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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: 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.)

Background: Detectable troponin below the 99th percentile may reflect an underlying cardiac abnormality which might entail prognostic consequences. This study aimed to investigate the prognosis of patients admitted to an emergency department (ED) with detectable troponin below the 99th percentile reference limit who did not present with an acute coronary syndrome (ACS)., Methods: We analysed the clinical data of all consecutive patients admitted to the ED during the years 2012 and 2013 in whom cardiac troponin was requested by the attending clinician (cTnI Ultra Siemens, Advia Centaur). Patients with troponin below the 99th percentile of the reference population (40 ng/L) and who did not have a diagnosis of ACS were selected, and their mortality was evaluated in a 2-year follow-up., Results: A total of 2501 patients had a troponin level below the reference limit, with 43.9% of those showing detectable levels (>6 ng/L and <40 ng/L). Patients with detectable levels were elderly and had a higher prevalence of cardiovascular history and more comorbidities. The total mortality in the 2-year follow-up was 12.4% in patients with detectable troponin and 4.5% in patients with undetectable troponin (p<0.001). In the Cox multivariate regression analysis, the detectable troponin was an independent marker of mortality at 2 years (HR 1.62, 95% CI 1.07-2.45, p=0.021)., Conclusions: Detectable troponin I below the 99th percentile is associated with higher mortality risk at 2-year follow-up in patients admitted to the ED who did not present with ACS.

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: 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.

We describe the adoption of high sensitive troponin I (hsTnI) in clinical practice in two hospital settings in Italy. Samples from 426 consecutive patients (mean age 68.8 ± 17.0) admitted to the Emergency Department with a suspected acute coronary syndrome (ACS) have been tested at admittance and after 3 and 6 hours by contemporary TnI and hsTnI. Results have been compared to the final clinical diagnosis. Troponin was detectable in 68.6% by TnI and 89.9% by hsTnI. Since hsTnI has a lower threshold for females, 38/41 patients with positive values only by hsTnI were women. The correlation between the assays was very high ( r = 0.92). A diagnosis of acute myocardial infarction (AMI) was made in 45 cases (10.5%). The negative and positive predictive values for a 50% troponin variation at 3 hours were 95.8% and 66.7% for hsTnI and 95.0% and 52.6% for TnI and at 6 hours 90.3% and 100% for hsTnI and 88.9% and 78.9% for TnI, respectively. Receiver operating characteristic (ROC) curve analysis demonstrated a greater efficiency by hsTnI at 3 hours versus 6 hours (AUC = 0.91 versus 0.72). The main benefits of hsTnI are the adoption of gender-specific 99th percentile and the shortening of time to decision.

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.

Background: Cardiac troponin (cTn) is the biomarker of choice for assessment of patients with acute coronary syndromes. Guidelines recommend the cTn 99th percentile derived from a cardiovascular healthy reference population as decision threshold. The importance of standardized criteria for the composition of such a reference population is well acknowledged. In this analysis, we investigated to which extent different statistical methods might have bearing on the calculated cTnI 99th percentile., Methods: cTnI (Abbott) 99th percentiles were determined in 521 cardiovascular healthy community-dwelling subjects using the nonparametric method, the Harrell-Davis bootstrap method and the robust method together with different tests to identify potential outliers (Dixon, Tukey, Reed) and different statistical softwares., Results: The cTnI 99th percentiles (nonparametric method) were 37ng/L (total population), 42ng/L (men) and 25ng/L (women). These estimates differed by -7.4% to +5.7% using the Harrell-Davis bootstrap method and were up to 64.1% lower using the robust method. For the robust method, cTnI 99th percentiles varied by 44.2% depending on the applied software. The method of Tukey classified nine subjects as outliers while no outlier was detected using the other methods. Excluding these nine subjects resulted in up to 60.2% lower cTnI 99th percentiles., Conclusions: Our results emphasize the need of a standardized statistical approach to calculate cTnI 99th percentiles. Our findings support the use of the nonparametric method and a conservative approach to detect outliers. This requires that the assessed population is sufficiently large and well selected on the basis of stringently applied clinical criteria., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Aim: While circulating biomarkers are critical tools for cardiovascular adult care, their relevance in childhood is unknown., Methods: We evaluated the behavior of plasma concentrations of clinically relevant cardiac biomarkers (NT-proBNP, hs-cTnI, sST2, Galectin-3) in 106 healthy children., Results: Subjects were divided into age subgroups: 24 newborns (0-30 days), 26 infants (1-12 months), 30 children (1-12 years) and 26 adolescents (13-18 years). Healthy adults were used as control. NT-proBNP (newborns: 504.3 [211.07-942.7] ng/L, median [25-75 percentiles]; infants: 200.64 [76.88-306.73]; children: 97.27 [49.24-271.80]; adolescents: 24.35 [13.14-58.83]; p < 0.001) and hs-cTnI (newborns: 9.3 [3.3-93.8] ng/L; infants: 13.8 [4.82-72.52]; children: 11.45 [4.0-48.10]; adolescents: 2.6[2.07-3.90]; p < 0.001) were highest in the first month of life, showing a decline in the next years. sST2 and Galectin-3 showed no differences., Conclusion: Changes in hs-cTnI and NT-proBNP suggest the design of age- and sex-based reference intervals that will have to be explored in a larger population.

