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Background: Many studies have assessed the biological variation (BV) of cardiac-specific troponins (cTn), reporting widely varying within-subject BV (CVI) estimates. The aim of this study was to provide meta-analysis-derived BV estimates for troponin I (cTnI) and troponin T (cTnT) for different sampling intervals and states of health., Methods: Relevant studies were identified by a systematic literature search. Studies were classified according to their methodological quality by the Biological Variation Data Critical Appraisal Checklist (BIVAC). Meta-analyses of BIVAC-compliant studies were performed after stratification by cTn isoform, exclusion of results below the limit of detection, states of health, and sampling interval to deliver reference change values (RCV), index of individuality (II) and analytical performance specifications (APS) for these settings., Results: Sixteen and 15 studies were identified for cTnI and cTnT, respectively, out of which 6 received a BIVAC grade A. Five studies had applied contemporary cTnI assays, but none contemporary cTnT. High-sensitivity (hs-) cTnI and cTnT delivered similar estimates in all settings. Long-term CVI estimates (15.1; 11.3%) derived from healthy individuals were higher than short-term (4.3%; 5.3%) for hs-cTnI and hs-cTnT, respectively, although confidence intervals overlapped. Estimates derived from diseased subjects were similar to estimates in healthy individuals for all settings., Conclusions: This study provides robust estimates for hs-cTnI and hs-cTnT applicable for different clinical settings and states of health, allowing for the use of RCV both to aid in the diagnosis of myocardial injury and for prognosis. BV-based APS appear too strict for some currently available technologies., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: A small shift in high-sensitivity cardiac troponin T (hs-cTnT) assays can lead to different result interpretation and consequent patient management. We explored whether a small bias could be detected using conventional internal quality control (QC) procedures, evaluated the performance of moving average (MA)-based QC procedures, and proposed a new QC procedure based on the moving rate (MR) of positive patient results of hs-cTnT assays., Methods: The ability of conventional QC to detect a 5 ng/L bias was examined using the13s/ 22s/R4s multi-rule procedure as deviation rules.We developed MA and MR procedures for the hs-cTnT assay using eight months of patient data. The performance of different MA or MR procedures was investigated by calculating the median number of patient samples affected until a bias introduced into the dataset was detected (MNPed). After comparing the MNPed across different procedures, we selected an optimal MA or MR procedure for validation. Validation graphs were plotted using the minimum, median, and maximum number of results affected until bias detection., Results: Our conventional QC procedures could not detect a positive bias of 5 ng/L. When a positive bias was introduced, MNPed was much higher using MA than using MR, with cut-off values of 5 ng/L and 14 ng/L, respectively. MR validation charts for optimal procedures provided insight into the MR performance., Conclusions: The MR procedure could detect different errors with few false alarms. In the hs-cTnT assay, the MR procedure with a smaller cut-off value outperformed MA and conventional QC procedures for small bias detection.

