Donato, Leslie J.

Abstract Objectives To evaluate the analytical and clinical performance characteristics of the fifth-generation troponin T reagent. Methods Troponin T was measured in 2,332 paired serum and plasma samples from emergency department and hospital patients using the fourth- and fifth-generation reagents. Testing was repeated after recentrifugation to determine the frequency of analytical outliers and percentage of patients with elevated values for each assay. We conducted separate experiments to determine the effects of biotin and hemolysis interference, as well as measure interinstrument variability, for fifth-generation troponin T. Results Analytic outliers occurred more frequently using the fifth-generation reagent (3.4%) compared with the fourth-generation reagent (1.0%). The frequency of elevated troponin T above the 99th percentile upper reference limit was 26% for the fourth-generation reagent and 52% for the fifth-generation reagent. Clinically significant assay interference by biotin was observed at 20 ng/mL, but hemolysis interference was not observed until an H index of 150. Instrument-to-instrument variability between e411 and e601/602 instrument platforms is predicted to confound clinical interpretation of troponin changes. Conclusions Analytical outliers and instrument-to-instrument variability are the two analytical variables most likely to confound interpretation of changes in fifth-generation troponin T results over time.

Abstract Background It is important for clinical laboratories to have protocols for investigating suspected biotin interference in patient samples. VeraPrep Biotin™ is a commercial product used to rapidly deplete biotin from serum/plasma samples. The objectives of this study were to verify that VeraPrep Biotin™: (a) does not impact immunoassay analyte recovery in control samples and (b) can effectively deplete biotin from samples (both biotin-spiked and samples from donors who ingested biotin supplements). Methods De-identified residual waste serum/plasma samples were combined to create 9 pools for each immunoassay. Plasma/serum samples (n = 23) were obtained from 6 healthy donors at varying times following ingestion of biotin (20 mg, 100 mg, or 200 mg). Nine Elecsys immunoassays were evaluated using the e 602 (Roche Diagnostics Inc.). Control, biotin-spiked (n = 10, ∼400 ng/mL), and donor samples were assayed pre- and post-VeraPrep treatment. Percentage analyte recovery [(posttreatment/pretreatment) × 100] was calculated for control samples. A laboratory-developed LC–MS/MS method was used to quantify biotin. Results In control samples (n = 81), 90–110% analyte recovery was observed post-VeraPrep treatment in over 95% of samples (77/81). The pre- and post-VeraPrep treatment biotin concentration [mean ± standard deviation (SD)] for specimens spiked with up to 500 ng/mL biotin was 357 ± 47 ng/mL and 1.0 ± 0.6 ng/mL, respectively. The mean (range) biotin concentration for the donor samples pre- and post-treatment was 166 (15–1029) ng/mL and 0.2 (<0.1–3) ng/mL, respectively (P = 0.004). Conclusions These data demonstrate that treatment with VeraPrep Biotin™ does not affect analyte recovery in biotin-negative samples and effectively depletes both spiked and endogenous biotin in serum/plasma.

ABSTRACTObjectives:Excessive fecal bile acids in adults have been associated with diarrhea‐predominant irritable bowel syndrome (IBS‐D), but their role in pediatric IBS‐D is unknown. Serum markers including 7α‐hydroxy‐4‐cholesten‐3‐one (C4) and fibroblast growth factor‐19 (FGF‐19) were validated in adults to detect bile acid diarrhea (BAD) compared to 48‐hour fecal bile acid collection (48FBA). Our aims were to assess fasting serum C4 and FGF–19 and 48FBA in a pediatric population, to compare measurements in IBS‐D patients and healthy controls (HC), and to determine the prevalence of BAD among children with IBS‐D.Methods:Using a cross‐sectional design, 26 patients with IBS‐D and 56 HC were recruited in two pediatric tertiary care centers. Fasting serum C4 and FGF‐19 and 48FBA were obtained. Participants completed a 7‐day bowel diary coinciding with stool collection. Associations were analyzed using Spearman correlations.Results:Mean age was 14.7 ± 2.5 years (42.3% female) in IBS‐D and 12.6 ± 2.4 years (39.3% female) in HC. There was a significant correlation of C4 with 48FBA (r = 0.48, P < 0.05) and an inverse association with FGF‐19 (r = –0.43, P < 0.05). No significant differences were noted in C4 (P = 0.32), FGF‐19 (P = 0.1), or 48FBA (P = 0.5) between IBS‐D and HC groups; however, 20% of IBS‐D patients had elevated C4 and 28% had low FGF‐19 values.Fecal primary BA was significantly correlated with stool frequency (r = 0.45, P < 0.002).Conclusions:Correlations of C4 with 48FBA and FGF‐19 are confirmed in a pediatric population. Twenty percent of pediatric patients with IBS‐D had abnormal fasting serum C4. This serum test could be applied to identify BAD in pediatric IBS‐D.

