Spironolactone metabolite causes falsely increased progesterone in the Abbott Architect immunoassay.

BACKGROUND: Immunoassays are important for routine clinical testing and medical diagnosis. However, they are limited by cross-reactivity especially at low analyte concentrations. There is a critical need to investigate compounds that can interfere with immunoassays. Herein, we describe the identification of canrenone, a spironolactone metabolite that falsely increases progesterone concentrations on the Abbott Architect i2000 Immunoassay. METHODS: Serum samples and assay diluents were spiked with spironolactone or canrenone and progesterone concentrations were measured on the Architect i2000 and Immulite XPi immunoassay platforms. Blood samples from patients taking spironolactone were analyzed with liquid chromatography-tandem mass spectrometry to evaluate the intrinsic response of progesterone concentrations to the presence of canrenone. RESULTS: We measured approximately 10-fold higher progesterone concentrations on the Abbott Architect i2000 compared to reference immunoassay analyzers (Siemens Immulite XPi and Roche Cobas e601/602), suggesting an analytical error which is unique to the Architect i2000 antibody and/or assay conditions. By measuring serum progesterone after addition of spironolactone or canrenone to serum samples, we found that canrenone falsely increased progesterone on the Architect i2000 immunoassay. However, this interference was more pronounced at low serum progesterone concentrations. Moreover, a strong positive correlation was seen between canrenone and measured serum progesterone concentrations. CONCLUSIONS: Our investigations are important for individuals who require progesterone measurements using the Architect i2000 immunoassay, especially because it is unlikely for clinicians to order canrenone measurements alongside progesterone measurements for individuals taking spironolactone. Further research is needed to determine whether canrenone can influence progesterone measurements on other immunoassay systems.

Cross-Institutional Evaluation of the Abbott ARCHITECT SARS-CoV-2 IgG Immunoassay.

OBJECTIVE: To describe a cross-institutional approach to verify the Abbott ARCHITECT SARS-CoV-2 antibody assay and to document the kinetics of the serological response. METHODS: We conducted analytical performance evaluation studies using the Abbott ARCHITECT SARS-CoV-2 antibody assay on 5 Abbott ARCHITECT i2000 automated analyzers at 2 academic medical centers. RESULTS: Within-run and between-run coefficients of variance (CVs) for the antibody assay did not exceed 5.6% and 8.6%, respectively, for each institution. Quantitative and qualitative results agreed for lithium heparin plasma, EDTA-plasma and serum specimen types. Results for all SARS-CoV-2 IgG-positive and -negative specimens were concordant among analyzers except for 1 specimen at 1 institution. Qualitative and quantitative agreement was observed for specimens exchanged between institutions. All patients had detectable antibodies by day 10 from symptom onset and maintained seropositivity throughout specimen procurement. CONCLUSIONS: The analytical performance characteristics of the Abbott ARCHITECT SARS-CoV-2 antibody assay within and between 2 academic medical center clinical laboratories were acceptable for widespread clinical-laboratory use.

A Novel Laboratory Assay to Monitor Unfractionated Heparin Dosing in Patients Taking Apixaban Prior to Hospital Admission.

INTRODUCTION: When monitoring heparin, anti-Xa assays are susceptible to interference from apixaban taken before admission and can result in inappropriate dose adjustments that can negatively affect patient care. METHODS: We derived a novel assay, termed corrected heparin (CH), using quantified values from a chromogenic anti-Xa assay with heparin calibrators before and after heparinase treatment to eliminate any interference from apixaban within the patient sample. We retrospectively assessed 469 specimens from 72 patients at our institution who had their unfractionated heparin infusion monitored using the CH assay because of known apixaban use. These patients were included in the study if they had detectable apixaban levels (>0.1 IU/mL by anti-Xa). RESULTS: The analytical performance of the assay was evaluated, and precision was found to be 8.8% within 1 day and 13.3% over multiple days, with acceptable linearity (R2 = 0.997). Evaluation of clinical performance was compared with the partial thromboplastin time (PTT), showing a lack of correlation similar to comparisons between the PTT and anti-Xa assay (Blood Coagul Fibrinolysis 1993;4:635-8). The mean time to a therapeutic result in this cohort was 10 hours and 10 minutes. The CH assay was used to determine how long the apixaban was detected by the anti-Xa assay. The majority of patients (80%) still had measurable anti-Xa assay interference from apixaban at 24 hours after the last apixaban dose. CONCLUSIONS: We have developed and evaluated an assay capable of quantifying heparin in the presence of apixaban. This assay showed acceptable performance in both analytical and clinical performance.

