Early kinetic profiles of troponin I and T measured by high-sensitivity assays in patients with myocardial infarction

Journal Pre-proofs Early kinetic profiles of troponin I and T measured by high-sensitivity assays in patients with myocardial infarction John W Pickering, Joanna M Young, Peter M George, Christopher J Pemberton, Antony Watson, Sally J Aldous, Toby Verryt, Richard W Troughton, A Mark Richards, Fred S Apple, Martin P Than PII: DOI: Reference:

S0009-8981(20)30064-4 https://doi.org/10.1016/j.cca.2020.02.009 CCA 16024

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Clinica Chimica Acta

Received Date: Revised Date: Accepted Date:

13 January 2020 7 February 2020 11 February 2020

Please cite this article as: J.W. Pickering, J.M. Young, P.M. George, C.J. Pemberton, A. Watson, S.J. Aldous, T. Verryt, R.W. Troughton, A. Mark Richards, F.S. Apple, M.P. Than, Early kinetic profiles of troponin I and T measured by high-sensitivity assays in patients with myocardial infarction, Clinica Chimica Acta (2020), doi: https://doi.org/10.1016/j.cca.2020.02.009

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Early kinetic profiles of troponin I and T measured by highsensitivity assays in patients with myocardial infarction Authors: John W Pickering1,2*, Joanna M Young1,2, Peter M George3, Christopher J Pemberton2 , Antony Watson1, Sally J Aldous1, Toby Verryt1, Richard W Troughton1,2, A Mark Richards2,4, Fred S Apple5, and Martin P Than1

Affiliations 1. Christchurch Hospital, Christchurch, New Zealand 2. Christchurch Heart Institute, Department of Medicine, University of Otago Christchurch, Christchurch, New Zealand 3. Assure Health, Christchurch, New Zealand 4. National University of Singapore, Singapore 5. Department of Laboratory Medicine and Pathology, Hennepin Healthcare/Hennepin County Medical Center and University of Minnesota, Minneapolis, MN, United States of America.

*Corresponding author Address for correspondence: Professor John Pickering Department of Medicine University of Otago Christchurch PO Box 4345 Christchurch 8140 New Zealand Email: [email protected] (JWP) Twitter: @kiwiskiNZ

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Highlights 

In patients with NSTEMI there was considerable variation in the kinetic profiles of cardiac troponin concentrations between individuals.

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Cardiac troponin-I concentrations increased at a much more rapid rate than cardiac troponin-T concentrations.

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Concentrations of one-third of patients took longer than the minimum recommended 3 hours from symptom onset to exceed rule-out decision concentration thresholds.

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Abstract The early concentration kinetic profiles of cardiac troponin in patients with non-ST-elevated myocardial infarction (NSTEMI) measured by high-sensitivity cardiac troponin I (hs-cTnI) and T (hs-cTnT) assays have not been described. In intermediate-to-high-risk of NSTEMI patients we measured serial cTn concentrations on ED arrival, at 1, 2, 3, 6-12, 24 and 48-hours with hs-cTnI and hs-cTnT assays. Log-normal curves were fitted to concentrations from time from symptom onset, and the time to rule-out decision thresholds estimated (hs-cTnI: 2ng/L and 5ng/L; hs-cTnT: 5ng/L). Among 164 patients there were 58 NSTEMI. The hs-cTnI to hs-cTnT ratio increased linearly over the first 6-12 hours following symptom onset. The estimated times from symptom onset to the 2ng/L and 5ng/L thresholds for hs-cTnI were 1.8h (0.1–3.1) and 1.9h (1.1–3.5) hours, and to the 5ng/L threshold for hs-cTnT 1.9h (1.1–3.8). The estimated time to exceed 5ng/L was ≥3 hours in 32.6% (95%CI: 20.0% to 48.1%) cases for hs-cTnI and 33.3% (19.6% to 50.0%) for hs-cTnT. cTnI concentrations increased at a much more rapid rate than cTnT concentrations in patients with NSTEMI. Concentrations of a high proportion of patients took longer than 3 hours from symptom onset to exceed the 5ng/L rule-out decision threshold.

Keywords high-sensitivity cardiac troponin, acute myocardial infarction, emergency department, emergency room, kinetics.

Abbreviations NSTEMI: non-ST-elevated myocardial infarction MI: myocardial infarction 3

hs-cTnI: high-sensitivity cardiac troponin I hs-cTnT: high-sensitivity cardiac troponin T ED: emergency department URL: upper-reference limit LoD: limit of detection EDACS: Emergency Department Assessment of Chest pain Score ADP: accelerated diagnostic pathway LoB: limit of blank ESC: European Society of Cardiology TASH: Transcoronary Ablation of Septal Hypertrophy PRESTO: Pre-hospital Evaluation of Sensitive Troponin

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1. Introduction Early risk stratification of patients presenting to the emergency department (ED) with symptoms of possible Non-ST elevated myocardial infarction (NSTEMI) is an important process for patients and health-systems alike.[1,2] The advent of assays high-sensitivity cardiac troponin (hs-cTn) assays has enabled more precise quantification of cTn concentrations below the 99th percentile upper-reference limit (URL). This, in turn, has led to the development of several novel risk-stratification algorithms which utilise these lower concentrations.[3,4] These include the European Society of Cardiology 0/1hour algorithm,[2,5–7], a proposed 0/2hour algorithm,[8] and algorithms to identify low-risk patients passed on a single blood measurement.[9] The concentration threshold assigned to risk stratify the patient to low-risk are usual at, or slightly above, the assay’s limit of detection (LoD). A concern with such algorithms is troponin measurements in patients who present very soon after onset of myocardial injury may register as undetectable or below this threshold. This could result in patients being inappropriately discharged. Therefore, some authors and guidelines express caution about using single sample rule-out algorithms in early presenters, usually taken to mean patients having a blood sample taken within 2 or 3 hours following symptom onset.[2,10–12] It is unclear whether these times truly represent a safe minimum time from symptom onset to risk-stratify patients using hs-cTn assays. Studies to-date have not attempted to derive a minimum symptom-onset-to-sampling-time. Instead, they have chosen to assess cTn threshold performance in the small cohort of those presenting either within 2 or 3 hours from symptom onset. Furthermore, most studies have too few time-points to quantify the early time-course profile of cTn concentrations following symptom onset. The hs-cTn assays measure plasma, serum or blood concentrations of either the cTnI or cTnT. These subunits are different sizes with cTnT being 50% larger than cTnI which suggests

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their release kinetics into the plasma and subsequent filtration and degradation might be different.[13] The aim of this study was to quantify and compare the early kinetic time-course of cTn concentrations measured by an hs-cTnI assay (Abbott) and the hs-cTnT assay (Roche) in patients at risk of NSTEMI. In particular, we aimed to quantify how long after symptom onset the URL and thresholds recommended for early low-risk stratification (rule-out) decisionmaking were reached. The low-risk stratification threshold for hs-cTnT considered was 5 ng/L as this is recommended by the European Society of Cardiology (ESC) guidelines and has been shown to be robust in meta-analyses.[2,10,14] For hs-cTnI we considered the ESC guidelines low-risk stratification threshold of 2 ng/L and 5 ng/L used in some algorithms and, again, shown to be robust in meta-analysis.[2,15]