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., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To determine gender-specific reference limits of high-sensitivity (hs) cardiac troponins (cTn) and validity of hs assay designation for both genders., Methods: After screening with a questionnaire, 827 presumably healthy individuals were further selected based on clinical criteria (n = 740), clinical criteria plus cardiac imaging including stress magnetic resonance imaging or stress echocardiography (n = 726), and extended cardio-pulmonary parameters (n = 626). Blood samples were measured with hs-cTnT (Roche Diagnostics) on a cobas e602 analyzer as well as hs-cTnI (Abbott Diagnostics) on an ARCHITECT i 2000 SR . The impact of health definition, statistical methods, instrument selection and limit of detection (LoD) on overall and gender-specific 99th percentiles was assessed., Results: Median age was 56 years (50.9% female) for the total study cohort. 99th percentiles for females and males ranged between 13.1 and 13.3 ng/L and 16.8-19.9 ng/L for hs-cTnT as well as 10.3-12.5 ng/L and 27.4-29.7 ng/L for hs-cTnI depending on health definition. Utilization of stricter health definition criteria reduced the difference of the gender-specific 99th percentiles between males and females for hs-cTnT to 3.7 ng/L (males 16.8 ng/L, females 13.1 ng/L), whereas the difference rather increased for hs-cTnI to 19.4 ng/L (males 29.7 ng/L, females 10.3 ng/L). Values > LoD could be measured in the majority of males and females using hs-TnT (81.4-83.3% and 96.5-96.9%, respectively). In contrast, values > LoD could not be observed in the majority of females using hs-cTnI (38.4-41.1%)., Conclusions: In a well-phenotyped healthy cohort, reference values for hs-cTnT were slightly higher, whereas hs-cTnI cut-offs were considerably lower than previously observed. Gender differences were more pronounced in hs-cTnI than in hs-cTnT and were further reduced for hs-cTnT by application of stricter health definition criteria. Contrary to hs-cTnI, hs-cTnT fulfilled criteria for hs designation for both genders., Competing Interests: Declaration of Competing Interest E.G., honoraria for lecturers from Roche Diagnostics, BRAHMS Thermo Scientific, Bayer Vital GmbH and Mitsubishi Chemical Europe, institutional research grant from Roche Diagnostics and Daiichi Sankyo, consultant for Roche Diagnostics and BRAHMS Thermo Scientific, outside the submitted work; M.M.-H., research support by the Medical Faculty of Heidelberg University, during conduct of the study, research support from Roche Diagnostics and BRAHMS Thermo Scientific, speaker honoraria from Roche Diagnostics, non-financial support by BRAHMS Thermo Scientific, Bayer Vital GmbH, Daiichi-Sankyo, Metanomics Health GmbH and Philips Electronics, outside the submitted work; M.B., research support from AstraZeneca and travel support from BRAHMS Thermo Scientific, outside the submitted work; M.V., financial support from Bayer Vital GmbH and Daiichi Sankyo, reimbursement for travel expenses and fees associated with attending seminars and conferences by Bayer Vital GmbH, Lilly Germany, GlaxoSmithKline, Roche Diagnostics and Brahms, outside the submitted work; K.M.S., research supported by Bayer Vital GmbH, outside the submitted work; S.B., non-financial support from Abbott Diagnostics, grants and personal fees from Abbott Diagnostics, Siemens, Thermo Fisher and Roche Diagnostics, outside the submitted work; H.K., honoraria from AstraZeneca, Eli Lilly, GlaxoSmithKline, Roche Diagnostics, and Bayer, holds a Troponin T Test Invention patent jointly with Roche and receives royalties for this patent, outside the submitted work. All other authors have nothing to disclose., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: How to select healthy reference subjects in deriving 99th percentiles for cardiac troponin assays still needs to be clarified. To assist with global implementation of high sensitivity (hs)-cardiac troponin (cTn) I and hs-cTnT assays in clinical practice, we determined overall and sex-specific 99th percentiles in 9 hs-cTnI and 3 hs-cTnT assays using a universal sample bank (USB)., Methods: The Universal Sample Bank (USB) comprised healthy subjects, 426 men and 417 women, screened using a health questionnaire. Hemoglobin A1c (>URL 6.5%), NT-proBNP (>URL 125 ng/L) and eGFR (<60 mL/min), were used as surrogate biomarker exclusion criteria along with statin use. 99th percentiles were determined by nonparametric, Harrell--Davis bootstrap, and robust methods., Results: Subjects were ages 19 to 91 years, Caucasian 58%, African American 27%, Pacific Islander/Asian 11%, other 4%, Hispanic 8%, and non-Hispanic 92%. The overall and sex-specific 99th percentiles for all assays, before and after exclusions (n = 694), were influenced by the statistical method used, with substantial differences noted between and within both hs-cTnI and hs-cTnT assays. Men had higher 99th percentiles (ng/L) than women. The Roche cTnT and Beckman and Abbott cTnI assays (after exclusions) did not measure cTn values at ≥ the limit of detection in ≥50% women., Conclusions: Our findings have important clinical implications in that sex-specific 99th percentiles varied according to the statistical method and hs-cTn assay used, not all assays provided a high enough percentage of measurable concentrations in women to qualify as a hs-assay, and the surrogate exclusion criteria used to define normality tended to lower the 99th percentiles., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Implementation of the 99th percentile as the upper reference limit for cardiac troponin (cTn) assays is a seemingly lucid recommendation, but, in reality, is incredibly complex. Lack of harmonization between cTn assays diminishes the ability to have a single medical decision point across manufacturer assay/instruments. Moreover, even within a single cTn assay there are several published values corresponding to the "99th percentile". Variability in the determined value is primarily a function of population selection including: sample size, age, sex, exclusion criteria, and statistical methods. Given the complexities associated with this value, some countries have taken an expert consensus approach to endorsing harmonized, assay-specific, cTn 99th percentile values. The purpose of this manuscript is to highlight the intricacies associated with selecting a cTn 99th percentile and to review the approach that Australia used to endorse a nationwide upper reference limit for the Architect STAT hs-cTnI assay.