The Relationship between the Initial Anti-factor Xa Measurement and the Duration of Direct Oral Anticoagulant Influence in Patients Transitioning to Heparin.

BACKGROUND: Anticoagulation monitoring during transition from direct oral anticoagulants (DOAC) to heparin infusions is a significant challenge. Factor Xa inhibitors influence the heparin calibrated antifactor Xa assay. The University of Virginia (UVA) Medical Center utilized a corrected antifactor Xa assay (c-AXA) during this transition period, which removes DOAC-mediated antifactor Xa activity (d-AXA) and reflects heparin-specific activity. Currently, the duration of this influence is not well described. STUDY OBJECTIVE: This study had two aims: to determine if the initial d-AXA is predictive of the duration of DOAC influence and to further characterize this influence among different patient populations. METHODS: This retrospective study included adult patients admitted to UVA Medical Center between September 2016 and March 2017, with c-AXA measurements, who received apixaban or rivaroxaban within 48 hours before heparin initiation. A Pearson correlation test, Kaplan-Meier Survival Analysis, and multivariate linear regression were used to assess the relationship between initial d-AXA and duration of influence. RESULTS: Sixty-eight patients met inclusion criteria and were maintained on either apixaban (85%) or rivaroxaban (15%) before heparin initiation. The initial d-AXA ranged from 0.11 to 3.27 IU/ml. The mean duration of influence was 69.3 ± 46.2 hours, with a median duration of 62.7 hours. No strong correlation was identified between initial d-AXA and duration of influence (R2 = 0.124). Presence of interacting medications significantly increased duration of influence (p=0.012). No significant difference in duration of influence existed between patients with normal renal function and those with dynamic renal function (p=0.84), or with body mass index (BMI) greater than 40 kg/m2 (p=0.16). CONCLUSIONS: The initial d-AXA was not predictive of duration of influence in patients transitioning from DOACs to heparin infusion; however, the median duration of influence suggests influence may be present for longer than currently stated in the literature, especially in those taking interacting medications.

Outbreak of Severe Hypoglycemia After Ingestion of a Male Enhancement Supplement - Virginia, August-November 2019.

In August 2019, the Virginia Poison Center (VPC) and the Blue Ridge Poison Center (BRPC) were contacted concerning patients experiencing repeated episodes of marked hypoglycemia following ingestion of a male enhancement supplement tablet marketed as "V8" in convenience stores in central Virginia. Over the following 3 months, the Virginia Department of Agriculture and Consumer Services (VDACS) and the Virginia Department of Health (VDH) conducted an investigation and identified 17 patients meeting the case definition (severe hypoglycemia within 48 hours of consuming an over-the-counter male enhancement supplement in a man with no history of use of insulin or other medication used to control blood glucose). Analysis of the V8 tablets revealed that most contained glyburide, a sulfonylurea oral hypoglycemic used in the treatment of diabetes and associated with prolonged hypoglycemia following overdose (1). To stem this outbreak, V8 was removed from stores when found, and public service announcements were released. The public health implications of V8 use include the potential for substantial morbidity from hypoglycemic episodes and the potential for mortality if health care services are not accessed in a timely manner when hypoglycemia occurs. The presence of V8 in the market poses a serious threat to public health because of its potentially life-threatening adverse effects.