Objective: Cardiovascular disease remains a leading cause of mortality worldwide. Ceramide scores have been associated with adverse outcomes in patients with established coronary artery disease. The prognostic value of ceramide score has not been assessed in the general population. We tested the hypothesis that ceramide scores are associated with major adverse cardiac events (MACE) in a community-based cohort with average coronary artery disease burden at enrollment. Approach and results: In a prospective community-based cohort, we performed passive follow-up using a record linkage system to ascertain the composite outcome of MACE, defined as acute myocardial infarction, coronary revascularization (bypass grafting or percutaneous intervention), stroke, or death. Ceramides were analyzed as log-transformed continuous variables, ratios or scores, and quartiles with adjustment for confounders. We analyzed 1131 subjects, 52% females, mean age±(SD) 64±9 years. After a median follow-up of 13.3 years (Q1, 12.7; Q3, 14.4), 486 patients experienced a MACE: myocardial infarction (80), coronary artery bypass surgery (34), percutaneous coronary intervention (62), stroke (94), and all-cause death (362). Ceramide ratios were significantly associated with MACE independently of LDL-c (low-density lipoprotein cholesterol) and conventional coronary artery disease risk factors. Those in the highest quartile of ceramide score had nearly 1.5-fold risk of MACE, hazard ratio, 1.47 (95% CI, 1.12–1.92). There was a dose-response association across quartiles of ceramide ratios and MACE. Conclusions: Elevated ceramide score is a robust predictor of cardiovascular disease and MACE in the community. The risk conferred by the ceramide score has a dose-response behavior and is independent of conventional risk factors.

Abstract Background and introduction Cardioversion is commonly used to terminate cardiac arrhythmias. Some previous reports have suggested that cardioversion results in myocardial injury as evidenced by increased levels of cardiac troponin. However, many of these studies were done years ago with less sensitive troponin assays and monophasic waveform defibrillators. Purpose To determine if external direct current (DC) cardioversion with biphasic rectilinear waveform shocks results in myocardial injury as assessed by high sensitivity cardiac troponin T (hs-cTnT) and I (hs-cTnI). Methods Patients scheduled for elective DC cardioversion for atrial fibrillation or atrial flutter were recruited. Plasma samples for measurement of hs-cTnT and hs-cTnI were obtained pre-cardioversion and as late as feasible but at least 6 hours post-cardioversion [median of 9 (7–11) hours]. Results A total of 96 patients were recruited. One patient was excluded because the pre-cardioversion sample was hemolysed. Median (25th–75th interquartile range) cumulative energy delivered was 121.6J (62.4–277.4J) and median highest energy individual shock was 121.0J (62.1–146.2J). A total of 39 (41.1%) patients received more than 1 shock, 23 (24.2%) patients received a cumulative energy of 300J or higher and 5 (5.3%) patients received a cumulative energy of 1,000J or more. The median pre-cardioversion hs-cTnT value was 11.48 (7.19–18.38) ng/L and the median hs-cTnI value was 5.1 (2.0–9.4) ng/L. Median post-cardioversion hs-cTnT value were 12.46 (7.98–20.28) ng/L and hs-cTnI value were 6.3 (3.5–10.0) ng/L. Wilcoxon-Signed ranks test showed a statistically significant change between the pre-and-post cardioversion hs-cTnT values (Z=−4.237, p&lt;0.001) and hs-cTnI values (Z=−4.822, p&lt;0.001). In only 5 (hs-cTnT) and 4 patients (hs-cTnI) was there an increase of &gt;5 ng/L. There were 5 cases where the post-cardioversion values of both hs-cTnT and hs-cTnI were above the 50% reference change value. There was no relation between the change in hs-cTn values and sex, number of shocks, total energy delivered (even in those who received more than 1,000J), highest energy per shock, total current delivered, highest current delivered per shock or transthoracic impedance. Conclusion(s) There is a statistically significant but very small change in median hs-cTnT and hs-cTnI values (1 ng/L and 1.2 ng/L respectively) after DC cardioversion. The results were similar even in patients where high energy shocks were delivered and did not vary based on the pre cardioversion baseline value. Patients who have marked troponin elevations after cardioversion should be assessed for other causes of myocardial injury. It should not be assumed that they have myocardial injury from the cardioversion alone. Figure 1 Funding Acknowledgement Type of funding source: Other. Main funding source(s): Dr. Allan Jaffe has substantial research funds from both grants and private industry. Funds were used to pay for blood sample collection and analysis of high sensitivity cardiac troponin T at Mayo Clinic. Abbott Laboratories donated reagents for the high sensitivity cardiac troponin I analysis.

