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Objectives In 2018, our clinical laboratory was alerted to back-to-back plasma sodium critical value callback failures on the same patient, occurring on different shifts and involving different technologists. Therefore, we set forth to investigate the root cause for the critical value callback failures. Design and methods We conducted a thorough investigation focused on the processes associated with critical value identification and notification for plasma sodium measurement performed on the Siemens Vista. Results Our investigation uncovered a flaw in the Siemens CentraLink middleware software. A default dark blue bar in the top row of the results review display was determined to obscure the red color which highlights critical values for lab staff identification. Sodium was disproportionately impacted by this flaw, as it is commonly ordered as part of metabolic panels, and is listed first among the panel analytes in the top row of the CentraLink display. Retrospective data review comparing critical callback failure rates for sodium to potassium and hemoglobin confirmed that sodium had significantly higher critical callback failure rates than these other analytes. After alerting the product manufacturer, Siemens programmed the CentraLink display so that the top row was blank and devoid of patient results, so that the blue color in the top row would no longer obscure the red visual cue of a patient’s critical result. Sodium critical value callback failures were reduced to 0% after this middleware display correction. Conclusions Middleware design flaws can have unexpected consequences on clinical laboratory operations. We encourage clinical laboratories to closely examine user interfaces utilized by laboratory staff, and be wary of potential impacts that the display format may have on results reporting.

Background: Antibody tests are critical in elucidating the epidemiology of the rapidly evolving Coronavirus Disease 2019 (COVID-19) global pandemic and in developing effective infection control measures. To avoid the significant risks posed by the use of tests with poor performance characteristics and associated supply chain disruptions, we developed an in-house SARS-CoV-2 total antibody test. The performance of our robustly validated test was compared with that of three commercial antibody tests, and our test was used to determine the seroprevalence of COVID-19 among healthcare workers and outpatients in Minnesota. Methods: To evaluate the clinical performance of our SARS-CoV-2 total antibody assay, 63 plasma and serum samples were analyzed from 34 SARS-CoV-2 PCR positive patients 4 - 35 days after symptom onset. Positive samples were analyzed for IgG titers in a follow up assay. Samples were analyzed from PCR negative (n=30) and presumed true negative pre-pandemic (n=207) patients. Among samples collected from patients >14 days after symptom onset, the assay had 100% diagnostic sensitivity (95% CI: 85·7 - 100·0%), 100% diagnostic specificity (95%CI: 98·5 - 100·0%), 100% positive predictive value (95% CI: 86·2 - 100·0%), and 100% negative predictive value (95%CI: 98·6 - 100·0%). The analytical specificity was 99·8% (95% CI: 98·92 - 99·97%), indicating minimal cross-reactivity. A screening study was conducted to ascertain the seroprevalence of COVID-19 among healthcare workers and outpatients in Minnesota. Findings: Serum collected from 1,282 healthcare workers (1,000 females, 280 males, 2 gender unreported; median age 41 years [range 18 - 73]) and 2,379 outpatients (1,460 females, 916 males, 3 gender unreported; median age 49 years [range 2 months - 93 years]) between April 13 and May 21, 2020 was analyzed using our SARS-CoV-2 total antibody assay. The COVID-19 seroprevalence was 2·96% (95% CI: 2·04 - 3·89%) among healthcare workers and 4·46% (95% CI: 3·63 - 5·28%) among outpatients from 35 clinics who had a previous COVID-19 exposure or symptoms. Interpretation: Rigorously validated serology tests with robust clinical and analytical performance, such as our in-house SARS-CoV-2 total antibody test, are of critical importance when conducting epidemiological studies to inform public health decisions during the COVID-19 pandemic. Funding: University of Minnesota Department of Laboratory Medicine and Pathology Declaration of Interests: The authors do not declare any competing interests. Ethics Approval Statement: The University of Minnesota Institutional Review Board (IRB) determined that this study was not considered human research.

