Single Troponin Measurement to Rule Out Myocardial Infarction

JACC Review Topic of the Week

Allan S. Jaffe, MD; Richard Body, MD; Nicholas L. Mills, MD; Kristin M. Aakre, PHD; Paul O. Collinson, MD;
Amy Saenger, PHD; Ole Hammarsten, MD; Ryan Wereski, MD; Torbjørn Omland, MD; Yader Sandoval,
MD; Jordi Ordonez-Llanos, MD, PHD; Fred S. Apple, PHD

J Am Coll Cardiol. 2023;82(1):60-69.

Abstract

The term "single-sample rule-out" refers to the ability of very low concentrations of high-sensitivity
cardiac troponin (hs-cTn) on presentation to exclude acute myocardial infarction with high clinical
sensitivity and negative predictive value. Observational and randomized studies have confirmed this
ability. Some guidelines endorse use of a concentration of hs-cTn at the assay's limit of detection, while
other studies have validated the use of higher concentrations, allowing this approach to identify a
greater proportion of patients at low risk. In most studies, at least 30% of patients can be triaged with
this approach. The concentration of hs-cTn varies according to the assay used and sometimes how
regulations permit reporting. It is clear that patients need to be at least 2 hours from the onset of
symptoms being evaluated. Caution is warranted, particularly with older patients, women, and patients
with underlying cardiac comorbidities.

Highlights

Rapid, safe, and accurate exclusion of acute myocardial infarction can facilitate triage of patients in the
emergency department.

Observational and randomized studies have found that a single low assay-dependent, hs-cTn
measurement taken >2 hours after symptom onset in a patient with a nonischemic electrocardiogram
can effectively exclude acute MI.

Additional studies are needed to assess the utility of this approach in patients undergoing evaluation
earlier after the onset of symptoms and to establish optimum blood level thresholds for women and
patients with specific comorbidities.

Introduction

A major advance associated with high-sensitivity cardiac troponin (hs-cTn) assays is the ability to exclude
acute myocardial infarction (MI) safely and rapidly.[1,2] This ability has the potential to rapidly identify
patients in which the likelihood of MI is low and short-term outcomes should be good. If the approach
has adequate sensitivity, it will allow patients to be safely discharged from emergency departments (ED)
earlier.[1,2] This ability will alleviate ED overcrowding. The data are persuasive that when there are too
many ED patients and wait times are long, all patients, regardless of their diagnoses, are at increased risk
for adverse events, including mortality.[3,4] ED overcrowding also has a negative impact on patient
satisfaction.[5]

hs-cTn assays detect low concentrations of cardiac troponin (cTn) with improved analytical precision.[6]
This allows thresholds below the 99th-percentile upper reference limit to be probed. By setting the
cutoff below the limit of detection (LoD) of the assay (ie, the lowest concentration measured that is
different from zero), it is possible to exclude myocardial injury and thus MI with a single blood test on
arrival to the ED.[2] This approach is used in patients who are clinically at low risk. This is a key
component of its success.

The initial studies in this area were observational. Many did not include consecutive patients, and in
some studies the time from symptom onset to sample acquisition was delayed for informed consent.[7]
The metrics for ruling out MI differ when one has complete ascertainment rather than a selected cohort
because the frequency of MI and thus the pretest probability of disease are less in the unselected
cohort.[8] In addition, the low values obtained may or may not result in patient discharge. Thus,
observational studies cannot evaluate the real-world impact of implementing these early rule-out
strategies. This can be done in randomized implementation trials, which we strongly endorse.[9]

The present review is part of an educational series from the International Federation of Clinical
Chemistry and Laboratory Medicine Committee on Clinical Applications of Cardiac Bio-Markers
concerning the use of cardiac biomarkers. The report focuses on the single-sample rule-out and its
potential benefits and limitations. Although we review available data, the report is not a guideline or a
meta-analysis. Instead it provides readers with an understanding of the approach and recommendations
to facilitate its optimal use.