Abstract Plasma concentrations of low-density-lipoprotein cholesterol (LDL-C) are directly associated with risk for coronary artery disease (CAD). Multisociety guidelines define LDL-C&gt;160mg/dL as a risk factor for CAD and LDL-C&gt;190mg/dL as an indication for lipid lowering medication, regardless of other clinical factors. Subfractionation of LDL according to size (LDL-s) enables differentiation between two LDL phenotypes: large-buoyant LDL and small-dense LDL. The small-dense LDL phenotype reportedly conveys increased risk for CAD. Major societies do not recommend LDL subfractions be used for clinical decision making and most payers do not cover LDL subfraction testing. Despite these restrictions, LDL subfraction is routinely requested by clinicians. Nuclear magnetic resonance (NMR) spectroscopy measures LDL-C and LDL-s. Following inquiries regarding interpretation of conflicting LDL-C and LDL-s results, we investigated associations between LDL-C and LDL-s measured by NMR in order to determine how often they provide contradicting or additive information. Verification of NMR LDL-C accuracy was confirmed by ß-quantification in a subset of patient samples (n=250). The average bias was -4.5mg/dL and the correlation coefficient was 0.92. High-risk was defined as LDL-C&gt;160mg/dL or LDL-s&lt;20.5 nm (small-dense LDL); and low-risk was defined as LDL-C&lt;70mg/dL or LDL-s&gt;20.5nm (large-buoyant LDL). In 26,710 clinical NMR analyses, the median LDL-C was 94.0mg/dL (range:5-436mg/dL) with median LDL-s of 20.8 nm (range:19.4–23.0nm). LDL-s moderately correlated with LDL-C (Ï#129;=0.51;p&lt;0.01). Small-dense-LDL was identified in only 18% (407/2,191) of patients with elevated LDL-C (&gt;160mg/dL) and was more common (73.2% of 6,093) in patients with low LDL-C (&lt;70mg/dL;p&lt;0.001). Associations with CAD were investigated among patients without cholesterol-lowering medication treatment referred for angiography (n=356). CAD (defined as stenosis &gt;50% in one or more coronary artery) was diagnosed in 14% (1/7) of subjects with low LDL-C (&lt;70mg/dL) compared to 59% (47/80) of subjects with elevated LDL-C (p=0.01). When stratifying by LDL-s, CAD was diagnosed in 50% (57/115) of subjects with small-dense LDL compared to 43% (104/241) of subjects with large-buoyant LDL (p=0.2). Small-dense LDL was identified in only 33% (26/80) of cases with elevated LDL-C. Limiting to subjects with elevated LDL-C, CAD was diagnosed in 50% (13/26) of subjects with concordant (high-risk) small-dense LDL compared to 61% (33/54) of subjects with discordant (low-risk) large-buoyant LDL (LDL-s&gt;20.5nm) (p=0.3). Our data confirm that LDL-s subfraction measured by NMR is reported discordantly in most cases when LDL-C is unequivocally high or low. Furthermore, CAD diagnosis was significantly associated with LDL-C, but not with LDL-s. Our data also show that in discrepant samples, elevated LDL-C correlates better with disease state compared to LDL-s. Therefore, LDL-s should not be used to justify treatment decisions in patients with elevated LDL-C. Laboratories should consider carefully whether or not to report LDL-s when it is known that misleading and discordant values will be reported in a majority of cases.