2. Methods This study was a prospective observational study in patients with symptoms suggestive of acute coronary syndrome who attended a single metropolitan and regional emergency department (Christchurch Hospital, Christchurch, New Zealand ED). Most patients self-present directly from the community (by ambulance or own transport), but there are also many referrals via primary care physicians. We assessed and compared the kinetic profiles of the Abbott ARCHITECT hs-cTnI (Abbott Diagnostics) and the Roche Elecsys 5th generation hs-cTnT (Roche Diagnostics) assays. The regional ethics committee approved the study and all patients provided written consent. Recruitment was from 5 July 2016 until 5 January 2018 as part of ongoing ED recruitment (ACTRN12611001076965). Recruitment methodology has been reported elsewhere[16]; briefly, eligible patients were ≥18y who presented acutely from the community to the ED with symptoms thought to

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suggest ACS in whom the attending physicians planned to investigate with serial cTn tests. In accord with American Heart Association guidelines[17], these included the presence of acute chest, epigastric, neck, jaw or arm pain or discomfort or pressure or breathlessness without apparent non-cardiac source. For this study only, patients were required to be considered as not low-risk for AMI. That is, they were to have an Emergency Department Assessment of Chest pain Score[18,19] (EDACS) ≥16 or an hs-cTnI concentration measured within the ED greater than or equal to the sex-specific 99th percentile URL of the assay or an electrocardiogram indicative of new ischemia[20], and they were expected to be admitted to a ward for further assessment. Exclusion criteria were ST elevation MI (STEMI) recorded in the ED, proven or suspected non-coronary pathology as the cause of chest pain and/or need for investigations for such possible non-coronary pathologies, need for admission regardless of a negative cTn result due to other medical conditions, symptoms began >24 hours prior to presentation (the time of symptom onset was recorded by a researcher during the ED assessment), previously enrolled in this study or unable or unwilling to provide informed consent. The clinical accelerated diagnostic pathway (ADP) for assessment of patients with possible ACS in operation at the time was the EDACS-ADP.[1,21] Adjudication for the presence of NSTEMI was made independently by two cardiologists according to the third Universal Definition.[22] A third cardiologist made an independent adjudication in case of disagreement. Cardiologist had access only to the clinical (hs-cTnI) and not the study troponin results.

2.1 Sampling and laboratory analysis Blood was drawn into lithium-heparin tubes, immediately centrifuged, and stored at 80C for later testing. hs-cTnI concentrations were measured by the Abbott ARCHITECT i2000 (Abbott Diagnostics, Chicago, Illinois). Planned sampling times were: on presentation, 7

and then one, two, three, six to twelve, twenty-four and forty-eight hours later. The hs-cTnI assay has a manufacturer reported limit of blank (LoB) of 0.7-1.3 ng/L, a limit of detection of 1.1-1.9 ng/L and sex specific URL of 16 ng/L for females and 34 ng/L for males (package insert). hs-cTnT concentrations were measured on a Cobas e411 (Roche diagnostics, Indiana). The hs-cTnT manufacturer-reported LoB is 3 ng/L, LoD 5 ng/L, and a single URL 14 ng/L. The sex specific URLs are 10 ng/L for females, 15 ng/L for males.[23] All measurements were made on the first thaw. A post-hoc experiment was performed to understand whether observed differenced in the rate of change of hs-cTnI and hs-cTnT concentrations was more likely due to different release kinetics of the I and T molecules or an artefact of the different calibration of the two assays across their detection range. We chose 6 samples from 3 patients and remeasured the cTn concentrations in undiluted and 4, 16, and in one case 128 times dilutions. Dilutants use were the Architect Multi-Assay Manual Diluent for hs-cTnI and Cobas Multi-Assay Diluent for hs-cTnT. If the ratios stayed the same, this suggests release kinetic differences, whereas if they change this suggests assay differences.

2.2 Outcome definitions and adjudication Index presentation NSTEMI (type-1 or type-2) was based on the Third Universal Definition for MI, requiring evidence of a rise or fall in cTn, with at least one concentration above the 99th percentile, together with evidence of myocardial ischemia (ischaemic symptoms, electrocardiogram changes or imaging evidence).[22] The clinical reference cTn assay used was the ARCHITECT hs-cTnI. Two physicians independently adjudicated the outcome with a third cardiologist tie-breaking where necessary.

2.3 Statistical Analysis

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The primary analysis was the construction of kinetic time-course profiles from the time from symptom onset of each hs-cTn in each patient diagnosed with an NSTEMI. We chose a priori to present individual profiles rather than present summary statistics (means and a measure of distribution) at multiple time points because individual time courses could provide additional information through demonstrating differences in the different shape, value of peak concentrations, and time to peak concentration, that cannot be found in summary statistics. The time from symptom onset was determined by clinicians who asked the patients when they first experienced symptoms. Curve fitting to the multiple measured hs-cTn concentrations in each patient was undertaken using non-linear least squares with a log-normal model. The adequacy of curve fitting was assessed by providing a 95% confidence interval (note unlike for linear least squares models the R2 metric is not an adequate measure of goodness of fit for non-linear models). Although from a clinical perspective within the emergency department measurements during the first ~12h post symptom onset are most important, the concentrations measured at latter time-points were necessary to obtain more accurate kinetic profiles over the first few hours. These profiles were used to estimate the hs-cTn concentrations both between measurements and before the first sample. We aimed to identify the time from symptom onset to the time at which the hs-cTn concentration first exceeded the proposed early rule-out thresholds and then the time it first exceeded the sex specific URL (with 95% confidence intervals). We produced summary statistics of these times for patients with type 1 and type 2 myocardial infarction separately because the mechanism of injury is different, and therefore the time courses may be different. We further determined and plotted the ratio of cTn concentrations measured to the hscTnI assay to that measured with the hs-cTnT assay at each sampling time point to determine if the relative rates of change of cTn concentrations were the same or different. Statistical calculations were made in R version 3.4.4 (The R Foundation for Statistical Computing).

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3 Results Of 164 patients recruited, 58 (35.4%) had an index NSTEMI (57 Type 1, 1 Type 2). Most patients were male (81%), the median age was 64 years, with large proportions of patient having hypertension, dyslipidemia, a history of previous myocardial infarction, angina, a prior percutaneous coronary angioplasty and/or family history of cardiovascular disease (Table 1). First blood samples were taken a median 4.5 (3.0-8.1) hours following symptom onset (Fig 1).

