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Biochemical markers may identify preterm infants with a patent ductus arteriosus at high risk of death or severe intraventricular haemorrhage
  1. A El-Khuffash1,
  2. D Barry1,
  3. K Walsh2,
  4. P G Davis3,
  5. E J Molloy1,2
  1. 1
    Department of Neonatology, National Maternity Hospital, Dublin, Ireland
  2. 2
    Department of Paediatric Cardiology, Our Lady’s Children Hospital, Crumlin, Ireland
  3. 3
    Neonatal Services, Royal Women’s Hospital, Carlton, Victoria, Australia
  1. Afif El-Khuffash, National Maternity Hospital, Holles Street, Dublin 2, Ireland; afif_faisal{at}hotmail.com

Abstract

Background: A patent ductus arteriosus (PDA) in preterm infants is associated with increased risk of intraventricular haemorrhage (IVH) and death. Cardiac troponin T (cTnT) and N-terminal-pro-B type natriuretic peptide (NTpBNP) are markers of cardiac function and can predict poor outcome in adults.

Aims: To determine whether echocardiography and cTnT/NTpBNP levels at 48 h predict death before discharge or severe IVH in preterm infants with a PDA.

Methods: Infants born <32 weeks' gestation or <1500 g underwent echocardiographic and cTnT/NTpBNP measurements at 12 and 48 h of life. Infants were divided according to their status at discharge: a closed PDA at 48 h, infants with a PDA at 48 h and IVH III/IV and/or death, and infants with a PDA at 48 h without IVH III/IV or death.

Results: Eighty infants with a median gestation of 28 weeks (IQR 26.1–29.5) and birth weight 1.06 kg (0.8–1.21) were included. At 48 h, infants with a PDA and IVH III/IV and/or death had significantly higher median cTnT/NTpBNP levels compared to infants with a PDA without IVH III/IV and/or death and those with spontaneous PDA closure (NTpBNP 9282, 5121 and 740 pmol/l, respectively, p = 0.008, and cTnT 0.66, 0.25 and 0.13 µg/l, respectively, p = 0.027). There were no differences in echocardiographic parameters of PDA size, left atrial to aortic ratio (LA:Ao), left and right ventricular outputs between the PDA groups.

Conclusions: NTpBNP and cTnT in conjunction with echocardiography may provide a basis for trials of targeted medical treatment in infants with a PDA.

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Patent ductus arteriosus (PDA) in preterm infants is associated with a higher incidence of intraventricular haemorrhage (IVH) and death.1 2 However it is difficult to predict which infants with a PDA go on to develop major complications.3 Conventional echocardiographic markers such as ductal diameter and left atrial to aortic ratio (LA:Ao) applied at 48 h of life do not predict outcome. In addition, treatment based on these parameters has not led to improved neurodevelopmental outcome at 2 years in preterm infants.47

Serum cardiac troponin T (cTnT) is a marker of cardiac injury and predicts mortality in adults with haemorrhagic and ischaemic stroke even in the absence of myocardial injury.8 9 Troponin T is a good predictor of myocardial injury in perinatally asphyxiated neonates,10 and levels are higher in premature infants with respiratory distress syndrome.11 The presence of a PDA may influence cTnT levels as diastolic steal reduces coronary blood flow leading to ischaemia. B-type natriuretic peptide (BNP) and its inactive by-product N-terminal Pro-BNP (NTpBNP) are released by the stressed neonatal myocardium in response to volume and pressure loading.12 BNP and NTpBNP levels are good markers of PDA and fall following successful treatment.13 14 NTpBNP is a more sensitive and specific marker of ventricular dysfunction than BNP and has a longer half-life (120 vs 20 minutes, respectively).15 We hypothesised that NTpBNP and cTnT may be useful in identifying infants with a PDA who would subsequently develop severe IVH and/or death. Our aim was to evaluate the accuracy of NTpBNP and cTnT levels compared to echocardiography in predicting death or severe IVH in preterm infants.