Streamlining Quality Review of Mass Spectrometry Data in the Clinical Laboratory by Use of Machine Learning.

CONTEXT.­: Turnaround time and productivity of clinical mass spectrometric (MS) testing are hampered by time-consuming manual review of the analytical quality of MS data before release of patient results. OBJECTIVE.­: To determine whether a classification model created by using standard machine learning algorithms can verify analytically acceptable MS results and thereby reduce manual review requirements. DESIGN.­: We obtained retrospective data from gas chromatography-MS analyses of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in 1267 urine samples. The data for each sample had been labeled previously as either analytically unacceptable or acceptable by manual review. The dataset was randomly split into training and test sets (848 and 419 samples, respectively), maintaining equal proportions of acceptable (90%) and unacceptable (10%) results in each set. We used stratified 10-fold cross-validation in assessing the abilities of 6 supervised machine learning algorithms to distinguish unacceptable from acceptable assay results in the training dataset. The classifier with the highest recall was used to build a final model, and its performance was evaluated against the test dataset. RESULTS.­: In comparison testing of the 6 classifiers, a model based on the Support Vector Machines algorithm yielded the highest recall and acceptable precision. After optimization, this model correctly identified all unacceptable results in the test dataset (100% recall) with a precision of 81%. CONCLUSIONS.­: Automated data review identified all analytically unacceptable assays in the test dataset, while reducing the manual review requirement by about 87%. This automation strategy can focus manual review only on assays likely to be problematic, allowing improved throughput and turnaround time without reducing quality.

The sample that would not clot.

BACKGROUND: Vitamin K is a vital component within both the intrinsic and extrinsic coagulation cascade as certain factors (II, VII, IX, X and protein C and S) utilize vitamin K as a cofactor during post translational modification. Deficiency of vitamin K can result in the inability to properly form blood clots, both in vivo and in vitro, due to reduced vitamin K dependent factor levels and function. Vitamin K deficiency can result from congenital causes, such as VKOR or CYP2C9 mutations, or acquired causes, such as nutritional deficiencies, antibiotic therapy, or supra-therapeutic warfarin dosing. RESULTS: In this case we present a patient with multifactorial vitamin K deficiency (due to nutritional defects and multiple genetic mutations in VKOR and CYP2C9) that was exacerbated by antibiotic and warfarin therapy during her hospital admission. CONCLUSION: This case displays the importance of genetic testing prior to warfarin dosing and the role antibiotics play in the coagulation cascade.

Improved Clinical Sensitivity of a Reflexive Algorithm to Minimize False-Negative Test Results by a Urine Benzodiazepine Immunoassay Screen.

BACKGROUND: Urine drug testing is an essential component of treating patients for chronic pain and/or anxiety and is used to monitor compliance during treatment. A common algorithm is to use an immunoassay as a urine drug screen (UDS), followed by mass spectrometry to confirm all presumptive positive samples. Many UDSs, however, have significant limitations, and false-negative test results can be common due to lack of antibody specificity. METHODS: Urine samples were screened by a benzodiazepine immunoassay followed by confirmatory testing using LC-MS/MS to determine an initial false-negative test rate for the screen. Attempts to improve the false-negative test rate included hydrolysis before screening and optimization of the absorbance cutoff required for a positive result. RESULTS: Hydrolysis corrected 41% of false-negative test results in samples containing parent benzodiazepines and/or metabolites but had no effect on samples containing only clonazepam. Of the confirmed false-negative test results, 85% (17 of 20) demonstrated absorbance values between 20 and 100, with 100 being the cutoff for a positive result. Implementing an optimized absorbance cutoff of 20, rather than 100, for a reflexive confirmation testing algorithm decreases the false-negative test rate of detecting benzodiazepine from 47% to 2%. CONCLUSIONS: Hydrolyzing samples before the benzodiazepine screen provided a modest improvement in the false-negative test rate; however, the screen still missed samples containing clonazepam. Optimization of the absorbance cutoff to reflex samples to LC-MS/MS markedly improved the false-negative test rate for all benzodiazepines.

