PAH (pulmonary arterial hypertension) is the leading cause of fatal right ventricular failure. However, rarely detectable, cTnT [cardiac TnT (troponin T)] is a significant prognostic marker. Therefore the aim of the present study was to evaluate the usefulness of a novel high-sensitive cTnT (hsTnT) assay as a parameter for functional and prognostic evaluation of PAH patients. In 55 PAH patients (idiopathic, n=20; chronic thromboembolic, n=30; and interstitial lung disease, n=5) with a mean pulmonary artery pressure of 45±18 mmHg, cTnT was measured by a fourth-generation conventional assay and a novel hsTnT assay with a lower detection limit at 2 pg/ml [total imprecision <10% at the 99th percentile value (13.4 pg/ml)]. In 90.9% of patients, cTnT was detectable using the hsTnT assay and in 30.9% using the fourth-generation assay. Concentrations >99th percentile were observed in 27.3% using hsTnT compared with 10.9% using the fourth-generation assay. A total of five out of six patients with cTnT values >30 pg/ml (fourth-generation assay) or >29.5 pg/ml (hsTnT assay) died during the 12-month follow-up. There was a correlation between hsTnT and 6-min walk distance (r=−0.92, P=0.0014), right ventricular systolic strain (r=0.95, P=0.0018) and strain rate (r=0.82, P=0.0021). In AUC (area under the curve) analysis, hsTnT predicted death at least as effectively as hFABP (heart-type fatty-acid-binding protein) or NT-proBNP (N-terminal pro-brain natriuretic protein). Moreover, hsTnT predicted a WHO (World Health Organization) functional class >II better than NT-proBNP or hFABP. In conclusion, in PAH patients, the novel biomarker hsTnT is associated with death and advanced WHO functional class, and is related to systolic right ventricular dysfunction and an impaired 6-min walk distance.
- cardiac troponin T (cTnT)
- high-sensitive cardiac troponin T (hsTnT)
- pulmonary arterial hypertension (PAH)
Chronic PAH (pulmonary arterial hypertension), a potential consequence of many distinct disease mechanisms, is characterized by progressive dyspnoea and limitation in exercise capacity . Although the clinical symptoms in PAH may result from peripheral airway dysfunction, skeletal muscle weakness and progressive RV (right ventricular) failure, RV dysfunction is the most important determinant of the outcome of patients [2,3]. In recent years, substantial progress has been achieved in the treatment of PAH, mostly due to improved pharmacotherapy . However, treatment is not only expensive, but also associated with severe side effects in a significant proportion of patients. Therefore non-invasive identification of high-risk subgroups may improve not only risk assessment, but also guidance for selection of treatment modalities and intensity.
Multiple clinical indexes have been applied to better assess the severity of disease and risk in PAH patients. WHO (World Health Organization) functional class, 6MWD (6-min walking distance), anatomical and functional echocardiographic parameters, cardiac biomarkers, and exercise testing by stress echocardiography or cardiopulmonary exercise testing are the most commonly used surrogate markers [5–8].
As RV dysfunction is the major predictor of adverse outcome, much attention has been directed to improve the assessment of the RV contractile function and reserve . Strain analyses by tissue Doppler have become the standard measures to study contractile function of LV (left ventricular) myocardium. Recent findings indicate that strain and strain rate are also useful in assessing RV function and may serve, in conjunction with NT-proBNP [N-terminal pro-BNP (brain natriuretic peptide)], as surrogate markers of contractile function in the overloaded right ventricle .
cTn (cardiac troponins) constitute the preferred markers for diagnosis of myocardial infarction and for identification of myocardial damage. Although elevated cTn levels have been found to be useful in predicting adverse haemodynamics and prognosis after acute PE (pulmonary embolism), their role is still controversial in patients with chronic PE, potentially due to the low prevalence of elevated cTn using less sensitive generations of assays.
In patients with PAH, increased serum levels of cTn have been found to correlate with higher heart rates, lower mixed oxygen saturation, and pronounced exercise limitation and blood levels of BNPs . Conversely, measurement of hFABP (heart-type fatty-acid-binding protein) was found to be more useful for risk assessment in patients with chronic thromboembolic PAH, although cTnT remained an ominous prognostic sign when detectable .
Recently, modifications of cTnT assays have been introduced that result in higher analytical sensitivity and the ability to measure concentrations at the 99th percentile of a reference population with an imprecision of <10%.
The primary aim of the present study was therefore to evaluate the usefulness of a novel hsTnT (high-sensitive TnT) assay as a parameter for clinical and functional evaluation, and for prognostic stratification of PAH patients in contrast with established biomarkers.