3.1 Measured concentrations in patients with NSTEMI The kinetic profiles shown in Fig 2 in patients with NSTEMI differed markedly between individuals for both assays. The peak concentrations were markedly different for both assays, with hs-cTnI varying between 35 ng/L and >50,000 ng/L and hs-cTnT between 17 ng/L and 5600 ng/L. Eleven (19.0%) patients with NSTEMI had hs-cTnI concentrations on presentation
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increase in cTn concentration over the first ~6 hours was exponential (linear on the log-scale; Fig 2). Amongst patients where a fit was possible for hs-cTnI, the median (lower quartile – upper quartile) time from symptom onset to the time at which the concentrations were estimated to exceed the sex-specific URL was 3.2 hours (1.7 – 5.1 hours) with a minimum of 0.6 hours and maximum of 13.4 hours (Table 2). The time from symptom onset to a threshold of 2 ng/L was 1.8 hours (0.1 – 3.1) and to 5 ng/L was 1.9 hours (1.1 – 3.5)(Table 2). The estimated time from symptom onset to >5ng/L was ≥3h in 32.6% (95%CI: 20.0% to 48.1%) and >5h in 15.2% (6.8% to 29.5%) of patients. At the more conservative threshold of >2ng/L this time was still ≥3h in 19.6% of patients. Amongst patients where a fit was possible for hs-cTnT the median (lower quartile – upper quartile) time from symptom onset to the time at which the concentrations were estimated to exceed the URL was 2.8 hours (1.6 – 4.8hours) with a minimum of 0.3 hours and maximum of 15.5 hours (Table 2). The time from symptom onset to a threshold of 5 ng/L was 1.9 hours (1.1– 3.8hours). The estimated time from symptom onset to >5ng/L was ≥3h in 33.3% (95%CI: 19.6% to 50.0% ) and >5h in 15.4% (6.4% to 31.2%) of patients. Fits were not possible in four patients with less than four cTn measurements and in four patients that demonstrated non-lognormal behaviour. Summary graphs of the concentrations binned from time of symptom onset indicate greatest concentrations >12 hours from symptom onset, Fig 3. There was a linear increase in the ratio of hs-cTnI to hs-cTnT over the first 3 to 6 hours in the ED, Fig 4, indicating hs-cTnI concentrations were rising faster than hs-cTnT concentrations. The post-hoc dilution experiment showed that for each sample there was a reduced ratio with 16-times dilution and all except one with 4-times dilution, Fig 5.

4. Discussion 11

The kinetic profiles of hs-cTnI and hs-cTnT from time from symptom onset in patients with NSTEMI varied considerably between patients. For both assays the first sample in patients ultimately diagnosed with NSTEMI exceeded the URL in more than 80% of cases and exceeded the proposed single-sample low-risk screening test (rule-out) threshold of 5 ng/L in all but one case for each assay. In both those ‘late-risers’ the time from symptom onset was greater than the recommended 3h. This means that if cTn alone had been used as the criterion for rule-out these patients would have been false negatives. The multiple sampling enabled fitting of curves to allow estimation of the time by which each individual patient’s cTn concentrations exceeded the recommended rule-out thresholds for the assay. This suggests that it took more than 3 hours from symptom onset for the concentrations to exceed these thresholds in a third and plausibly 80% (upper 95% confidence interval limit) of patients. The most striking result was that there was a linear increase in the ratio of hs-cTnI to hs-cTnT concentrations over the first few hours post-symptom onset indicating the more rapid rise of hs-cTnI concentrations than hs-cTnT concentrations. Understanding the release profiles from profiles constructed from aggregates of many patients where the mean (or median) concentrations of biomarkers are plotted can be misleading as individual patients may vary considerably as we have illustrated here. Indeed, some patients did not even exhibit the classic rise and fall of concentration over a log-normal shaped curve. Unfortunately, there are very few studies of cTn where multiple individuals have been presented and these do not include NSTEMI patients. The release kinetics of cTn in STEMI patients is not necessarily the same as in NSTEMI patients. Studies in STEMI patients have not measured cTn soon after symptom onset or as frequently as in our study. Nevertheless, Solecki and colleagues observed that the cTn concentrations measured by a contemporary cTnI assay and the hs-cTnT assay were maximally elevated after about 12 hours from admission and rapidly decayed after that.[24] They also observed with hs-cTnT a secondary peak at about 90 hours from admission that

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had previously been reported with contemporary, 4th generation cTnT assay.[24–26] Laugaudin and colleagues also noted the bi-phasic nature of the cTnT release in STEMI patients. They also measured cTnI with the Abbott Architect hs-cTnI assay. For both assays the median time to peak from admission was 11.8h.[27] We have previously published multiple measurements over two hours following controlled, clinically induced myocardial injury in 31 patients undergoing Transcoronary Ablation of Septal Hypertrophy (TASH).[28] cTn concentrations measured with the same two assays as here rose exponentially over two hours in all patients. Nearly all patients had concentrations above the URL within 30 minutes. This is quicker than our study probably reflecting the severity of the injury due to TASH and, notably, the fact that at baseline prior to surgery more than 50% of patients had hs-cTnT greater than the URL. Studies which have reported on diagnostic metrics in early presenters have simply chosen a time from symptom onset, usually 2 or 3 hours, to define “early” and to report on in this context rather than reporting on multiple time points and attempting to derive a minimum based on a pre-defined statistical metric (such as 99% sensitivity in early presenters). Clearly there must be for any individual and any infarct, a duration following injury-onset during which an assay cannot detect significant concentrations of cTn. Most false negatives using a single sample rule-out cTn threshold may occur because the measurement was temporally too soon after injury-onset. This is supported by data from our collaborative meta-analysis of 9241 patients which assessed the hs-cTnT <5ng/L rule-out strategy. There were only 14 NSTEMI with hs-cTnT <5ng/L of whom 7 (50%) had samples taken within 3-hours of symptom onset.[10] The results from our study indicate the difficulty of “one size fits all” approach. For both assays the estimated the time needed before the concentrations exceeded the decision rule-out thresholds was longer than 3 hours in a large proportion of patients. This suggests that efforts to measure the cTn concentration earlier, such as in ambulances, must be treated cautiously as 3h may not be a sufficiently conservative margin where a

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substantial proportion of patients have early measurements. However, as yet there are no high-sensitivity point-of-care cTn assays for use in these circumstances. The Pre-hospital Evaluation of Sensitive Troponin (PRESTO) study (NCT:03561051) which aims to recruit 790 patients with blood samples taken first in the ambulance may be able to test the safety of rule-out strategies in very early presenters. Furthermore, as most patients who have very low cTn concentrations do not have NSTEMI , and therefore the negative predictive value will be very high simply because the prevalence of NSTEMI in such patients will be very low, we suggest that sensitivity rather than negative predictive value be used as the safety metric in such studies.[29] The exact time of myocardial injury is not easily established, and so the less than accurate “time from symptom onset” is used. This timeframe can sometimes be difficult to determine precisely. Additionally, though this represents the likely time of onset of myocardial ischemia, infarction may not necessarily occur at the time of symptom onset. The individual kinetic profiles indicate that there were several patients for whom the injury occurred possibly earlier than the time from symptom onset recorded (SP0816, SP0841, SP1011) and others where injury may have occurred several hours after the reported symptom onset (SP0952, SP0985, SP1035). In order to establish a “safe” time threshold a very large data set with several hundreds of NSTEMI patients who have had cTn concentrations measured within just a few hours of onset of injury is needed.[29] Such a study would also need to have a very clearly defined and globally reproduceable methodology for estimating the time from symptom onset. The differential rate of increase over the first few hours in cTn I and T concentrations is a notable finding. It means that in this time period there was a more rapid increase in cTnI concentration than cTnT concentration. Where concentrations were higher, so was the ratio of the concentrations measured with hs-cTnI to those measured with hs-cTnT. This observation in itself is not unique as it could be deduced from studies of differential concentrations of hs-

