylpyrrolidone) after exposure to natural organic matter (NOM, i.e., Suwannee River Humic and Fulvic Acid Standards and Pony Lake Fulvic Acid Reference). We show that NOM interaction with the nanoparticle surface depends on (i) the NOM's chemical composition, where sulfur- and nitrogen-rich NOM more significantly increases colloidal stability, and (ii) the affinity of the capping agent for the AgNP surface, where nanoparticles with loosely bound capping agents are more effectively stabilized by NOM. Adsorption of NOM is shown to have little effect on AgNP dissolution under most experimental conditions, the exception being when the NOM is rich in sulfur and nitrogen. Similarly, the toxicity of AgNPs to a bacterial model (Shewanella oneidensis MR-1) decreases most significantly in the presence of sulfur- and nitrogen-rich NOM. Our data suggest that the rate of AgNP aggregation and dissolution in aquatic environments containing NOM will depend on the chemical composition of the NOM, and that the toxicity of AgNPs to aquatic microorganisms is controlled primarily by the extent of nanoparticle dissolution.