Safety Metrics Required for the Single-sample Rule-out

There are several important concepts. Emergency medicine physicians advocate that the risk for missing
major adverse cardiac events in patients in whom MI is "ruled out" should be <1% at 30 days.[10] This is
a high bar, but it is guideline supported.[11,12] There is controversy over how to implement this
approach. Many studies define the optimal cutoff as the highest cTn concentration that enables the
greatest proportion of patients to be ruled out (effectiveness) with a negative predictive value (NPV) of
>99.5% (safety) for MI or cardiac death at 30 days.[13] This equates to 1 false negative in every 200
patients. However, unlike sensitivity, NPV is influenced by the frequency of the event.[14,15] When the
frequency of MI is low, any cutoff will provide a high NPV. It is therefore essential that the cutoff also
provide high sensitivity. It has been proposed by the National Institute for Health and Care Excellence in
the United Kingdom that the optimal cutoff should provide sensitivity of at least 97%, ideally >99%,
when incorporated into a clinical pathway that includes electrocardiography and clinical presentation.
[16] This works well when sample sizes are large, there is complete clinical ascertainment of events, and
the frequency of MI is not exceptionally low. In small studies, NPV can be misleading, and reporting
sensitivity is essential. Confirmation of the metrics for these assays ideally should depend on randomized
implementation trials and be compared with usual care.[9]

We recommend that studies defining the optimal cutoff using the NPV also report sensitivity, along with
95% CIs, and the frequency of MI and cardiac death at 30 days. Typically, this approach is implemented in
patients without evidence of myocardial ischemia on electrocardiography who are tested at least 2 hours
after symptom onset.[2] Clinicians can select an approach that has been evaluated in a population with a
similar frequency of MI as their local population and is best suited to their health care system to
maximize efficiency (the largest proportion suitable for outpatient management) or to be more
conservative (hospital admissions to minimize false negatives). A graphical illustration of this approach is
shown in Figure 1. Recent data suggest that an approach using values less than the LoD of the assay
might help in those who present <2 hours after symptom onset.[17] This approach may also be of help in
patients with nonspecific electrocardiographic changes.[18]

Another important area is the endpoint measured to support safety. Some studies focus on type 1 MI
and/or cardiovascular death, and others include all MI subtypes. The original High-STEACS (High-
Sensitivity Troponin in the Evaluation of Patients With Acute Coronary Syndrome) study had a primary
outcome of a composite of index type 1 MI and cardiac death at 30 days.[13] Other guidelines have
included emergent coronary revascularization as part of major adverse cardiac events.[18] We advocate
that studies report the MI subtypes evaluated to support safety.

Finally, it should be appreciated that single measurements are subject to uncertainty on the basis of the
rounding of concentrations, which is guideline mandated.[1,6] The imprecision associated with this
factor can be calculated (Table 1).

Figure 1. Cardiac Troponin Concentration at Presentation and Risk for MI

(Top) Negative predictive value of a range of high-sensitivity cardiac troponin I concentrations

(ARCHITECT STAT, Abbott) on presentation for the composite outcome of index myocardial infarction (MI)
and MI or cardiac death at 30 days. (Bottom) Proportion of patients with suspected acute coronary
syndrome with troponin concentrations below each threshold. Reprinted with permission from Body et
al.20

The Single-sample Rule-out

The single-sample rule-out relies on hs-cTn assays to measure low hs-cTn concentrations around the
assay's LoD. With low concentrations on arrival in the ED, the likelihood of acute MI is small using the
criteria from the fourth universal definition of MI.[19] The first study comprehensively evaluating this
approach used the limit of blank (LoB) as a cutoff.[20] The LoB is the lowest concentration that can be
distinguished from noise analytically.[6] Other studies have used the LoD, but many have used higher
optimized concentrations that identify a larger proportion of the population as suitable for safe
discharge. The assumptions undergirding the approach are as follows.