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cTnI and hs-cTnT in single sample studies.[30] Nor does it indicate that one assay is superior to the other. The different rate of increase may be due to differences in release kinetics, protein binding and rates of proteolysis of the I and T molecules during the NSTEMI event. While myocardial damage will result in equimolar amounts released, the different size molecules, different mechanisms involved in the movement of the cTn complex sub-unit from the cardiac muscle to the circulation, and different rates of degradation and clearance may explain the differential rates of increase (and decrease) of the concentrations measured with the two assays.[13] It is also possible that differences in how the assays have been calibrated across their measurement range may explain the findings. The reduction in ratio with dilution (Fig 5) suggests that assay calibration does play a role as the ratios would not have changed with dilution if the assays each measured the same proportion of circulating cTn epitope at every concentration. The use of both the assays in conjunction to risk stratify patients within the ED has been examined.[31] The ratio of the two concentrations was considerably inferior as a predictor of the diagnosis of NSTEMI than either concentration alone. However, the sum, product or a combination using thresholds did increase the number of patients eligible for early rule-out. Our study suggests that if the ratio has any clinical application it may be only in seeing if there is a substantial change in the ratio over serial samples. Further work is required to see if this would have any real clinical benefit. One limitation of this study is that the measurement of “time from symptom onset”, whilst a pragmatic measurement that can be estimated clinically, is not necessarily accurate nor a reflection of the time from onset of myocardial injury. The estimated time to thresholds is dependent on the log-normal fitting algorithm. Whilst log-normal reflects the process of release and degradation or filtration of a molecule, it is not possible to state categorically that it reflects the true state of rise in concentration. However, even if we were to consider that the fitting is not very accurate at concentrations at or below the rule-out threshold, any

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inaccuracy is only likely to make marginal changes to the estimated time from symptom onset to threshold. Even if we overestimated this time by two hours for each patient 15% of them would still be “late risers”. Second, the study was limited to measurements with one assay for each cTn type. It is unknown if, and by how much, the results may have varied with different assays. The cobas e411 hs-cTnT assay is known to be less analytical sensitive compared to the newer cobas e601/2 and e801/2 analyzers are able to measure hs-cTnT concentrations more precisely with lower LoDs to 3.0 ng/L. As calibration between different generation assays is not identical[4,32] it is possible that some of the results from this study may vary with different analysers. It is possible that release kinetics depend in part on renal function. We did not observe any relationship between creatinine and either the time to hs-cTn crossing a threshold or the maximum hs-cTn reached. However, our patients do not have a sufficient range of renal function to properly address this possibility. In conclusion, cTnI concentrations increase at a much more rapid rate than cTnT concentrations in patients with NSTEMI. However, with the Roche e411 hs-cTnT and Abbott ARCHITECT i2000 hs-cTnI, the assay specific limit of detection is reached earlier with the hs-cTnT assay and the URL latter. There was a substantial proportion of patients for whom the time from symptom onset to LoD was greater than the recommended 2 or 3 hours.

5. Funding This research was supported by a grant from the New Zealand Heart Foundation grant (1664). JWP is supported by a fellowship from the Canterbury Medical Research Foundation, Emergency Care Foundation, and Canterbury District Health Board. This study was supported by research grants from Abbott Point of Care and from Roche Diagnostics in order to measure the cTn concentrations. Neither company was involved in the design and conduct

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of the study; collection, management, analysis, and interpretation of the data; preparation or approval of the manuscript; and decision to submit the manuscript for publication.

6. References [1] E.A. Amsterdam, J.D. Kirk, D.A. Bluemke, D. Diercks, M.E. Farkouh, J.L. Garvey, M.C. Kontos, J. McCord, T.D. Miller, A. Morise, L.K. Newby, F.L. Ruberg, K.A. Scordo, P.D. Thompson, C.R. on behalf of the American Heart Association Exercise and Prevention Committee of the Council on Clinical Cardiology, Council on Cardiovascular Nursing, and Interdisciplinary Council on Quality of Care and Outcomes Research, Testing of Low-Risk Patients Presenting to the Emergency Department With Chest Pain: A Scientific Statement From the American Heart Association, Circulation. 122 (2010) 1756–1776. https://doi.org/10.1161/CIR.0b013e3181ec61df. [2] M. Roffi, C. Patrono, J.-P. Collet, C. Mueller, M. Valgimigli, F. Andreotti, J.J. Bax, M.A. Borger, C. Brotons, D.P. Chew, B. Gencer, G. Hasenfuss, K. Kjeldsen, P. Lancellotti, U. Landmesser, J. Mehilli, D. Mukherjee, R.F. Storey, S. Windecker, H. Baumgartner, O. Gaemperli, S. Achenbach, S. Agewall, L. Badimon, C. Baigent, H. Bueno, R. Bugiardini, S. Carerj, F. Casselman, T. Cuisset, Ç. Erol, D. Fitzsimons, M. Halle, C. Hamm, D. Hildick-Smith, K. Huber, E. Iliodromitis, S. James, B.S. Lewis, G.Y.H. Lip, M.F. Piepoli, D. Richter, T. Rosemann, U. Sechtem, P.G. Steg, C. Vrints, J. Luis Zamorano, 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC)., Eur. Heart J. 37 (2016) 267–315. https://doi.org/10.1093/eurheartj/ehv320. [3] A.H.B. Wu, R.H. Christenson, D.N. Greene, A.S. Jaffe, P.A. Kavsak, J. OrdonezLlanos, F.S. Apple, Clinical laboratory practice recommendations for the use of cardiac troponin in acute coronary syndrome: Expert opinion from the Academy of the American Association for Clinical Chemistry and the Task Force on Clinical Applications of Cardiac Bio-Markers of the International Federation of Clinical Chemistry and Laboratory Medicine, Clin. Chem. 64 (2018) 645–555. https://doi.org/10.1373/clinchem.2017.277186. [4] F.S. Apple, Y. Sandoval, A.S. Jaffe, J. Ordonez-Llanos, for the IFCC Task Force on Clinical Applications of Cardiac Bio-Markers, Cardiac troponin assays: Guide to understanding analytical characteristics and their impact on clinical care, Clin. Chem. 63 (2017) 73–81. https://doi.org/10.1373/clinchem.2016.255109. [5] M. Rubini Gimenez, R. Twerenbold, C. Jaeger, C. Schindler, C. Puelacher, K. Wildi, T. Reichlin, P. Haaf, S. Merk, U. Honegger, M. Wagener, S. Druey, C. Schumacher, L. Krivoshei, P. Hillinger, T. Herrmann, I. Campodarve, K. Rentsch, S. Bassetti, S. Osswald, C. Mueller, One-hour rule-in and rule-out of acute myocardial infarction using high-sensitivity cardiac troponin I., Am. J. Med. 128 (2015) 861-870.e4. https://doi.org/10.1016/j.amjmed.2015.01.046. [6] T. Reichlin, C. Schindler, B. Drexler, R. Twerenbold, M. Reiter, C. Zellweger, B. Moehring, R. Ziller, R. Hoeller, M. Rubini Gimenez, P. Haaf, M. Potocki, K. Wildi, C. Balmelli, M. Freese, C. Stelzig, H. Freidank, S. Osswald, C. Mueller, One-hour rule-out

