Jonathan R. Genzen

Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease.

Summary Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca2+ signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

A Retrospective Analysis of Urine Drugs of Abuse Immunoassay True Positive Rates at a National Reference Laboratory

Urine drug screens are commonly performed to identify drug use or monitor adherence to drug therapy. The purpose of this retrospective study was to evaluate the true positive and false positive rates of one of our in-house urine drug screen panels. The urine drugs of abuse panel studied consists of screening by immunoassay then positive immunoassay results were confirmed by mass spectrometry. Reagents from Syva and Microgenics were used for the immunoassay screen. The screen was performed on a Beckman AU5810 random access automated clinical analyzer. The percent of true positives for each immunoassay was determined. Agreement with previously validated GC-MS or LC-MS-MS confirmatory methods was also evaluated. There were 8,825 de-identified screening results for each of the drugs in the panel, except for alcohol (N = 2,296). The percent of samples that screened positive were: 10.0% for amphetamine/methamphetamine/3,4-methylenedioxy-methamphetamine (MDMA), 12.8% for benzodiazepines, 43.7% for opiates (including oxycodone) and 20.3% for tetrahydrocannabinol (THC). The false positive rate for amphetamine/methamphetamine was ∼14%, ∼34% for opiates (excluding oxycodone), 25% for propoxyphene and 100% for phencyclidine and MDMA immunoassays. Based on the results from this retrospective study, the true positive rate for THC drug use among adults were similar to the rate of illicit drug use in young adults from the 2013 National Survey; however, our positivity rate for cocaine was higher than the National Survey.

Liver function testing on the Abaxis Piccolo Xpress: Use in Ebola virus disease protocols.

BACKGROUND Laboratories that choose a point-of-care approach for liver function testing in patients undergoing evaluation for Ebola virus disease (EVD) have few options to choose from. The primary objective of this study was to conduct a performance characterization of a Clinical Laboratory Improvement Amendments (CLIA)-waived liver function panel on the Abaxis Piccolo® Xpress chemistry analyzer. The secondary objectives were to evaluate multiple specimen types, characterize whole blood specimen stability, and validate disposable exact transfer pipettes. Our final objective was to assess instrument airflow from a biosafety perspective. METHODS An instrument performance characterization, including precision, linearity, accuracy, reference interval verification, and specimen type evaluation was conducted using Liver Panel Plus reagent discs on the Piccolo® Xpress. RESULTS All assays demonstrated acceptable linearity (slopes, 0.938-1.061; observed error, 0.8-6.3%). Assay precision was 0.0-3.6% (%CV; within-day studies) and 0.9-5.6% (between-day studies). Method comparison experiments (versus Roche cobas c502/c702 chemistry analyzers) showed excellent correlation for most assays, although a few notable differences were observed (Piccolo versus Roche): alkaline phosphatase, -18.6%; amylase, -29.0%; total bilirubin, +0.3mg/dl. Pre-programmed reference intervals were verified except for the alkaline phosphatase (male and female) and alanine aminotransferase (female), which had greater than 10% of results fall below the programmed ranges. Piccolo instrument results were largely consistent across specimen types tested (lithium-heparin whole blood, lithium-heparin plasma, and serum), although some statistical differences were observed for aspartate aminotransferase, gamma glutamyltransferase, and total protein. Whole blood time course studies demonstrated that some analytes (albumin, amylase, and total protein) showed remarkable stability, while others (such as aspartate aminotransferase) showed a slight trend toward decreased activity over time. Exact volume transfer pipettes provided an effective disposable option for disc loading. Finally, airflow studies suggested that, in the context of EVD protocols, instrument placement in a biosafety level (BSL) 2 cabinet or greater is justified. CONCLUSIONS Given its analytical performance and ease of operation, the Piccolo Xpress was transferred to a BSL-2 cabinet in our BSL-3 suite for use in our hospitals diagnostic protocol for providing liver function testing in patients undergoing evaluation for EVD.

Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice: A Survey of Participants in the College of American Pathologists Syphilis Serology Proficiency Testing Program

CONTEXT -Syphilis serology screening in laboratory practice is evolving. Traditionally, the syphilis screening algorithm begins with a nontreponemal immunoassay, which is manually performed by a laboratory technologist. In contrast, the reverse algorithm begins with a treponemal immunoassay, which can be automated. The Centers for Disease Control and Prevention has recognized both approaches, but little is known about the current state of laboratory practice, which could impact test utilization and interpretation. OBJECTIVE -To assess the current state of laboratory practice for syphilis serologic screening. DESIGN -In August 2015, a voluntary questionnaire was sent to the 2360 laboratories that subscribe to the College of American Pathologists syphilis serology proficiency survey. RESULTS -Of the laboratories surveyed, 98% (2316 of 2360) returned the questionnaire, and about 83% (1911 of 2316) responded to at least some questions. Twenty-eight percent (378 of 1364) reported revision of their syphilis screening algorithm within the past 2 years, and 9% (170 of 1905) of laboratories anticipated changing their screening algorithm in the coming year. Sixty-three percent (1205 of 1911) reported using the traditional algorithm, 16% (304 of 1911) reported using the reverse algorithm, and 2.5% (47 of 1911) reported using both algorithms, whereas 9% (169 of 1911) reported not performing a reflex confirmation test. Of those performing the reverse algorithm, 74% (282 of 380) implemented a new testing platform when introducing the new algorithm. CONCLUSION -The majority of laboratories still perform the traditional algorithm, but a significant minority have implemented the reverse-screening algorithm. Although the nontreponemal immunologic response typically wanes after cure and becomes undetectable, treponemal immunoassays typically remain positive for life, and it is important for laboratorians and clinicians to consider these assay differences when implementing, using, and interpreting serologic syphilis screening algorithms.