First, there is a relationship between the molecular weight of a protein and the rapidity with which it
reaches the circulation.[21] The rapid release of cTn, a molecule of low molecular weight (33.5 kDa for
cTnT, 23.5 kDa for cTnI), is supported by a study showing increased systemic concentrations of after
occluding a coronary artery for 90 seconds.[22] One hs-cTnI assay increased within 15 minutes. Second,
release of biomarkers following cardiac injury depends on blood flow. If coronary blood flow is absent, it
will take longer for cTn to be released. This is a problem with ST-segment elevation MI, in which the
incidence of total occlusion is high.[23] With non–ST-segment elevation MI, the frequency of total
occlusion is about 30%, and such patients come to the hospital later than those with ST-segment
elevation MI.[24] For the subset of patients who present early or with total occlusions, this approach is
not advocated.[7,13,25] The European Society of Cardiology guidelines suggest the single-sample rule-
out only in patients without ST-segment elevation who are at least 3 hours after symptom onset.[26] This
may have evolved because many studies required informed consent, delaying patient evaluations until
after 3 hours.[27] A real-world evaluation of this timing occurred with the High-STEACS study, in which
the NPV of the single-sample rule-out approach was lower in those within 2 hours of symptom onset
compared with those presenting at >2 hours (97.6% [95% CI: 95.8%-99.2%] vs 99.8% [95% CI: 99.6%-
100.0%]).[13]

Third, cardiovascular risk factors associated with atherosclerosis and cardiovascular comorbidities cause
a graded increase in hs-cTn concentration within the reference range.[28] Thus, low concentrations
suggest that few risk factors for underlying atherosclerotic disease or cardiovascular comorbidities exist.
This factor augments the sensitivity of the approach by identifying a group with a low pretest probability
of disease. Accordingly, patients who might potentially be poorly triaged with this approach are those
with nonatherothrombotic reasons for MI and few traditional risk factors, for example, those with
microvascular dysfunction, vasospasm, spontaneous coronary artery spasm, and/or coronary embolic
disease.[19]

Clinical Studies

The metrics for the use of this approach vary tremendously.[2] Although the initial study used the LoB,
[6] this threshold has not been adopted because of the high degree of assay imprecision at LoB
concentrations. Therefore, the LoD, for which imprecision is <20% to 25%, is preferred.[6] The LoD does
not guarantee high precision, and it is for that reason that the U.S. Food and Drug Administration (FDA)
has restricted the reporting of hs-cTn concentrations to the limit of quantitation (LoQ), which is the
lowest concentration with a coefficient of analytical variation of 20%.[29]

Result reporting is further complicated because for some hs-cTn assays, there is variability in precision
depending upon the analytical platform and the sample matrix.[30] For many hs-cTn assays, it is not
necessary to use concentrations as low as the LoD or even the LoQ. Studies have documented that for
many hs-cTn assays, concentrations greater than the LoD can establish an NPV of >99.5%. These higher
concentrations allow the rule-out of a larger number of patients and increase effectiveness (Figure 1).
The International Federation of Clinical Chemistry and Laboratory Medicine Committee on Clinical
Applications of Cardiac Bio-Markers and the American Association for Clinical Chemistry Academy
advocate that reporting below the 20% coefficient of analytical variation concentration is likely of value
and should not negatively affect safety.[31] This issue most directly affects the hs-cTnT assay from Roche
Diagnostics, whose LoD is 3 or 5 ng/L depending on the analyzer. Yet the FDA allows reporting only to a
concentration of the LoQ of 6 ng/L. Only recently have data been published to suggest that this
concentration can be relied on for the single-sample rule-out.[32]

Many observational studies have validated the single-sample rule-out, as have systematic reviews and
individual patient–level meta-analyses.[2,33] Recently, 3 randomized trials have provided more robust
validation. The first, a stepped-wedge randomized controlled trial called HiSTORIC (High-Sensitivity
Cardiac Troponin on Presentation to Rule Out Myocardial Infarction), used the Abbott hs-cTnI assay.[34]
In this noninferiority trial, standard care with serial sampling was compared with the single-sample rule-
out using a validated optimized cutoff of <5 ng/L,[13] well above the 1.9 ng/L LoD, in >31,000 patients.
The incidence of MI or cardiac death was very low in both groups. Length of ED stay was significantly
lower in the single-sample group. Furthermore, the proportion of patients discharged increased from
50% with standard care to 71% with the single-sample rule-out. This strategy was deployed only in those
who presented ≥3 hours after symptom onset. The incidence of MI or cardiac death at 30 days was low
(0.3% with the early rule-out strategy and 0.4% with standard care). This small difference did not meet
the prespecified criteria to conclude that it was noninferior to standard care. Nonetheless, the frequency
of events was so low that it met the metrics desired for adequate identification of these patients. Follow-
up at 1 year also demonstrated no increase in cardiac events, suggesting that discharge did not
compromise care.[9]