MATERIALS AND METHODS
This prospective study enrolled 55 consecutive patients with PAH. The diagnosis was established by right heart catheterization, pulmonary angiography, spiral computer tomography and echocardiography. Significant coronary artery disease was excluded by stress testing or coronary angiography.
Patients were treated according to current guidelines for PAH . They were studied while on stable doses of their medication over the previous 4 weeks and showed no clinical or radiological signs of cardiopulmonary decompensation.
Given the interference between kidney function and biomarker concentrations, patients with severe renal failure (creatinine clearance <60 ml·min−1·1.73m−2) were excluded.
The protocol was approved by the local ethics committee of the University of Heidelberg, and all patients gave written informed consent prior to inclusion.
Assessment of cardiopulmonary function
The 6MWT  and two-dimensional transthoracic echocardiography  were performed using standard protocols. Two-dimensional transthoracic echocardiography for non-invasive imaging was used to obtain information on RV and LV function, including assessment of RV end-diastolic area, RV end-systolic area, RV fractional area change, the RV end-diastolic area/LV end-diastolic area ratio and LV ejection fraction . Moreover, sPAP (systolic pulmonary arterial pressure) and the Tei-Index (RV myocardial performance index) were measured as described previously . For RV systolic functional assessment, TAPSE (tricuspid annular systolic excursion) was measured from the apical four-chamber view as recommended previously .
Systolic strain and strain rate were derived from tissue Doppler velocities . A strain region of interest was placed in the basal segment of the RV free wall. Measurements of three to five heart cycles were recorded and averaged for all analyses.
Blood sampling for biomarker analysis
Blood samples were drawn from a peripheral vein and were analysed for serum NT-proBNP, hFABP, hsTnT and cTnT.
hsTnT was measured using the latest pre-commercial version of the hsTnT assay (Roche Diagnostics). The lower detection limit of this assay is 2 pg/ml (=0.002 μg/l). Improvement of sensitivity and precision was achieved by increasing the sample volume to 50 μl, optimizing the degree of ruthenylation of the signal antibody, and optimizing the buffer composition to reduce background signal. As described by the manufacturer in the assay kit (Roche Diagnostics), the inter-assay CV (coefficient of variation) was 8% at 10 pg/ml and 2.5% at 100 pg/ml. The intra-assay CV was 5% at 10 pg/ml and 1% at 100 pg/ml. Normal reference values were established from three reference studies including 1934 apparently healthy volunteers and blood donors between 20 and 71 years of age. The 99th percentile value was determined at 13.4 pg/ml.
cTnT was measured using the fourth-generation commercial one-step electrochemiluminescence immunoassay based on electrochemiluminescence technology (Roche Diagnostics). The lower detection limit is 0.01 μg/l with a recommended diagnostic threshold of 30 pg/ml (corresponding to 0.03 μg/l).
NT-proBNP was measured using a highly sensitive and specific electrochemiluminescence immunoassay (Elecsys proBNP; Roche Diagnostics). The inter-assay CV is 3.2–2.4% from 175–4962 pg/ml and 5.7% at 64 pg/ml. The measurement range extends from 5 to 35000 pg/ml.
hFABP was measured with the turbidimetric latex immunoassay as described previously . The intra-assay CV was 5.3% at 7.7 μg/l (=7700 pg/ml) and 1.2% at 41.6 μg/l (=4160 pg/ml). The inter-assay CV was 9.3% at 7.3 μg/l (=7300 pg/ml) and 2.7% at 41.4 μg/l (=4140 pg/ml). The minimal detection limit of the assay was 1.1 μg/l (=1100 pg/ml).
All results are expressed as means±S.D., or as medians with 25th and 75th quartiles. Correlations between biomarkers and functional test results were calculated using Pearson product-moment correlation coefficient. Levels of biomarkers were log-transformed before analysis to stabilize the variance. An ANOVA was used to test the difference between biomarker levels in WHO functional classes using the Student–Newman–Keuls test for all pair-wise comparisons. A two-tailed P value of 0.05 was regarded significant. Statistical analysis was performed using Medcalc (version 10.3.2).
A total of 55 patients were included with a diagnosis of PAH, which was idiopathic in 20 cases, thromboembolic in 30 cases and associated with interstitial lung disease in five cases. Among the 55 patients, 21 patients (38%) were in WHO functional classes III and IV, and 20 patients had a 6MWD of less than 150 m, indicating severe right heart failure. Consistently, NT-proBNP levels were elevated above 125 pg/ml in 48 PAH patients (87%). The baseline characteristics of the entire cohort are given in Table 1.
