Clin Chem Lab Med 2022; 60(2): 169–182

Guidelines and Recommendations from Scientific Societies

Aldo Clerico*, Martina Zaninotto, Alberto Aimo, Ruggero Dittadi, Domenico Cosseddu,
Marco Perrone, Andrea Padoan, Silvia Masotti, Lucia Belloni, Marco Migliardi,
Antonio Fortunato, Tommaso Trenti, Lucia Malloggi, Piero Cappelletti, Gianni Antonio Galli,
Sergio Bernardini, Laura Sciacovelli and Mario Plebani

Use of high-sensitivity cardiac troponins in the

emergency department for the early rule-in and
rule-out of acute myocardial infarction without
persistent ST-segment elevation (NSTEMI) in Italy
Expert opinion document from the Study Group of Cardiac Biomarkers associated to the
Italian Societies ELAS (European Ligand Assay Society, Italy Section), SIBIoC (Società Italiana
di Biochimica Clinica), and SIPMeL (Società Italiana di Patologia Clinica e Medicina di
Laboratorio)

https://doi.org/10.1515/cclm-2021-1085 values for the most rapid algorithms (0 h/1 h or 0 h/2 h) to

Received October 7, 2021; accepted November 26, 2021; rule-in and rule-out NSTEMI. Finally, another important
published online December 20, 2021 point is the possible differences between demographic and
clinical characteristics of patients enrolled in multicenter
Abstract: Serial measurements of cardiac troponin are rec- trials compared to those routinely admitted to the Emergency
ommended by international guidelines to diagnose myocar- Department in Italy. The Study Group of Cardiac Biomarkers,
dial infarction (MI) since 2000. However, some relevant supported by the Italian Scientific Societies Società Italiana
differences exist between the three different international di Biochimica Clinica, Italian Society of the European Ligand
guidelines published between 2020 and 2021 for the man- Assay Society, and Società Italiana di Patolgia Clinica e
agement of patients with chest pain and no ST-segment Medicina di Laboratorio decided to revise the document
elevation. In particular, there is no agreement on the cut-offs previously published in 2013 about the management of pa-
or absolute change values to diagnose non-ST-segment tients with suspected NSTEMI, and to provide some sugges-
elevation MI (NSTEMI). Other controversial issues concern tions for the use of these biomarkers in clinical practice, with
the diagnostic accuracy and cost-effectiveness of cut-off a particular focus on the Italian setting.

*Corresponding author: Prof. Aldo Clerico, MD, Laboratory of Andrea Padoan, Laura Sciacovelli and Mario Plebani, Department of
Cardiovascular Endocrinology and Cell Biology, Department of Laboratory Medicine, University Hospital of Padova, Padova, Italy.
Laboratory Medicine, Fondazione CNR Toscana G. Monasterio, Scuola https://orcid.org/0000-0003-1284-7885 (A. Padoan).
Superiore Sant’Anna, Via Trieste 41 56126, Pisa, Italy, https://orcid.org/0000-0003-3156-1399 (L. Sciacovelli).
E-mail: [email protected] https://orcid.org/0000-0002-0270-1711 (M. Plebani)
Martina Zaninotto, Department of Laboratory Medicine, University Lucia Belloni, Dipartimento di Medicina di laboratorio, Arcispedale
Hospital of Padova, Padova, Italy; and Department of Medicine- Santa Maria Nuova - IRCCS, Reggio Emilia, Italy
DIMED, University of Padova, Padova, Italy Antonio Fortunato, U.O.C. Patologia Clinica, Ascoli Piceno, Italy
Alberto Aimo and Silvia Masotti, Fondazione CNR Regione Toscana G. Tommaso Trenti, Azienda Ospedaliero - Universitaria Policlinico
Monasterio e Scuola Superiore Sant’Anna, Pisa, Italy di Modena c/o Ospedale Civile di Baggiovara, Modena,
Ruggero Dittadi, Ospedale dell’Angelo ULSS 3 Serenissima, Italy
Laboratorio di Analisi Cliniche, Mestre, Italy Lucia Malloggi, Laboratorio Analisi, Azienda Ospedaliera-
Domenico Cosseddu and Marco Migliardi, S.C. Laboratorio Analisi, Universitaria di Pisa, Pisa, Italy
A.O. Ordine Mauriziano di Torino, Torino, Italy Piero Cappelletti, SIPMeL, Castelfranco Veneto, Italy
Marco Perrone and Sergio Bernardini, Division of Cardiology and Gianni Antonio Galli, Estote Misericordes, Borgo San Lorenzo,
Clinical Biochemistry, University of Rome Tor Vergata, Rome, Italy Firenze, Italy
170 Clerico et al.: Use of cardiac troponins in emergency department

