Antibody-mediated interferences affecting cardiac troponin assays: recommendations from the IFCC Committee on Clinical Applications of Cardiac Biomarkers.

The International Federation of Clinical Chemistry Committee on Clinical Applications of Cardiac Biomarkers (IFCC C-CB) provides educational documents to facilitate the interpretation and use of cardiac biomarkers in clinical laboratories and practice. Our aim is to improve the understanding of certain key analytical and clinical aspects of cardiac biomarkers and how these may interplay. Measurements of cardiac troponin (cTn) have a prominent place in the clinical work-up of patients with suspected acute coronary syndrome. It is therefore important that clinical laboratories know how to recognize and assess analytical issues. Two emerging analytical issues resulting in falsely high cTn concentrations, often several fold higher than the upper reference limit (URL), are antibody-mediated assay interference due to long-lived cTn-antibody complexes, called macrotroponin, and crosslinking antibodies that are frequently referred to as heterophilic antibodies. We provide an overview of antibody-mediated cTn assay interference and provide recommendations on how to confirm the interference and interpret the results.

Analytical Considerations in Deriving 99th Percentile Upper Reference Limits for High-Sensitivity Cardiac Troponin Assays: Educational Recommendations from the IFCC Committee on Clinical Application of Cardiac Bio-Markers.

The International Federation of Clinical Chemistry Committee on Clinical Application of Cardiac Bio-Markers provides evidence-based educational documents to facilitate uniform interpretation and utilization of cardiac biomarkers in clinical laboratories and practice. The committee's goals are to improve the understanding of certain key analytical and clinical aspects of cardiac biomarkers and how these may interplay in clinical practice. Measurement of high-sensitivity cardiac troponin (hs-cTn) assays is a cornerstone in the clinical evaluation of patients with symptoms and/or signs of acute cardiac ischemia. To define myocardial infarction, the Universal Definition of Myocardial Infarction requires patients who manifest with features suggestive of acute myocardial ischemia to have at least one cTn concentration above the sex-specific 99th percentile upper reference limit (URL) for hs-cTn assays and a dynamic pattern of cTn concentrations to fulfill the diagnostic criteria for MI. This special report provides an overview of how hs-cTn 99th percentile URLs should be established, including recommendations about prescreening and the number of individuals required in the reference cohort, how statistical analysis should be conducted, optimal preanalytical and analytical protocols, and analytical/biological interferences or confounds that can affect accurate determination of the 99th percentile URLs. This document also provides guidance and solutions to many of the issues posed.

Sex-specific cut-off values for soluble suppression of tumorigenicity 2 (ST2) biomarker increase its cardiovascular prognostic value in the community.

BACKGROUND: Suppression of tumorigenicity 2 (ST2) has important cardiovascular prognostic value in community patients; however, previous analyses have utilized non-sex specific cut-off values. We assessed whether sex-specific ST2 cut-off values would improve the prognostic utility of ST2 in the asymptomatic community. METHODS: A total of 2042 participants underwent clinical assessment and echocardiographic evaluation. Baseline measurements of high sensitivity troponin, natriuretic peptides and ST2 were obtained in 1681 individuals. ST2, cardiac biomarkers and associated co-morbidities were evaluated by sex-specific ST2 quartile analysis. ST2 concentrations were also analysed as dichotomous variables defined as being above the sex-specific cut-off for each the outcomes of heart failure (HF), major adverse cardiac event (MACE) and mortality. RESULTS: Median ST2 concentration was 29.4 ng/mL in male subjects and 24.1 ng/mL in female subjects. Higher ST2 concentrations were associated with incident HF (p<0.001; preserved ejection fraction (EF) p<0.001, reduced EF p=0.23), MACE (p=0.003) and mortality (p<0.001) across sex-specific quartiles. Event-based, hazard ratio (HR) analysis revealed sex-specific ST2 cut-offs were significantly more predictive of incident HF, MACE and mortality compared to non-sex-specific analysis even following adjustment for cardiac co-morbidities and traditional biomarkers. CONCLUSIONS: These data suggest that sex-specific cut-offs, greater than non-sex specific cut-offs, significantly impact the prognostic value of the biomarker ST2 in the asymptomatic community cohort.Clinical SignificanceSuppression of tumorigenicity 2 (ST2) is a biomarker which has known associations with heart failure (HF), major adverse cardiac events (MACEs) and mortality in the general population.Recent data support the concept of sex-specific cut off values and individualized approaches based on sex to predict cardiovascular disease. Given the difference in pathobiology between the sexes, the fact that such approaches improve risk stratification is understandable. Thus, when sex-specific treatments are developed, this may similarly lead to improved outcomes.The use of sex-specific ST2 cut-off values significantly improved the prognostic value in predicting HF, MACE, and mortality in an asymptomatic community. This prognostication was particularly strong for HF with preserved ejection fraction and remained clinically significant following adjustment for cardiac co-morbidities and other traditional cardiac biomarkers (NTproBNP and hscTnI).