Homogeneous assays are advantageous because of their simplicity and rapid kinetics but typically their performance is severely compromised compared to heterogeneous assay formats. Here, we report a homogeneous immunoassay utilizing switchable lanthanide luminescence for detection and site-specifically labeled recombinant antibody fragments as binders to improve the assay performance. Switchable lanthanide luminescence enabled elimination of assay background due to division of the luminescent lanthanide chelate into two non-luminescent label moieties. Simultaneous biomolecular recognition of model analyte cardiac troponin I by two antibody fragments brought the label moieties together and resulted in self-assembly of luminescent mixed chelate complex. The assay was very rapid as maximal signal-to-background ratios were achieved already after 6 min of incubation. Additionally, the limit of detection was 0.38 ng/mL (16 pM), which was comparable to the limit of detection for the heterogeneous reference assay based on the same binders (0.26 ng/mL or 11 pM). This is the first study to apply switchable lanthanide luminescence in immunoassays and demonstrates the versatile potential of the technology for rapid and sensitive homogeneous assays., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: A rise or fall of cardiac troponin is a prerequisite for the diagnosis of acute myocardial infarction. Defining significant changes requires knowledge of both biological and analytical variation. The short-term biological variation of cardiac troponin in healthy individuals is 3%-48%. However, healthy individuals may not be representative for patients in whom cardiac troponin measurement is often of clinical importance. Therefore, we studied the individual variation of cardiac troponin in patients with symptoms of stable coronary artery disease., Methods: Twenty-four patients scheduled for elective coronary angiography were included. Blood samples were drawn once at enrollment and serially at six 4-h intervals on the day before coronary angiography. Cardiac troponin was measured with hs-cTn assays from Abbott Laboratories (premarket cTnI assay) and Roche Diagnostics (Elecsys(®) cTnT assay with two different lots)., Results: The short-term individual variation in cardiac troponin I (cTnI) was 14%, the reference change value (RCV) 49%, and RCV-log-normal (rise/fall) 54%/-35%. The corresponding values for cTnT were 7%, 23%, and 26%/-21%. The long-term variation for cTnI was 24%, RCV 69%, and RCV-log-normal (rise/fall) 97%/-49%. The corresponding values for cTnT were 11%, 32%, and 37%/-27%., Conclusions: The short-term individual variation of cardiac troponin in patients with symptoms of stable coronary artery disease is similar to the biological variation previously demonstrated in healthy individuals. Our results suggest that a change in cardiac troponin concentrations of >50% can be used in attempting to diagnose acute myocardial injury. To detect significant long-term changes in cardiac troponin concentrations, larger changes will be required., (© 2012 American Association for Clinical Chemistry)

Background: Cardiac tropoin I (cTnI) measurements show an approximately 20- to 40-fold difference between assays, and better standardization and harmonization are needed. Toward this goal, the AACC cTnI Standardization Committee collaborated with the National Institute of Standards and Technology (NIST) in an earlier study to select 2 candidate reference materials (cRMs)., Methods: Two troponin cRMs, a troponin C-troponin I-troponin T (CIT) complex from human heart tissue and a CIT complex from recombinant technology, were supplied to NIST for assessment of composition and purity, and cTnI value assignment. These cRMs and 6 cTnI-positive human serum pools were shipped to manufacturers of 15 cTnI assays. Commutability of the materials was examined by determining the numerical relationship for the cRM preparations between each manufacturer-specified field method and each of the other 14 field methods. These relationships were then compared with the corresponding numerical relationships for the human serum pools. Harmonization of methods was accomplished by determining regression parameters relative to the analytical system yielding values closest to the median for each serum pool. These regression parameters were used to recalculate pool values to harmonize the assays. Interassay CVs before and after harmonization were determined., Results: Characterization of the CIT and CI cRMs showed that these materials were of specified composition. The proportion of cTnI methods that demonstrated commutability for the CIT cRM was 45%; for the CI cRM, 39% of methods demonstrated commutability. Interassay cTnI variability for the field methods ranged from 82% to 97%, median 88%. After harmonization, variability of the serum pools for the cTnI methods was decreased to between 9.0% and 23%, median 15.5%., Conclusions: The proportion of methods demonstrating commutability was too low for use as a common calibrator for the cTnI field methods. However a simple strategy using serum pools can improve harmonization of field cTnI methods by more than 5-fold. The CIT cRM was selected by the AACC cTnI standardization committee, and a new lot has been classified as the cTnI certified reference material Standard Reference Material 2921 by NIST.