The second randomized trial was called RAPID-TnT (Rapid Assessment of Possible Acute Coronary
Syndrome in the Emergency Department With High-Sensitivity Troponin T).[35] It was designed with so-
called masked and unmasked groups. The masked-group clinicians were not informed of hs-cTnT
concentrations that were very low and therefore treated patients as if they were using a less sensitive
cTn assay. In the unmasked group, the concentrations were shared, and single-sample rule-out was
permitted. The analysis was more complicated because the early rule-out strategy involved both a 0/1-
hour protocol as well as the single-sample rule-out. It should be noted that 13% of patients came back to
the ED because of chest pain, and readmission was more common in the unmasked (4.0%) than the
masked arm (2.7%). During long-term follow-up, there was an increased signal for mortality in patients
who had elevations of hs-cTnT in the unmasked group.[36] One explanation might be that when
clinicians did not have hs-cTn concentrations, they were more conservative.

These conflicting data caused ambivalence about the strategy in the United States, where the ability of
some hs-cTn assays to report the concentrations associated with success of the single-sample strategy
was precluded by the FDA's prohibition. This has led to controversy over whether this approach should
be used with hs-cTnT. Four studies have probed the use of hs-cTnT concentrations of <6 ng/L. Two small
trials used hs-cTnT with 0-hour and 3-hour sampling. Both suggested that the approach was successful.
[37,38] Both studies used a higher 99th percentile (19 ng/L, as recommended in the U.S. package insert)
for diagnosing MI compared with those used in Europe (common cutoffs of 14 ng/L, 9 ng/L in women,
and 17 ng/L in men) and in the Universal Sample Bank.[39] A third trial, in Canada, used lower 99th-
percentile concentrations (<8 ng/L in men and <7 ng/L in women) and showed sensitivity of 98.5% for 7-
day outcomes.[40] Finally, a recent large study with a very small number of MIs (2.1%) also
demonstrated that single-sample rule-out was worthwhile.[41] However, the low incidence of MI raises
the question of whether such an effect would be seen if the incidence of MI were higher.

The LoDED (Limit of Detection in the ED) randomized trial included 632 low-risk patients with suspected
MI and normal electrocardiographic findings at 8 hospitals in the United Kingdom.[42] In that trial,
patients were individually randomized to MI rule-out on the basis of an initial hs-cTnI or hs-cTnT
concentration less than the LoD of the assay in use or to standard care. No patients who met the criteria
for single-sample rule-out had major adverse cardiac events within 30 days. However, there was not a
statistically significant increase in early discharges from the ED (46% within 4 hours in the intervention
group vs 37% with standard care; adjusted OR: 1.58; 95% CI: 0.84–2.98). Although the difference in the
proportion discharged between arms was almost identical to that observed in RAPID-TnT, the CIs were
wide, and the study may have been underpowered. LoDED suggests that the approach using the LoD
cutoff is safe, but the effectiveness of the strategy cannot be taken for granted. Hospitals must work to
ensure protocol adherence and alter their clinical work flow to maximize the benefits.