99th Percentile Upper-Reference Limit of Cardiac Troponin and the Diagnosis of Acute Myocardial Infarction.

BACKGROUND: Concerns exist regarding how the 99th percentile upper reference limit (URL) of cardiac troponin (cTn) is determined and whether it should be derived from normal healthy individuals. CONTENT: The 99th percentile URL of cTn is an important criterion to standardize the diagnosis of myocardial infarction (MI) for clinical, research, and regulatory purposes. Statistical heterogeneity in its calculation exists but recommendations have been proposed. Some negativity has resulted from the fact that with some high-sensitivity (hs) cTn assays, a greater number of increases above the 99th percentile are observed when transitioning from a contemporary assay. Increases reflect acute or chronic myocardial injury and provide valuable diagnostic and prognostic information. The etiology of increases can sometimes be difficult to determine, making a specific treatment approach challenging. For those reasons, some advocate higher cutoff concentrations. This approach can contribute to missed diagnoses. Contrary to claims, neither clinical or laboratory guidelines have shifted away from the 99th percentile. To support the diagnosis of acute MI, the 99th percentile URL remains the best-established approach given the absence of cTn assay standardization. Importantly, risk stratification algorithms using hs-cTn assays predict the possibility of MI diagnoses established using the 99th percentile. SUMMARY: The 99th percentile of cTn remains the best-established criterion for the diagnosis of acute MI. While not perfect, it is analytically and clinically evidence-based. Until there are robust data to suggest some other approach, staying with the 99th percentile, a threshold that has served the field well for the past 20 years, appears prudent.

Circulating electrolytes and the prevalence of atrial fibrillation and supraventricular ectopy: The Atherosclerosis Risk in Communities (ARIC) study.

BACKGROUND AND AIMS: Evaluating associations of circulating electrolytes with atrial fibrillation (AF) and burden of supraventricular arrhythmias can give insights into arrhythmia pathogenesis. METHODS AND RESULTS: We conducted a cross-sectional analysis of 6398 participants of the Atherosclerosis Risk in Communities (ARIC) study, ages 71-90, with data on serum electrolytes (magnesium, calcium, potassium, phosphorus, chloride, sodium). Prevalence of AF was determined from electrocardiograms and history of AF hospitalizations. A subset of 317 participants also underwent electrocardiographic recordings for up to 14 days using the Zio® patch. Burden of other supraventricular arrhythmias [premature atrial contractions (PACs), supraventricular tachycardia] was determined with the Zio® patch. We used logistic and linear regression adjusting for potential confounders to determine associations of electrolytes with arrhythmia prevalence and burden. Among 6394 eligible participants, 614 (10%) had AF. Participants in the top quintiles of magnesium [odds ratio (OR) 0.82, 95% confidence interval (CI) 0.62, 1.08], potassium (OR 0.82, 95%CI 0.68, 1.00), and phosphorus (OR 0.73, 95%CI 0.59, 0.89) had lower AF prevalence compared to those in the bottom quintiles. No clear association was found for circulating chloride, calcium or sodium. Higher concentrations of circulating calcium were associated with lower prevalence of PACs in the 12-lead electrocardiogram, while higher concentrations of potassium, chloride and sodium were associated with higher PAC prevalence. Circulating electrolytes were not significantly associated with burden of PACs or supraventricular tachycardia among 317 participants with extended electrocardiographic monitoring. CONCLUSION: Concentrations of circulating electrolytes present complex associations with selected supraventricular arrhythmias. Future studies should evaluate underlying mechanisms.