Background We report a unique case of transient hyperphosphatasemia in a pediatric patient with a history of hepatic and skeletal abnormalities. Patient and Methods A 2-month old male was diagnosed with progressive familial intrahepatic cholestasis type-2 and osteoporosis after marked increases in liver function tests were noted at 1 month of age. He underwent a second liver transplantation at 1 y. The increased liver function test trend resolved a few weeks post-transplantation. Four months after successful liver transplantation, unexplained significant increases in alkaline phosphatase (ALP) were observed, and they persisted for almost 9 months. Among the etiologies under consideration for the isolated increased ALP activity were viral infections and macro-ALP. Results A persistent trend in abnormally increased ALP for 9 months was investigated leading to a confirmed diagnosis of transient hyperphosphatasemia (TH). Conclusion Pediatric post-liver transplant patients with skeletal and hepatic abnormalities including isolated markedly increased ALP activities represent a previously undescribed TH patient population. The 4.3% prevalence of TH in pediatric liver transplant recipients within our healthcare system is considerably higher than the previously reported prevalence of 2.1% for patients within the United States.

S-nitrosothiols (SNOs) are endogenous signaling molecules that have numerous beneficial effects on the airway via cyclic guanosine monophosphate-dependent and -independent processes. Healthy human airways contain SNOs, but SNO levels are lower in the airways of patients with cystic fibrosis (CF). In this study, we examined the interaction between SNOs and the molecular cochaperone C-terminus Hsc70 interacting protein (CHIP), which is an E3 ubiquitin ligase that targets improperly folded CF transmembrane conductance regulator (CFTR) for subsequent degradation. Both CFBE41o- cells expressing either wild-type or F508del-CFTR and primary human bronchial epithelial cells express CHIP. Confocal microscopy and IP studies showed the cellular colocalization of CFTR and CHIP, and showed that S-nitrosoglutathione inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the mature and immature forms of F508del-CFTR. In fact, SNO inhibition of the function and expression of CHIP not only improved the maturation of CFTR but also increased CFTR's stability at the cell membrane. S-nitrosoglutathione-treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than cells that were not treated, suggesting that the S-nitrosylation of CHIP prevents the ubiquitination of CFTR by inhibiting CHIP's E3 ubiquitin ligase function. Furthermore, the exogenous SNOs S-nitrosoglutathione diethyl ester and S-nitro-N-acetylcysteine increased the expression of CFTR at the cell surface. After CHIP knockdown with siRNA duplexes specific for CHIP, F508del-CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del-CFTR expression on airway epithelial cell surfaces. Together, these findings indicate that S-nitrosylation of CHIP is a novel mechanism of CFTR correction, and we anticipate that these insights will allow different SNOs to be optimized as agents for CF therapy.

We assessed the feasibility of a highly sensitive immunoassay method based on single molecule array (Simoa) technology to detect IgG and IgA antibodies against SARS-CoV-2 spike protein receptor binding domain (RBD) in saliva from individuals with natural or vaccine-induced COVID-19 immunity. The performance of the method was compared to a laboratory-developed SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay (ELISA). Paired serum and saliva specimens were collected from individuals (n = 40) prior to and 2 weeks after receiving an initial prime COVID-19 vaccine dose (Pfizer/BioNTech BNT162b2 or Moderna mRNA-1273). Saliva was collected using a commercially available collection device (OraSure Inc.) and SARS-CoV-2 RBD IgG antibodies were measured by an indirect ELISA using concentrated saliva samples and a Simoa immunoassay using unconcentrated saliva samples. The IgG results were compared with paired serum specimens that were analyzed for total RBD antibodies using the ELISA method. The analytical sensitivity of the saliva-based Simoa immunoassay was five orders of magnitude higher than the ELISA assay: 0.24 pg/mL compared to 15 ng/mL. The diagnostic sensitivity of the saliva ELISA method was 90% (95% CI 76.3–97.2%) compared to 91.7% (95% CI 77.5–98.2%) for the Simoa immunoassay without total IgG-normalization and 100% (95% CI 90.3–100%) for the Simoa immunoassay after total IgG-normalization when compared to the serum ELISA assay. When analyzed using the SARS-CoV-2 RBD IgG antibody ELISA, the average relative increase in antibody index (AI) between the saliva of the post- and pre-vaccinated individuals was 8.7 (AIpost/pre). An average relative increase of 431 pg/mL was observed when the unconcentrated saliva specimens were analyzed using the Simoa immunoassay (SARS-CoV-2 RBD IgGpost/pre). These findings support the suitability of concentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG antibodies via ELISA, and unconcentrated saliva specimens for the measurement of SARS-CoV-2 RBD IgG and IgA using an ultrasensitive Simoa immunoassay.