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[7]

[8]

[9]

[10]

[11]

[12] [13] [14] [15]

and rule-in of acute myocardial infarction using high-sensitivity cardiac troponin T., Arch. Intern. Med. 172 (2012) 1211–1218. https://doi.org/10.1001/archinternmed.2012.3698. J.W. Pickering, J.H. Greenslade, L.A. Cullen, D. Flaws, W.A. Parsonage, S. Aldous, P. George, A.W.M. Worster, P.A. Kavsak, M.P. Than, Assessment of the European Society of Cardiology 0-Hour/1-Hour algorithm to rule-out and rule-in acute myocardial infarction., Circulation. 134 (2016) 1532–1541. https://doi.org/10.1161/CIRCULATIONAHA.116.022677. J. Boeddinghaus, T. Reichlin, L.A. Cullen, J.H. Greenslade, W.A. Parsonage, C. Hammett, J.W. Pickering, T. Hawkins, S. Aldous, R. Twerenbold, K. Wildi, T. Nestelberger, K. Grimm, M. Rubini-Gimenez, C. Puelacher, V. Kern, K. Rentsch, M.P. Than, C. Mueller, Two-Hour Algorithm for triage towards rule-out and rule-in of acute myocardial infarction using high-sensitivity cardiac troponin I, Clin. Chem. 62 (2016) 494–504. https://doi.org/10.1373/clinchem.2015.249508. R. Body, E. Carlton, M. Sperrin, P.S. Lewis, G. Burrows, S. Carley, G. McDowell, I. Buchan, K. Greaves, K. Mackway-Jones, Troponin-only Manchester Acute Coronary Syndromes (T-MACS) decision aid: single biomarker re-derivation and external validation in three cohorts., Emerg. Med. J. EMJ. 34 (2017) 349–356. https://doi.org/10.1136/emermed-2016-205983. J.W. Pickering, M.P. Than, L.A. Cullen, S. Aldous, E.T. Avest, R. Body, E.W. Carlton, P. Collinson, A.M. Dupuy, U. Ekelund, K.M. Eggers, C.M. Florkowski, Y. Freund, P. George, S. Goodacre, J.H. Greenslade, A.S. Jaffe, S.J. Lord, A. Mokhtari, C. Mueller, A. Munro, S. Mustapha, W.A. Parsonage, W.F. Peacock, C.J. Pemberton, A.M. Richards, J. Sanchis, L.P. Staub, R. Troughton, R. Twerenbold, K. Wildi, J. Young, Rapid rule-out of acute myocardial infarction With a single high-sensitivity cardiac troponin T measurement below the limit of detection: A collaborative meta-analysis., Ann. Intern. Med. 166 (2017) 715–724. https://doi.org/10.7326/M16-2562. A. Shah, A. Anand, Y. Sandoval, K.K. Lee, S.W. Smith, P.D. Adamson, A.R. Chapman, T. Langdon, D. Sanderman, A. Vaswani, F.E. Strachan, A. Ferry, A.G. Stirzaker, A. Reid, A.J. Gray, P.O. Collinson, D.A. McAllister, F.S. Apple, D.E. Newby, N.L. Mills, High-sensitivity cardiac troponin I at presentation in patients with suspected acute coronary syndrome: a cohort study, The Lancet. 386 (2016) 2481–2488. https://doi.org/10.1016/S0140-6736(15)00391-8. K. Thygesen, J.S. Alpert, A.S. Jaffe, B.R. Chaitman, J.J. Bax, H.D. White, ESC Scientific Document Group, Fourth universal definition of myocardial infarction (2018), Eur. Heart J. 40 (2019) 237–269. https://doi.org/10.1093/eurheartj/ehy462. M.S. Parmacek, R.J. Solaro, Biology of the troponin complex in cardiac myocytes, Prog. Cardiovasc. Dis. (2004). https://doi.org/10.1016/j.pcad.2004.07.003. N. Bandstein, R. Ljung, M. Johansson, M.J. Holzmann, Undetectable high-sensitivity cardiac troponin T Level in the emergency department and risk of myocardial infarction, J. Am. Coll. Cardiol. 63 (2014) 2569–2578. https://doi.org/10.1016/j.jacc.2014.03.017. A.R. Chapman, K.K. Lee, D.A. McAllister, L.A. Cullen, J.H. Greenslade, W.A. Parsonage, A.W.M. Worster, P.A. Kavsak, S. Blankenberg, J.T. Neumann, N.A. Soerensen, D. Westermann, M.M. Buijs, G.J. Verdel, J.W. Pickering, M.P. Than, R. Twerenbold, P. Badertscher, Z. Sabti, C. Mueller, A. Anand, P.D. Adamson, F. Strachan, A. Ferry, D. Sandeman, A. Gray, R. Body, B. Keevil, E.W. Carlton, K. Greaves, F.K. Korley, T.S. Metkus, Y. Sandoval, F.S. Apple, D.E. Newby, A.S.V. Shah, N.L. Mills, Association of High-Sensitivity Cardiac Troponin I Concentration with Suspected Acute Coronary Syndromes, JAMA J. Am. Med. Assoc. 318 (2017) 1913– 1924. https://doi.org/10.1001/jama.2017.17488.