Educational Recommendations on Selected Analytical and Clinical Aspects of Natriuretic Peptides with a Focus on Heart Failure: A Report from the IFCC Committee on Clinical Applications of Cardiac Bio-Markers.

The IFCC Committee on Clinical Applications of Cardiac Bio-Markers (C-CB) has directives and initiatives focused on providing evidence-based educational resources to aid and improve understanding around key analytical and clinical aspects of cardiac biomarkers used in clinical practice and the research setting. As a task force, we have previously published position statements and recommendations focused on use and analytical aspects of high-sensitivity cardiac troponin assays. The current educational document is the first from the C-CB highlighting important biochemical, analytical, and clinical aspects as they relate to the natriuretic peptides (NPs), including B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), with a focus on heart failure.

High sensitivity, contemporary and point-of-care cardiac troponin assays: educational aids developed by the IFCC Committee on Clinical Application of Cardiac Bio-Markers.

The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) formed a Task Force on the Application of Cardiac Bio-markers (TF-CB) in 2008, re-designated in 2018 as a committee (C-CB), to produce educational materials on cardiac biomarkers. Established in June 2017, definitive tables covering the majority of high-sensitivity, contemporary and point-of-care (POC) cTn assays have been developed by the C-CB and are available on the IFCC website. These tables provide extensive information about assays' analytical characteristics and encompass information on diagnostic discriminants, particularly the 99th percentiles, as provided by the manufacturers.

Cardiac troponin and natriuretic peptide analytical interferences from hemolysis and biotin: educational aids from the IFCC Committee on Cardiac Biomarkers (IFCC C-CB).

Two interferences recently brought to the forefront as patient safety issues include hemolysis (hemoglobin) and biotin (vitamin B7). The International Federation for Clinical Chemistry Committee on Cardiac Biomarkers (IFCC-CB) obtained input from a majority of cTn and NP assay manufacturers to collate information related to high-sensitivity (hs)-cTnI, hs-cTnT, contemporary, and POC cTn assays, and NP assays interferences due to hemolysis and biotin. The information contained in these tables was designed as educational tools to aid laboratory professionals and clinicians in troubleshooting cardiac biomarker analytical results that are discordant with the clinical situation.

Establishment of Community-Based Reference Intervals for Fructosamine, Glycated Albumin, and 1,5-Anhydroglucitol.

BACKGROUND: There is growing interest in fructosamine, glycated albumin, and 1,5-anhydroglucitol (1,5-AG) as alternative measures of hyperglycemia, particularly for use in settings where traditional measures (glucose and HbA1c) are problematic or where intermediate (2-4 weeks) glycemic control is of interest. However, reference intervals for these alternative biomarkers are not established. METHODS: We measured fructosamine, glycated albumin, and 1,5-AG in a community-based sample of US black and white adults who participated in the Atherosclerosis Risk in Communities (ARIC) Study. We calculated reference intervals, evaluated demographic differences, and derived cutoffs aligned with current diagnostic cutpoints for HbA1c and fasting glucose. RESULTS: In a healthy reference population of 1799 individuals (mean age, 55 years; 51% women; 15% black), the 2.5 and 97.5 percentiles, respectively, were 194.8 and 258.0 µmol/L for fructosamine, 10.7% and 15.1% for glycated albumin, and 8.4 and 28.7 µg/mL for 1,5-AG. Distributions differed by race, sex, and body mass index. Equivalent concentrations of fructosamine and glycated albumin corresponding to an HbA1c of 6.5% (96.5 percentile) were 270.2 µmol/L and 15.6%, respectively. Equivalent concentrations of fructosamine and glycated albumin corresponding to a fasting glucose of 126 mg/dL (93.9 percentile) were 261.7 µmol/L and 15.0%, respectively. CONCLUSIONS: The reference intervals for these biomarkers should inform their clinical use. Diagnostic cutpoint equivalents for fructosamine and glycated albumin could be useful to identify persons with hyperglycemia in settings where fasting glucose or HbA1c are not available or where the interpretation of these traditional measures is problematic.

Specificity of B-Type Natriuretic Peptide Assays: Cross-Reactivity with Different BNP, NT-proBNP, and proBNP Peptides.