BACKGROUND: Implantable medical devices and hardware are prolific in medicine, but hardware associated infections remain a major issue. OBJECTIVE: To develop and evaluate a novel, biologic antimicrobial coating for medical implants. METHODS: Electrochemically compacted collagen sheets with and without crosslinked heparin were synthesized per a protocol developed by our group. Sheets were incubated in antibiotic solution (gentamicin or moxifloxacin) overnight, and in vitro activity was assessed with five-day diffusion assays against Pseudomonas aeruginosa. Antibiotic release over time from gentamicin-infused sheets was determined using in vitro elution and high performance liquid chromatography (HPLC). RESULTS: Collagen-heparin-antibiotic sheets demonstrated larger growth inhibition zones against P. aeruginosa compared to collagen-antibiotic alone sheets. This activity persisted for five days and was not impacted by rinsing sheets prior to evaluation. Rinsed collagen-antibiotic sheets did not produce any inhibition zones. Elution of gentamicin from collagen-heparin-gentamicin sheets was gradual and remained above the minimal inhibitory concentration for gentamicin-sensitive organisms for 29 days. Conversely, collagen-gentamicin sheets eluted their antibiotic load within 24 hours. Overall, heparin-associated sheets demonstrated larger inhibition zones against P. aeruginosa and prolonged elution profile via HPLC. CONCLUSION: We developed a novel, local antibiotic delivery system that could be used to coat medical implants/hardware in the future and reduce post-operative infections.

The global prevalence of type-two diabetes mellitus (T2DM) makes it a disease of public health concern. T2DM is strongly linked with insulin resistance caused by increased levels of visceral fat. Visceral fat secretes several adipocytokines that regulate body metabolism. Resistin is one of these adipocytokines which is encoded by the RETN gene. Herein, we tested the association of the RETN +299(G>A) and −420(C>G) single nucleotide polymorphisms (SNPs) with T2DM. T2DM patients (n=282) and healthy subjects (n=125) were included in the study. Subjects with metabolic syndromes other than diabetes were excluded. Genotyping of subjects was performed using PCR-RFLP. The +299(G>A) SNP was associated with T2DM (P=0.038). The AA genotype was higher in T2DM (17%) compared to controls (8%) with an odd ratio of 2.16 and 95% CI of 1.34 to 4.56. With respect to −420(C>G) SNP, no significant association was found with the risk of T2DM (P=0.128). The haplotype analysis of the examined SNPs indicated that the CA haplotype of the −420 and +299 SNPs in RETN was associated with T2DM risk (P=0.004; odd ration 4.0, 95% CI: 1.56–10.0). In conclusion, the present findings suggest a role of the RETN locus in modulating the risk of T2DM.