18

[16] M.P. Than, L.A. Cullen, S. Aldous, W.A. Parsonage, C.M. Reid, J.H. Greenslade, D. Flaws, C.J. Hammett, D.M. Beam, M.W. Ardagh, R. Troughton, A.F.T. Brown, P. George, C.M. Florkowski, J.A. Kline, W.F. Peacock, A.S. Maisel, S.H. Lim, A. Lamanna, A.M. Richards, 2-Hour accelerated diagnostic protocol to assess patients with chest pain symptoms using contemporary troponins as the only biomarker: the ADAPT trial., J. Am. Coll. Cardiol. 59 (2012) 2091–2098. https://doi.org/10.1016/j.jacc.2012.02.035. [17] R.V. Luepker, F.S. Apple, R.H. Christenson, R.S. Crow, S.P. Fortmann, D. Goff, R.J. Goldberg, M.M. Hand, A.S. Jaffe, D.G. Julian, D. Levy, T. Manolio, S. Mendis, G. Mensah, A. Pajak, R.J. Prineas, K.S. Reddy, V.L. Roger, W.D. Rosamond, E. Shahar, A.R. Sharrett, P. Sorlie, H. Tunstall-Pedoe, Case definitions for acute coronary heart disease in epidemiology and clinical research studies: a statement from the AHA Council on Epidemiology and Prevention; AHA Statistics Committee; World Heart Federation Council on Epidemiology and Prevention; the European Society of Cardiology Working Group on Epidemiology and Prevention; Centers for Disease Control and Prevention; and the National Heart, Lung, and Blood Institute., Circulation. 108 (2003) 2543–2549. https://doi.org/10.1161/01.CIR.0000100560.46946.EA. [18] M.P. Than, J.W. Pickering, S.J. Aldous, L.A. Cullen, C.M.A. Frampton, W.F. Peacock, A.S. Jaffe, S.W. Goodacre, A.M. Richards, M.W. Ardagh, J.M. Deely, C.M. Florkowski, P. George, G.J. Hamilton, D.L. Jardine, R.W. Troughton, P. van Wyk, J.M. Young, L. Bannister, S.J. Lord, Effectiveness of EDACS versus ADAPT accelerated diagnostic pathways for chest pain: A pragmatic randomized controlled trial embedded within practice., Ann. Emerg. Med. 68 (2016) 93-102.e1. https://doi.org/10.1016/j.annemergmed.2016.01.001. [19] M.P. Than, D. Flaws, S. Sanders, J. Doust, P. Glasziou, J. Kline, S. Aldous, R. Troughton, C. Reid, W.A. Parsonage, C.M. Frampton, J.H. Greenslade, J.M. Deely, E. Hess, A.B. Sadiq, R. Singleton, R. Shopland, L. Vercoe, M. Woolhouse-Williams, M. Ardagh, P. Bossuyt, L. Bannister, L.A. Cullen, Development and validation of the Emergency Department Assessment of Chest pain Score and 2 h accelerated diagnostic protocol., Emerg. Med. Australas. EMA. 26 (2014) 34–44. https://doi.org/10.1111/1742-6723.12164. [20] L.A. Cullen, M.P. Than, A.F.T. Brown, M. Richards, W.A. Parsonage, D. Flaws, J.E. Hollander, R.H. Christenson, J.A. Kline, S. Goodacre, A.S. Jaffe, Comprehensive standardized data definitions for acute coronary syndrome research in emergency departments in Australasia., Emerg. Med. Australas. EMA. 22 (2010) 35–55. https://doi.org/10.1111/j.1742-6723.2010.01256.x. [21] M.P. Than, J.W. Pickering, J.M. Dryden, S.J. Lord, S.A. Aitken, S.J. Aldous, K.E. Allan, M.W. Ardagh, J.W.N. Bonning, R. Callender, L.R.E. Chapman, J.P. Christiansen, A.P.J. Cromhout, L.A. Cullen, J.M. Deely, G.P. Devlin, K.A. Ferrier, C.M. Florkowski, C.M.A. Frampton, P.M. George, G.J. Hamilton, A.S. Jaffe, A.J. Kerr, G.L. Larkin, R.M. Makower, T.J.E. Matthews, W.A. Parsonage, W.F. Peacock, B.F. Peckler, N.C. van Pelt, L. Poynton, A.M. Richards, A.G. Scott, M.B. Simmonds, D. Smyth, O.P. Thomas, A.C.Y. To, S.A. Du Toit, R.W. Troughton, K.M. Yates, Improving care processes for patients with suspected acute coronary syndrome (ICareACS): A study of cross-system implementation of a national clinical pathway, Circulation. 137 (2018) 354–363. https://doi.org/10.1161/CIRCULATIONAHA.117.031984. [22] K. Thygesen, J.S. Alpert, A.S. Jaffe, M.L. Simoons, B.R. Chaitman, ESC/ACCF/AHA/WHF Expert Consensus Document: Third universal definition of myocardial infarction, Circulation. 126 (2012) 2020–2035. https://doi.org/10.1161/CIR.0b013e31826e1058/-/DC1.

19

[23] Y. Sandoval, A.S. Jaffe, Using high-sensitivity cardiac troponin T for acute cardiac care, Am. J. Med. 130 (2017) 1358-1365.e1. https://doi.org/10.1016/j.amjmed.2017.07.033. [24] K. Solecki, A.M. Dupuy, N. Kuster, F. Leclercq, R. Gervasoni, J.-C. Macia, T.-T. Cung, B. Lattuca, F. Cransac, S. Cade, J.-L. Pasquié, J.P. Cristol, F. Roubille, Kinetics of highsensitivity cardiac troponin T or troponin I compared to creatine kinase in patients with revascularized acute myocardial infarction., Clin. Chem. Lab. Med. 53 (2015) 707–714. https://doi.org/10.1515/cclm-2014-0475. [25] H.A. Katus, A. Remppis, T. Scheffold, K.W. Diederich, W. Kuebler, Intracellular compartmentation of cardiac troponin T and its release kinetics in patients with reperfused and nonreperfused myocardial infarction., Am. J. Cardiol. 67 (1991) 1360– 1367. [26] E.C.H.J. Michielsen, J.H.C. Diris, V.W.V.C. Kleijnen, W.K.W.H. Wodzig, M.P. Van Dieijen-Visser, Investigation of release and degradation of cardiac troponin T in patients with acute myocardial infarction, Clin. Biochem. 40 (2007) 851–855. https://doi.org/10.1016/j.clinbiochem.2007.04.004. [27] G. Laugaudin, N. Kuster, A. Petiton, F. Leclercq, R. Gervasoni, J.C. Macia, T.T. Cung, A.M. Dupuy, K. Solecki, B. Lattuca, S. Cade, F. Cransac, J.P. Cristol, F. Roubille, Kinetics of high-sensitivity cardiac troponin T and I differ in patients with ST-segment elevation myocardial infarction treated by primary coronary intervention, Eur. Heart J. Acute Cardiovasc. Care. 5 (2016) 354–363. https://doi.org/10.1177/2048872615585518. [28] C. Liebetrau, L. Gaede, J.S. Wolter, J. Homann, A. Meyer, O. Dorr, H.M. Nef, C. Troidl, C.W. Hamm, H. Mollmann, A.M. Richards, C.J. Pemberton, Release kinetics of high-sensitivity cardiac troponins I and T and troponin T upstream open reading frame peptide (TnTuORF) in clinically induced acute myocardial infarction, Biomark. Biochem. Indic. Expo. Response Susceptibility Chem. 22 (2017) 304–310. https://doi.org/10.1016/j.ahj.2011.04.007. [29] J.W. Pickering, The need to improve derivation and description of algorithms to rule-out patients with possible myocardial infarction, Circulation. 139 (2019) 1351–53. https://doi.org/10.1161/CIRCULATIONAHA.118.038418. [30] J.P.J. Ungerer, L. Marquart, P.K. O’Rourke, U. Wilgen, C.J. Pretorius, Concordance, variance, and outliers in 4 contemporary cardiac troponin assays: implications for harmonization., Clin. Chem. 58 (2012) 274–283. https://doi.org/10.1373/clinchem.2011.175059. [31] N. van der Linden, K. Wildi, R. Twerenbold, J.W. Pickering, M. Than, L. Cullen, J. Greenslade, W. Parsonage, T. Nestelberger, J. Boeddinghaus, P. Badertscher, M. Rubini Giménez, L.J.J. Klinkenberg, O. Bekers, A. Schöni, D.I. Keller, Z. Sabti, C. Puelacher, J. Cupa, L. Schumacher, N. Kozhuharov, K. Grimm, S. Shrestha, D. Flores, M. Freese, C. Stelzig, I. Strebel, Ò. Miró, K. Rentsch, B. Morawiec, D. Kawecki, W. Kloos, J. Lohrmann, A.M. Richards, R. Troughton, C. Pemberton, S. Osswald, M.P. van DieijenVisser, A.M. Mingels, T. Reichlin, S.J.R. Meex, C. Mueller, Combining high-sensitivity cardiac troponin I and cardiac troponin T in the early diagnosis of acute myocardial infarction, Circulation. 138 (2018) 989–999. https://doi.org/10.1161/CIRCULATIONAHA.117.032003. [32] D.S. Herman, P.A. Kavsak, D.N. Greene, Variability and error in cardiac troponin testing, Am. J. Clin. Pathol. 148 (2017) 281–295. https://doi.org/10.1093/ajcp/aqx066. Table 1: Demographics and presenting features

