Highly sensitive troponin immunoassays: navigating between the scylla and charybdis

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From: Giuseppe Lippi, Martina Montagnana, Rosalia Aloe and Gianfranco Cervellin, Highly Sensitive Troponin Immunoassays: Navigating between the Scylla and

Charybdis. In Gregory S. Makowski, editor: Advances in Clinical Chemistry, Vol. 58, Burlington: Academic Press, 2012, pp. 1-29.

ISBN: 978-0-12-394383-5 © Copyright 2012 Elsevier Inc.

Academic Press

ADVANCES IN CLINICAL CHEMISTRY, VOL. 58

Author's personal copy

HIGHLY SENSITIVE TROPONIN IMMUNOASSAYS:NAVIGATING BETWEEN THE SCYLLA AND CHARYBDIS

Giuseppe Lippi,*,1 Martina Montagnana,† Rosalia Aloe,*and Gianfranco Cervellin‡

*U.O. Diagnostica Ematochimica, Dipartimento di Patologiae Medicina di Laboratorio, Azienda Ospedaliero-Universitaria

di Parma, Parma, Italy†Sezione di Chimica Clinica, Dipartimento di Scienze dellaVita e della Riproduzione, Universita di Verona, Verona, Italy

‡U.O. Pronto Soccorso e Medicina d’ Urgenza, AziendaOspedaliero-Universitaria di Parma, Parma, Italy

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orresponding author: Giuseppe Lippi, e-mail: [email protected]; [email protected]; gius

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2. I
ntroduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. A
cute Coronary Syndrome and Myocardial Infarction . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1.
E lectrocardiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.2.
B iomarkers of Myocardial Necrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. B
iochemistry and Biology of Troponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. C
linical Significance of the Measurement of Cardiospecific Troponins . . . . . . . . . . . 10
5.1.
T raditional and ‘‘Dynamic’’ Approach to Interpret Troponin Values . . . . . . . 13
5.2.
I nfluence of Clinical and Demographical Variables on Troponin Values . . . . 16
6. C
onclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
R
eferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1. Abstract

The recent development and introduction into clinical and laboratory

practice of the novel highly sensitive cardiac troponin assays has contributed

to improve the diagnosis and risk stratification of patients presenting with

suspected myocardial injury and acute coronary syndrome at the emergency

pi@

Inc.rved.

2 LIPPI ET AL.


department (ED). The enhanced ability to detect very low amount of tropo-

nin in blood with the novel tests has also raised the challenge of intercepting

values above the 99th percentile of the reference population in apparently

healthy subjects as well as several patients with comorbidities different from

myocardial infarction. As such, the diagnostic approach and the triage of

chest pain patients need to be readjusted and, most probably, the role of

clinical judgment will assume greater importance than with the old genera-

tion immunoassays. In this perspective, a strategy based on the ‘‘delta

biomarker approach’’ rather than that based on the traditional ‘‘peak’’

value might be more viable, since the observation of a dynamic pattern of

troponin values might help discriminating the timing of injury as well as

distinguishing acute from chronic etiologies and thereby increasing both the

specificity and the positive predictive value (PPV) of the test. Nevertheless,

this approach would require an accurate definition of the reference ranges as

well as the identification of the magnitude of increase or decrease during

serial sampling. We are thereby navigating between the Scylla (i.e., definition

of appropriate 99th percentile of the reference population) and Charybdis

(i.e., identification of a reliable delta threshold) of the novel highly sensitive

immunoassays. The aim of this article is to provide an overview on the

clinical impact of the highly sensitive cardiac troponin immunoassays in

the differential diagnosis of patients presenting to ED with thoracic pain

suggestive for acute coronary syndrome.

2. Introduction

As recently emphasized by Institute of Medicine’s (IOM) Committee on

the Future of Emergency Care in the United States Health System (USHS),

the emergency care system is facing an exponentially increasing epidemic of

crowded EDs, patients boarding in hallways waiting to be admitted, and

daily ambulance diversions. As an example, nearly 114 million visits were

made to hospital EDs in the US in 2003, that is, more than one for every three

people. The most common medical diagnoses among ED patients, excluding

injuries, were acute upper respiratory infections (5.7%), abdominal pain

(3.9%), chest pain (3.7%), and spinal disorders (2.5%) [1].

Hospital-based emergency care is facing the emerging challenge of balanc-

ing the roles of hospital-based emergency and trauma care, not simply urgent

and lifesaving care, but also safety net care for uninsured patients, public

health surveillance, disaster preparation, and adjunct care in the face of

increasing patient volume and limited resources. In particular, 91% of EDs

responding to a national survey already reported overcrowding as a problem,

and almost 40% reported that overcrowding occurred on a daily basis [1].

HIGHLY SENSITIVE TROPONIN IMMUNOASSAYS 3


Increase in disease prevalence in the forthcoming decade is also expected to

worsen ED occupation and most part of these ED visits will be predictably

related to neurological and cardiovascular disease, especially acute coronary

syndrome (ACS), and acute myocardial infarction (AMI). Therefore, the

Committee on the Future of Emergency Care in the USHS recommends

that hospital chief executive officers adopt enterprise wide operations man-

agement and related strategies to improve the quality and efficiency of

emergency care. Interdisciplinary working groups including experts in emer-

gency care, inpatient critical care, hospital operations management, nursing,

and other relevant clinical disciplines such as laboratory medicine should

also be established to develop standards of practice, as well as guidelines and

recommendations to implement efficient strategies for the most appropriate

triage of the patients in the ED, for the timely and reliably identification

of acute conditions that need urgent treatment and for discharging patients

who do not require hospitalization or monitoring in observation units [1].

Biomarkers of myocardial ischemia, injury, and necrosis can undoubtedly

support this process. The improved analytic sensitivity of the highly sensitive

troponin immunoassays has remarkably increased the capability to detect

even modest and clinically questionable signs of myocardial injury, thereby

limiting the potential usefulness of these novel immunoassays when an

appropriate strategy for their use is not established. The more challenging

issues are the accurate definition of the reference ranges as well as the

identification of the magnitude of increase or decrease during serial sampling,

to limit the burden of ‘‘false positive’’ cases of AMI and thereby prevent

overcrowding in the EDs. The aim of this article is to provide a systematic

overview of the current scientific literature about the clinical use of the highly

sensitive troponin assays. Both PubMed and Google Scholar were systemati-

cally searched for the terms ‘‘highly sensitive,’’ ‘‘troponin,’’ ‘‘reference

change value (RCV),’’ and ‘‘reference range’’ and the most relevant articles

were selected and discussed.

