Heterogeneity Index in Neonatologist-performed Lung Ultrasound in Neonates Receiving Respiratory Support - a Pilot Study
Conditions: Intensive Care Units, Neonatal, Infant, Newborn, Infant, Premature, Point-of-Care Systems, Ultrasonography, Lung Ultrasound, Lung Ultrasound Score, Image Interpretation, Computer Assisted, Reproducibility of Results, Respiratory Distress Neonatal, Respiratory Support
Sponsor: Medical University of Graz

 
 
 
Study Protocol 
 
 
 
 
Heterogeneity Index in Neonatologist-performed Lung 
Ultrasound in Neonates Receiving Respiratory Support 
- a pilot study 
 
 
 
 
 
Melina Winkler, MD 
Division of Neonatology, Department of Pediatrics, Medical 
University of Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria 
 
Thomas Suppan, PhD, MSc 
Division of Neonatology, Department of Pediatrics, Medical 
University of Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria 
 
Alana Seigner 
Division of Neonatology, Department of Pediatrics, Medical 
University of Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria 
 
Bernhard Schwaberger, MD, PhD 
Division of Neonatology, Department of Pediatrics, Medical 
University of Graz, Auenbruggerplatz 34/2, 8036 Graz, Austria 
 
 
 
 
Version 1.0 
17.03.2025 
 
 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 1/ 7 
1. Introduction 
Lung ultrasound is rapidly gaining recognition as a valuable clinical tool for dynamic lung 
assessment.1–3 
Recently, its application in neonatal intensive care units (NICU) has received increasing 
attention. Neonatologist-performed lung ultrasound (NPLUS) has proven to be a highly 
reliable, quick and safe diagnostic tool in neonatal intensive care.4,5 The clinical utility of 
NPLUS extends to diagnosing and monitoring a range of respiratory disorders, including 
acute respiratory distress syndrome, transient tachypnoea of the newborn, pneumonia, 
pleural effusion, atelectasis, lung edema, meconium aspiration syndrome, and 
pneumothorax.6–11 Unlike conventional X-ray imaging, NPLUS provides a radiation-free 
alternative with high specificity and sensitivity, making it an attractive option for neonatal 
lung assessment. 3,12,13  
2. Background 
Lung ultrasound relies on interpreting ultrasound artifacts, analysing the pleura, and 
visualizing consolidations and pleural effusions, thereby improving the differential 
diagnosis of respiratory distress in neonates. 
Neonates can progress from a white lung pattern (i.e. complete aeration loss due to fluid-
filled alveoli) through B-lines (vertical, dynamic artifacts arising at the fluid-air interface, 
commonly linked to extravascular lung water or alveolar-interstitial edema) to A-lines 
(horizontal reverberation artifacts suggesting improved aeration and fluid clearance), 
marking a gradual transition toward lung aeration. 
The impairment of lung aeration can be quantified based on the number and type of 
visualized artefacts. 14,15 
Several studies have investigated the role of NPLUS in neonatal respiratory assessment. By 
employing the lung ultrasound score (LUS) - a semi-quantitative tool for evaluating lung 
aeration and pathology - neonatologists can identify infants requiring surfactant therapy 
or those at risk for developing bronchopulmonary dysplasia. 5,8,16–18 
There is emerging evidence that NPLUS is not only a reliable tool for predicting the need 
for surfactant administration but also for differentiating between the underlying causes of 
respiratory distress in neonates. To date, different scoring systems have been developed 
for various clinical contexts. 19–21 These scores aim to provide a comprehensive assessment 
of lung condition by systematically screening the lung fields. We aim to use the LUS 
proposed Rodriguez-Fanjul 7,8,16, dividing the chest into six regions (anterior, lateral and 
posterior region on each side), assigning a score from 0 – 3 to each region.  
 
Among the many advantages of this accessible semi-quantitative method, the subjectivity 
of the scoring system remains a limitation that may influence the evaluation of a patient’s 
lung condition. Moreover, the standard 0–3 scoring scale of each lung area may not always 
capture subtle yet clinically significant changes in lung aeration over time.  
 
