The Role of Transthoracic Echocardiography in Diagnosis of Pulmonary Hypertension of Infants With Bronchopulmonary Dysplasia
Condition: Pulmonary Hypertension; Bronchopulmonary Dysplasia
MeSH terms: Hypertension, Pulmonary; Bronchopulmonary Dysplasia; Hypertension
Study type: Observational

Summary: 
      Pulmonary hypertension may develop in premature newborn infants due to impaired lung
      development. The diagnosis of this disease can actually be made with interventional methods.

      In this study, we evaluated the importance of echocardiographic examination and blood
      laboratory tests in diagnosing this disease.
    

Detailed description: 
      Bronchopulmonary dysplasia diagnosis was done by the same neonatologist according to the
      criteria published by the American National Institutes of Health (NIH) in 2001.

      Demographic data of patients like: birth weight, gender, gestational age; neonatal morbidity
      like: surfactant administration, PDA and its treatment, duration of mechanical ventilation,
      diuretic usage, intraventricular hemorrhage and its grade, necrotizing enterocolitis (NEC)
      and its severity, any surgical procedures and late-onset sepsis were recorded.

      Moreover; maternal and perinatal risk factors like presence of chorioamnionitis, maternal
      hypertension and administration of antenatal steroids were also recorded.

      Echocardiography:

      Echocardiographic measurements were performed with a GE Vivid-7 pro equipped with a 3-7 MHz
      for 2-dimensional and color flow Doppler mapping by the same pediatric cardiologist.

      Investigations were done in parasternal long, short axis, four chamber, five chamber views
      using M mode and Doppler wave. Two-dimensional measurements were done according to the
      guidelines of American Society of Echocardiography. M mode views were obtained in parasternal
      long axis by putting cursor between mitral valve and apical to tips of the MV leaflets.
      Ejection fraction and end diastolic or end systolic volumes were calculated using the
      Teicholtz (Teich) method. Estimated Pulmonary artery systolic pressure can be determined by
      using the modified Bernoulli equation: 4V2, where V is the maximum velocity of the tricuspid
      valve regurgitation jet, measured by continuous wave Doppler, added to the estimated right
      atrial pressure.

      The echocardiography parameters showing pulmonary hypertension are tricuspid velocity, mean
      pulmonary artery pressure (PAP), pulmonary vascular resistance, pulmonary acceleration time,
      pulmonary velocity time integral, fraction of area changes of Right ventricle (RV), RV
      myocardial performance index (TEI). Severe pulmonary hypertension affects not only RV but
      also LV systolic functions. Therefore we measured Left Ventricle (LV) eccentricity index and
      LV TEI parameters in order to see whether they were affected in patients w BPD and PHT.

      Pulmonary vascular resistance (PVR) can be measured using Tricuspid Velocity (m/s) and
      Velocity Time IntegralRVOT (cm) PVR (Wood units) = 10 × (TRV/VTIRVOT) + 0.16. Here, a
      TRV/VTIRVOT<0.2 corresponds approximately to a PVR of <2 Wood Units [8].

      Left Ventricular Eccentricity index (EI), measured at end-systole and end-diastole, from the
      parasternal short axis 2D image at the mid-papillary muscle level. The formula (EI=D2/D1) was
      used where D1 is the ventricular diameter perpendicular to the interventricular septum
      bisecting D2: the diameter parallel to the interventricular septum [9].

      RV systolic dysfunction occurs in PHT and shown by RV Fraction of Shortening (FS), tricuspid
      annular plane systolic excursion (TAPSE) and Inferior vena cava distensibility index.

      Inferior vena cava distensibility index is calculated by measuring the Dmax and Dmin of IVC
      from the subcostal view, and IVCDI exceeding 18% has been reported to be predictive of fluid
      responsiveness in adults, and it is often extrapolated in children as well.
      IVCDI=Dmax-Dmin/Dmin [10].

      First echocardiograms were performed at 36 to 38 weeks' post-menstrual age (PMA) for
      screening later repeated at 1, 3, 6 months after the first echocardiogram. Measurements were
      made during 3 consecutive cycles and mean values were used in statistical analysis.

      Pulmonary hypertension diagnosis was done according to presence of at least one of the
      following criteria [11]: 1) the velocity of tricuspid valve regurgitation of ≥3 m/s in the
      absence of pulmonary stenosis or 2)estimated right ventricular systolic pressure (RVSP)
      greater than 40 mm Hg, or RVSP/systemic systolic blood pressure greater than 0.5, any 3)
      cardiac shunt with bidirectional or right-to-left flow, 4) flat or left-deviated
      interventricular septal configuration and right ventricular hypertrophy with chamber
      dilation.