3. Acute Coronary Syndrome and Myocardial Infarction

Chest pain represents the third leading cause of ED occupation, whose

prevalence is expected to increase steadily in the forthcoming years and that

will thereby pose a great organizational and economical burden on hospital-

based emergency care [1]. Moreover, extended ED length of stay for chest

pain patients imposes substantial ED opportunity costs and decreased po-

tential revenue [2]. It is also noteworthy that patients with ACS/AMI have

an increased risk of short-term adverse cardiovascular outcomes (i.e., death,

cardiac arrest, delayed AMI, development of congestive heart failure,

4 LIPPI ET AL.


ventricular tachycardia or fibrillation, supraventricular dysrhythmias, symp-

tomatic bradycardia, hypotension) when they are admitted during time

of high ED occupancy (odds ratio [OR]¼3.1, 95% confidence interval

[CI]¼1.0–9.3) [3]. All those interventions aimed at improving the diagnosis

of ACS/AMI in the ED and consequently reducing delays in hospital admis-

sions have thus a great potential to improve patient’s outcomes as well as

hospital revenues.

The American College of Cardiology/American Heart Association (ACC/

AHA) [4,5], the American College of Cardiology Foundation/AHA Task

Force [6], and European Society of Cardiology (ESC) [7] consensus guide-

lines recognize the importance of early risk stratification in the management

of chest pain patients. Nevertheless, although the vast array of risk stratifi-

cation tools available such as the Goldman rule, PURSUIT (Platelet glyco-

protein IIb/IIIa in unstable angina (UA): Receptor Suppression Using

Integrilin) risk score, GRACE (Global Registry of Acute Coronary Events)

risk score, ACI-TIPI (Acute Cardiac Ischemia-Time Insensitive Predictive

Instrument), and TIMI (Thrombolysis In Myocardial Infarction) risk score

help distinguish high-risk from low-risk patients, they might not allow phy-

sicians to reliably identify patients who are safe for discharge from the ED

without serial cardiac markers or provocative testing [8], since the area under

the curve (AUC) for predicting death or AMI at 1 year of these stratification

tools is typically lower than 0.750 [9].

Patients with suspected ACS/AMI must be evaluated in a very short time

at the ED, since decisions made on the basis of the initial assessment have

substantial clinical and economic implications [10]. The first decision about

the triage is typically made by the patient, who must decide whether and

when to access the healthcare system. Media campaigns such as ‘‘Act in

Time’’ in United States, as well as additional local campaigns across Europe,

provide broad patient education and advise the patient who feel heart attack

symptoms or observe the signs in others to wait no longer than few minutes

(i.e., 5 min) before calling the numbers 9-1-1 in United States or 112 in

Europe, respectively [11,12]. Due to the large number of patients with symp-

toms suggestive of ACS, the heterogeneity of the population, and the risk of

short-term adverse events, a strategy for the initial evaluation and manage-

ment is thereby essential. The most successful strategies are those designed to

identify AMI patients and, sometimes, screen for UA and underlying coro-

nary artery disease (CAD). Most of them use a combination of cardiac

biomarkers, short-term observation, diagnostic imaging, and provocative

testing. An ECG should be performed immediately after presentation and

evaluated by an experienced emergency medicine physician, with a goal of

10 min within ED arrival. When STEMI (ST-segment elevation myocardial

infarction) is diagnosed (see below), the decision about reperfusion strategy



(either fibrinolysis or percutaneous coronary intervention) should be accom-

plished within the following 10 min [13]. Strong evidence has been provided

of a direct relationship between delay in treatment and death [14–16], so that

immediate assessment including a 12-lead ECG is crucial and efforts should

be made to achieve the goal of reperfusion within 90 min since presentation

(door-to-balloon time) [17] The five most important factors deducible from

the initial history that contribute to estimate the likelihood of ischemia due to

CAD include, ranked in the order of importance: the nature of the anginal

symptoms, prior history of CAD, gender, age, and the number of traditional

risk factors [18–22]. Among these, the older age appears the most important

in patients with suspected ACS and without preexisting clinical CAD. The

characteristics of pain/discomfort, which are thoroughly described in the

ACC/AHA 2002 and 2007 Guideline Update for theManagement of Patients

With Chronic Stable Angina [5], include deep, poorly localized chest or arm

discomfort, which is reproducibly associated with physical exertion or emo-

tional stress and is relieved promptly (i.e., in less than 5 min) with rest and/or

the use of sublingual nitroglycerin. Patients with UA/non-STEMI (NSTEMI)

may suffer from a type of discomfort that has all of the characteristics of

typical angina except that the episodes are more severe and prolonged, may

occur at rest, or may be precipitated by less exertion than in the past.

Although the use of the simple term ‘‘chest pain’’ is commonplace to refer

to the typical discomfort of ACS, patients do not often perceive these symp-

toms to be a true pain, especially when they are mild or atypical.

3.1. ELECTROCARDIOGRAPHY

The ECG is crucial in the triage of patients with suspected ACS/AMI not

only to add support to the clinical suspicion of CAD, but also to provide

prognostic information based on the pattern and magnitude of abnormalities

[23–26]. ECG recording performed during the presenting symptoms is partic-

ularly useful. Importantly, transient ST-segment changes (greater than or

equal to 0.05 mV [i.e., 0.5 mm]) developing during a symptomatic episode at

rest and resolving when the patient becomes asymptomatic are highly sugges-

tive of acute ischemia and reflect a high likelihood of an underlying severe

CAD. Patients whose current ECG is suggestive for ischemia can be assessed

with greater diagnostic accuracy when compared with a prior ECG, if avail-

able [27]. The 12-lead ECG lies at the center of the decision pathway for the

evaluation and management of patients with acute ischemic discomfort.