METHODS

All preterm neonates with birth weights between 500 and 1500 g and gestation between 24 and 32 weeks born at the National Maternity Hospital, Dublin, Ireland between July 2006 and August 2007 were eligible. Infants with major congenital abnormalities, cardiac lesions other than PDA, and IVH occurring before 48 h of life were excluded. Maternal details including pre-eclampsia, histological chorioamnionitis, antenatal steroid administration, and the presence of antepartum haemorrhage were recorded. Apgar score at 5 minutes and respiratory distress syndrome (RDS) were noted. RDS was defined by clinical signs of respiratory distress and a rising partial pressure of carbon dioxide (pCO2) on an arterial blood gas sample. In addition, radiological diagnosis was used to further support clinical evidence.16 A complete course of antenatal steroids was defined as betamethasone 12.5 mg given twice, 12 h apart and a single dose was termed partial treatment. Cranial ultrasound scans were performed at 12 and 48 h, and days 5 and 7 of life by the same consultant radiologist. Infants were grouped according to their worst ultrasound scan during their stay using the Papile classification17 into 1) No IVH, IVH grades I and II, and 2) IVH grades III and IV. Echocardiographic examination with simultaneous NTpBNP and troponin T measurements were carried out at 12 and 48 h of age by a single echocardiographer (AK). Both the radiologist and echocardiographer were masked from the NTpBNP and cTnT results. Periventricular leukomalacia (PVL), and death before discharge were recorded.

Biochemical parameters

NTpBNP and cTnT measurements were taken immediately following the echocardiogram. A sample of 1 ml of blood was collected in a lithium–heparin bottle and centrifuged and the plasma frozen at −20°C for later batch analysis using the Elecsys 2010 quantitative assay (www.roche.com). The Roche test is a non-competitive chemiluminescent technology for the determination of NTpBNP in human serum and plasma. This is a two-site (sandwich) assay incorporating a polyclonal NTpBNP-specific antibody and a polyclonal NTpBNP-specific antibody labelled with a ruthenium complex. The variance is 4.6% (5.6 pmol/l) and 1.9% (107.5 pmol/l), respectively, for low- and high-concentration patient samples, and the respective day-to-day variance is 5.5% (6.4 pmol/l) and 2.6% (113.6 pmol/l).18

The cTnT assay is an electrochemiluminescent sandwich enzyme-linked immunosorbant assay, which has a lower limit of detection of 0.01 pg/l, with minimal cross-reactivity with cardiac troponin I (0.002%) and skeletal troponin T (0.001%). This third-generation assay is unaffected by bilirubin levels up to exchange values, sample haemolysis, or renal insufficiency. The repeatability coefficient for a paired sample is 10% and the variability coefficient for precision analysis is 6.4% (Anon 1999, troponin T STAT data sheet, Roche diagnostics, www.roche.com).

Echocardiographic measurements

We used the Siemens Acuson Sequoia ultrasound machine and a 10v4 cardiology multi-frequency probe. Studies were performed by a single echocardiographer (AK) using standard neonatal windows including apical, parasternal, subcostal, and high parasternal windows. Two-dimensional, M-mode imaging, pulse and colour Doppler information were recorded. We obtained measurements at 12 and 48 years of age and the echocardiographer and the supervising clinician were blinded to the NTpBNP and cTnT results. The following echocardiographic variables were determined in each study:

  • Ductal diameter: ductal diameter ⩾1.5 mm was considered significant.19

  • LA:Ao: we used a cut-off ratio of ⩾1.5 to define significance.19

  • Descending aortic end-diastolic velocity (DAo EDV): to assess the degree of diastolic steal caused by the duct during diastole.20

  • Left and right ventricular outputs (LVO and RVO): measured using established methods.21 22

Treatment of a PDA was at the discretion of the consultant on service who was blinded to the results of the echocardiographic examinations. The results of echocardiographs were not used to determine PDA treatment which was based on clinical signs of a PDA. Treatment was commenced on or after day 3 of life. Prophylactic treatment was not used during the study period.

Statistical analysis

The cohort was divided into infants whose ducts closed spontaneously at 48 h and infants with a PDA at 48 h. Infants with a PDA at 48 h were subdivided into those without IVH III/IV or death, and infants with a PDA and IVH III/IV, death or both. The echocardiographic and plasma NTpBNP/cTnT measurements were non-parametric continuous variables. Medians were compared using the Mann–Whitney U test for non-parametric data. The Kruskal–Wallis test was used for multiple groups, and the chi-squared test was used for categorical data. Subgroup analysis was performed based on the presence or absence of death and/or IVH III/IV. Receiver operating characteristic curves were constructed, and sensitivity, specificity, positive predictive value and negative predictive value of the bioassay (NTpBNP and cTnT) for the presence of significant ductal left-to-right shunting and complications were calculated. Results were expressed as median (interquartile range (IQR)) unless stated otherwise. We considered a p value of <0.05 as significant.