      Serum Biomarkers:

      Serum biomarkers like kallistatin, NT-ProBNP, gelsolin, homocysteine, cystatin C levels were
      measured only in the beginning of the study and not repeated in the following controls.

        1. Serum Kallistatin Levels Blood for kallistatin level was taken to the tubes centrifuged
           at 4000 rpm for 10 min at 4°C. The serum was kept at -80°C as frozen until required.
           Kallistatin levels were determined by ELISA (R&D Systems, Inc. Minneapolis, USA) as
           previously described [12].

        2. Plasma NT-ProBNP Levels Five ml blood sample was drawn from both patient and control
           groups for N terminal pro Brain natriuretic peptide (NT-ProBNP). Blood samples were
           centrifuged at +4°C and 1500 rpm for 5 minutes. Plasma part of the upper phase was taken
           in to another tube for NT-ProBNP calculation. Samples were preserved at -80°C till the
           study date. Later on, they were studied with ELISA method using Biomedica N-terminal pro
           BNP commercial kits (NT- ProBNP enzyme immuno assay kit Biomedica, Bratislava, Slovakia)
           and Elecsys® 1010 aoutoanalyzer (Roche Diagnostics, Basel, Switzerland). Results were
           expresses as fmol/ml (1fmol/ml=16.1pg/ml).

        3. Serum Gelsolin Levels:

           Venous blood samples of the healthy individuals and patients were drawn on admission.
           The blood samples were immediately placed in sterile etylene diamin tetra asetic acid
           test tubes and centrifuged at 2000-3000 rpm for 20 minutes at 4°C to collect plasma.
           Plasma was stored at -80°C until assayed. The concentration of gelsolin in plasma was
           analysed by enzyme-linked immunosorbent assay using commercial kits (Hangzhou
           Eastbiopharm Co., Ltd, Hangzhou, Zhejiang, China) in accordance with the manufacturer's
           instructions.

        4. Plasma Homocysteine Levels:

           Plasma Homocysteine was measured using an auto-biochemical analyzer (AU5800, Beckman
           Coulter Company, U.S.A.) by the enzymatic method. This method uses the S-adenosyl
           homocysteine (SAH) hydrolase reaction principle, in which SAH is hydrolyzed by
           hydrolytic enzymes into adenosine and Homocysteine; adenosine is immediately hydrolyzed
           into ammonia and hypoxanthine, and nicotinamide adenine dinucleotide (NADH) is converted
           to NAD by ammonia and glutamic dehydrogenase. The concentration of Homocysteine in the
           sample is proportional to the NADH transformation rate. The detection reagents were
           provided by DiaSys Diagnosis System GmbH (Shanghai) Co., Ltd.

        5. Serum Cystatin-C Levels:

      Cystatin C levels were determined with an immune nephelometry method on an ProSpec analyzer
      (BN ProSpec, Siemens, Frimley, United Kingdom) using latex enhanced particles coated with
      anti-cystatin C antibodies. The reference values for young healthy persons range from 0.53 to
      0.95 mg/l. The coefficient of variation was 1.8% within one run and 2.0% during
      reproducibility tests. Freezing and long-term storage up to 25 years has a small impact on
      stability of cystatin C [13].

      The study was approved by the local research Ethics Committee. All the parents were informed
      written consent were taken from each of them about participating in the study.

      Statistical analysis:

      Data analysis was performed using IBM SPSS Statistics Standard Concurrent User V 25 (IBM
      Corp., Armonk, New York, USA). Continuous variables were expressed either as mean ± standard
      deviation or median 25th-75th percentiles. Distribution of numerical data is evaluated by
      Shapiro Wilk normality test Q -Q graphics. Categorical variables were expressed as counts,
      proportions, and percentages. A Kruskal-Wallis test was used to determine whether or not
      there is a statistically significant difference between the medians of three or more
      independent groups with non-parametric values. If statistical significance was detected
      (p<0.05) Post Hoc Analysis - Tukey test was performed for pairwise comparisons. For the
      repeated measures analysis of a dependent group; Friedman test was used.
    

Observational model: Other
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Preterm newborns followed in the neonatal intensive care unit
      
Biospecimen description: 
      Biomarkers like kallistatin, NT-ProBNP, gelsolin, homocysteine, cystatin C levels were
      measured.

      Serum was taken for Kallistatin, gelsolin and cystatin C Plasma was taken for NT-proBNP and
      homocysteine
    
Keywords: Bronchopulmonary dysplasia; Pulmonary hypertension; Echocardiography