Patients presenting with an elevation of ST-segment greater than or equal to

1 mm (0.1 mV) in at least two contiguous leads are candidate for acute

reperfusion therapy. Patients who present with ST-segment depression are

initially considered to have either UA or NSTEMI; the distinction between

6 LIPPI ET AL.


these two diagnoses is ultimately based on biomarkers of myocardial necrosis

[28–30]. In patients with a clinical suspicion of ACS, marked (i.e., greater than

or equal to 2 mm [0.2 mV]) symmetrical precordial T-wave inversion strongly

suggests acute ischemia, particularly that due to a critical stenosis of the left

anterior descending coronary artery [31]. Patients with this ECG pattern often

exhibit hypokinesis of the anterior wall and are at high risk [32]. Revasculari-

zation will often reverse both the T-wave inversion and wall-motion disorder

[33]. Nonspecific ST-segment and T-wave changes, usually defined as ST-

segment deviation of less than 0.5 mm (0.05 mV) or T-wave inversion of less

than or equal to 2 mm (0.2 mV), are less diagnostically helpful than the previ-

ous findings. Established Q waves greater than or equal to 0.04 s are also less

helpful in the diagnosis of UA, although they do reflect a high likelihood of

significant CAD since they are suggestive of a prior AMI. Isolated Q waves

in lead III may be a normal finding, especially in the absence of repolarization

abnormalities in any of the inferior leads. Noteworthy, a completely normal

ECG in a patient with chest pain does not exclude the possibility of ACS,

because 1–6% of these patients are incidentally diagnosed with AMI, and

up to 4% also suffer from UA [26,34,35]. Additional and common causes

of ST-segment and T-wave changes must always be considered. In patients

with ST-segment elevation, left ventricular (LV) aneurysm, pericarditis, myo-

carditis, Prinzmetal’s angina, early repolarization (e.g., in young black males),

apicalLVballooning syndrome (Takotsubo cardiomyopathy) aswell asWolff–

Parkinson–White syndrome are pathologies to be carefully considered and

eventually ruled out. Cerebrovascular events and drug therapy with tricyclic

antidepressants or phenothiazines can also cause deep T-wave inversion.

A gradient of risk of death and cardiac ischemic events can be established,

based on the nature of the ECG abnormality [25,36,37]. Patients with ACS

and confounding ECG patterns such as bundle branch block, paced rhythm,

or LV hypertrophy are at the highest risk for death, followed by patients with

ST-segment deviation (either elevation or depression). Patients with isolated

T-wave inversion or normal ECG patterns can instead be considered at the

lowest risk. Importantly, the prognostic information of the ECG pattern

remains an independent predictor of death even after adjustment for clinical

findings and cardiac biomarker measurements [36–39]. Because a single

12-lead ECG recording provides only a snapshot of a dynamic process [40],

the usefulness of obtaining serial ECG tracings or performing continuous

ST-segment monitoring has been extensively assessed [24,41]. Although serial

ECGs increase the ability to diagnose UA and AMI [42,43], the diagnostic

efficiency is reportedly higher with serial cardiac biomarker measurements

[43,44]. Nevertheless, the identification of new injury on serial 12-lead ECG

remains the principal eligibility criterion for emergency reperfusion therapy,

regardless of cardiac biomarkers.



As regards patients triage, special units have been established, variously

referred to as ‘‘chest pain units’’ and ‘‘emergency department observation

units’’, to allow a more definitive evaluation and thereby avoid unnecessary

hospital admission of patients with possible ACS and low-risk ACS, as well

as the inappropriate discharge of patients with active myocardial ischemia.

The personnel of these units use critical pathways or protocols designed to

help decide about the presence or absence of myocardial ischemia and, if

present, to characterize it further as UA or NSTEMI and to define the

optimal next step in the care of the patient (e.g., admission, acute interven-

tion) [44]. The obvious target is to achieve a diagnosis after a limited amount

of time, that is, usually between 12 and 24 h, depending on the local policies.

Typically, the patient undergoes a predetermined observation period with

serial cardiac biomarkers and ECGs. At the end of the observation period,

the patient is reevaluated and generally undergoes functional cardiac testing

(e.g., resting nuclear scan or echocardiography) and/or provocative testing

(e.g., treadmill, stress echocardiography, or stress nuclear testing) or nonin-

vasive coronary imaging study. Those patients who have a recurrence of

chest pain strongly suggestive of ACS, a positive biomarker value, a signifi-

cant ECG change, or a positive functional/stress test or Coronary CT Angi-

ography are generally admitted for inpatient evaluation and treatment.

3.2. BIOMARKERS OF MYOCARDIAL NECROSIS

The diagnosis of acute, evolving, or recent MI is typically based on the

detection of increased blood concentrations of biomarkers of myocardial

necrosis in the setting of a clinical syndrome consistent with myocardial

ischemia [45]. According to the ‘‘Universal Definition of Myocardial Infarc-

tion’’ issued by the Joint ESC/ACCF/AHA/WHF Task Force, ‘‘the term

myocardial infarction should be used when there is a rise and/or fall of

cardiac biomarkers along with evidence of myocardial ischemia with at

least one of the following: (a) symptoms of ischemia; (b) ECG changes

indicative of new ischemia (new ST–T changes or new left bundle branch

block); (c) development of pathological Q waves in the ECG; and/or (d)

imaging evidence of new loss of viable myocardium or new regional wall-

motion abnormality’’ [46]. According to this definition, biomarkers measure-

ment has become essential to supplement ECG findings, clinical signs and

symptoms as well as patient history. A variety of cardiac biomarkers such as

myoglobin, heart-type fatty acid-binding protein, ischemia modified albu-

min, myeloperoxidase, circulating pregnancy-associated protein-A and

others have proposed so far, but most provide little to null information

over cardiac troponin testing and, accordingly, none is in routine use for

the diagnosis of ACS/AMI [47,48]. Therefore, since a marker with a high

8 LIPPI ET AL.


sensitivity and high negative predictive value is ideal to allow expeditious

evaluation and discharge from the ED, the current recommendations of

the National Academy of Clinical Biochemists (NACB) and International

Federation of Clinical Chemistry and Laboratory Medicine (IFCC) are in

support of the use of cardiospecific troponins as the preferred biomarkers

for risk stratification, recommending their assessment in all patients with

suspected ACS/AMI. The initial sample should be collected on hospital

presentation, followed by serial sampling at time points depending on the

clinical circumstances, most frequently at 6–9 h. It is also recommended that

the optimal precision of the assay at the 99th percentile of the upper reference

limit (URL) should be established at a total coefficient of variation (CV)

<10% [46].