According to recent international guidelines, including the 2012 Third Universal Definiton of Myocardial Infarction by the Joint ESC/ACCF/AHA/WHF Task Force, an increase in cardiac troponin (cTn) levels over the 99th percentile upper reference limit (99th URL) should be considered clinically relevant, this cut-off being measured with an imprecision ≤10 CV%. In theory 99th URL values strongly depend not only on demographic and physiological variables (i.e. criteria for considering the reference population "healthy"), but also on the analytical performance of cTn methods and mathematical algorithms used for the calculation. The aim of the present article was therefore to review the methodological and pathophysiological factors affecting the evaluation and calculation of the 99th URL for cTn assay. The critical analysis made showed that no uniform procedure is followed, and nor have experts or regulatory bodies provided uniform guidelines for researchers or cTn assays manufacturers as an aid in "their quest to define normality". In particular, little attention has been paid to the way in which a healthy reference population is to be selected, or the criteria for calculating the 99th URL value for cTn assays, thus highlighting the need for international recommendations not only for demographic and physiological variables criteria for defining a healthy reference population, but also for calculating mathematical algorithms for establishing/calculating clinical decision values. An expert consensus group, comprising laboratory and clinical scientists, biomedical statisticians, industrial and regulatory representatives, should be responsible for drawing up these guidelines.

Background: A multitude of troponin assays for the point-of-care (POC) have been developed showing a lack of analytical sensitivity and precision. We present a new platform solution for the high-sensitivity detection of cardiac troponin T (cTnT) in a 30 μL whole blood sample with a turnaround time of 11 min., Methods: The immunoassay was completely run in a ready-to-use plastic disposable, a centrifugal microfluidic disc with fully integrated reagents. After the sample application, the assay was automatically processed by separating the cellular blood components via centrifugation, followed by incubation of a defined volume from the generated plasma with the immunoreagents. The fluorescence in the signal zone of a membrane was measured after its washing for the cTnT quantitation., Results: A calibration curve, measured in whole blood samples spiked with native human cTnT, was generated covering a range up to a concentration of approximately 8300 ng/L. The lower detection limit was determined to be 3.0 ng/L. At a concentration of 14 ng/L, the 99th percentile value from the high-sensitivity cardiac troponin T (hs-cTnT) assay in the Elecsys® system, the imprecision (CV) was 3.8%. A CV profile indicated that the functional sensitivity for a CV <10% was 6.8 ng/L. The assay did not show any significant cross-reaction with human skeletal troponin T. We observed an excellent correlation with the hs-TnT Elecsys® assay for 49 clinical plasma samples (r=0.9744)., Conclusions: The described technology shows that an analytical performance for a highly sensitive determination of cTnT can be achieved in a POC setting.

Background: This study evaluates the association between high sensitivity troponin I (hsTnI) and T (hsTnT) and the morphology of coronary artery plaques detected by coronary computed tomography angiography (CCTA) in patients with suspected coronary artery disease (CAD)., Methods: Patients undergoing CCTA were prospectively enrolled. CCTA was indicated by a low to intermediate pretest probability for CAD during routine clinical care. Within 24 hours of CCTA examination, peripheral blood samples were taken to measure hsTnI, hsTnT, and N-terminal probrain natriuretic peptide (NT-proBNP)., Results: A total of 99 patients were enrolled with 43% without CAD, 9% with noncalcified plaques, 28% with calcified plaques, and 19% with mixed type plaque lesions. Both hsTnI and hsTnT levels were able to discriminate significantly between the groups, especially in the presence of mixed coronary plaques (AUC range: 0.741-0.752; p = 0.0001). In multivariate logistic regression models, hsTnT, but not hsTnI, was still significantly associated with mixed coronary plaque morphology (odds ratio = 8.968; 95% CI 1.999-40.241; p = 0.004)., Conclusions: Both hsTnI and hsTnT are able to discriminate between different coronary artery plaques morphologies, whereas hsTnT was significantly associated with mixed coronary plaques in patients with suspected CAD. This trial is registered with NCT03074253.

High sensitive cardiac troponin assays have become the gold standard in the diagnosis of an acute type 1 myocardial infarction (MI) in the absence of ST-segment elevation. Several acute or chronic conditions that impact cardiac troponin levels in the absence of a MI might lead to a misdiagnosis of MI. For example, patients with impaired renal function as well as elderly patients often present with chronically increased cardiac troponin levels. Therefore, the diagnosis of MI type 1 based on the 99th percentile upper limit of normal threshold is more difficult in these patients. Different diagnostic approaches might help to overcome this limitation of reduced MI specificity of sensitive troponin assays. First, serial troponin measurement helps to differentiate chronic from acute troponin elevations. Second, specific diagnostic cut-offs, optimized for a particular patient group, like elderly patients, are able to regain specificity. Such an individualized use and interpretation of sensitive cardiac troponin measurements improves diagnostic accuracy and reduces the amount of misdiagnosed MI type 1.

The usefulness of cardiac troponins for detection of myocardial cell necrosis and risk stratification has been established beyond doubt. Cardiac troponin testing is a key diagnostic element for the diagnosis and management of patients with acute coronary syndromes without ST segment elevation and is increasingly used in non-coronary diseases to indicate prognostically important cardiac damage. Given the biochemical and analytical differences of cTnT and cTnI there is ongoing controversy regarding the comparability and clinical performance of cTnT and cTnI. cTnT and cTnI are both expressed in cardiomyocytes but differ with respect to biochemical and analytical characteristics. While minor differences of analytical precision or biochemical properties are not relevant for diagnosis and management of patients with acute coronary syndromes and most diseases with non-coronary related elevations of cardiac troponins, these differences may be amplified in patients with chronic renal failure. In fact, recent studies in patients with end-stage renal disease under chronic hemodialysis have readdressed the issue whether cTnT and cTnI are really comparable. The present review will provide a state-of-the-art overview on the performance of cardiac troponins in acute coronary disease and other clinical conditions.