RESULTS

Eighty preterm infants underwent 160 echocardiographic examinations paired with simultaneous NTpBNP and cTnT determinations (fig 1). Their median (IQR) gestation was 28 weeks (26.1–29.5) and median (IQR) birth weight 1.06 kg (0.87–1.21). Thirty-five infants closed their PDA spontaneously by 48 h of life and in 45 infants the PDA remained open. There were no significant differences between the groups in gender, Apgar score at 5 minutes or maternal antenatal factors (table 1). Infants with a PDA had a higher incidence of IVH III/IV, PVL, death and combined death or IVH III/IV. Infants with a PDA and poor outcome had lower gestation and birth weights (table 2).

Figure 1 Breakdown of the study cohort.
Table 1 Characteristics of the study groups
Table 2 Outcomes in infants with and without a PDA

NTpBNP and cTnT at 12 h of life

At 12 h of life all infants had a PDA with left to right shunting. The median NTpBNP value for the cohort was 1273 pmol/l (664–2798) and median cTnT was 0.20 µg/l (0.11–0.40). NTpBNP and cTnT were unaffected by gender, antepartum haemorrhage, chorioamnionitis, prolonged rupture of membranes, antenatal steroids, or mode of delivery. Infants that closed their PDA spontaneously at 48 h had lower 12 h cTnT levels (0.14 vs 0.30 μg/l, p = 0.04). Infants with RDS had significantly higher NTpBNP (1391 vs 815 pmol/l, p = 0.04) and cTnT (0.24 vs 0.09 μg/l, p = 0.008) at 12 h. After adjusting for RDS and PDA, gestation and birth weight had no impact on NTpBNP and cTnT levels. When the PDA group was subdivided into infants with IVH III/IV and/or death (n = 20), and infants without these complications (n = 25), there were no significant differences in the echocardiographic measurements or the cTnT and NTpBNP levels between them and the spontaneous PDA closure groups at 12 h (table 3). In addition, there was no association between 12 h NTpBNP and cTnT levels and outcomes.

Table 3 Echocardiographic and biochemical markers in the cohort at 12 h

NTpBNP and cTnT at 48 h of life

NTpBNP was significantly higher in infants with a PDA at 48 h compared to those with spontaneous PDA closure (6059 vs 740 pmol/l, p<0.001). In addition, infants with a PDA at 48 h had significantly higher cTnT levels (0.43 vs 0.13 μg/l, p<0.001). Table 4 illustrates the association of NTpBNP and cTnT with outcomes in the whole cohort. NTpBNP was significantly higher in infants with IVH III/IV, infants that died and in infants with death/IVH III/IV. cTnT was significantly higher in infants with IVH III/IV, death and combined death/IVH III/IV. Infants with spontaneous PDA closure at 48 h had significantly lower LVO, and DAo EDV compared to infants with a PDA (table 5). However, the echocardiographic measurements of PDA significance (PDA diameter and LA:Ao ratio), LVO, RVO, and DAo EDV were not significantly different between the PDA subgroups. Infants with a PDA and IVH III/IV and/or death had significantly higher NTpBNP and cTnT levels compared to infants with a PDA without IVH III/IV and death and spontaneous PDA closure groups (9282, 5121 and 740 pmol/l, respectively, for NTpBNP, p = 0.008, and 0.66, 0.25 and 0.13 μg/l, respectively, for cTnT, p = 0.027) (table 5, fig 2).

Figure 2 48 hours cTnT and NTpBNP levels and outcomes in the groups. A Mann–Whitney U test was used to compare medians.
Table 4 Comparison of NTpBNP and cTnT between different outcomes
Table 5 Echocardiographic and biochemical markers in the cohort at 48 h

More infants in the PDA with IVH III/IV and death group received inotropes at 48 h compared with the other groups (table 1). However, when controlling for inotrope usage, NTpBNP and cTnT remained independently significantly associated with death and severe IVH (r = 0.5, p<0.001 for NTpBNP and r = 0.39, p = 0.002 for cTnT).