Recognition of rare hemoglobin variants by hemoglobin A1c measurement procedures.

BACKGROUND: Unrecognized hemoglobinopathies can lead to measured hemoglobin A1c (Hb A1c) concentrations that are erroneous or misleading. We determined the effects of rare hemoglobin variants on capillary electrophoresis (CE) and HPLC methods for measurement of Hb A1c. METHODS: We prospectively investigated samples in which Hb A1c was measured by CE during a 14-month period. For samples in which the electropherograms suggested the presence of rare hemoglobinopathies, hemoglobin variants were identified by molecular analysis or by comparison with electropherograms of known variants. When sample volume permitted, Hb A1c was measured by 2 HPLC measurement procedures and by boronate affinity HPLC. RESULTS: Hb A1c was measured by CE in 33,859 samples from 26,850 patients. 15 patients (0.06%) were identified as having rare hemoglobinopathies: Hbs A2 prime, Agenogi, Fannin-Lubbock I, G Philadelphia, G San Jose, J Baltimore, La Desirade, N Baltimore, Nouakchott, and Roanne. Among 6 of these samples tested by 2 ion-exchange HPLC methods, the rare Hb was detected by both HPLC methods in only one sample, and none were detected by boronate affinity HPLC. The mean of the Hb A1c results of 2 HPLC methods differed from the result of the CE method by 0.7-2.2% Hb A1c in samples with variant hemoglobins versus <0.2% Hb A1c in samples without variants. CONCLUSION: Measurement procedures differ in the ability to detect the presence of rare Hb variants and to quantify Hb A1c in patients who harbor such variants.

The ARTµS: a novel microfluidic CD4+ T-cell enumeration system for monitoring antiretroviral therapy in HIV patients.

We report on a novel and cost-effective microfluidic platform that integrates immunomagnetic separation and cell enumeration via DNA-induced bead aggregation. Using a two-stage immunocapture microdevice, 10 µL of whole blood was processed to isolate CD4+ T-cells. The first stage involved the immuno-subtraction of monocytes by anti-CD14 magnetic beads, followed by CD4+ T-cell capture with anti-CD4 magnetic beads. The super hydrophilic surface generated during polydimethylsiloxane (PDMS) plasma treatment allowed for accurate metering of the CD4+ T-cell lysate, which then interacted with silica-coated magnetic beads under chaotropic conditions to form aggregates. Images of the resulting aggregates were captured and processed to reveal the mass of DNA, which was used to back-calculate the CD4+ T-cell number. Studies with clinical samples revealed that the analysis of blood within 24 hours of phlebotomy yielded the best results. Under these conditions, an accurate cell count was achieved (R(2) = 0.98) when compared to cell enumeration via flow cytometry, and over a functional dynamic range from 106-2337 cells per µL.

An outbreak of acute delirium from exposure to the synthetic cannabinoid AB-CHMINACA.

BACKGROUND: Synthetic cannabinoid containing products are a public health threat as reflected by a number of outbreaks of serious adverse health effects over the past 4 years. The designer drug epidemic is characterized by the rapid turnover of synthetic cannabinoid compounds on the market which creates a challenge in identifying the particular etiology of an outbreak, confirming exposure in cases, and providing current information to law enforcement. RESULTS: Between 28 May 2014 and 8 June 2014, 35 patients were evaluated and treated at the University of Florida Health Medical Center in Gainesville following reported exposure to a synthetic cannabinoid containing product obtained from a common source. Patients demonstrated acute delirium (24) and seizures (14), and five required ventilator support and ICU-level care; none died. The presence of N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (AB-CHMINACA), or one of its predicted metabolites was confirmed in 15 of 21 cases. A rapid public health response and aggressive public messaging prevented further morbidity, identified the source, and led to law enforcement seizure of the implicated product. DISCUSSION: The significance of this outbreak lies as much in the rapid occurrence of unpredictable, life-threatening adverse health effects from a newly identified synthetic cannabinoid compound as it does in the multidisciplinary investigation and novel partnership between local public health, the laboratory, and the chemical industry, resulting in termination of the outbreak. CONCLUSION: A coordinated response and collaboration between law enforcement, the local public health, emergency medical services and Health Center staff, were all key interventions in preventing a more substantial public health outbreak resulting from use of a novel synthetic cannabinoid compound. Real time collaborations between toxicology laboratories, suppliers of analytical standards and the public health system may be useful in the face of future novel chemical exposures.