Keywords: acute coronary syndrome; cardiac troponins; international documents are mostly based on results from
cardiovascular risk; high-sensitivity methods; myocardial multicenter clinical studies carried out in highly specialized
infarction; myocardial injury. centers. These results may be not readily applied to the
everyday clinical practice in Italy, where elderly patients
with multiple comorbidities often represent the majority of
patients admitted to ED. Furthermore, as emphasized even
Aim of the new document and recently by guidelines and documents by international sci-
methodology entific societies [5, 8, 10, 13, 14], a close collaboration be-
tween clinicians and clinical biochemists is needed to
In 2013, an intersociety group including experts of Emer- validate the decisional levels and identify the best diag-
gency/Urgency Medicine, Cardiology and Clinical Biochem- nostic algorithms for each institution. In particular, the re-
istry published some recommendations on the use of sults reported in this document refer to studies carried out by
high-sensitivity cardiac troponins (hs-cTn) in the Emergency the Inter-Society Study Group on Cardiac Biomarkers, which
Department (ED) to diagnose non-ST segment elevation evaluated both the analytical characteristics of hs-cTnI and
myocardial infarction (NSTEMI) [1]. These recommendations hs-cTnT assays most used in Italy and the distribution of
were meant to be tailored on the specific needs of the Italian values of these biomarkers in an Italian population with the
healthcare system [1], had a wide diffusion and were applied calculation of the 99th percentile upper reference level
by many institutions across Italy. (URL). A draft of the document was revised by a group of
The last five years have witnessed a rapid development external experts belonging to different disciplines: Emer-
of high-sensitivity assay methods for cardiac troponin I and gency/Urgency Medicine, Cardiology and Laboratory Med-
T (hs-cTnI and hs-cTnT) [2]. The results obtained with these icine. Finally, the approval of the Boards of the Italian
new assays have allowed a better understanding of the scientific societies involved was asked.
pathophysiology of myocardial injury as well as a more
accurate interpretation of circulating levels of cTn in
healthy adults, both at rest and during physical exercise Definition, analytical
[2, 3]. This has warranted an in-depth revision of guidelines
published in the previous years by the most important in-
characteristics and clinical results
ternational scientific societies in Cardiology and Labora- of hs-cTnI and hs-cTnT methods in
tory Medicine. These recommendations have revolutioned
both the definition of myocardial damage and the diag-
healthy subjects and in patients
nostic algorithms for myocardial infarction (MI) [4–9]. In with cardiovascular disorders
particular, we must remember some recent contributions of
Italian researchers, such as the documents published in hs-cTnI and hs-cTnT measurement is regarded by all
2020 by the Study Group on Myocardial Biomarkers of the guidelines as the optimal strategy to detect myocardial
Società Italiana di Patologia Clinica e Medicina di Labo- injury and to diagnose MI [4–9]. The last 10 years have
ratorio (SIPMeL) on “Myocardial Biomarkers for the Diag- witnessed a significant and progressive improvement of
nosis and Risk Prediction of NSTEMI”, which considered the analytical performance of immunometric methods for
both organizational and clinical issues about the use of cTnI and cTnT [3, 15]. These hs methods can measure
hs-cTn assays, particularly in the ED [10–12]. circulating biomarker levels in the majority of healthy
Based on these premises, the Inter-Society Study subjects of both sexes [2, 3, 15]. The use of hs-cTnI and hs-
Group on Cardiac Biomarkers deemed it essential to update cTnT methods has changed not only our understanding of
2013 recommendations considering the new evidence. The MI pathophysiology, but also our approach to the diag-
general scheme of the 2013 document was retained: nosis and monitoring of MI [1–11]. In particular, the
(1) Appropriate and correct definitions 2018 document by the American Association for Clinical
(2) Proper indications Chemistry (AACC) and International Federation of Clinical
(3) Use to rule in MI Chemistry (IFCC) established two criteria that are crucial to
(4) Use to rule out MI. define hs-cTn methods [5]. The first criterion is that the
method must measure the threshold value to diagnose MI,
The document was written considering international defined as the 99th percentile URL, with an imprecision
guidelines published from 2018 to 2021. The focus on the (expressed as coefficient of variation, CV) ≤10% [5]. The
Italian setting is justified by the consideration that second criterion is that a hs-cTn method must measure
 Clerico et al.: Use of cardiac troponins in emergency department 171

biomarker levels above a limit of detection (LoD) in a Table : Analytical sensitivity parameters of some hs-cTnI and hs-
reference population including apparently healthy sub- cTnT methods.

jects of both sexes with at least 300 women and 300 men
Methods LoB, LoD, LoQ % LoQ % References
[3, 5, 15, 16]. The requirement for at least 600 individuals is
ng/L ng/L CV, ng/L CV, ng/L
critical to calculate the 99th percentile URL with an
hs-cTnI
acceptable confidence interval [3, 5, 15, 16]. As women have
ARCHITECT . . . . [, ]
on average significantly lower hs-cTn than men of the same
ACCESS DxI . . . . [, ]
age, this second criterion basically requires that hs-cTn can ADVIA XPT . . . . [, ]
be measured in a subgroup of at least 300 apparently VITROS . . . . []
healthy women [3, 5, 15, 16]. hs-cTnT
hs-TnI and hs-TnT methods have LoD values usually ECLIA  –   [, ]