4. Biochemistry and Biology of Troponins

The proteins of the troponin complex are low-molecular mass molecules

involved in the regulation of calcium-mediated interaction of actin and

myosin, which are expressed in skeletal and cardiac muscle, but not in

smooth muscle. The cardiac troponin complex consists of three single chain

polypeptides [49]. Troponin T (TnT, 37 kDa) binds the other troponin

components as well as to tropomyosin and facilitates contraction; troponin

I (TnI, 22.5 kDa) binds to actin and inhibits actin-myosin interaction by

controlling the position of tropomyosin on actin filaments in response to

Ca2þ [50]; and troponin C (TnC, 18 kDa) binds calcium ions [51,52]. Long

range allosteric interactions occur between troponin molecules [53]. More-

over, TnI, by mean of its C-terminal domain (residues 193–210), actively

participates in proper stabilization of tropomyosin in both the ‘‘blocked

state’’ and the ‘‘Ca2þ-activated state’’ [54].

Cardiac TnT (cTnT) and TnI (cTnI) are encoded by two specific genes,

TNNI3 (located at 19q13.4) and TNNT2 (located at 1q32), whereas TnC is

codified by a single gene (TNNC1) in both the cardiac and skeletal muscle

[55]. Several differences characterize the biochemistry, the release in the

bloodstream from thin filaments and cytoplasmatic compartment, as well

as the clearance of cTnT and cTnI [56,57]. The gene TNNT2 encodes a

transcript of 288-amino acid, 37 kDa, although combinatorial alternative

splicing of two 50 exons may yield four human isoforms of different molecu-

lar weight (from cTnT-1 to cTnT-4). In the human heart, the alternative

splicing of these two 50 exons generates four isoforms. The isoforms cTnT-1

and cTnT-2 contain both peptides encoded by the 30- and 15-nt exons or the

peptide encoded by the 30-nt exon alone, and are mainly expressed in the

fetal myocardium, whereas cTnT-4, which lacks these sequences, is expressed



in fetal myocardium and reexpressed in the failing adult heart. The isoform

cTnT-3 which, contains the 5-residue peptide, is, however, the dominant

form in the adult heart [56]. The TNNI3 gene transcript also undergoes

alternative splicing, which determines tissue-specific isoforms. The mature

cardiac isoform is a 210-amino acid, 22.5 kDa protein with a posttransla-

tional tail of 32 amino acids on the N-terminus [56].

At variance with TnC, which is characterized by an identical structure in

cardiomyocytes, skeletal, and smooth muscle cells, the amino acid sequences

of skeletal and cardiac isoforms of TnT and TnI are sufficiently different to

allow recognition by specific monoclonal antibodies [58]. However, while

cTnT differs by only 6–11 amino acid residues from its skeletal muscle

counterpart, cTnI has an extra 31 amino acid residue at the N-terminus

and its amino acid sequence shows nearly 40% dissimilarity from extracar-

diac isoforms [59,60]. cTnI is not expressed in any stage of development in

skeletal muscle, whereas low levels of cTnT have been identified in some

disorders of skeletal muscle in which there is a release and protein expression

normally lacking in adults [61,62]. Within the myocyte, cTnT is mostly linked

to TnC and cTnI (i.e., a ternary complex cTnT–I–C), and the free cytoplas-

mic form only represents �6–8% of the total intracellular pool [63]. cTnI,

which is more hydrophobic, is instead mostly present in a binary complex

(cTnI–TnC, molecular mass of 42 kDa) and only minimally in the ternary

complex (77 kDa). Overall, the intracellular concentration and the free cyto-

plasmic pool of cTnT is higher than that of cTnI (i.e., 6–8% vs. 3%) [64].

Although the various forms of intracellular troponins are all released and

hence detectable in the blood of patients after an irreversible and prolonged

damage such as AMI, reversible myocardial injuries that impair the perme-

ability of the plasma membrane determine modest and transitory release of

cardiac troponins from the free cytosolic pool [56]. In patients with AMI,

both troponins undergo posttranslational modification in the injured myocar-

dium as well as extensive postremodeling in blood catalyzed by endogenous

proteases which are released after cardiac injury or that are physiologically

present in blood cells (i.e., calpains, caspases, catepsin L, and gelatinase A).

This aspect, along with the evidence that these proteins are mainly released as

binary complexes cTnI–cTnC and ternary complexes cTnT–cTnI–cTnC in

AMI patients, account for a great part of the interassay variability since the

different cocktails of antibodies of the current immunoassays display hetero-

geneous immunoreactivity with the complexed troponin as well as with their

degradation products [55].

The kinetic curves for cTnI and cTnT are rather similar. In the moderate to

large AMI, but not in patients with microinfarctions, the kinetic is well

defined. Both cTnI and cTnT appear in blood 3–6 h after the onset of symp-

toms and peak in parallel at 12–24 h except for patients without reperfusion, in

10 LIPPI ET AL.


whom cTnI peaks at nearly 24 h whereas the cTnT peakmight be delayed, that

is, approximately 72–96 h after AMI occurrence. Both troponins are steadily

increased for at least 4–5 days after an AMI, although cTnT values usually

return to the baseline not earlier than 10–14 days versus 7–10 days for cTnI

[65–68] (Fig. 1). The clearance is very fast [69,70], so that the half life of

troponins and their complex in the blood is nearly 2 h [71]. The continual

release from the myofibrillar pool, at least until the contractile apparatus

undergoes total degradation, support the prolonged detection of troponins

in blood after a myocardial injury [72]. The relatively high molecular weights

of cardiac troponins (i.e., 37 kDa for cTnT and 22.5 kDa for cTnI), along with

the increased levels observed in patients with impaired renal function, support

the hypothesis that renal clearance might have a role in the metabolism of

cardiac troponins, especially for cTnT and for the degradation fragments

produced by cardiac and extracardiac proteolysis [73–76].

5. Clinical Significance of the Measurementof Cardiospecific Troponins

Albeit being highly cardiospecific [77], increased troponin values especially

measured with the new highly sensitive immunoassays reflect, however, a

kaleidoscope of direct and indirect cardiac injury besides AMI [78,79], as well

as preanalytical [52,80] and analytical issues [81,82], such as those listed in

Table 1. In some of these conditions, the modest rise of troponin in blood has

00 1 3 4 5 6 8 9 10 11

cTnIcTnTURL

FIG. 1. Kinetics of cardiac troponin I (cTnI) and T (cTnT) in blood after an acute myocardial

infarction. URL: 99th percentile of the upper reference limit.