Correction: Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Correction for 'Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene' by Bankim J. Sanghavi et al., Lab Chip, 2015, DOI: 10.1039/c5lc00840a.

Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels.

Steps to diagnosis of a case of surreptitious intake of one of the newer direct oral anticoagulants: a case report and literature review.

Little is known about the effects of newer oral anticoagulants on various coagulation factors. When presented with a case of intentional or suspected overdose with an abnormal coagulation profile, it is imperative to have a working diagnostic algorithm to narrow the cause to a specific drug or drug class. This may become more crucial and time sensitive when dealing with a case of acute hemorrhage. Here we discuss the first reported case of what appears to be a surreptitious intake of newer oral anticoagulants and the steps leading to the diagnosis.

Quantitation of 25-hydroxyvitamin D2 and D3 in serum and plasma by LCMS/MS.

OBJECTIVE: We developed and validated an analytical method for quantifying 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in serum and plasma. METHODS: Samples, pretreated with zinc sulfate and methanol, were extracted with hexane. Separation was achieved via UHPLC and 25OHD quantification was accomplished by a triple quadrupole mass spectrometer. RESULTS: Imprecision was 3.6-15.1%CV and bias 88.0-126.0%. Extraction efficiency was 76.5-110.5%, whereas the matrix effect ranged from -46.7 to -32.0%. The method was applied to authentic specimens. The results showed no significant difference between serum and plasma; strong correlation with paired values from an external laboratory; and analyte stability for 15 days. CONCLUSION: This method provides reliable and accurate measurement of 25OHD for use in clinical practice.

Trends in licit and illicit drug-related deaths in Florida from 2001 to 2012.

BACKGROUND: Florida, the epicenter of the recent prescription drug epidemic in the United States, maintains a statewide drug mortality surveillance system. We evaluated yearly profiles, demographic characteristics, and correlation between drug trends to understand the factors influencing drug-induced mortality. METHODS: All drug-related deaths reported to the Florida Medical Examiners Commission during 2001-2012 were included (n=92,596). A death was considered "drug-related" if at least one drug was identified in the decedent. Depending on its contribution to death, a drug could be listed as a causative agent or merely present, but not both. RESULTS: Rate of drug-caused deaths was 8.0 per 100,000 population in 2001, increasing to 17.0 in 2010 and then decreasing to 13.9 in 2012. Benzodiazepines had the highest mortality rate in 2010, although <10% were solely due these drugs. Opioid-caused mortality rate also peaked in 2010 and started to decline (-28%) in 2010-2012. The heroin-caused mortality rates were negatively correlated with opioids and benzodiazepines (ρ's ≥ -0.670; P≤0.034). Ethanol- and cocaine-mortality rates stabilized to 3.0-3.1 and 2.8-3.0 per 100,000 over 2009-2012, respectively. Amphetamines, zolpidem, and inhalants-caused deaths were on the rise with rates of ≤0.6 per 100,000. CONCLUSIONS: Overall declines in benzodiazepine- and opioid-caused deaths in 2011-2012 may have been related to Florida's attempts to regulate prescription drug abuse. This period, however, was also marked by a rise in heroin-caused mortality, which may reflect growing use of heroin as an alternative. Increases in amphetamines, zolpidem, and inhalants-induced mortality are an additional public health concern.