ranging from 1 to 3 ng/L (Table 1), corresponding to the The analytical sensitivity parameters reported in the Table have been
troponin content of around 5–8 mg of myocardium, evaluated in the same reference laboratory using standardized
while the 99th percentile URL (i.e., the threshold to protocols, as previously reported in detail refs. [, , –]. LoB,
limit of blank; LoD, limit of detection; LoQ % CV, limit of
diagnose myocardial injury) corresponds to the content of
quantification at % CV; ARCHITECT, Architect Highly Sensitive TnI
30–40 mg of myocardium [2, 3, 15–18]. A myocardial method for the Architect iSR platform (Abbott Diagnostics, Roma,
damage involving a few mg of tissue is well below the Italy) [, ]; CCESS DxI, Access hsTnI method for the DxI platform
detection limit of echocardiography or other cardiac im- (Beckman Coulter, Inc. Brea, CA  USA) [, ]; ADVIA, ADVIA
aging techniques [2, 3, 15–18]. Centaur High-Sensitivity Troponin I (TNIH) method for the Centaur XPT
platform (Siemens Healthineers, Milano, Italy) [, ]. VITROS,
In 2018, the Fourth Universal Definition of MI has
VITROS High-Sensitivity Troponin I Assay method (Ortho Clinical
established that a myocardial injury is present when there
Diagnostics, Illkirch CEDEX, France) for the platform VITORS  [];
is at least one hs-cTnI or hs-cTnT value above the 99th ECLIA, Elecsys Troponin T-hs method for the Cobas platform (Roche
percentile URL [6]. This document recommends that hs-cTn Diagnostics Italia, Monza) [, ].
methods are used in all subjects with suspected cardiac
widely skewed distribution of hs-cTnI and hs-cTnT in
disease, and not only of acute coronary syndrome (ACS)
healthy populations, the daily renewal of cardiomyocytes
[6, 11, 18, 19].
must increase on average of 10–15 times compared to the
Many authors have suggested that hs-cTnI and hs-cTnT
median of distribution (about 1–4 ng/L) before trespassing
levels in a healthy adult subject must be considered a reliable
the 99th percentile URL (Table 2) [3, 12, 22].
indicator of the physiological daily renewal of myocardial
tissue [2, 3, 15–18]. The 99th percentile URL values in healthy
adults (17–35 ng/L) (Table 2) are compatible with a daily
turnover of about 30–40 mg of myocardial tissue, in agree- Automated platforms and point-of-care
ment with experiments on humans and other mammals testing (POCT)
[2, 3, 15–18].
Recently, a large number of studies and three meta- Until 2020, the POCT methods for cTn could not be used to
analyses have demonstrated that hs-cTnI or hs-cTnT below diagnose NSTEMI because they did not met the quality
the 99th percentile URL, but within the third tertile of dis- requirements specified in international guidelines [7–9].
tribution in the reference population (and then within The introduction of POCT methods to measure hs-cTn
normal levels), are associated with a significantly higher represents an important progress given that these tests
cardiovascular risk than biomarker levels in the first tertile allow an earlier diagnosis by reducing the turnaround time
[20, 21]. The very good performance of hs-cTn for risk (TT) [7, 30–34]. Furthermore, POCT methods for hs-cTn
stratification is due to their high analytical sensitivity could enable an effective screening of patients with sus-
(Table 1) and their small intra-individual variability, which pected ACS at home, in the ambulatory setting or in the
is on average only 9% [22]. ambulance, with a great reduction of the times to diag-
Some recent studies reported that within the range nosis, treatment and admission to specialized structures
from LoD to the 99th percentile URL, a >30% variation [7, 30–34]. A reduction in the ischemic time translates in a
between two measurements of hs-cTnI or hs-cTnT in smaller area of necrosis, leading to smaller infarct size, less
different times in a same individual can be deemed sig- arrhythmias, and a lower likelihood of adverse ventricular
nificant [3, 21–25]. Accordingly, the >30% difference can be remodeling [7–9].
used to assess changes between two hs-cTn values also in In 2016, the Study Group of Myocardial Biomarkers of
patients with suspected ACS in the ED [3, 22]. Given the the SIPMeL proposed diagnostic and therapeutic
172 Clerico et al.: Use of cardiac troponins in emergency department

Table : Distributions of plasma cardiac troponins I concentrations measured with some commercially available high-sensitivity methods in
an Italian reference population (age ranging from  to  years, mean age . years).

Population th perc., Median, th perc., .th perc., th perc.,
(number of subjects) ng/L ng/L ng/L ng/L ng/L

ARCHITECT
Women (n=) . . . . .a (.–.)
Men (n=) . . . . .a (.–.)
ACCESS DxI
Women (n=) . . . . .a (.–.)
Men (n=) . . . . .a (.–.)
ADVIA XPT
Women (n=) . . . . .a (.–.)
Men (n=) . . . . .a (.–.)