TABLE 1

LEADING SOURCES OF ELEVATIONS OF HIGHLY SENSITIVE TROPONINS IN PLASMA

Demographical

� Increasing age� Male gender

Analytical

� Heterophile antibodies� Human anti-mouse antibodies� Rheumatoid factor� Complement� Presence of fibrin in serum or plasma after centrifugation of the sample� Unsuitable samples (e.g., hemolyzed, lipaemic, icteric)� Analytical errors (e.g., instrument malfunctioning)

Cardiac

� Coronary artery disease� Acute Coronary Syndrome and/or Myocardial infarction� Revascularization procedures� Cardiac contusions such as trauma, ablation, pacing, cardioversion, and

endomyocardial biopsy� Myocarditis and pericarditis� Rhabdomyolysis with cardiac injury� Atrial fibrillation, tachy-arrhythmias, and other severe arrhythmias� Valvular heart disease� Aortic dissection� Hypertrophic cardiomiopathy� Severe chronic heart failure� Sepsis� Infiltrative diseases such as amyloidosis, hemochromatosis, sarcoidosis, and scleroderma� Cardiac rejection posttransplantation� Toxicity from drugs, cardiotherapics, toxins, carbon monoxide

Extracardiac

� Activation of the sympathoadrenal system� Pulmonary embolism and acute pulmonary edema� Chronic obstructive pulmonary disease (COPD)� Pulmonary hypertension� Acute neurological disease including stroke and subarachnoid hemorrhages� Severe hypotension and hypertension� Chronic renal failure� Hypothiroidism� Sickle cell disease� Strenuous physical activity



been attributed to pathophysiological process different from myocardial

necrosis, that is, physiological renewal of the human myocardium [83] or

increase cellular permeability and early troponin leakage from cytosolic pool

or from a different readily accessible cell pool occurring during strenuous

12 LIPPI ET AL.


physical exercise [84,85]. As specifically regards this last condition, the pre-

cise mechanisms of cellular leakage involves a series of events, whereby

cardiac ischemia interferes with the normal function of the plasmamembrane

and bubbles (also known as ‘‘blebs’’) develop at the cell surface and gradually

grow. A transitory or moderate cardiac ischemia, such as that occurring

during aerobic physical exercise, only triggers an irreversible membrane

injury, and blebs are partially reabsorbed or shed into the circulation with

their protein content. This clinically insignificant phenomenon is mirrored by

a low (typically <1000 ng/L) and short lasting (<24–36 h) amount of detect-

able troponin in the blood of endurance athletes [85].

The ‘‘Universal Definition of Myocardial Infarction’’ document as well as

a variety of further studies and publications, have highlighted that troponin

values exceeding the 99th percentile of the URL not only help diagnose an

AMI, but also underlie an increased risk of death and recurrent ischemic

events, thereby assisting the risk stratification [73,86]. Detectable troponin

concentration exceeding the 99th percentile URL using the new sensitive

immunoassays are also associated with adverse prognostic outcomes [81].

In a study on a cohort of subjects presenting to the ED with symptoms

suggestive of ACS, cTnI was measured with a highly sensitive assay (Beckman

Coulter; limit of detection [LOD] of 2.06 ng/L and 99th percentile URL of

8.00 ng/L) [87]. The patient population was then classified into four groups

according to the cTnI value (i.e., <5.00, 5.00–9.99, 10.00–40.00, and

>40.00 ng/L). The endpoints were defined as subsequent readmission for

AMI and/or date of death at 30 days, 6 months, 1, 2, 5, and 10 years

postpresentation. At 30 days, patients with cTnI >40.00 ng/L were at higher

risk for the combined end point (Hazard ratio [HR] 7.20; 95% CI 1.66–31.21).

Then, both the >40.00 ng/L and the 10.00–40.00 ng/L groups were at higher

risk for the combined end point (death/AMI) at 6 months (>40.00 ng/L

HR: 5.82; 95% CI 2.02–16.75; 10.00–40.00 ng/L HR: 3.77; 95% CI 1.26–

11.27), 1 year (>40.00 ng/L HR: 4.58; 95% CI 190–11.04; 10.00–40.00 ng/L

HR: 3.83; 95% CI 1.37–8.33), 2 years (>40.00 ng/L HR: 4.32; 95% CI 2.00–

9.32; 10.00–40.00 ng/L HR: 4.01; 95% CI 1.85–8.70), 5 years (>40.00 ng/L

HR: 1.94; 95% CI 1.18–3.18; 10.00–40.00 ng/L HR: 1.89; 95% CI 1.15–3.11),

and 10 years (>40.00 ng/L HR: 1.85; 95% CI 1.23–2.77; 10.00–40.00 ng/L

HR: 1.66; 95% CI 1.10–2.52).

Bonaca et al. assessed cTnI with a sensitive assay (TnI-Ultra assay,

ADVIA Centaur, Siemens) in 4513 patients with NSTEMI and found that

applying a decision limit set at the 99th percentile URL (i.e., 40 ng/L, the unit

transition from mg/L to ng/L is recommended with the routine use of new

generation cTnI and cTnT immunoassays), patients with values above this

threshold had a nearly threefold higher risk of death/AMI at 30 days (95%

confidence interval: 2.0–4.4) [88]. Moreover, patients values comprised



between the cut-off defined for the prior generation assay (i.e., 100 ng/L) and

40 ng/L were at significantly higher risk of death/AMI at 30 days and death

at 12 months than patients with cTnI <40 ng/L. The independent and useful

prognostic predictive value of a plasma concentration of troponin above the

99th percentile URL has also been confirmed in other clinical setting such as

chronic heart failure [89], atrial fibrillation [90], thrombotic [91] and non-

thrombotic pulmonary embolism [92], infiltrative disorders such as amyloid-

osis [93] and dialysis-dependent chronic renal failure [94].

5.1. TRADITIONAL AND ‘‘DYNAMIC’’ APPROACH TO

INTERPRET TROPONIN VALUES

Due to the incessant introduction of the novel highly sensitive immunoas-

says, the analytical ability to measure troponin in blood continues to move

lower, so that we are increasingly facing the new challenge of apparently

healthy subjects as well as several patients with comorbidities different from

AMI who display detectable troponin concentration (i.e., values above the

99th percentile URL) at ED presentation. As highlighted by Judd EHollander

[8] ‘‘none of us possesses a magic crystal ball,’’ so that the diagnostic approach

as well as the triage of chest pain patients presenting at the ED need to be

flexibly readjusted and, most probably, the role of clinical judgment will

assume much greater importance than with traditional assays considering

that the more sensitive the immunoassay, the lower the specificity for ischemic

injury. In this perspective, a reliable strategy to use results of highly sensitive

immunoassays is that entailing the ‘‘delta biomarker approach’’ rather than

that based on the traditional ‘‘peak’’ value [95], since the observation of a

dynamic pattern of values might help discriminating the timing of injury as

well as distinguishing acute from chronic etiologies and thereby increasing

both the specificity and the PPV of the test [96].