20

Not MI (n=106)

MI (n=58)

Overall (n=164)

Sex (Female)

21 (19.8%)

11 (19.0%)

32 (19.5%)

Age (years) (Median [LQ-UQ])

64 (57 - 76)

64 (59 - 74)

64 (57 - 74)

Systolic Blood Pressure (mmHg) (Median [LQUQ])

140 (130 170)

150 (130 170)

150 (130 170)

Diastolic Blood Pressure (mmHg) (Median [LQUQ])

80 (70 - 92)

84 (76 - 92)

82 (72 - 92)

Creatinine ( mol/L)(Median [LQ-UQ])

90 (82 - 98)

95 (85 - 100)

91 (82 - 99)

7 (6.6%)

12 (20.7%)

19 (11.6%)

History of Diabetes

19 (17.9%)

7 (12.1%)

26 (15.9%)

History of Hypertension

64 (60.4%)

32 (55.2%)

96 (58.5%)

History of Dyslipidemia

80 (75.5%)

39 (67.2%)

119 (72.6%)

Prior Myocardial Infarction

38 (35.8%)

26 (44.8%)

64 (39.0%)

History of Heart Failure

6 (5.7%)

3 (5.2%)

9 (5.5%)

History of Peripheral Vascular Disease

9 (8.5%)

5 (8.6%)

14 (8.5%)

69 (65.1%)

27 (46.6%)

96 (58.5%)

Prior CABG

7 (6.6%)

6 (10.3%)

13 (7.9%)

Prior PTCA

44 (41.5%)

26 (44.8%)

70 (42.7%)

Family History of Cardiovascular Disease

52 (49.1%)

29 (50.0%)

81 (49.4%)

New Ischaemic Changes on ECG

19 (17.9%)

21 (36.2%)

40 (24.4%)

EDACS (Mean [SD])

21 (± 4.0)

21 (± 5.3)

21 (± 4.5)

Current Smoker

History of Angina

Presented are: mean +/- standard deviation (SD), median (LQ:lower quartile – UQ:upper quartile), or n(%). CABG: Coronary Artery Bypass Graft. PTCA: Percutaneous Transluminal Coronary Angiography. ECG: Electrocardiograph. EDACS: Emergency Department Assessment of Chest pain Score.

21

Table 2 Patient level data and estimates for patients with myocardial infarction Time from symptom onset and concentrations of the first measured sample ID SP0 711 SP0 714 SP0 724 SP0 734 SP0 740 SP0 750 SP0 754 SP0 762 SP0 765 SP0 766 SP0 768 SP0 769 SP0 781 SP0 782 SP0 790 SP0 792

hsTnT (ng/L)

hsTnI (ng/L)

Estimated times to thresholds for hsTnI 2 ng/L 5 ng/L URL Max threshold threshold threshold threshold (h) (h) (h) (h)

Max hsTnI (ng/L)

Estimated times to thresholds for hsTnT 5 ng/L URL Max threshold threshold threshold (h) (h) (h)

Max hsTnT (ng/L)

Age

Sex

Cr

Time (h)

78

F

104

3.1

94

246

1

66

M

116

4.1

160

1500

1

71

M

90

6.3

10

3

6.2

7.0

9.1

36.8

3048

5.6

7.4

44.6

411

1

57

M

101

7.7

40

85

3.2

3.8

5.9

23.1

445

3.8

5.3

21.2

119

1

52

M

90

23.4

535

4332

50

M

89

4.3

35

34

2.8

3.2

4.3

35.0

18510

2.9

3.6

9.7

212

1

65

F

72

4.0

48

59

1.2

1.5

2.0

18.5

294

0.3

0.5

13.4

64

1

52

M

104

2.3

59

175

1.1

1.3

1.8

11.1

6538

1.3

1.7

10.2

559

1

51

F

71

16.8

747

16730

2.5

2.8

3.2

26.3

31909

3.8

4.4

35.4

1889

1

83

M

99

4.3

11

15

2.6

3.3

6.0

35.4

414

3.1

5.6

25.8

48

1

62

M

94

2.8

51

83

1.3

1.5

2.3

16.6

2201

1.0

1.5

18.9

233

1

64

M

84

6.6

216

2020

0.4

0.5

0.7

13.0

2595

0.5

0.7

19.8

308

1

70

M

87

2.8

60

386

0.5

0.5

1.0

13.4

1394

0.2

0.5

68.5

204

1

50

M

95

17.0

276

1110

43

F

79

3.0

51

192

1.2

1.4

1.7

15.2

7740

1.4

1.7

19.8

661

1

67

M

81

7.2

56

214

3.1

3.5

4.9

25.3

3123

2.9

4.2

41.9

326

1

MI type

1

1

22

SP0 795 SP0 799 SP0 802 SP0 810 SP0 811 SP0 815 SP0 816 SP0 822 SP0 827 SP0 837 SP0 841 SP0 842 SP0 847 SP0 863 SP0 872 SP0 876 SP0 877 SP0 887 SP0 889 SP0 897 SP0 899 SP0 901