A more definitive observational validation for hs-cTnT has recently been published.[32] In approximately
86,000 patients studied at multiple sites, the frequency with which those who had concentrations of hs-
cTnT <6 ng/L developed myocardial injury (a subsequent concentration greater than the sex-specific
99th-percentile upper reference limit) was roughly 1%. It was lower in men, and the only signal of
possible concern was in women who were older (>65 years) and had comorbidities for cardiac disease,
who had a lower NPV (97%). When this approach was applied to an extensively adjudicated cohort of
nearly 2,000 patients with nonischemic electrocardiographic findings, it was more robust. Thus, it is clear
the single-sample approach works with hs-cTnT as well as most of the hs-cTnI assays.[32,43,44] The
proportion of patients in whom this strategy is applicable varies by study but ranges from 29% to 74%
depending on the study cited and the approach used.[32–35,43,44]

It is notable however, that the concern with regard to older patients is similar to that reported from
Israel.[45] These patients had lower NPVs when they were older and had higher GRACE (Global Registry
of Acute Coronary Events) scores.[45] No study has specifically addressed whether sex-specific
thresholds would improve this strategy in women. The concept that those with a high pretest probability
of cardiovascular disease are those in whom this approach may fail is supported by a recent evaluation
comparing patients with and those without known coronary artery disease, for which the European
Society of Cardiology 0/1 hour algorithm had an NPV of only 96.6% and sensitivity of 93.2% using hs-
cTnT. It is unclear whether the problems were with the single-sample rule-out using a value <6 ng/L or
the change in values over 1 hour or both. The data call attention to the need for clinical oversight when
implementing this strategy.[46]

Point-of-care (POC) assays have been touted as useful for the single-sample rule-out, but most have
relied on stored plasma samples and not fresh whole blood.[47–51] Recently, a novel hs-cTnI POC assay
(Atellica VTLi, Siemens Healthineers; <8 minutes) has been validated on whole blood. It derived (fresh
whole blood) and validated (stored plasma) a cutoff concentration (<4 ng/L) to identify patients at low
risk for index MI and low risk at 30 days (<1%). Up to 40% of patients presenting with symptoms
suggestive of ischemia might be discharged.[51] We are likely to see more reports on POC hs-cTn assays
as they are evaluated and validated for single-sample rule-out strategies for MI. Given the rapid
turnaround time of these assays, this could provide further efficiencies for crowded EDs.

Central Illustration. Single-Sample Rule-Out for Myocardial Infarction With Cardiac Troponin

The metrics and application of the single-sample rule-out approach for myocardial infarction (MI). The
figure emphasizes the need for safety first while attempting to maximize efficiency, thus the application
of clinical judgement and the presence of nonischemic electrocardiographic findings. Areas where
additional information would be helpful are listed in boxes on the left. *Negative predictive value (NPV)
can be misleading in smaller datasets or when the prevalence of MI is low. Accordingly, all studies should
report both NPV and sensitivity and their respective CIs. CVa = coefficient of analytical variation; NPV =
negative predictive value.

Conclusions

The data presented herein and summarized in the Central Illustration lead to the following
recommendations.

First, the evidence-based studies demonstrate that the single-sample rule-out strategy on the basis of
low concentrations of hs-cTn and nonischemic electrocardiographic findings is a safe way to exclude MI.
Recent data suggest that perhaps a low HEART (history, electrocardiogram, age, risk factors, and initial
troponin) score may help as well.[52]

Second, at present, this strategy should be used only in patients presenting >2 hours after symptom
onset.

Third, clinical judgment, not hs-cTn concentration alone, must be used to safely implement the single-
sample rule-out strategy. Particular care in older patients, women, and those with cardiac comorbidities
is advised.

Fourth, for each hs-cTn assay and the analytical platform that is used, individualized cutoff
concentrations should be derived and validated that optimize the performance of the assay by
maintaining an NPV of >99.5%. NPV can be misleading in smaller datasets or when the prevalence of MI
is low. Accordingly, all studies should report both NPV and sensitivity and their respective CIs. When
incorporated into a clinical pathway for 30-day risk for MI or death, this will maximize the proportion of
patients eligible for early discharge (29%-74%) (Table 2).

Fifth, POC hs-cTn assays must be validated using fresh whole blood for single-sample rule-out strategies
for MI. They should meet the same clinical safety and efficacy standards as central laboratory hs-cTn
assays. POC testing may solve cTn measurement turnaround time issues when present.