There was a close relationship between WHO functional class and biomarker results, 6MWT and echocardiographic parameters. As WHO functional class increased, there was a significant increase in biomarker concentrations and a consistent decrease in 6MWT, and strain and strain rate in echocardiography.
The prevalence of patients with positive cTnT results varied widely and was closely related to the cTnT assay and the cut-off used. Accordingly, cTnT was detectable in 50 of 55 patients (90.9%) using the hsTnT assay, whereas only 17 patients (30.9%) had concentrations above the lower detection limit (10 pg/ml) using the fourth-generation cTnT assay. Using the 10% CV as the decision cut-off, cTnT was positive in six patients (10.9%), whereas 15 patients (27.3%) had values exceeding the 99th percentile value using the hsTnT assay.
Within 12 months after enrolment, a total of five patients died due to progressive RV failure without an indication of acute PE. All of these patients were in WHO functional class IV and had cTnT values >30 pg/ml. Conversely, no patient died with an hsTnT level <0.013 μg/ml, which is the 10% CV and 99th percentile value (P<0.0001) respectively.
Regarding the ability to predict death within 12 months, cTnT >30 pg/ml (using the fourth-generation assay) predicted all deaths (Table 2), whereas NT-proBNP and hFABP were less predictive. hsTnT was also superior in its relationship with functional capacity (WHO functional class) as compared with cTnT (using the fourth-generation assay), NT-proBNP and hFABP (Table 3). hsTnT levels were significantly higher in patients with WHO functional classes III and IV compared with patients with mild exercise limitation (Figure 1).
Moreover, the echocardiographic indices of RV contractile function systolic strain and strain rate showed a significant correlation with hsTnT (Figures 4A and 4B respectively). In contrast, there was no significant relationship between the Tei-Index, reflecting diastolic function (results not shown).
Although cTnT is regarded as indicator of myocardial injury, NT-proBNP often reflects myocardial overload. The relationship between hsTnT and NT-proBNP levels is shown in Figure 5.
The major findings of the present study are as follows. First, the use of a high-sensitive cTnT assay enables detection of cTnT in more than 90% of all patients with PAH compared with 30.9% using the currently used fourth-generation assay from the same manufacturer. Concentrations above the 99th percentile of a healthy reference population are encountered in 27.3% of cases with the new hsTnT assay, but only in 10.9% of cases using the fourth-generation assay when using the cut-off with an imprecision of <10% CV. This improved detection is achieved by higher analytical sensitivity with a lower limit of detection at 2 pg/ml compared with 10 pg/ml with the currently used fourth-generation cTnT assay, as well by an improved assay precision at the 99th percentile of a healthy reference population.
Secondly, a cTnT value >30 pg/ml as measured by the fourth-generation cTnT and hsTnT assays are excellent predictors of death within 12 months and perform at least as well as hFABP or NT-proBNP.
Thirdly, cTnT concentrations rose with increasing severity of WHO functional class, and there was a clear inverse correlation between detectable cTnT concentrations and echocardiographic parameters of systolic RV dysfunction, as well as reduced exercise capacity as measured by the 6MWD. Serum levels of hsTnT proved superior to cTnT determined using the fourth-generation assay, hFABP and NT-proBNP in identifying patients with more severe PAH in WHO functional class >II.
Previous studies have demonstrated that detectable cTnT levels in patients with acute PE are associated with unfavourable prognosis [20–22]. In patients with confirmed PE, the prevalence of cTnT or cTnI above the 99th percentile or the 10% CV has been reported to be as high as 32 and 21% respectively .
In contrast with acute PE, the prevalence of elevated cTn concentrations in PAH is much lower. Torbicki et al.  first reported the potential prognostic role of detectable cTnT in PAH. Although the prevalence of detectable cTnT was only 14%, elevated levels appeared to be associated with a poor prognosis. In contrast with acute PE, this low prevalence of elevated cTn, together with the finding that increases in cTn were exclusively found in patients with advanced stages of PAH, raised the contention that cTn may not be useful in PAH. Therefore several other biomarkers, including hFABP and BNPs, were tested in a few small cohorts [8,22–26] and in a single prospective trial . Lankeit et al.  reported the largest cohort that included 93 patients with chronic thrombembolic PAH. In this study, hFABP concentrations were significantly higher in those patients with an adverse outcome. Disappointingly, cTnT was not helpful, due to a very low prevalence of only 4% of patients with levels above the lower detection limit (>0.01 μg/l). However, all patients with detectable cTnT concentrations died, rendering cTnT an ominous indicator of outcome when present .