To correlate fetal lung tissue characteristics with lung pathologies, gynaecologists have 
introduced the Heterogeneity Index (HI) as a quantitative assessment tool. 22–24  

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 2/ 7 
The HI represents a quantitative ultrasound texture analysis method that has 
demonstrated promising diagnostic performance, with a sensitivity of 88% and specificity 
of 92% for evaluating fetal lung maturity and predicting respiratory distress in the 
newborn. 22,25–27 This technique analyses ultrasonographic fetal lung heterogeneity at the 
pixel level. By assessing the patterns and variations within the lung tissue, this method 
provides a numerical representation of lung texture, rather than a categorical score as 
used in current lung ultrasound scoring systems. 28,29 Although the fetal lung has different 
physical properties compared to the neonatal lung, as it is not yet aerated, the same 
method could be applied to assess the lung condition in neonates after birth. As aeration 
progresses, the neonatal lung generates ultrasound artifacts that, despite the significant 
differences in physical properties, could still be quantitatively evaluated using the HI. 
This objective approach enhances the accuracy of lung ultrasound assessments and may 
contribute to improved decision-making in neonatal respiratory management. We aim to 
introduce a quantitative ultrasound analysis that can be easily implemented into clinical 
practice, as it might provide valid information from standard lung ultrasound.  
To the best of our knowledge, this approach has not yet been applied in the field of NPLUS.  
3. Objectives 
This study aims to perform NPLUS in neonates receiving non-invasive or invasive 
respiratory support in the NICU and analyse the raw ultrasound image data to derive the 
HI. The HI will then be compared to the LUS to assess its clinical relevance. 
 
Hypothesis I: It is feasible to obtain the HI through NPLUS in the study population (‘proof 
of concept’).  
 
Hypothesis II: The HI correlates with the LUS. A higher HI reflects a more severe respiratory 
condition, characterized by greater loss of aeration, and is associated with a higher LUS. 
Conversely, a lower HI corresponds to better lung aeration and a lower LUS, indicating a 
healthier lung condition. 
 
Hypothesis III: During the first week of life, in neonates receiving respiratory support, the 
HI demonstrates a dynamic change between two time points at which NPLUS is performed. 
A decrease in HI would indicate an improvement in the neonate’s lung condition. The 
decrease in the HI between the two assessment time points may be more pronounced 
compared to the LUS, suggesting a higher discriminative ability of the HI in detecting 
changes in lung condition. 
 
Hypothesis IV: There is a correlation between HI and both the duration of respiratory 
support required and the applied mode of ventilation. 
4. Design, setting and participants 
Study design: The study is designed as an observational, prospective pilot study.  
 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 3/ 7 
Study population: This study will include neonates with a gestational age between 28+0 
weeks and full term who require either non-invasive or invasive respiratory support in the 
NICU. 
 
Study procedure: A trained medical professional will perform NPLUS at predefined time 
points after birth: NPLUS 1 within 72 hours of birth and NPLUS 2 within 72 hours (minimum 
24 hours) after the first examination. To minimize interference with routine care, NPLUS 
will be performed by an independent physician who is not involved in the patient’s routine 
clinical care. Participation in the study will not alter the standard clinical care in the NICU. 
Clinicians will be blinded to the NPLUS results except in cases of pneumothorax and pleural 
effusion. 
 
NPLUS is performed bilaterally with longitudinal scans of the chest on the midclavicular, 
anterior, and posterior axillary line. The scans can be performed either with the patient in 
supine or prone position as the posterior axillary line is accessible from both sides of the 
neonate. A 12-15 MHz linear transducer is used, preferably with the preset “small parts” 
and an ultrasound penetration depth of 3-4 cm. The raw ultrasound images are saved as 
Tag Image File Format (TIFF).  
 
Heterogeneity Index (HI): The raw ultrasound images will be reviewed by a trained 
medical professional. A region of interest (ROI) will be manually selected in each lung area 
that should be analysed. Each ROI will be a square that includes the largest possible area 
with lung artefacts, starting from the pleura and excluding subcutaneous tissue.  
The grayscale-based analysis will be performed using a custom-made program developed 
in MATLAB (R2024b, Version 11.19.6.2). Texture features in the lung tissue will be related 
to pathophysiologic processes. The HI will then be determined by measuring the intensity 
of each pixel and calculating the average of all pixels within the ROI.  
 
Lung ultrasound score (LUS): The score from 0-3 will be assigned to each scan area based 
on the detected pattern (0 = A-pattern: only A-lines; 1 = B-pattern: three or more B-lines, 
well-spaced; 2 = severe B-pattern: crowded and/ or fusing B lines with/ without 
consolidations limited to subpleural space; 3 = extended consolidations). 7,8,16 
To ensure inter-observer reliability, two neonatologists, blinded to the patient’s identity 
and clinical conditions, will independently score the anonymized ultrasound images.  
 
Inclusion criteria:  
• Preterm neonates born between 28+0 and 36+6 weeks of gestation and full-term 
neonates (> 37+0 weeks of gestation) admitted to the NICU of the Division of 
Neonatology, Medical University of Graz. 
• Presence of respiratory distress requiring respiratory support at the time of NPLUS 
1.  
• Written informed consent obtained from the parents prior to the measurement. 
 