75

M

99

3.1

11

6

2.2

3.0

9.0

16.6

45

1.0

8.0

19.8

17

1

61

M

101

2.9

23

31

1.8

2.0

2.7

16.2

8684

1.7

2.3

18.5

552

1

70

M

123

5.1

89

538

2.3

2.5

3.2

19.4

23732

2.1

2.8

23.1

630

1

72

M

99

3.1

15

13

2.2

2.8

4.8

45.5

1110

1

48

M

87

3.8

25

26

0.5

0.8

4.2

5.6

35

1

75

M

122

16.7

1856

18281

0.4

0.4

0.6

22.1

18164

53

M

84

1.5

13

48

0.7

68.0

92

65

M

82

7.2

69

109

0.5

0.7

1.7

8.8

128

70

F

72

12.2

227

907

4.1

4.6

5.3

23.5

2488

63

M

97

3.1

125

239

1.5

1.7

2.2

16.2

14231

64

M

96

4.1

18

48

0.1

71

76

M

108

4.3

23

49

0.8

1.0

2.8

9.7

79

0.6

1.7

10.2

31

1

65

M

102

4.3

384

4695

1.0

1.1

1.5

20.3

71498

1.2

1.6

38.2

4156

1

50

M

86

1.8

18

42

0.5

0.6

1.6

12.5

158

0.6

1.4

9.3

46

1

81

M

117

3.3

68

61

0.5

0.7

1.7

6.1

93

0.6

99

1

80

M

136

4.8

74

22

62

M

88

16.7

2323

42903

2.4

2.7

3.5

29.0

66552

4.0

4.8

38.2

5571

1

76

M

96

2.3

33

32

1.5

1.7

2.5

18.5

3639

1.3

1.8

16.6

375

1

82

F

113

3.3

184

1101

0.5

0.6

0.8

13.9

4661

0.5

0.7

15.2

499

1

46

M

90

3.2

54

442

0.2

0.3

0.7

68.5

9840

66

M

94

5.0

40

64

62

M

95

8.3

51

244

1.1

1.5

35.0

2303

1 1

0.3

4.2

79

1

1.9

2.4

24.9

308

1

0.9

1.3

28.6

1134

1 1

1

1 1

0.6

0.9

2.5

68.5

1904

2.4

4.1

57.0

170

1

23

SP0 923 SP0 938 SP0 939 SP0 940 SP0 952 SP0 960 SP0 967 SP0 983 SP0 985 SP0 986 SP1 001 SP1 006 SP1 011 SP1 014 SP1 031 SP1 035 SP1 039 SP1 053 SP1 056 SP1 057 Su mm ary Stat

55

M

92

13.7

11

40

10.2

11.1

13.4

28.1

871

11.1

15.3

36.8

55

1

71

M

97

13.5

64

119

7.9

8.6

10.7

17.1

184

7.6

9.0

16.2

77

1

76

F

82

2.3

330

2075

50

M

116

10.8

84

330

5.4

5.9

7.2

28.6

15315

5.6

6.9

38.2

931

1

63

M

78

21.0

66

1160

9.0

9.9

12.4

45.1

6392

12.8

15.5

44.6

228

1

54

M

73

3.4

12

17

0.9

1.3

3.6

11.1

66

1.9

4.3

8.8

21

1

64

M

90

4.6

16

43

1.1

1.5

3.9

15.2

94

1.2

4.2

24.4

26

1

84

M

162

18.2

65

109

1.5

392

2.2

4.1

28.1

70

1

61

M

82

22.1

643

19107

5.2

5.7

7.1

34.5

34037

10.9

12.3

38.2

2054

1

59

M

109

1.9

12

51

0.9

1.1

1.8

68.5

91530

1.3

2.1

68.5

4681

1

71

M

90

4.1

45

75

2.0

2.4

3.7

68.5

18428

1

88

M

100

3.4

101

160

0.7

0.9

1.7

14.8

490

1

72

F

69

3.6

4

20

87

M

118

4.1

34

36

2.1

2.5

4.0

9.7

149

1.4

2.4

9.7

55

1

83

M

98

3.8

86

273

1.9

2.2

2.7

17.1

29012

1.5

2.1

20.8

809

1

65

F

99

7.8

39

56

4.2

4.9

5.8

24.4

599

3.8

4.7

19.8

106

1

59

F

71

6.1

16

15

3.6

4.4

3.8

13.0

43

3.3

4.4

9.7

23

1

69

F

80

12.1

28

45

6.8

7.7

8.9

19.8

104

3.9

5.1

14.8

31

1

77

M

93

2.3

47

84

0.2

0.7

8.4

131

0.3

7.0

59

1

72

M 19.0 % Fema le

98

1.5

21

16

0.6

0.8

2.3

7.0

66.0

0.5

0.9

6.1

47.0

2

95 (85 -

4.2 (3.17.8)

51 (2393)

85 (41428)

1.7 (0.73.0)

1.7 (0.93.4)

18 (13-28)

1649 (1369551)

1.8 (1.1-3.8)

2.6 (1.54.8)

20 (12-36)

212 (57595)

98% Type 1

64 (59 74)

1

1

3 (1.7-5)

24

istic 100 s ) SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure; Cr: Creatinine; Smok: Smoker (current); DM: Diabetes Mellitus; HTN: Hypertension; Dyslip: Dyslipidemia; MI: Myocardial Infarction; HF: Heart Failure; PVD: Peripheral Vascular Disease; Ang: Angina; CABG: Coronary Artery Bypass Graft; PTCA: Percutaneous Transluminal Coronary Angiography; CVD: Cardiovascular Disease; URL: Upper Reference Limit (sex specific); Max: Maximum

25

Figure 1. Histograms of patient numbers from time from symptom onset until first blood sample. MI: Myocardial Infarction. The bin width is 30 minutes.

Figure 2. cTn concentrations as a function of time from the onset of symptoms in patients with a final diagnosis of myocardial infarction. hs-cTnI (white circles and black curve) and hs-cTnT (black circles and grey curve) concentrations (log base 2 scale). Curves are a log-normal non-linear least squares fit with dotted lines showing fits at the 95% confidence interval. The title on each graph is the study ID, Sex (M or F) and Age of the patient. SP1057 was a type 2 MI; all the rest were type 1 MI.

Figure 3. Summary graph of cTn concentrations as a function of time from the onset of symptoms in patients with a final diagnosis of myocardial infarction. (A) hs-cTnI. (B) hs-cTnT. Boxes indicate the interquartile range and medium; whiskers are at 1.5 times the interquartile range. Jittering (horizontally) has been used so as to better indicate individual concentrations.

Figure 4. The ratio of hs-cTnI to hs-cTnT concentrations as a function of time from the onset of symptoms in patients with a final diagnosis of myocardial infarction. The dotted line are a fit through the presentation, 1h, 2h, and 3h samples. The horizontal line is at a ratio of 1. The title on each graph is the study ID, Sex (M or F) and Age of the patient.

Figure 5. The ratio of hs-cTnI to hs-cTnT concentrations as a function of sample dilution for 3 selected patients at two timepoints each. The numbers are the measured hs-cTnI concentrations.

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27

28

29

30

31

Highlights 

Kinetic profiles of troponin concentrations varied considerably between MI patients



Troponin-I concentrations increased more rapidly than troponin-T concentrations



Concentrations of 1/3rd of patients took >3 hours to exceed MI rule-out thresholds

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Credit Author Statement

Early kinetic profiles of troponin I and T measured by high-sensitivity assays in patients with myocardial infarction John W Pickering: Conceptualization, Methodology, Formal analysis, Visualization, Writing - Original Draft, Supervision, Writing - Review & Editing Joanna M Young: Investigation, Data Curation, Project administration, Validation, Writing - Review & Editing Peter M George: Methodology, Writing - Original Draft, Writing - Review & Editing, Supervision Christopher J Pemberton: Resources, Writing - Review & Editing Antony Watson: Investigation, Data Curation, Writing - Review & Editing Sally J Aldous: Investigation, Writing - Review & Editing Toby Verryt: Investigation, Writing - Review & Editing Richard W Troughton: Methodology, Resources, Validation, Writing - Review & Editing, Funding acquisition A Mark Richards: Resources, Methodology, Writing - Review & Editing, Funding acquisition Fred S Apple: Writing - Review & Editing Martin P Than: Resources, Methodology, Writing - Original Draft, Supervision, Funding acquisition, Writing - Review & Editing, Supervision

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