More recently, more refined generations of cTn assays with higher analytical sensitivity, i.e. lower detection limit and adequate precision to measure low troponin concentrations at the 99th percentile of a healthy reference population, have become available and have stimulated the hypothesis that the use of such a high-sensitive cTn assay may improve risk stratification and may allow insights into disease pathophysiology. In the present study, the use of hsTnT enabled identification of more patients with a cTnT concentration above the 99th percentile. The cTnT concentrations were detectable in all patients and only 27.3% of all patients had cTnT concentrations above the 99th percentile value. In the present study, elevated cTnT values were no longer restricted to WHO functional class IV, but can also be found in less advanced WHO functional classes. The present study found that hsTnT performed at least comparably with hFABP or NT-proBNP for prediction of death within 12 months. Moreover, hsTnT performed significantly better regarding prediction of more advanced WHO functional class than cTnT determined using the fourth-generation assay, hFABP and NT-pro BNP. Although the number of patients and events was low, we could clearly demonstrate the adverse prognosis associated with elevated cTnT concentrations above the 99th percentile.
The underlying pathological mechanisms for an increase in cTn after acute PE or in patients with chronic PAH remain unclear. Several mechanisms have been proposed, including myocardial ischaemia and necrosis from acute increase in RV afterload, or myocardial stress from increased wall tension. In PE, elevated cTn levels are associated with the presence of RV dysfunction [20,22].
In patients with chronic left heart failure, elevated cTn levels have been observed to correlate with functional impairment and unfavourable prognosis [27–29]. This is in concert with the present findings in PAH with chronic RV dysfunction. In the present study, there was a correlation between echocardiographic parameters of RV function and elevated cTn.
The hsTnT-positive patients had significant RV dysfunction and reduced regional contractility during echocardiography. Consistently, RV strain and strain rate were inversely related with hsTnT values, indicating that hsTnT is an indicator of systolic RV dysfunction. In contrast, no association was found between diastolic RV dysfunction, as measured by the Tei-index, and cTn level. These findings are particularly important, as previous observations have promoted echocardiography as a tool for the functional evaluation of patients with PAH [10,30]. In this context, strain echocardiography has been reported as a surrogate of pulmonary haemodynamics in smaller heterogeneous populations, including patients with PAH due to ischaemic LV failure or chronic thromboembolism .
The present study has some limitations. On the one hand, the small sample size and the low number of fatal events might have attenuated the statistical and clinical performance of the biomarkers tested. In particular, a larger number of fatal events and a longer observational period in a future study will substantiate the role of hsTnT as a novel marker of prognosis. Therefore the present observations will prompt the validation of the novel biomarker hsTnT in a larger multicentre PAH cohort by us and others.
On the other hand, hsTnT and NT-proBNP were measured from a single frozen blood sample obtained on presentation. Notably, cTnT was measured serially after 6 h and usually on the day following admission, as a typical increase/fall in cTn is required for the diagnosis of acute myocardial infarction and in order to distinguish an acute from a chronic troponin increase. Although cTn concentrations above the 99th percentile value may also be associated with stable coronary heart disease, acute or chronic LV heart failure, myocarditis, drug toxicity and other cardiac diseases, an increase in cTn is almost always associated with an unfavourable prognosis [27–29,32]. The measurement of biomarker levels in only a single blood sample for functional and prognostic evaluation of PAH patients appears to comply with the recommended monitoring during visits in a specialized outpatient clinic .
The present pilot study on 55 well-characterized patients with PAH indicates the usefulness of cTnT as a biomarker of disease severity when measured with a high-sensitive assay. It is tempting to speculate from other observations studying patients with acute increase in RV afterload due to acute PE that a combination of biomarker levels and echocardiographic parameters of RV function will improve risk stratification by increasing the positive predictive value of either test. In order to test this hypothesis, larger studies are needed to test the predictive power of hsTnT and its contribution to therapeutic decisions.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Abbreviations: 6MWD, 6-min walk distance; BNP, brain natriuretic peptide; cTn, cardiac troponin(s); CV, coefficient of variation; hFABP, heart-type fatty-acid-binding protein; NT-proBNP, N-terminal pro-BNP; PE, pulmonary embolism; PAH, pulmonary arterial hypertension; RV, right ventricular; sPAP, systolic pulmonary arterial pressure; TAPSE, tricuspid annular systolic excursion; hsTnT, high-sensitive cTnT; WHO, World Health Organization
- © The Authors Journal compilation © 2010 Biochemical Society