BackgroundThe LIAISON® QuantiFERON®-TB Gold Plus (QFT-Plus) assay, a fully automated chemiluminescence immunoassay (CLIA) system, has recently received FDA approval for the detection of interferon-γ (IFN-γ) on the LIAISON XL analyzer. Here, we evaluated the diagnostic performance of the LIAISON’s CLIA method in comparison to the widely used ELISA method using the Qiagen QuantiFERON-TB Gold Plus Blood Collection Tubes.MethodsHeparinized blood samples from 329 participants were categorized into 3 cohorts, including 15 with confirmed tuberculosis (TB) (active TB cohort), 129 non-TB (low-risk cohort), and 185 potential TB (mixed risk cohort). Samples were analyzed with both assays and results were interpreted according to the manufacturers’ criteria.ResultsThe LIAISON CLIA assay showed an overall agreement with the Qiagen ELISA assay in 13/14 (92.8%) samples among the active cohort, 93/95 (97.9%) among the low-risk cohort, with a Cohen’s kappa value of 0.76 and 0.74, respectively. Test results for 185 mixed risk cohort participants showed 97.8% agreement between the LIAISON and the Qiagen, with 17 positive, 163 negative, and 1 indeterminate. Four samples were discrepant; 3 of these were negative on the ELISA but positive on the CLIA assay.ConclusionOverall, the results were comparable (>92% agreement) in our study cohorts. In addition, our mixed risk cohort results showed an excellent agreement of 0.88 (Cohen’s kappa value) between the 2 assays. These findings suggest that the automated LIAISON QFT-Plus assay has a comparable diagnostic performance to the Qiagen assay and can be used for latent TB infection (LTBI) diagnosis.

Context Sudaxine is a novel respiratory stimulant that increases ventilatory drive via NO+ -thiolate signaling and is under development for reversal of opioid-induced respiratory depression and other critical care indications. Objective This study investigates the pharmacokinetic characteristics after intravenous administration of Sudaxine by using a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Materials and methods A sensitive LC-MS/MS method was validated to determine the concentration of Sudaxine in beagle dog plasma after intravenous administration of Sudaxine at (3, 10, 30, and 100 mg/kg). Blood samples (1 mL) were collected at designated time points and SDX concentration was measured for pharmacokinetic study. Results The calibration curve was linear within the range of 50-5,000 ng/mL with the lower limit of quantification at 50 ng/mL. The CTmax for all doses was reached at 10 minutes (Tmax ). Over the dose range studied, average concentration - time curves and systemic exposure (CTmax and AUC0-t ) increased to Sudaxine dose. The terminal half-life of Sudaxine in dogs ranged from 10 to 30 minutes and about 17.3 ± 1.0% of Sudaxine was protein-bound in dog plasma. Discussion and conclusions We developed a novel LC-MS/MS method of Sudaxine detection and quantification and determined its pharmacokinetic profiles after intravenous administration in canine subjects. Sudaxine followed first-order kinetics with rapid dose-dependent clearance rates and short half-life making it an ideal candidate for use in a critical care setting with intramuscular or IV administration.

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOSoxy. The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOSoxy. Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOSoxy is ∼65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain.

Background The ADAMS™ HA-8180V is the 8th generation of a fully automated ion-exchange HPLC system from ARKRAY, and the first to be released onto the US market. We evaluated the HA-8180V, for routine hemoglobin A1c measurement in comparison with the Roche Cobas c501, the Tosoh G8 analyzer for normal hemoglobin, and with the Trinity analyzer for hemoglobin variants. Methods The analytical performance (linearity, precision, carryover, and sample stability) was assessed based on the Clinical and Laboratory Standards Institute (CLSI) and manufacturer guidelines. A comparison of the HA-8180V against two major analytical methods was performed for 100 whole blood samples. HA-8180V variant mode was also compared against the Trinity ultra2 A1c analyzer for 50 samples containing hemoglobin variants (HbC 14, HbS 14, HbD 12, and HbE 10). Results The within-run and total CVs were Conclusion The eighth generation ADAMS HA-8180V A1c analyzer demonstrated high analytical performance adequate for routine clinical use.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