More information on the demographic characteristics of the reference population were previously reported [, , , ]. ath percentile
(% confidence interval) calculated by bootstrap method, as previously described [, , , ].

pathways for patients admitted to the ED with suspected The methods above for cTnI meet the analytical re-
NSTEMI [34]. Clinical studies evaluating POCT methods quirements for a hs assay (Table 3). They allow a rapid
with analytical performance meeting the requisites time to response and have excellent sensitivity and repro-
for hs-cTn methods have been published from 2019 ducibility, without a reduction in specificity. Nonetheless,
onwards (Table 3). Sorensen et al. [35] compared the validation of POCT methods for hs-cTnI has not been
the diagnostic accuracy of this method with the hs-cTnI completed. As recently remarked by the National Institute
Architect method using both the rapid 0 h/1 h algorithm for Health and Care Excellence (NICE) guidelines [9], the
and the standard 0 h/3 h algorithm in a first group of 669 most important limitation of these studies is that POCT
patients, with a validation cohort of 610 patients. In methods were employed on plasma samples stored at very
2020, Boeddinghaus et al. [36] compared the diagnostic low temperatures for months to years [35–37]. Therefore,
efficiency of a POCT hs-cTnI-TriageTrue method values measured could not correspond to those in fresh
compared to several hs-cTnI and hs-cTnT methods used plasma samples. More recently, Gopi et al. [38] analyzed
in different centers, assessing 1,261 patients (178 of the analytical performance and results of the POCT
whom with MI, 14%). The POCT hs-cTnI-TriageTrue PATHFAST hs-cTnI method in 224 samples of plasma from
method displayed similar analytical performance (Ta- 191 patients admitted to the ED with suspected NSTEMI.
ble 3) and diagnostic accuracy to hs-cTnI or hs-cTnT This study demonstrated that whole-blood samples can be
methods used in each center to diagnose MI [36]. In 2021, used interchangeably with plasma [38].
Apple et al. [37] evaluated the analytical performance In agreement with NICE guidelines [9], we can conclude
and calculated the 99th percentile URL of the POCT that, at the present time, some of the most recent POCT
Atellica VTLi hs-cTnI method. This study [37] showed methods for hs-cTnI measurement have a similar analytical
that this method meets the requirements for a hs-cTnI performance than hs-cTn methods using completely auto-
method [5]. mated platforms. Nevertheless, further studies are needed to

Table : Parameters of analytical sensitivity and th percentile values of some POCT methods for cTnI assays.

Author (refs.) Method LoD, LoQ % th percentile URL,

ng/L CV, ng/L ng/L

Sorensen et al. [] PATHFAST cTnI-II assay (LSI Medience Corporation; . . W . (.–.)a
Mitsubishi Chemical Europe, Düsseldorf, Germany) M . (.–.)a
Boeddinghaus et al. [] TriageTrue high sensitivity troponin I test, Quidel .–. .–. W . (.–.)b
Corporation, San Diego, California) M . (.–.)b
Apple et al. [] POC Atellica VTLi immunoassay (Siemens Healthineers, . W . (.–.)b
Eindhoven,The Netherlands) M . (.–.)b

LoD, limit of detection; LoQ % CV, limit of quantification at % CV; W, women; M, men. a% confidence interval, b% confidence interval.
 Clerico et al.: Use of cardiac troponins in emergency department 173

establish the accuracy of the cut-offs used and particularly if but only for a first screening when hs-cTn methods are
the cost/benefit ratio of these new POCT methods can be not available.
compared to hs-cTnI and hs-cTnT assays currently used in
clinical laboratories.

Algorithms for the diagnosis of

Take-home messages NSTEMI: what the most recent
– International guidelines consider mostly hs methods. international guidelines say
– Clinical studies are generally performed in centers
with a high degree of specialization, and patient se- The 2018 Fourth Universal Definition of MI [6] recommends
lection often differs across studies. the use of sex-specific cut-offs to diagnose MI and stresses
– Results from these studies cannot be readily translated that significant differences exist between 99th percentile
to the real situation of Italian institutions, therefore a URL values of different hs-cTnI methods. The diagnosis of
close collaboration between clinicians and Laboratory MI relies on the kinetics of hs-cTnI and hs-cTnT circulating
Medicine specialists is needed to identify decisional levels, evaluated through standardized diagnostic algo-
levels and the most effective diagnostic pathways for rithms, and should be made when there are increasing or
each institutions. decreasing biomarker levels, together with evidence of
– Increasing circulating levels and above the critical myocardial ischemia based on signs, symptoms, imaging
difference (>30%) of hs-cTnI or hs-cTnT over hours, data or autopsy [6].
days or months, even within the normal range, are The NICE guidelines [9] published in August 2020
associated with a worse outcome, not only in patients specified the analytical requirements (including the sex-
with heart failure or ACS, but also in apparently specific 99th percentile URL) and results of clinical studies
healthy, asymptomatic subjects. using 9 hs-cTnI and 2 hs-cTnT methods that can be used to
– Although some recent POCT methods for the mea- diagnose MI [9]. These guidelines also identified two
surement of hs-cTnI have an analytical performance challenging scenarios. The first one is the finding of a
comparable to hs-cTnI methods currently used in hs-cTnI or hs-cTnT value lower than or equal to the LoD on
clinical laboratories and extensively validated, there is admission to the ED to exclude MI (rule-out). The second
currently no multicenter study assessing the cost/ one is a strategy with repeated measurements to confirm or
benefit ratio of these new methods in patients with exclude the diagnosis of MI. NICE guidelines recommend
suspected NSTEMI. mostly the 0/3 h algorithm for both rule-in and rule-out [9].
Recommended threshold values for rule-in and rule-out
and 99th percentile URL values should be differentiated
based on the specific method and patient sex. Strategies
Recommendations based on multiple measurements have usually a better
diagnostic accuracy than those based on a single mea-
– hs assays for the measurement of cTnI and cTnT are surement [9]. Furthermore, these guidelines recommend
immunometric methods able to measure the 99th that biomarker values be interpreted according to the
percentile URL of a reference population with a CV≤10. clinical presentation. Most notably, a patient with negative
– hs methods can also detect circulating cTn in at least hs-cTn measurement should not be hastily discharged
one half of a reference population including at least without further clinical exams, particularly when symp-
300 men and 300 women. toms last from less than 2 h, which could account for the
– Methods that measure the 99th percentile URL in the lack of a significant increase of the biomarker [9]. NICE
reference population between 10 and 20% can still be guidelines also emphasize that the diagnostic accuracy of
used in the clinical practice. They must be defined as the ECLIA Elecsys hs-cTnT and the Architect hs-cTnI
contemporary sensitivity methods, and cannot be methods have been evaluated by multiple studies
defined as hs. Guidelines strongly recommend that (30 and 9, respectively), while all other hs-cTnI methods
clinical laboratories adopt as soon as possible hs-cTn have been validated in few or no studies [9]. The same
methods. guidelines notice that limited evidence is available on the
– Methods measuring the 99th percentile URL with a comparison between the diagnostic accuracy of different
CV≥20% should not be used anymore to diagnose MI, methods and particularly the cost/benefit ratios of the
174 Clerico et al.: Use of cardiac troponins in emergency department