BACKGROUND: B-type natriuretic peptides (BNPs) are used clinically to diagnose and monitor heart failure and are present in the circulation as multiple proBNP-derived fragments. We investigated the specificity of BNP immunoassays with glycosylated and nonglycosylated BNP, N-terminal proBNP (NT-proBNP), and proBNP peptides to probe the cross-reactivity of each assay. METHODS: Nine B-type natriuretic peptides were studied,including synthetic and recombinant BNP (Shionogi, Scios, Mayo), human and synthetic glycosylated and nonglycosylated NT-proBNP (HyTest, Roche Diagnostics), and human glycosylated and nonglycosylated proBNP (HyTest, Scios). Five BNP [Abbott, Abbott POC, Alere, Beckman Coulter, Siemens (Centaur)], 9 NT-proBNP [Ortho-Clinical Diagnostics, Roche, Response, bioMerieux, Siemens (Dimension, Immulite, Stratus CS), Mitsubishi] and 3 research-use-only proBNP immunoassays [Biosite (Alere), Bio-Rad, Goetze] were evaluated. Specificity was assessed by calculating the recovery between baseline and peptide-spiked human plasma pools at target concentrations of 100 ng/L BNP, 300 ng/L proBNP, or 450 ng/L NT-proBNP. All assays were performed in duplicate. RESULTS: BNP and NT-proBNP assays demonstrated substantial cross-reactivity with proBNP peptides. NT-proBNP assays do not detect glycosylated forms of either NT-proBNP or proBNP. proBNP assays preferentially detect the BNP 1-32 peptide and have minimal cross-reactivity with BNP peptides and glycosylated proBNP. CONCLUSIONS: BNP or NT-proBNP results are not transferable among the current existing immunoassays owing to their differences in cross-reactivity and ability to detect various glycosylated forms of proBNP-derived fragments. Opportunities remain to standardize and harmonize BNP and NT-proBNP assays, as well as to develop specific proBNP assays, to widen their clinical scope of use.

Implementation of Clinical Decision Support Rules to Reduce Repeat Measurement of Serum Ionized Calcium, Serum Magnesium, and N-Terminal Pro-B-Type Natriuretic Peptide in Intensive Care Unit Inpatients.

BACKGROUND: We assessed the impact of clinical decision support (CDS) rules within the electronic health record for ionized calcium (iCa), serum magnesium (Mg), and N-terminal pro-B-type natriuretic peptide (NT-proBNP) in intensive care unit (ICU) inpatients at a large academic center. METHODS: A repeat order for measurement of iCa or Mg placed within 24 (iCa) or 48 (Mg) h of a previously nonactionable result, or additional orders for NT-proBNP beyond 1 within a single hospitalization, triggered a CDS pop-up alert showing the prior result and offering the opportunity to cancel the order or to place the order after entering an indication for repeat testing. The number of tests performed for each of these analytes and incidence of adverse clinical outcomes potentially associated with hypocalcemia or hypomagnesemia were compared between the 90-day period before CDS implementation and two 90-day periods immediately following. RESULTS: iCa test volumes decreased by 48%, Mg by 39%, and NT-proBNP by 28% in the 90-day period immediately following implementation and remained decreased by 54%, 49%, and 22%, respectively, during the following 90-day period (all P values <0.0002). Adverse clinical outcomes potentially associated with hypocalcemia or hypomagnesemia did not increase (all P-values >0.17). CONCLUSIONS: Implementation of CDS dramatically decreased repeat testing of iCa, Mg, and NT-proBNP without adversely impacting clinical outcomes in the ICU. Expansion of the rules from the ICU units to include the entire hospitalized patient population and expansion to additional analytes is expected to lead to further reductions in testing.

Prognostic Value of Serial Measurements of Soluble Suppression of Tumorigenicity 2 and Galectin-3 in Ambulatory Patients With Chronic Heart Failure.