The NACB has formerly established that a relative troponin variation

(also referred as ‘‘change’’ or ‘‘delta’’) of >20% from the baseline value

6–9 h after presentation represents a significant (>3 standard deviation,

SD) variation, on the basis of a 5–7% analytical imprecision (analytical

CV, CVa) which is typical for most assays. Although this threshold of

variation has also been cosidered useful for discriminating the timing of

injury as well as being suggestive of AMI that is either evolving (value

increase) or resolving (value decrease) [97], it has been simply settled by

calculating three times the imprecision at the cut-off concentrations [98],

rather than evaluating biological variation of troponin, (no studies had

examined the implications of the delta biomarker approach with the newer

generation of highly sensitive immunoassays at that time). Along with

the definition, the equation for calculating a significant difference was

14 LIPPI ET AL.


provided as follows: ‘‘1.96�2½�SD¼2.77�SD’’. Considering that the

SD can be replaced with imprecision (i.e., CVa), Fraser calculated that

assays with the optimal CVa of 10% would require a higher change (i.e., up

to 28%) to achieve clinical significance and, in turn, a delta of 20% would

require an analytical imprecision (CVa) of the immunoassay lower than

10% (i.e., 7.2%) [99]. Although a minimum theoretical delta cut-off can be

easily calculated according to the previous formula on the basis of the

imprecision of each assay, as shown in Fig. 2, this would not consider

the biological variability of the biomarker and it is hence not surprising

that the assessment of inter- and intraindividual troponin variability has

become the focus of a further series of epidemiological investigations.

Apple et al. reassessed by means of receiver operating characteristics

(ROC) curve analysis the optimal cut point for classification of AMI and a

troponin variation between specimens obtained at the time of presentation at

the ED, and at a follow-up time a minimum of 4 h (and maximum of 10 h).

A delta of 30% with a newer generation cTnI immunoassay (VITROSÒ; 99th

percentile URL: 0.034 mg/L) had the best diagnostic performances for diag-

nosing AMI, displaying sensitivity of 75% (95% CI 61%–86%) and specificity

of 91% (95% CI 87%–94%) [95]. An interesting approach was used by

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

0% 5% 10% 15% 20% 25% 30%

Assay imprecision (%)

The

oret

ical

del

ta (

%)

FIG. 2. Theoretical delta threshold for establishing significant variations of highly sensitive

troponin values calculated according to the imprecision of each assay (CV, %).



Introcaso et al., who assessed serial cTnI measurements (Accu troponin-I,

Beckman Coulter; 99th percentile URL: 40 ng/L) in patients admitted to the

ED by means of critical value (Cr) of RCV [100]. First, healthy subject were

enrolled to evaluate intra-(n¼10) and interindividual (n¼70) biological

variation of cTnI. The analytical, intra-, and interindividual CVs were re-

spectively 4.4%, 86%, and 81%, yielding an estimated RCV of 240%. Then,

variation of cTnI values calculated from a first sample collected on patients

admission to the ED for suspected ACS and that obtained on a second

sample drawn 4–6 h afterward was compared with the CrRCV, calculated

as follows: [first TnI result]þ [(first TnI result)�RCV]. As such, the diagnos-

tic performance of CrRCV for diagnosing ACS was as follows: 62% sensitiv-

ity, 83% specificity, 88% negative predictive value (NPV) and 52% PPV.

Scharnhorst assessed the diagnostic efficiency of a highly sensitive cTnI

assay (TnI-Ultra for Advia Centaur, SiemensMedical Solutions Diagnostics)

for detecting AMI at the ED with two alternative strategies. The traditional

approach, based on an AMI cut-off value (i.e., >100 ng/L) at arrival in the

ED and 2, 6, and 12 h later yielded a PPV of 100% at all points and a NPV of

92% at arrival (sensitivity 70%, specificity 100%), increasing to 96% at 2 h

(sensitivity 87%, specificity 100%), and 99% at both 6 and 12 h (sensitivity

97%, specificity 100%). With the latter approach, using a more sensitive

strategy based on a troponin concentration within the first 2 h >60 ng/L

(i.e., the 99th percentile URL) or an increase of 30% in the troponin concen-

trations between arrival and 2 h, the sensitivity increased to 100%, the

specificity decreased to 87%, but the NPV and PPV were 100% and 70%,

respectively. As such, the second ‘‘more sensitive approach’’ allowed to

identify all patients with a final diagnosis of AMI (NPV: 100%) [101].

Aldous et al. also evaluated the diagnostic performance of a highly sensi-

tive cTnT immunoassay (Roche, Elecsys 2010) using a change in troponin

from baseline to follow-up (i.e., samples taken at a median time of 9.4 h,

interquartile range 6.3–13.3 h) after the baseline sample of either 20% or 50%

in combinationwith�1 result of troponin�99th percentileURL (i.e., 14 ng/L).

The standard approach (i.e., troponin peak values �99th percentile

URL) yielded 90.9% sensitivity and 80.6% specificity, whereas sensitivity

and specificity using the ‘‘delta approach’’ were 71.8% and 93.7% (þ20%

delta) and 61.8% and 96.8% (þ50% delta) [102]. One basic aspect in assessing

the significance of changes in both cTnI and cTnT is that the distributions of

values of these biomarkers in the general population displays a little ‘‘right-

skewness’’ [103], so that the significance of differences in serial results, that is, the

RCV, should be preferably calculated using a log-normal approach, such as that

suggested by Fokkema et al. [104].

These preliminary investigations paved the way to a series of subsequent

studies which have assessed the inter- and interindividual troponin variability

16 LIPPI ET AL.


as well as the estimated RCV of this marker, whose results are synthesized in

Table 2 [103,105–108]. Interestingly, Schultze et al. also developed an ultra-

sensitive cTnI immunoassay (Erenna, Singulex; LOD¼0.2 pg/mL, Lower

limit of quantification with �10% CV¼0.8 pg/mL) to assess TnI concentra-

tion and variation in rats [109]. The baseline reference range (mean 4.94 pg/mL,

range 1–15 pg/mL) of cTnI in rats was similar to that previously reported in

humans (1–12 pg/mL), dogs (1–4 pg/mL) and monkeys (4–5 pg/mL) using the

same assay. Moreover, log-normal RCV values for a statistically significant

increase or decrease in cTnI values were 206.7% and 67.4%, respectively.