different diagnostic algorithms employing different hs- Another relevant aspect is the time from onset of
cTnI and hs-cTnT methods [9]. The differences between ischemic symptoms. The Fourth Universal Definition of MI
different methods in risk stratification of patients with [6] identifies a special group of patients presenting late to
possible ACS have been specifically evaluated [39]. the ED (“late presenters”). These patients could be evalu-
The 2020 European Society of Cardiology (ESC) guide- ated when the peak concentration has already been reached
lines [7] recommend the most rapid algorithms for the and so the circulating levels of biomarker are decreasing
diagnosis of MI in patients admitted to the ED with sus- [6, 8, 57]. cTn decrease is much lower than the rapid increase
pected ACS. The first option is the 0 h/1 h algorithm, which during the first hours of ischemia. Therefore, variations in
involves blood sampling on admission and after 1 h. As a biomarker values can be difficult to detect across a few
second option, the 0 h/2 h algorithm is proposed, with blood hours (as in the 0 h/1 and 0 h/2 h algorithms), especially
sampling on admission and after 2 h [7]. ESC guidelines also when infarcts areas are small [6, 8, 57]. Therefore, clinicians
propose algorithms with different cut-offs for each method. must be careful of these “late presenters”, who may also
The assessment of biomarker kinetics in these guidelines is present elevated hs-cTnI or hs-cTnT (and then a confirmed
based on cut-offs that express the absolute difference be- myocardial damage), but small variations in biomarker
tween concentrations at baseline and after 1 or 2 h, defined levels during the observation period (from 1 to 3 h) after
as “ delta (Δ) change” and expressed as ng/L. admission to the ED, possibly leading to an underdiagnosis
The preference for more rapid algorithms is justified by of MI [8, 10, 57]. According to some studies, this group of
the consideration that reducing the time to diagnosis pro- patients could account for 26% of cases of MI [8].
motes an earlier anti-ischemic treatment and then the Importantly, the cut-off values based on absolute dif-
salvage of larger areas of myocardium still reversibly ferences (Δ change) recommended by 2020 ESC guidelines
damaged [7]. Furthermore, ESC guidelines emphasize that [7] for use in the rapid algorithms (0 h/1 h or 0 h/2 h) for
cut-off values to rule out or rule in MI were validated in ECLIA hs-cTnT method were validated in multicenter
many multicenter studies [40–50]. hs-cTn concentrations clinical trials. However, both NICE guidelines [9] and the
recommended as critical thresholds to exclude MI gener- IFCC document [8] report that there are still insufficient
ally correspond to a minimal sensitivity value associated data on the cut-off values based on absolute differences
with a ≥99% negative predictive value. These guidelines [7] (Δ change) for some hs-cTnI methods.
also state that rapid algorithms have demonstrated in
many clinical studies a balance between efficacy and safety
that is not significantly different from the standard 0/3 h Considerations on guideline
algorithm recommended by 2015 ESC guidelines [4].
In February 2021, the document on the diagnosis of recommendations
NSTEMI by the International Federation of Clinical Chemistry
(IFCC) Committee on Clinical Application of Cardiac Bio- Application of rapid diagnostic algorithms in
markers was published [8]. This document critically reap- the clinical practice in Italy
praised some recommendations by 2020 ESC guidelines [7].
The first controversial point was that 2020 ESC guidelines do Only about 30% of patients admitted to the ED with chest
not recommend the use of sex-specific cut-offs, particularly pain receives a final diagnosis of MI [6, 7]. A rapid evaluation
regarding the 99th percentile URL [7], as a threshold for of patients with a low probability of MI allows a more effi-
myocardial damage and to diagnose MI, as explicitly cient and rapid management of patients admitted to the ED,
requested by the Fourth Universal Definition of MI [6]. Both also reducing costs, and decreases the time to diagnosis,
the IFCC document [8] and NICE guidelines [9] recommend enabling an early and specific treatment that improves out-
sex- and method-specific cut-offs because many studies have comes [7, 58, 59]. Accordingly, 2020 ESC guidelines strongly
demonstrated that differentiation based on sex allows a more recommend rapid diagnostic algorithms to promptly identify
accurate diagnosis, particularly for hs-cTnI methods and in patients at lowest risk, who can be monitored on an outpa-
women [51–56]. On the contrary, the utility of sex-specific cut- tient basis or outside of cardiac intensive care units [7].
offs has not been definitely demonstrated for hs-cTnT Nonetheless, some considerations about the organization of
methods, given that the mean difference between values in clinical activity are warranted to better understand some
men and women in the different reference populations is obstacles to the introduction of rapid diagnostic algorithms
around 5 ng/L, which approaches the LoD of the method in clinical practice. While the diagnostic and prognostic
(Table 1), while for example this difference is 11 ng/L for the utility of the 0/3 h algorithm has been extensively validated,
hs-cTnI Architect method [16]. and this algorithm has also a favorable cost/benefit ratio
 Clerico et al.: Use of cardiac troponins in emergency department 175