Regulation of Mg2+ Reabsorption and Transient Receptor Potential Melastatin Type 6 Activity by cAMP Signaling

The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg(2+) channels participate in transcellular Mg(2+) transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg(2+) reabsorption in the kidney. The molecular details of this process are, however, unknown. Adenylate cyclase 3 (Adcy3) has been shown to colocalize with the Na(+)/Cl(-) cotransporter, a marker of the distal convoluted segment of the kidney, the principal site of TRPM6 expression. Given the critical role of TRPM6 in Mg(2+) reabsorption, an inducible kidney-specific Adcy3 deletion mouse model was characterized for blood and urinary electrolyte disturbances under a normal--and low--Mg(2+) diet. Increased urinary Mg(2+) wasting and Trpm6 mRNA levels were observed in the urine and kidney of Adcy3-deleted animals compared with wild-type controls. Serum Mg(2+) concentration was significantly lower in Adcy3-deleted animals at day 7 on the low Mg(2+) diet. Using patch clamp electrophysiology, cell surface biotinylation, and total internal reflection fluorescence live cell imaging of transfected HEK293 cells, we demonstrated that cAMP signaling rapidly potentiates TRPM6 activity by promoting TRPM6 accumulation at the plasma membrane and increasing its single-channel conductance. Comparison of electrophysiological data from cells expressing the phosphorylation-deficient S1252A or phosphomimetic S1252D TRPM6 mutants suggests that phosphorylation at this intracellular residue participates in the observed stimulation of channel activity. Altogether, these data support a physiologically relevant magnesiotropic role of cAMP signaling in the kidney by a direct stimulatory action of protein kinase A on the plasma membrane trafficking and function of TRPM6 ion channels.

N-acetylcysteine interference of Trinder-based assays.

OBJECTIVES The primary objective of this study was to evaluate potential interference of Trinder-based chemistry assays by N-acetylcysteine (NAC). A secondary objective was to look for evidence of interference in patients treated with NAC for acetaminophen (APAP) overdose. DESIGN AND METHODS Dilutions of NAC in plasma were tested for interference using the following Roche Diagnostics Trinder-based assays on a cobas 8000 system: enzymatic creatinine (Cr), cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C), triglycerides (TRIG), and uric acid (UA). Two non-Trinder Roche assays - urea nitrogen (BUN) and glucose (GLUC) - were tested as controls. Sekisui N-geneous® low-density lipoprotein cholesterol (LDL-C) reagent was also evaluated. Retrospective chart review of APAP overdose cases over 49months was conducted to look for differences in plasma Cr before and after intravenous (IV) NAC administration. RESULTS NAC concentrations (shown in parentheses) that caused ≥10% inhibition for individual assays were (in order of sensitivity to interference): TRIG (570mg/L)>CHOL (740mg/L)≈Cr (790mg/L)>UA (1100mg/L)>HDL-C (1760mg/L)>LDL-C (2900mg/L). Neither BUN nor GLUC achieved significant inhibition up to 10,000mg/L NAC. Evidence for relatively minor inhibition of Cr was observed in patients after NAC administration. CONCLUSIONS NAC inhibition of the assays investigated typically occurs at concentrations higher than expected during IV and oral NAC therapy.

Benchmarking to Identify Practice Variation in Test Ordering: A Potential Tool for Utilization Management

BACKGROUND Appropriate test utilization is usually evaluated by adherence to published guidelines. In many cases, medical guidelines are not available. Benchmarking has been proposed as a method to identify practice variations that may represent inappropriate testing. This study investigated the use of benchmarking to identify sites with inappropriate utilization of testing for a particular analyte. METHODS We used a Web-based survey to compare 2 measures of vitamin D utilization: overall testing intensity (ratio of total vitamin D orders to blood-count orders) and relative testing intensity (ratio of 1,25(OH)2D to 25(OH)D test orders). RESULTS A total of 81 facilities contributed data. The average overall testing intensity index was 0.165, or approximately 1 vitamin D test for every 6 blood-count tests. The average relative testing intensity index was 0.055, or one 1,25(OH)2D test for every 18 of the 25(OH)D tests. Both indexes varied considerably. CONCLUSIONS Benchmarking can be used as a screening tool to identify outliers that may be associated with inappropriate test utilization.