Background: To address the continuing need for the standardization of clinical human cardiac troponin I (cTnI) assays, NIST, with the assistance of the AACC/IFCC Cardiac Troponin I Standardization Committee, has developed a new certified reference material, Standard Reference Material (SRM) 2921: Human Cardiac Troponin Complex., Methods: The concentration of cTnI in SRM 2921 was determined through a combination of reversed-phase liquid chromatography (LC) with ultraviolet detection and amino acid analysis. Characterization of the intact troponin subunits was accomplished through reversed-phase LC coupled with mass spectrometry. Posttranslational modifications to the cTnI in SRM 2921 were investigated by combining proteolytic digestion with matrix-assisted laser desorption/ionization mass spectrometry. Additionally, reference concentration values for cTnT and cTnC were also determined., Results: The concentration of human cTnI in SRM 2921 is 31.2 (1.4) mg/L (where 1.4 mg/L is the uncertainty at a 95% level of confidence), as certified through a method that provides traceability to the International System of Units (SI). Reference concentration values of the cTnT and cTnC subunits were determined to be 36.9 (3.8) mg/L and 24.2 (1.3) mg/L, respectively., Conclusions: This first cTnI reference material should provide SI traceability to clinical cTnI assays once commutability has been validated, and could assist in the international harmonization of cTnI assays as a tool for understanding the underlying causes of interassay variability.

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: Cardiac troponin I (cTnI) results vary 100-fold among assays. As a step toward standardization, we examined the performance of 10 candidate reference materials (cRMs) in dilution studies with 13 cTnI measurement systems., Methods: Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 microg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 microg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs., Results: The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution., Conclusions: The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.

Troponin I is a sensitive and specific marker for the diagnosis of myocardial infarction. Several commercially available immunoassays measure the concentration of troponin I in serum. The microparticle enzyme immunoassay (MEIA) for troponin I (Abbott Laboratories, Abbott Park, IL) is widely used in clinical laboratories, including our hospital laboratory. We studied the effect of bilirubin and hemolysis on the MEIA for troponin I and compared our assay with a newly available chemiluminescent assay (CLIA) for troponin I (Bayer Diagnostics, Tarrytown, NY). We also measured CK-MB concentration using the MEIA CK-MB assay. One serum pool was prepared by combining several specimens of one patient with elevated troponin I and with a diagnosis of myocardial infarction. Other serum pools were prepared by combining sera with similar troponin I values. All serum pools showed normal bilirubin concentrations and had no hemolysis. Then we supplemented aliquots of serum pools with various concentrations of bilirubin (5.0, 10.0, 15.0, and 20.0 mg/dL). After supplementation, troponin I concentrations were measured again using the MEIA and CLIA. We observed a statistically significant decrease in troponin I concentration in the presence of bilirubin with the MEIA. For example, in serum pool 1, the troponin I concentration was 16.3 (bilirubin: 0.8 mg/dL). In the presence of 5.0, 10.0, 15.0 and 20.0 mg/dL of added bilirubin, the cardiac troponin I concentrations were 13.9, 13.4, 13.3 and 13.0 ng/ml respectively. We observed similar negative interference of bilirubin in troponin I measurement by the MEIA in other pools. The troponin I value decreased slightly (not statistically significant) in one pool and did not change in two other pools in the presence of bilirubin when we measured troponin I concentration using the CLIA. Interestingly, bilirubin did not interfere with the MEIA CK-MB assay. Moderate hemolysis did not have any effect on the troponin I assay using either the MEIA or CLIA. However, gross hemolysis (hemoglobin > 40 mg/dL) interfered with both assays for troponin I.

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.

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.

We examined the release of cardiac troponin T (cTnT) and I (cTnI) into the blood of patients after acute myocardial infarction (AMI). Three postAMI serum samples were applied in separate analytical runs onto a calibrated gel filtration column (Sephacryl S-200), and the proteins were separated by molecular weight. Using commercial cTnT and cTnI assays measured on collected fractions, we found that troponin was released into blood as a ternary complex of cTnT-I-C, a binary complex of cTnI-C, and free cTnT, with no free cTnI within the limits of the analytical methodologies. The serum samples were also examined after incubation with EDTA and heparin. EDTA broke up troponin complexes into individual subunits, whereas heparin had no effect on the assays tested. We added free cTnC subunits to 24 AMI serum samples and found no marked increase in the total cTnI concentrations, using an immunoassay that gave higher values for the cTnI-C complex than free cTnI. To characterize the cross-reactivity of cTnT and cTnI assays, purified troponin standards in nine different forms were prepared, added to serum and plasma pools, and tested in nine quantitative commercial and pre-market assays for cTnI and one approved assay for cTnT. All nine cTnI assays recognized each of the troponin I forms (complexed and free). In five of these assays, the relative responses for cTnI were nearly equimolar. For the remainder, the response was substantially greater for complexed cTnI than for free cTnI. Moreover, there was a substantial difference in the absolute concentration of results between cTnI assays. The commercial cTnT assay recognized binary and ternary complexes of troponin on a near equimolar basis. We conclude that all assays are useful for detection of cardiac injury. However, there are differences in absolute cTnI results due to a lack of mass standardization and heterogeneity in the cross-reactivities of antibodies to various troponin I forms.