compared to previous longer algorithms (6–12 h) [1, 4, 6, 9], is used, because circulating levels of hs-cTnI and hs-cTnT
the rapid algorithms have not been accurately validated depend on sex, age, time from ischemia onset, extent of the
using all hs-cTnI methods [8, 9]. Additionally, as also ischemic and necrotic area and comorbidities [9, 11, 17, 18,
observed in the document by the IFCC [8], the rapid algo- 64–70]. In particular, severe chronic kidney disease can
rithms are difficult to implement in the majority of hospitals significantly alter biomarker kinetics [9, 17, 18, 67, 70].
around the world. In particular, a 2019 study including 1,902 The protein chain of cardiac troponins (molecular
centers in 23 countries across five continents reported that in weight of about 30–40 kDa) is rapidly degraded in the
Europe only 60% of hospitals had adopted hs-cTn methods, circulation, particularly in their terminal portions, there-
and could then potentially employ rapid algorithms [60]. fore fragments with a lower molecular weight (around
While specific data for Italy are not available, even the 14–20 kDa) can already be detected during the first hours
implementation of the 0/3 h algorithm rather than longer after an MI and can persist in the bloodstream for several
algorithms has required a profound reorganization of ED days, becoming the prevalent circulating forms of the
activity in many centers [1, 61–63]. Considering that even the biomarker at the end of the kinetic curve [3, 11, 17, 18,
most recent hs-cTnI and hs-cTnT methods using automated 64–70]. Furthermore, the three cardiac troponins of the
platforms have a mean time to analysis of 15–25 min, sarcomere complex (TnI, TnC and TnT) can bond with
obtaining the result and sending it to the ED within 60 min each other and with other plasma proteins [71]. Some
seems almost impossible in almost all clinical laboratories in patients can also develop autoantibodies against cTn
Italy, including those located in close proximity of the ED molecules, forming circulating complexes that affect the
and that can devote a specific section to biomarker mea- measurement of cTn with immunometric methods [3,
surement to this analysis. 72–75]. Therefore, the presence of different circulating
2020 ESC guidelines suggest that blood sampling for forms and the formation of complexes with other circu-
hs-cTn measurement must always be performed at base- lating proteins not only alter cTn kinetics, but also
line and after 1 h, whether the result of baseline mea- reduce the reliability of cTn measurement, since
surement has been reached or not. Furthermore, these different immunometric methods show a heterogeneous
guidelines recommend that patients not ruled out nor specificity for degraded forms of troponins and com-
ruled in after 1 h undergo a third blood sampling 3 h after plexes between troponins and other plasma proteins or
admission [7]. The 0 h/2 h algorithm seems more reason- autoantibodies [3, 12, 72–75].
able for Italian institutions compared to the 0 h/1 h algo- Árnadóttir et al. [76] recently evaluated the kinetics of
rithm. Indeed, in most cases the 1-h sampling would be hs-cTnI (measured through two different methods) and
performed without knowing the result of the baseline hs-cTnT over 240 min in 34 humans after myocardial
measurement, with the possibility of an useless blood ischemia induced through an intracoronary balloon infla-
sampling (when baseline value already allowed to exclude tion during an elective coronary angiography. These
MI) or alternatively because the 1-h measurement would patients were divided into four groups according to
still not be sufficient to rule out or rule in MI, thus requiring ischemia duration (0, 30, 60 and 90 s). Circulating
a further sampling at 3 h or later. The 0 h/2 h algorithm biomarker levels increased significantly from 15 to 240 min
should be more accurate than the 0 h/1 h algorithm, with all hs-cTnI and hs-cTnT methods, but significant dif-
particularly to rule in MI. If the 2-h measurement does not ferences in biomarker kinetics were observed across the
show a significant variation of the biomarker, a further three methods [76]. In particular, after a 60-s ischemia,
measurement at 3 h or later would be required, but the hs-cTnI (measured through the ADVIA Centaur method)
doubled after 45 min, while with the hs-cTnI Architect
same would have occurred if the 0 h/1 h algorithm were
method after 90 min and with the hs-cTnT methods after
pursued. This possibility could not be infrequent in late
135 min [76]. Furthermore, changes in hs-cTnI over time
presenters, as discussed above.
were greater than those of hs-cTnT and the kinetics were
more accurately represented by an exponential than a linear
curve [76]. These results support the hypothesis that cardiac
Pathophysiological and clinical troponins can be released also following a reversible
considerations on diagnostic algorithms for myocardial damage, without cardiomyocyte necrosis;
NSTEMI indeed, hs-cTnI and hs-cTnT reached values above the 99th
percentile URL already with ischemia durations of 30 and
Patients with NSTEMI show different biomarker concen- 90 s, which according to the Authors should not induce
trations and kinetics, even when the same hs-cTn method cardiomyocyte necrosis [76].
176 Clerico et al.: Use of cardiac troponins in emergency department