Ordering of the Serum Angiotensin-Converting Enzyme Test in Patients Receiving Angiotensin-Converting Enzyme Inhibitor Therapy: An Avoidable but Common Error

BACKGROUND Serum angiotensin-converting enzyme (ACE) levels may be decreased by use of ACE inhibitor (ACEI) medication. In this study, we determined how often ACE levels were measured in patients receiving ACEI therapy. METHODS ACE levels analyzed over a 54-month preintervention time period at an academic medical center were reviewed retrospectively for tests performed during ACEI therapy. These data were compared with a large, deidentified dataset of ACE levels measured at a national reference laboratory; in vitro studies of ACEI inhibition; and liquid chromatography time-of-flight mass spectrometry detection of lisinopril in a subset of clinical specimens. RESULTS Over a 54-month period, 1,292 patients had ACE levels measured, with 108 patients (8.4%) receiving ACEI therapy at the time of testing. ACE levels measured for patients receiving ACEI therapy were substantially lower. In general, clinical teams did not recognize a medication effect on ACE levels. Introduction of a warning prompt in the electronic health record reduced the ordering of ACE levels in patients receiving ACEIs by > 60% in a 17-month postintervention time period. The deidentified dataset of ACE levels at a reference laboratory showed a bimodal distribution, with a peak of very low ACE levels. Using liquid chromatography time-of-flight mass spectrometry, the presence of lisinopril was confirmed in a subset of specimens with low ACE activity. In vitro studies of two different ACE assays showed significant inhibition of activity at clinically relevant concentrations. CONCLUSIONS Assessment of ACE activity is often measured for patients receiving ACEIs, potentially leading to low ACE concentrations and inaccurate interpretations.

Clinical performance evaluation of total protein measurement by digital refractometry and characterization of non-protein solute interferences

Objectives Refractometric methods to measure total protein (TP) in serum and plasma specimens have been replaced by automated biuret methods in virtually all routine clinical testing. A subset of laboratories, however, still report using refractometry to measure TP in conjunction with serum protein electrophoresis. The objective of this study was therefore to conduct a modern performance evaluation of a digital refractometer for TP measurement. Design and methods Performance evaluation of a MISCO Palm Abbe™ digital refractometer was conducted through device familiarization, carryover, precision, accuracy, linearity, analytical sensitivity, analytical specificity, and reference interval verification. Comparison assays included a manual refractometer and an automated biuret assay. Results Carryover risk was eliminated using a demineralized distilled water (ddH2O) wash step. Precision studies demonstrated overall imprecision of 2.2% CV (low TP pool) and 0.5% CV (high TP pool). Accuracy studies demonstrated correlation to both manual refractometry and the biuret method. An overall positive bias (+5.0%) was observed versus the biuret method. On average, outlier specimens had an increased triglyceride concentration. Linearity was verified using mixed dilutions of: a) low and high concentration patient pools, or b) albumin-spiked ddH2O and high concentration patient pool. Decreased recovery was observed using ddH2O dilutions at low TP concentrations. Significant interference was detected at high concentrations of glucose (>267 mg/dL) and triglycerides (>580 mg/dL). Current laboratory reference intervals for TP were verified. Conclusions Performance characteristics of this digital refractometer were validated in a clinical laboratory setting. Biuret method remains the preferred assay for TP measurement in routine clinical analyses.

Thrombin-mediated degradation of parathyroid hormone in serum tubes

BACKGROUND Intact parathyroid hormone (PTH) tests are frequently sandwich immunoassays. Enzymes that cleave PTH may cause falsely lower PTH results. The objective of this study was to determine whether bovine thrombin in Becton Dickinson (BD) Vacutainer rapid serum tubes™ (RSTs) may lead to PTH results that are lower than in plasma separator tube™ (PST) or serum separator tube™ (SST) collections. METHODS Tubes of blood (PST, SST, and RST) were collected from donors. PTH concentrations were measured on a Roche Cobas e602 analyzer in aliquots held at room temperature or 4°C across time. Instrument comparison studies were also conducted on an Abbott Architect i1000SR and a Siemens Immulite 2000 XPi. Previously collected serum specimens were also incubated in exogenous bovine thrombin, the direct thrombin inhibitor hirudin, or both. Freshly collected RST specimens were also spiked with hirudin after clotting and centrifugation. RESULTS Significant decreases in PTH degradation rate constants were observed according to tube type, with degradation rates faster in RSTs than SSTs, and SSTs faster than PSTs. PTH degradation rate was temperature dependent. PTH decreases induced by exogenous bovine thrombin, as well as endogenous human thrombin, were reduced by hirudin. CONCLUSIONS Bovine thrombin is responsible for the decrease in PTH results observed in RSTs. Endogenous human thrombin, activated during clot formation, is likely responsible for the smaller decreases observed in non-RST sera versus plasma.