The delta biomarker approach has also been proposed for risk stratifica-

tion after AMI. The first evidence that the 2007 AMI definition criteria for

change (i.e., >3 SD or >20%) might be effective for improving risk stratifica-

tion was provided by Apple et al. who determined the prognostic value of a

newer generation cTnI immunoassay (VITROSÒ; 99th percentile URL:

0.034 mg/L) and by means of ROC curve analysis also established that a 30%

variation from samples obtained at the time of presentation at the ED and at a

follow-up time a minimum of 4 h (and maximum of 10 h) even improved the

prediction of 60-day cumulative RR of events from 8.2 (using the conventional

change >20%) to 10.5 [95]. Interestingly, both follow-up cTnI >0.034 mg/Land change >30% were independently predictive of risk, but the combination

of these two parameters was much more powerful. More recently, Kavsak

et al. demonstrated that the 2007 AMI definition criteria for change (i.e.,

>3 SD or >20%) might be effective for improving risk stratification at 30

days, 6 months, and 1 year using either the delta between lowest and highest

troponin concentrations (HRs comprised between 4.1 and 12.3), or that be-

tween the first and the second specimens collected (HRs comprised between 3.0

and 4.5) [110]. The same change definition applied to a novel highly sensitive

assay provided, however, much lower HRs at all endpoints with either the

delta between lowest and highest troponin concentrations (HRs comprised

between 3.4 and 4.1; p<0.05 at 6 months and 1 year) or that between the first

and the second specimens (HRs comprised between 1.7 and 1.8; all p¼ns).

After performing a further ROC curve analysis to assess optimal change for

the novel highly sensitive immunoassay, the AUC and HRs for AMI/death at

a much higher delta (i.e., 235%) were 0.70 and 3.5 (p¼0.006), respectively.

5.2. INFLUENCE OF CLINICAL AND DEMOGRAPHICAL

VARIABLES ON TROPONIN VALUES

The challenge of selecting the ‘‘normal’’ reference population for calculating

the 99th percentile URL is enormously magnified with the novel highly

sensitive immunoassays, since the improved analytical performance allows

to identify a large number of subjects, up to 95%, without ACS but yet

TABLE 2

STUDIES THAT HAVE ASSESSED THE INTRA- AND INTERINDIVIDUAL TROPONIN VARIABILITY AS WELL AS THE REFERENCE CHANGE VALUE

Method Platform

LOD

(ng/L)

CV<10%

(ng/L)

URL

(ng/L)

Study

population Term

Analytical

variability (%)

Biological variability

Index of

individuality

Lognormal

RCV (%) Ref.

Intraindividual

(%)

Interindividual

(%)

Troponin

I (Backman)

Access 2.06 8.66 8.00 Healthy

subjects

0–4 h 3.5 3.4 45.3 0.1 �15.8, 45.2 [105]

0–8 weeks 2.7 2.6 41.6 0.1 �10.6, 14.0

Troponin

I (Singulex)

384-Well

ELISA

plate

0.2 4 10 Healthy

subjects

0–4 h 8.3 9.7 57 0.2 �32, þ46 [103]

0–8 weeks 15.0 14.0 63 0.4 �45, þ81

Troponin

T (Roche)

Elecsys 2010 5.0 13 13.5 Healthy

subjects

Hourly 9.7 21 N/A N/A �47, þ90 [106]

Weekly 9.7 30 N/A N/A �58, �135

E 170 5.0 13 13.5 Healthy

subjects

Hourly 7.8 15 N/A N/A �39, þ64

Weekly 7.8 30 N/A N/A �58, �138

Troponin

T (Roche)

Elecsys 2010 5.0 13 40.0 ARIC study 6 weeks 6.9 16.6 N/A N/A 68.5a [107]

Troponin

T (Roche)

Elecsys 2010 5.0 13 13.5 Heart

failure

patients

14 days <2.5 7.2 N/A 0.1 80–125a [108]

28 days <2.5 22.6 N/A 0.3 78–128a

62 days <2.5 28.9 N/A 0.3 80–125a

90 days <2.5 15.6 N/A 0.2 74–136a

Variability is expressed in terms of coefficient of variation (CV, %).

LOD, Lower limit of detection; CV<10%, lowest concentration with an imprecision (CV) <10%; URL, upper limit of the reference range.

The specific characteristic of each immunoassay are available from the Website of the International federation of Clinical Chemistry and Laboratory

Medicine (IFCC) (http://www.ifcc.org/PDF/ScientificActivities/IFCC_Troponin_Table_vDec_2010_FINAL_ng_L_28Jan11.pdf).aLognormal RCV for increase.


http://www.ifcc.org/PDF/ScientificActivities/IFCC_Troponin_Table_vDec_2010_FINAL_ng_L_28Jan11.pdf

18 LIPPI ET AL.


displaying detectable troponin concentration [76,111]. Accordingly, an inad-

equate screening for comorbidities as well as selection of a heterogeneous

cohort of subjects of both genders and across wide age ranges might result in

an inappropriate definition of the 99th percentile URL.

Fred Apple originally proposed a two-tier analysis for overcoming the

challenge of inaccurate interpretation of troponin values in clinical practice,

that is, by using both the 99th percentile URL and imprecision values at the

99th percentile based on a young, healthy reference population diversified by

some demographical variables such as gender race, and ethnicity [112]. As

such, the IFCC currently provides and regularly updates a scorecard where

the main characteristics of the various immunoassays are listed [113]. While

this efforts is indeed helpful for both assisting the worldwide process of assay

clearance as well as the transition to the newer generations of highly sensitive

immunoassays, the definition of a healthy reference or ‘‘normal’’ population

remains a challenge, since a variety of demographical, biological, and clinical

variables might potentially modify the 99th percentile URL and thereby

influence the diagnostic performance of highly sensitive immunoassays in

patients admitted to the ED. Regardless of myocardial ischemia and addi-

tional causes of myocardial injury already mentioned [78,79] (Table 1), sig-

nificant variations of the 99th percentile URL values might also be

determined by gender [114], the regular practice of moderate to strenuous

aerobic physical activity [84,85], hypertension [115], chronic kidney disease

[116], the presence of myocardial impairment and coronary artery stenosis

[117], chronic clinically silent rupture of noncalcified plaque [118], activation

of the sympathoadrenal system [119], exacerbations of chronic obstructive

pulmonary disease [120] as well as pulmonary arterial hypertension [121].