The results of this study are in agreement with those by be promptly referred to coronary angiography. The same
Pickering et al. [77], who found that hs-cTnI increases more reasoning can apply to patients who display an increment,
rapidly than hs-cTnT in patients with NSTEMI and that there after 1 or 2 h, much higher than 30% (such as 40 or 50%),
are significant differences between patients in biomarker but even in this case do not reach the threshold of 99th
kinetics even when the measure is performed with the same percentile URL (considering the sex- and method-specific
method. Furthermore, the kinetic of both troponins during URL). Contrary to 2020 ESC guidelines [7], NICE guidelines
the first 6 h is log-linear (and then exponential) [77]. Similar [9] and the IFCC document [8] suggest that these patients
findings were reported in a more recent multicenter study be reevaluated with a further sampling at 3 h or later to
that evaluated only the variations within the first 2 h with check if the 99th percentile URL is reached, as requested for
two hs-cTnI methods (hs-cTnI Architect and ADVIA Centaur) the diagnosis of myocardial injury and then MI [6].
and the hs-cTnT method [78]. Asymptomatic individuals from the general population
Considering the large differences in many studies be- with hs-cTnI and hs-cTnT values in the third tertile of refer-
tween hs-cTnI and hs-cTnT levels in patients with NSTEMI ence values (and then still below the 99th percentile URL)
during the first hours after admission to the ED [3, 8, 11, 12, have a significantly higher risk of death and major cardio-
18, 76–78], it is reasonable to expect that Δ changes sug- vascular events even in the median term (from 6 months to 2
gested by 2020 ESC guidelines [7] for rule in and rule out in years), compared to subjects in the first tertile [20, 21, 79, 89].
rapid algorithms, expressed as absolute differences across In general, the cardiovascular risk in the general population
1 or 2 h, are characterized by large confidence intervals, increases first linearly and then exponentially with increasing
which nonetheless are not reported by these guidelines. It values of cardiac-specific biomarkers hs-cTn and also B-type
is crucial that these cut-offs are accurately validated in natriuretic peptides [20, 21].
multicenter studies based on large patient populations. The case of patients with suspected NSTEMI who pre-
These studies have not been actually carried out for several sent a significant change in hs-cTnI or hs-cTnT values,
hs-cTnI methods, as highlighted by NICE guidelines [9] and expressed as absolute or percentage change, over 1–3 h, but
the IFCC document [8]. that still does not reach the threshold of 99th percentile
Another important aspect from an analytical perspec- URL, deserves consideration in clinical studies assessing
tive is that the reference change value between two mea- the cost/benefit ratio of the different diagnostic approached
sures of hs-cTnI and hs-cTnT is on average 30% for based on rapid algorithms for the diagnosis of NSTEMI. At
biomarker concentrations ≥5 ng/L [3, 12, 23–25] (Figure 1). present, considering the results of studies on the cardio-
In other words, although the absolute difference between vascular risk in the general population [20, 21, 79–89] and
two measures is strictly dependent on demographic and in agreement with 2020 ESC guidelines [7], we should
clinical characteristics and the analytical performance of consider patients with suspected NSTEMI who present a
the method, the percentage that denotes a significant significant increase in hs-cTnI and hs-cTnT over a few hours
change between two measures of hs-cTnI or hs-cTnT in a as individuals with a higher risk of acute cardiovascular
same individual is on average the same (i.e., >30%). It is events. Moreover, 2020 ESC guidelines [7] recommend that
then easy to calculate if a biomarker increase or a decrease also Global Registry of Acute Coronary Events (GRACE) risk
can be considered as significant, namely if there is a >95% score should be considered to predict the outcome of patients
probability that the variation is not due to an analytical with a high cardiovascular risk (Class of recommendation I,
error and to the intraindividual biological variability, but level of evidence B). The suggestion by 2020 ESC guidelines
more likely to an ongoing disease process such as a [7] of starting an antithrombotic treatment and referring these
myocardial necrosis [3, 12, 23–25]. patients as soon as possible to coronary angiography seems
Another aspect that might be confusing for clinicians than reasonable, although a careful evaluation of clinical,
who use rapid algorithms is that some patients with sus- ECG and echocardiographic findings remains essential.
pected NSTEMI can show hs-cTnI and hs-cTnT concentra- Another important clinical issue is the influence of
tions higher than cut-off values, calculated based on Δ reduced glomerular filtration rate (GFR) on the kinetics of
changes, after 1 or 2 h, and then be ruled in, even though hs-cTnI and hs-cTnT in patients with AMI. Indeed, patients
not having any value higher than the 99th percentile URL. with renal disease can have increased circulating levels and
According to the Fourth Universal Definition of MI [6], cut-off values of hs-cTnI and hs-cTnT due to the reduced
these patients do not display a myocardial damage, which GFR [90–92]. Accordingly, assay-specific optimal cutoff
is considered by this document as an essential prerequisite levels for hs-cTnI and hs-cTnT methods adjusted for GFR
for the diagnosis of MI. According to 2020 ESC guidelines values should be considered [90–92]. Furthermore, the
[7], these patients should be ruled in for NSTEMI and then diagnostic performance of hs-cTnI and hs-cTnT methods in
 Clerico et al.: Use of cardiac troponins in emergency department 177