Background: In severe acute coronary syndromes (ACS) elevation of markers of inflammation and acute phase reaction (APR) like C-reactive protein (CRP) as well as a release of troponin have been reported. Using a high sensitivity troponin T (TnT) test we investigated whether an APR occurs in ACS only in the presence of ischemic myocardial damage., Methods: In 85 patients with ACS C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, thrombin antithrombin III complexes (TAT) and kallikrein were determined vs. high sensitive TnT (> or =0.02 ng/ml) initially and 2 d later vs. 45 patients with stable angina pectoris and 42 controls., Results: In stable angina pectoris, markers of inflammation and coagulation were slightly elevated (p < 0.05). Initially in ACS elevations of CRP to 1.2 +/- 0.3 mg/dl, SAA to 4.8 +/- 2.6 mg/dl and fibrinogen to 448 +/- 21 mg/dl (all p < 0.01 vs. controls) were found followed by a significant APR (p < 0.01). In the subgroup of TnT positive ACS patients, an APR with increased CRP (4.1 +/- 1.3 mg/dl), SAA (20.4 +/- 8.3 mg/dl), and fibrinogen (641 +/- 45 mg/dl) was detectable (all p < 0.05 vs. TnT negative patients). In contrast, patients without TnT release showed APR markers comparable to patients with stable angina pectoris., Conclusion: Our findings demonstrate an association between myocardial injury in ACS and acute phase reaction as evidenced by several molecular markers. A highly sensitive TnT-test identified myocardial injury in about all patients with APR while a standard TnT cut-off (0.1 ng/ml) missed 32% of these patients. Thus, the APR in patients with ACS is strongly associated with at least minor ischemic myocardial damage and prior findings of an APR independent from myocardial injury are probably based on less sensitive troponin tests.

The rapid troponin T assay CARDIAC T Quantitative was recalibrated using Elecsys Troponin T 3rd Generation as a new reference method. This paper presents the method comparisons at six centres using the new reference method. Method comparison between CARDIAC T Quantitative versus Elecsys Troponin T 3rd Generation were performed using 319 samples from patients with acute coronary syndromes. The quality of the CARDIAC T Quantitative was controlled by a daily single determination of CARDIAC Control Troponin T, and for the Elecsys Troponin T 3rd Generation, the Elecsys controls were included in each run. The results for the control materials for the CARDIAC T Quantitative were between 93% and 107% of the target values. The CV ranged from 7% to 16%. From the regression analysis, according to Bablok and Passing (y=1.07x) and the Bland and Altman plot, the bias between CARDIAC T Quantitative and Elecsys Troponin T 3rd Generation is from +6% to +7%. The correlation coefficient is 0.93, and a 3x3 comparison of the clinical efficiency yielded 92% clinical concordance between CARDIAC T Quantitative and Elecsys Troponin T 3rd Generation. In conclusion, CARDIAC T Quantitative was in good agreement with the reference and calibration method Elecsys Troponin T 3rd Generation.

Background: Cardiac troponin I and T (cTnI and cTnT) are specific biochemical serum markers for acute myocardial infarction (AMI). However, cTnI diagnostic assays are plagued by difficulties, resulting in >/=20-fold differences in measured values. These discrepancies may result from the release of the numerous cTnI modification products that are present in ischemic myocardium. The resolution of these discrepancies requires an investigation of the exact forms of cTnI present in the bloodstream of patients after myocardial injury., Methods and Results: A western blot-direct serum analysis protocol was developed that allowed us to detect intact cTnI and a spectrum of up to 11 modified products in the serum from patients with AMI. For the first time, we document both a cTnI degradation pattern and the existence of phosphorylated cTnI in serum. The number and extent of these modifications reflect patterns similar to the time profiles of the routine clinical serum markers of total creatine kinase, creatine kinase-MB, and cTnI (determined by ELISA). Data from in vitro experiments, which were undertaken to study the degradation of human recombinant cTnI and cTnT when spiked in serum, indicate that some modification products present in patient serum existed in the myocardium and that recombinant cTnI alteration dramatically reduces the detectability of cTnI by the Immuno1 assay over time (our assay was unaffected)., Conclusions: This pilot study defines, for the first time, what forms of cTnI and cTnT appear in the bloodstream of AMI patients, and it clarifies the lack of standardization between different cTnI diagnostic assays.