Abstract Lipoprotein(a) [Lp(a)] is a pro-atherogenic and pro-thrombotic LDL-like particle recognized as an independent risk factor for cardiovascular disease (CVD) that is resistant to typical lipid-lowering treatments. The cholesterol within Lp(a) (Lp(a)-C) contributes to the reported LDL-cholesterol (LDL-C) concentration by nearly all available methods including beta-quantification, direct homogenous assays, and all estimating equations. Accurate LDL-C measurements are critical for identification of genetic hyperlipidemia conditions such as familial hypercholesterolemia (FH). FH risk estimators such as the Dutch Lipid Clinic Network (DLCN) criteria utilize LDL-C concentration cut-offs and other clinical inputs to assess the likelihood of FH. Therefore, failure to adjust for Lp(a)-C can impact accurate FH classification, appropriate follow-up testing and treatments, and interpretation of cholesterol-lowering treatment efficacy. Lp(a)-C can be estimated from Lp(a) mass as measured by immunoassay using an average cholesterol content per particle. However, Lp(a)-C size and composition varies significantly within individuals resulting in inaccurate Lp(a)-C estimates. In this study, we use direct Lp(a)-C measurements to assess the potential misclassification of FH risk due to the contribution of Lp(a)-C to LDL-C in patient samples submitted for advanced lipoprotein profiling. A total of 28,200 samples submitted for lipoprotein profiling were included. The profiling included lipid testing in a CDC-certified laboratory on Roche cobas 501 (cholesterol and triglycerides by enzymatic method, high-density lipoprotein cholesterol by MgCl2/dextran sulfate precipitation). LDL-C was measured by beta-quantification, and Lp(a)-C by quantitative lipoprotein electrophoresis (SPIFE Vis Cholesterol, Helena Laboratories). The DLCN LDL-C cut-offs (155, 190, 250, and 330mg/dL) were applied to LDL-C results before and after accounting for Lp(a)-C contribution. Lp(a)-C was detected in 3,728 (13.2%) samples. The median (range) concentrations of Lp(a)-C and LDL-C were 11mg/dL (5-108mg/dL) and 121mg/dL (27-678mg/dL), respectively. Overall, subtracting Lp(a)-C would reclassify 6.5% of all samples into a lower LDL-C category within the DLCN algorithm. Within the LDL-C scoring categories, 7.0% (n=222) of subjects with LDL-C 155-189mg/dL, 5.6% (n=66) of subjects with LDL-C 190-249mg/dL, 5.2% (n=10) of subjects with LDL-C 250-329mg/dL, and 3.4% (n=4) of subjects with LDL-C &gt;330mg/dL would be down-classified after adjusting for Lp(a)-C. Limiting to subjects with measurable Lp(a)-C, reclassification to a lower diagnostic threshold occurred in 47.4% of subjects with LDL-C 155-189mg/dL, 37.5% with LDL-C 190-249mg/dL, 41.6% with LDL-C 250-329mg/dL, and 33.3% with LDL-C &gt;330mg/dL after adjustment. Current guidelines recommend screening for elevated Lp(a) in patients with family history of CVD. Our data show that a high percentage of samples evaluated for advanced lipid testing contain measurable Lp(a)-C that could cause mis-classification in FH prediction algorithms. If labeled high probability of FH, these mis-classifications could trigger inappropriate work-up for suspected FH. As clinical follow-up and therapeutic strategies differ between FH and elevated Lp(a), proper distinction between LDL-C and Lp(a)-C is needed to guide appropriate patient management.

Emerging data supports a potential role of neurotensin (NT) in the development of obesity, obesity-associated comorbidities, and certain cancers. The association of NT with colon cancer risk has not been explicitly explored. We determined plasma levels of pro-NT, a stable NT precursor fragment, in 223 incident colon cancer patients and 223 age-, gender-, BMI-matched population controls participating in a population-based case–control study of colon cancer. On average, the cases have significantly higher levels of pro-NT than the controls (median = 205.6 pmol/L vs 183.1 pmol/L, respectively; P = 0.02). Multivariate logistic regression models, adjusted for age, gender, BMI, family history of colorectal cancer, smoking, diabetes mellitus, alcohol, and non-steroidal anti-inflammatory drugs use, show statistically significant risk associations: for continuous measure of pro-NT, the OR estimate was 1.30 (95% CI =1.03–1.64; P = 0.026) for each increment of 175 pmol/L; for dichotomized measure of pro-NT, the OR estimate was 1.75 (95% CI = 1.12–2.74; P = 0.025) for those in the top quartile comparing to the other participants. Our results support circulating levels of pro-NT as a novel risk biomarker for colon cancer.