Figure 1: Imprecision profile of 4 hs-cTnI methods and the hs-cTnT method.

The imprecision profile was evaluated in the same laboratory (Fondazione CNR Regione Toscana G. Monasterio, Pisa) using a standardized
protocol, as described in details in previous papers [15, 23–29]. Plasma samples (usually 10 to 14 for each method) of healthy adult subjects or
patients with cardiac disorder and different biomarker concentrations (from about 2 ng/L to around 50 ng/L) were measured several times over
at least 2 months using at least two different lots of the same method. The values from hs-cTnI and hs-cTnT methods are reported with different
colors and symbols. The equation best fitting the values from different methods is reported, together with the R coefficient of correlation. The
dashed line indicates the critical value of analytical imprecision (10% coefficient of variation) recommended by guidelines for the 99th
percentile upper reference limit [5]. The methods evaluated were: (i) cTnT ECLIA: Elecsys Troponin Gen five STAT Immunoassay
electrochemiluminescence immunoassay (ECLIA) (Roche Diagnostics). (ii) cTnI Access: Access hsTnI with the DxI platform (Beckman Coulter
Inc.). (iii) cTnI ADVIA: ADVIA centaur high-sensitivity troponin I, with the Centaur XPT platform (Siemens Healthineers Diagnostics). (iv) cTnI
Architect: ARCHITECT STAT High Sensitive Troponin-I, with the ARCHITECT i1000SR platform (Abbott Diagnostics Division).

patients with renal disease and suspected NSTEMI can be by 2015 ESC guidelines [4]. A further sampling at 6 h could be
improved by use of algorithms taking into account both considered, according to clinical judgment, when no sig-
admission troponin and dynamic changes in biomarker nificant change was observed during the first 3 h. Biomarker
concentrations [90–92]. kinetic was deemed suggestive for myocardial necrosis if
circulating levels increased by at least 50% from baseline [1].
MI could be diagnosed in the presence of significant
biomarker variations together with signs and/or symptoms
Proposals for the use of diagnostic suggesting myocardial ischemia [1].
algorithms for hs-cTnI and hs-cTnT methods These recommendations seem still valid in the current
in patients with suspected NSTEMI in the setting of Italian institutions, given that the transition from
Italian setting algorithms lasting ≥6 h to the 0/3 h algorithm is still
ongoing in many hospitals [62, 63]. Moreover, rapid algo-
In the 2013 document, the Inter-Society group proposed that rithms endorsed by 2020 ESC guidelines [7], particularly
the 0/3 h algorithm be adopted as soon as possible in Italian the 0 h/1 h algorithm, do not seem to be applicable in
institutions [1]. The same algorithm was then recommended almost all Italian centers.
178 Clerico et al.: Use of cardiac troponins in emergency department

All the most recent guidelines recommend that Tn methods [93]. Considering the large differences in
NSTEMI is ruled out when admission value is lower than or organization and patient admission to ED in Italy
equal to the LOD of the method [4, 7–9]. Similarly, if the [1, 10–12, 61–63, 94, 95], the rapid algorithms could be
admission value is higher than around 5 times the 99th mostly easily implemented in highly specialized car-
percentile URL, as indicated in a specific table of 2020 ESC diovascular centers where there is a laboratory dedi-
guidelines [7], but also by other guidelines [4, 6, 8, 9], we cated to urgencies and a well-organized network for
can reasonably rule in NSTEMI. When patients are ruled the management of ACS [94, 95].
out or ruled in based on a single sample, given that
values ≤LOD or well above the 99th percentile URL are
considered, sex- (or even age-) specific cut-offs are not Research funding: None declared.
deemed necessary. Considering the rapid 0 h/1 h and Author contributions: All authors have accepted responsibility
0 h/2 h algorithms, the 2020 ESC guidelines [7] state that for the entire content of this manuscript and approved its
optimal thresholds for rule-out were selected to allow for a submission.
minimal sensitivity and Negative Predictive Value (NPV) of Competing interests: Authors state no conflict of interest.
99%, while optimal thresholds for rule-in were selected to Informed consent: Not applicable.
allow for a minimal positive predictive value (PPV) of 70%. Ethical approval: Not applicable.
In May 2021, Westwood et al. [93] performed a meta-
analysis on behalf of NICE. The authors evaluated hs-cTn
assays for the management of adults presenting with acute
chest pain, in particular for the early rule-out of MI using a
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