Only one infant in the spontaneous closure group developed a severe IVH. The 12- and 48 h NTpBNP/cTnT were 2997 pmol/l/0.104 μg/l and 502 pmol/l/0.027 μg/l, respectively. Only one infant in the spontaneous PDA closure group died before discharge. The 12- and 48 h NTpBNP/cTnT were 2023 pmol/l/1.52 μg/l and 6605 pmol/l/6.07 μg/l, respectively.

A receiver operating characteristics curve was constructed for the Elecsys 2010 assays’ ability to predict death alone, and the combined outcome of IVH III/IV and/or death in preterm infants. NTpBNP yielded an area under the curve (AUC) of 0.90 (95% CI 0.80 to 0.99, p<0.001) for predicting death before discharge, with a value of 6500 pmol/l providing a sensitivity of 82% and a specificity of 92%. cTnT yielded an AUC of 0.86 (95% CI 0.78 to 0.96, p<0.001), with a value of 0.45 μg/l providing a sensitivity of 82% and a specificity of 76%. For the combined outcome of severe IVH and/or death, the AUC for NTpBNP was 0.84 (95% CI 0.72 to 0.96, p<0.001) and a level of 5500 pmol/l had a sensitivity of 80% and a specificity of 80%. AUC for cTnT was 0.81 (95% CI 0.69 to 0.92, p = 0.001), with a level of 0.42 μg/l having a sensitivity of 70% and a specificity of 75%.

DISCUSSION

PDA is associated with adverse outcomes in a subgroup of infants.23 24 We have also shown that death and IVH III/IV was significantly higher in infants with a PDA compared to infants with spontaneous PDA closure by 48 h. The main aim of this study was to assess the ability of NTpBNP and cTnT to predict outcome in the subgroup of infants with a PDA. Echocardiography alone cannot identify the high-risk PDA group. However, the addition of biochemical markers can delineate infants with a PDA and severe IVH and/or death. At 12 h there were no significant differences in any of the echocardiographic parameters between the three groups indicating that PDA-associated shunting may not be significant at this early stage. At 48 h, there were no differences in ductal size, LA:Ao ratio, LVO or RVO in infants with a PDA and good or poor outcomes. Although different from the spontaneous PDA closure group, echocardiographic parameters were similar in the two PDA subgroups at 48 h.

We demonstrated the association between elevated NTpBNP and cTnT and poor outcome in infants with a PDA. cTnT and NTpBNP were unaffected by gestation, birth weight, sex, chorioamnionitis, and mode of delivery. This has been previously shown by other groups for cTnT and NTpBNP.2529 NTpBNP and cTnT levels were higher at 12 h in infants with RDS. cTnT levels at 12 h were higher in infants with a patent ductus at 48 h, possibly secondary to ductal steal affecting coronary arteries leading to myocardial ischaemia.30 Trevisanuto et al measured cTnT levels in 11 preterm infants with a PDA and 12 controls with similar gestation, birth weight and background. They found no difference in TnT levels between the PDA group and the controls, and concluded that cTnT levels are not influenced by PDA.31 However the study was not adequately powered to detect clinically important differences and therefore the relationship between troponin T and PDA remains largely unexplored in premature infants.

None of our cohort had a severe IVH at the time of the 48 h cranial ultrasound scan. NTpBNP and cTnT accurately predicted the subsequent occurrence of severe IVH and death in infants with a PDA. Infants that developed the complications were of lower birth weight and gestation. However the rise in NTpBNP and cTnT could not be attributed to these factors alone as there was a lack of correlation between these biochemical assays, gestation and birth weight when adjusted for RDS and PDA.

NTpBNP is elevated in 60% of adults with acute ischaemic stroke, with higher values associated with increased mortality. Similarly NTpBNP is independently associated with an unfavourable outcome following stroke in another series despite a normal ejection fraction.32 33 A similar association exists between stroke and troponin T, with elevated levels being an independent predictor of mortality.34 NTpBNP was shown to be useful as a screening tool for the presence of a PDA in preterm neonates.14 NTpBNP and cTnT may help to pinpoint a specific subgroup of infants at high risk for severe IVH who may benefit from early intervention such as targeted medical treatment of a PDA.