Objective: Vasopressin increases water permeability and activity of the epithelial sodium channel ENaC in the distal nephron of the kidney, favoring increased water and sodium retention. We hypothesized that genetically mediated differences in circulating vasopressin concentrations could contribute to features of hypertension that vary between non-Hispanic blacks and whites. To test this hypothesis we measured copeptin, a fragment of pre-pro arginine vasopressin with a prolonged half-life that is more easily quantified. Methods: The Genetic Epidemiology of Responses to Antihypertensives (GERA) I study was designed to identify predictors of response to diuretic therapy in non-Hispanic black and non-Hispanic white adults with essential hypertension. Blood samples were obtained after a 4 week washout of anti-hypertensive medication while on a sodium intake of 2 mmol/kg. Copeptin was measured from thawed EDTA plasma samples using the copeptin proAVP homogeneous automated immunofluorescent assay on the BRAHMS Kryptor Compact PLUS (Thermo Fisher). Results: Copeptin was significantly higher in blacks (median: 7.4 pmol/L, IQR: 4.4-10.2 pmol/L) than in whites (median: 5.4 pmol/L, IQR: 3.6-8.0 pmol/L) (p <0.001), while renin was lower (Table 1). Racial differences remained significant (p = 0.001) in multivariate models that included 24 hour urine sodium excretion and calculated plasma osmolality. Conclusions: Higher copeptin (vasopressin) concentrations in non-Hispanic blacks compared to whites may contribute to the distinct differences in the hypertensive phenotypes between these racial groups. The underlying mechanism(s) require further study.

INTRODUCTION: Patients with inflammatory bowel disease (IBD), whether Crohn's disease (CD) or ulcerative colitis (UC), have known alterations in the enterohepatic circulation of bile acids (BA) that may contribute to chronic diarrhea, but the fecal BA patterns are not well defined. We aimed to audit all patients with IBD who had fecal BA measurements as part of evaluation of chronic diarrhea at a tertiary care center. METHODS: Methods: 65 patients with IBD (18 with UC and 47 with CD) were evaluated at a single center and underwent measurements of fecal total and individual BAs based on a 48-hour collection performed at the end of a 4-day, 100-gram fat diet. The electronic medical records were interrogated to obtain type of IBD, disease activity, location of disease, prior surgery, IBD related therapies, presenting symptoms, and response to treatment with BA sequestrants. Bile acid diarrhea (BAD) diagnosis was based on >10% primary BAs, >4% primary BAs + >1,000 µmol/48 h, or total fecal BAs >2,337 µmol/48 h. IBD disease activity was based on radiologic, endoscopic and histologic parameters. Data are reported as mean ± standard error of the mean (SEM). RESULTS: Table 1 summarizes the patterns of BAD in our cohort with IBD. Figure 1 demonstrates the mean ± SEM of total fecal BAs and % primary BAs in each group of IBD patients. Among patients with UC, 10 had poorly controlled disease and 8 had well controlled disease, and both cohorts had elevated primary BAs (mean 28.9% ± 12.5%; 34.6% ± 21%, respectively). All patients with an ileal pouch anal anastomosis (IPAA) had elevated total fecal BAs (BADt) compared to those with an intact colon. In patients with CD, those with ileal resections had elevated BADt and % primary BAs (6263 µmol/48 h ± 1012 µmol/48 h; 79% ± 5.9%, respectively). Patients with an intact ileum, had elevated % primary BAs regardless of disease activity: controlled CD 34.7% ± 9.3%; poorly controlled CD 57.8% ± 9.5%; 15 out of 47 patients with CD were started on BA sequestrants with a positive response in 10/15. CONCLUSION: Discussion: BAs may play a role in the pathophysiology of chronic diarrhea in patients with IBD, regardless of inflammatory disease activity. Future studies are required to assess the impact of fecal bile acid testing and treatment with BA sequestrants on inflammation and chronic diarrhea in patients with IBD.