BACKGROUND: B-Type natriuretic peptides (BNP) and cardiac troponin T (cTnT) predict cardiovascular events in heart failure (HF) patients, but additional refinement in risk stratification may be possible by targeting pathways leading to fibrosis. We aimed to assess the value of serial measurements of soluble suppression of tumorigenicity 2 (sST2) and galectin-3 to identify risk for adverse pathophysiologic processes. METHODS: New York Heart Association (NYHA) functional class III-IV HF patients (n = 180; LVEF ≤40%) were prospectively evaluated with biomarkers collected every 3 months over 2 years and analyzed regarding a primary end point of death/cardiac transplantation and a secondary end point of HF-related hospitalization or death/transplantation. RESULTS: Time-dependent univariate analyses demonstrated that elevations of sST2 (≥49.3 ng/mL male, ≥33.5 ng/mL female) and galectin-3 (≥22.1 ng/mL) were predictive of the primary and secondary end points. In multivariate models adjusted for BNP, cTnT, and clinical variables, sST2 but not galectin-3 remained an independent predictor (hazard ratio 3.22, 95% confidence interval 1.76-5.89; P < .001). With serial measurements, only sST2 demonstrated incremental value in reclassifying patients to higher risk. CONCLUSIONS: Serial monitoring of sST2 (indicating myocardial fibrosis and remodeling) and cTnT (reflecting myocardial injury) identifies highest-risk HF outpatients and may be valuable to guide patient tailored therapy during follow-up evaluations. Serial galectin-3 monitoring in ambulatory HF patients may not be of benefit.

Validation of a proposed novel equation for estimating LDL cholesterol.

BACKGROUND: Aggressive LDL cholesterol (LDL-C)-lowering strategies are recommended for primary and secondary prevention of cardiovascular events. A newly derived equation for LDL-C estimation was recently published that addressed limitations in the commonly used Friedewald LDL-C calculation method. The novel method was reported to classify patients with superior concordance to measured LDL-C compared to the Friedewald method, particularly in patients with LDL-C <70 mg/dL. METHODS: We evaluated the performance of the novel method within an independent cohort of 23 055 patients with LDL-C measured by the gold standard ß-quantification reference method. RESULTS: Overall Friedewald underestimated and the novel method overestimated measured LDL-C. Both estimations significantly deviated from the reference method when LDL-C was <70 mg/dL. Overall, the Friedewald and novel calculations correctly classified 77% and 78% of patients, respectively. The largest discrepancy in classification was observed in individuals with measured LDL-C <70 mg/dL. For this group the novel calculation would reclassify 8.7% of patients as >70 mg/dL compared to the Friedewald equation. CONCLUSIONS: We compared both novel and Friedewald estimated LDL-C against the LDL-C reference method; in contrast, the prior study relied on validation of a subset of samples by ß-quantification to allow the use of the vertical autoprofile method for LDL-C measurement. We conclude that the novel method has some benefits but it is unclear whether improvements over the Friedewald calculation are substantive enough to justify making the change in routine clinical practice and to improve patient outcomes.

Genetic and biochemical analyses in dyslipidemic patients undergoing LDL apheresis.

OBJECTIVE: Familial hypercholesterolemia (FH) can be due to mutations in LDLR, PCSK9, and APOB. In phenotypically defined patients, a subset remains unresponsive to lipid-lowering therapies and requires low density-lipoprotein (LDL) apheresis treatment. In this pilot study, we examined the genotype/phenotype relationship in patients with dyslipidemia undergoing routine LDL apheresis. DESIGN: LDLR, APOB, and PCKS9 were analyzed for disease-causing mutations in seven patients undergoing routine LDL apheresis. Plasma and serum specimens were collected pre- and post-apheresis and analyzed for lipid concentrations, Lp(a) cholesterol, and lipoprotein particle concentrations (via NMR). RESULTS: We found that four patients harbored LDLR mutations and of these, three presented with xanthomas. While similar reductions in LDL-cholesterol (LDL-C), apolipoprotein B, and LDL particles (LDL-P) were observed following apheresis in all patients, lipid profile analysis revealed the LDLR mutation-positive cohort had a more pro-atherogenic profile (higher LDL-C, apolipoprotein B, LDL-P, and small LDL-P) pre-apheresis. CONCLUSION: Our data show that not all clinically diagnosed FH patients who require routine apheresis have genetically defined disease. In our small cohort, those with LDLR mutations had a more proatherogenic phenotype than those without identifiable mutations. This pilot cohort suggests that patients receiving the maximum lipid lowering therapy could be further stratified, based on genetic make-up, to optimize treatment.