A provider notified the laboratory of a missed sodium critical value notification on a patient; during further investigation, it was noted that the laboratory had missed a second sodium critical value notification several hours later on the same patient. The two missed critical callbacks occurred on different shifts with different technologists, pointing to a systematic issue. A thorough investigation was undertaken involving review of critical callback procedures, staff interviews, and retrospective data review to determine the etiology. It was confirmed that the middleware, Siemens Centralink, had captured and displayed the critical values for review. The Centralink display highlights rows with critical results in red for easy visual identification by the laboratory technologist, and a numerical code of “8” appears in the Normal Severity (NS) column as an additional cue. Upon discussion with involved laboratory staff, a supervisor noted that sodium consistently appears in the top row of the Siemens Centralink result screen when held for review. Our Siemens Centralink display was noted to have a blue bar that by default appears in the top row and was noted to obscure the red highlight and NS Flag that provides a visual cue to staff that it is a critical value. Therefore, we hypothesized that the sodium critical values were missed due to being obscured by the default blue bar, and hypothesized that they could be preferentially affected relative to other analytes due to sodium’s default position in the top row when displayed. To investigate further, we pulled retrospective critical value callback data from our university hospitals for 2018. Our overall critical callback failure rates were ~2.0%. When broken down by individual analyte, we found that 7.2% of sodium critical values were not called back to providers, whereas only 1.0% of potassium critical values were missed. Additionally, critical callback failure rates for hemoglobin were 0.1%; our hematology analyzer has a hard stop for all critical values, whereas the chemistry analyzer does not. In conclusion, we discovered that the default display on our Siemens Centralink middleware screen places a blue bar in the top row that obscures the visual cues associated with a critical value. Because sodium results are placed in the top row on the display screen, missed sodium critical callbacks occurred seven times more often than missed potassium callbacks. To remedy the issue, a blank row has been added to the display as the top row so that no clinical data are obscured by the default blue bar. Additionally, the very low critical callback failure rate on the hematology analyzer points to a need for a hard stop on the chemistry analyzer. We will prospectively monitor critical callback data for sodium to determine whether the display change improves sodium callback rates.

Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC proteins by confocal microscopy. Increased intracellular cAMP level significantly attenuated RSV-induced disassembly of AJC. These barrier-protective effects of cAMP were due to the activation of PKA signaling and did not involve Epac activity. Increased cAMP level reduced RSV-induced reorganization of the actin cytoskeleton, including apical accumulation of an essential actin-binding protein, cortactin, and inhibited expression of the RSV F protein. These barrier-protective and antiviral-function of cAMP signaling were evident even when cAMP level was increased after the onset of RSV infection. Taken together, our study demonstrates that cAMP/PKA signaling attenuated RSV-induced disruption of structure and functions of the model airway epithelial barrier by mechanisms involving the stabilization of epithelial junctions and inhibition of viral biogenesis. Improving our understanding of the mechanisms involved in RSV-induced epithelial dysfunction and viral pathogenesis will help to develop novel anti-viral therapeutic approaches.

Peroxynitrite has emerged as a major cytotoxic agent implicated in a host of pathophysiological conditions triggered by its multifaceted nitroxidative reactions. Peroxynitrite's very short half-life under physiological conditions reflects both its fast intrinsic decomposition and its notorious reactivity with many cellular targets. Sensitive and accurate measurement of peroxynitrite is crucial to investigations that aim at shedding light on the illusive pathophysiologic roles of this metabolite. The development process of probes and sensors capable of selectively detecting and measuring any analyte depends primarily on the availability of pure authentic amounts of it in order to evaluate sensor performance. There are numerous methods of peroxynitrite synthesis or generation in situ. However, they all come with varying levels of byproducts that may interfere with the characterization of performance of peroxynitrite sensing probes and devices. This chapter gives the background and a brief review of major methods of peroxynitrite synthesis and in situ generation. Particularly, we discuss advantages and drawbacks of the various methods in the context of the development and characterization of peroxynitrite sensors and probes.