We found that infants who developed a severe IVH had elevated troponin levels at 48 h. The pathophysiology of IVH in preterm infants differs from stroke in adults. The release of catecholamines associated with adult stroke may lead to myocardial stress and foci of necrosis resulting in a rise in TnT and NTpBNP after the occurrence of the event.35 36 In infants, haemorrhage originates from the friable germinal matrix which only involutes after 34 weeks’ gestation. Bleeding from these vessels may occur following haemodynamic instability.37 Conversely, a rise of cTnT and NTpBNP may be associated with sympathomimetic activity, and cardiovascular compromise preceding the evolution of the haemorrhage.

Two infants in the spontaneous closure group developed severe complications (1 death and 1 severe IVH). Their 48 h NTpBNP and cTnT values were comparable to their counterparts in the PDA group. However, due to the low rate of severe complication in the spontaneous closure group we were unable to assess the accuracy of these biochemical markers in predicting outcome in the absence of a PDA.

Gagliardi et al assessed the discriminatory ability of the clinical risk index for babies (CRIB), CRIB-II, and simplified version of the score for neonatal acute physiology (SNAPPE)-II in detecting death before discharge in 720 preterm infants.38 Following the exclusion of babies weighing 400–499 g (n = 15), the AUCs for CRIB, CRIB-II and SNAPPE-II were 0.898, 0.905, and 0.835, respectively. These results were comparable to the AUCs for NTpBNP and cTnT and death in our cohort.

Infants with a PDA and IVH III/IV and/or death received more inotropes. The association between elevated cTnT levels and inotrope use has been previously shown by Clark et al.39 However, after adjusting for inotrope usage, the biochemical markers remained independently associated with severe IVH and death in the PDA subgroup, suggesting that this association is independent of inotrope use. The cTnT levels reported in this study are higher than those in previous reports.25 39 40 This may be explained by the higher incidence of PDA and IVH in our population as the assay used to measure cTnT was the same in all studies. McNamara et al has proposed that PDA shunting may also involve the coronary vessels therefore contributing to myocardial ischaemia and a rise in cTnT.30 Whether the rise of NTpBNP and cTnT is a result of a PDA, poor myocardial function associated with a PDA or a direct result of the processes leading to IVH evolution and mortality needs further study.

What is already known on this topic

  • NTpBNP is higher in infants with a PDA at 48 h compared to infants without a PDA. cTnT levels are elevated in asphyxiated infants and preterm infants with RDS.

What this study adds

  • Echocardiography alone at 48 h cannot identify infants with a PDA that go on to develop severe IVH and/or death.

  • NTpBNP and cTnT are higher in infants with a PDA that subsequently develop IVH III/IV or death compared to those with a PDA and without complications.

We demonstrated an association between cTnT and NTpBNP with severe IVH and death in infants with a PDA. These biochemical markers may prove to be a useful adjunct to a recent clinical and echocardiographic PDA staging system proposed by McNamara et al.30 The numbers in our study are relatively small and more research is needed to confirm the association of NTpBNP and cTnT with adverse outcomes in preterm infants. The incidence of IVH in our study was relatively high. The predictive value of cTnT and NTpBNP should be tested in populations with different rates of IVH.

Medical therapy for PDA has well-recognised adverse effects and neither prophylaxis nor treatment on the basis of clinical and echocardiographic signs has been shown to improve long-term outcomes. Accurately identifying infants with PDA who are at highest risk of poor outcome using cTnT and NTpBNP may allow more successful trials of targeted medical therapy of PDA.

Acknowledgments

We would like to thank Dr John Murphy and Dr Claudine Vavasseur for all their support and encouragement, and the Unit 8 nursing staff for being so helpful and accommodating of this project.

REFERENCES

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Footnotes

  • Competing interests: None.

  • Funding: National Maternity Hospital Research Fund.

  • Ethics approval: The study was approved prior to commencement by the National Maternity Hospital’s Ethics Committee, Dublin, Ireland.

  • Patient consent: Informed parental consent was requested within the first 24 h of life.

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