Although there is currently no agreement on the use of gender or age-specific

reference intervals, both these variables might influence the definition of the

99th percentile URL [111]. Mild highly sensitive troponin elevations are

frequently observed in elderly non-AMI patients in both nondiabetics

[114,122] and type 2 diabetic patients [123]. In a multicenter study including

1098 consecutive patients presenting with symptoms suggestive of AMI, base-

line troponin levels were measured with three highly sensitive immunoassay

in >70-years-old non-AMI patients. Levels above the 99th percentile URL

were recorded in 51% (cTnT, Elecsys 2010, Roche; 99th percentile URL: 14 ng/

L), 17% (cTnI, ADVIA Centaur, Siemens; 99th percentile URL: 40 ng/L), and

13% (cTnI, Architect, Abbott; 99th percentile URL: 28 ng/L), while the

corresponding figures in <70-years-old non-AMI patients were 14, 8 and 7%,

respectively. As such, that best diagnostic performances in the elderly were

obtained increasing the optimal cut-off levels of all highly sensitive troponin

assays as compared with non-AMI younger patients (54 vs. 17 ng/L for cTnT

Roche; 32 vs. 8 ng/L for highly sensitive cTnI Abbott, and 45 ng/L vs. 39 ng/L

for cTnI Siemens) [124].



Taking into account all these variables would imply the adoption of differ-

ential cut-offs or development of test-specific algorithms for patients with

acute chest pain according to a variety of demographical and clinical variables.

Due to the increasing use of cardiac troponins for identifying and monitoring

myocardial injury in patient populations outside of the ACS/AMI setting,

outcome studies are hence needed for establishing the optimal concentration

and/or cut-off in a variety of patients with different types of cardiac disease

other than AMI [125], such as toxicity from chemoteraphy [126].

6. Conclusions

According to the definition of Apple [112], troponin immunoassays are

current classified according to percentage of measurable normal values below

the 99th percentile URL, as follows: level 1 (or ‘‘contemporary’’): <50%;

level 2 (first generation highly sensitive): 50% to <75%; level 3 (second

generation highly sensitive): 75% to <95%; and level 4 (third generation

highly sensitive): �95%. The introduction of highly sensitive immunoassays

has thereby caused a paradigm shift in the diagnostic approach of patients

admitted to the ED with suspected ACS/AMI, whereby the remarkably

increased diagnostic sensitivity and the NPV of these tests not only will

allow rule out of patients much earlier than with traditional assays (e.g., 2–

3 h after the onset of the symptoms), but they will also predictably outweigh

the advantages offered by combining the ‘‘old generation’’ troponin testing

with early and sensitive but less specific biomarkers such as myoglobin,

ischemia modified albumin, heart-type fatty acid-binding protein, choline,

myeloperoxidase, and pregnancy-associated plasma protein A [75,127–129].

The additional predictive information (i.e., risk stratification) provided by

highly sensitive troponin testing also raises some doubts as to whether

predictive and expensive markers such as copeptin and others would carry

supplementary and meaningful clinical information to troponin testing alone

[130]. As such, any theoretical benefit of a multimarker approach in this

setting would require the appropriate inclusion of biomarkers whose isolate

elevation reflect different disease subprocesses [131]. As brilliantly high-

lighted by Allan S Jaffe, it is, however, undeniable that the introduction of

highly sensitive troponin immunoassays ‘‘. . .have (probably) outstripped the

ability of clinicians to keep up with how to use them clinically’’ [82], and

thereby new challenges are emerging [121].

First, while we recognize that it is indeed difficult—if not impractical—to

adopt of a wide range of 99th percentile URLs stratified according to the age,

gender, physical activity, health status and potential comorbidities of the

patient, the emergency medicine physicians should place major focus on

history and clinical presentation, in order to properly interpret troponin values

20 LIPPI ET AL.


at ED admission, prevent overdiagnosis of both ACS and AMI, troubleshoot

the ‘‘new’’ potential causes of elevations observed with these novel immunoas-

says and carefully consider the prognostic implications (i.e., adverse prognosis

not specifically attributable to CAD) associated with detectable levels of

troponin in blood [82,96]. Predictably, the definition of appropriate critical

pathways or protocols will become more crucial than ever, since the EDs are

increasingly under a huge pressure to evaluate patients expeditiously and

blood samples are collected on an escalating number of patients at ED

presentation.

Then, the identification of the appropriate change threshold to be used for

the serial analysis of troponin values remains a challenge that requires further

clinical and prospective studies [132]. At present, the identification of a com-

mon delta threshold that would fit for all the commercially available immu-

noassays seems inappropriate and potentially misleading, since the calculation

of the RCV is inherently based on the combined analytical imprecision of both

the method and the instrumentation. It might be hence advisable to calculate

the threshold locally or, when this is unfeasible, to refer to the available data

previously published and synthesized in Table 2. Nevertheless, when the delta

Highly sensitivetroponin

immunoassays

99th percentileURL

Deltathreshold

FIG. 3. The challenges of the novel highly sensitive troponin immunoassays: navigating

between the Scylla (i.e., definition of appropriate 99th percentile Upper Limit of the reference

range, URL) and Charybdis (i.e., identification of a reliable delta threshold). Reproduction of

James Gillray’s print of ‘‘Scylla & Charybdis.’’



of change is below that established for diagnosing anAMI, it seems cautious to

suggest that the emergency medicine physicians should rely once more on the

clinical judgment before discharging the patients.

In conclusion, we are currently navigating between the Scylla (i.e., defini-

tion of appropriate 99th percentile URL) and Charybdis (i.e., identification

of a reliable delta threshold) of the novel highly sensitive immunoassays

(Fig. 3). Additional problems to be solved include the low interassays com-

parability of cTnI results, which is partially attributable to the use of anti-

bodies recognizing different epitopes on the molecule, and the current lack of

standardization of troponin immunoassays. A Standard Reference Material

(SRM) 2921 for Human Cardiac Troponin Complex has been characterized

in 2006 [133]. This reference material, which is expected to provide several

advantages such as traceability and commutability, has not, however, been

made available to the diagnostics companies so far.

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