Exclusion criteria:  
• Cardiopulmonary malformations  
• Pleural effusion 
• Pneumothorax 
 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 4/ 7 
Sample Size: A sample size of 40 patients (20 preterm and 20 full-term neonates) will be 
analysed in this study. Sample size calculations cannot be performed in advance, as no 
data from previous studies are available. Therefore, this pilot study aims to generate data 
for the sample size estimation in the consecutive main study. 30 
5. Statistical analysis 
Demographic data, clinical characteristics, and lung ultrasound features will be 
summarized descriptively. First, the Shapiro-Wilk test will be used to assess the normality 
of the data. Depending on the distribution, data will be presented as mean (standard 
deviation, SD) or median (interquartile range, IQR). 
 
Differences in primary and secondary outcome parameters are calculated using Chi-square 
and Fisher's exact tests for categorical variables, and t-test or Mann-Whitney U test for 
continuous variables. 
 
Additionally, correlations between HI and LUS, as well as duration of respiratory support 
required and the applied mode of ventilation will be analysed using either the Spearman’s 
rank correlation coefficient or the Pearson’s correlation, depending on which is more 
appropriate.  
 
The correlation analyses are considered in an explorative sense; therefore, no multiple 
testing corrections will be performed. A value of p < 0.05 will be considered as statistically 
significant. 
 
MATLAB (R2024b, version 11.19.6.2) will be used for statistical analysis. 
6. Outcome measures 
Primary Outcome Measures 
• Determination of the heterogeneity index of NPLUS 1 and NPLUS 2 
• Determination of the LUS by Rodriguez-Fanjul 16 of NPLUS 1 and NPLUS 2 
 
Secondary Outcome Measures 
• Duration of respiratory support (in hours) 
• Mode of respiratory support (non-invasive versus invasive ventilation) 
• Routinely obtained capillary blood gas analysis of the neonate within 6 hours 
before or after NPLUS 1 or NPLUS 2 
• Routinely obtained monitoring parameters including arterial oxygen saturation 
(SpO2), heart rate (either by pulse oximetry or electrocardiography), and – if 
available – cerebral tissue oxygen saturation (near-infrared spectroscopy) 
• SpO2/FiO2 ratio (S/F ratio) 
• Chest X Ray findings (if performed within 6 hours before or after NPLUS 1 or NPLUS 
2) 
• pH of the umbilical artery 
• APGAR score 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 5/ 7 
• Pregnancy risk factors including intra-amniotic infection, and premature rupture of 
the membranes, pre-eclampsia 
• Prenatal corticosteroids 
7. Research Plan and timetable 
The study will start in spring/summer 2025. 
The study is designed for an approximate running time of two years.  
 
 
 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 6/ 7 
8. References 
1. 
Kurepa, D., Zaghloul, N., Watkins, L. & Liu, J. Neonatal lung ultrasound exam 
guidelines. Journal of Perinatology 38, 11–22 (2018). 
2. 
Singh, Y. et al. Recommendations for hemodynamic monitoring for critically ill 
children—expert consensus statement issued by the cardiovascular dynamics 
section of the European Society of Paediatric and Neonatal Intensive Care (ESPNIC). 
Crit Care 24, (2020). 
3. 
Singh, Y. et al. International evidence-based guidelines on Point of Care Ultrasound 
(POCUS) for critically ill neonates and children issued by the POCUS Working Group 
of the European Society of Paediatric and Neonatal Intensive Care (ESPNIC). Crit 
Care 24, 1–16 (2020). 
4. 
Alonso-Ojembarrena, A., Gregorio-Hernández, R. & Raimondi, F. Neonatal point-of-
care lung ultrasound: what should be known and done out of the NICU? European 
Journal 
of 
Pediatrics 
vol. 
183 
1555–1565 
Preprint 
at 
https://doi.org/10.1007/s00431-023-05375-5 (2024). 
5. 
Aichhorn, L. et al. children The Role of Lung Ultrasound in the Management of the 
Critically Ill Neonate-A Narrative Review and Practical Guide. (2021) 
doi:10.3390/children8080628. 
6. 
Martino, L. De et al. Lung ultrasound score predicts surfactant need in extremely 
preterm neonates. Pediatrics 142, (2018). 
7. 
Raimondi, F. et al. Neonatal Lung Ultrasound and Surfactant Administration: A 
Pragmatic, Multicenter Study. Chest 160, 2178–2186 (2021). 
8. 
Corsini, I. et al. Lung UltrasouNd Guided surfactant therapy in preterm infants: an 
international multicenter randomized control trial (LUNG study). Trials 24, 1–11 
(2023). 
9. 
Raimondi, F. et al. Lung ultrasound score progress in neonatal respiratory distress 
syndrome. Pediatrics 147, (2021). 
10. 
Poerio, A. et al. Lung ultrasound features predict admission to the neonatal 
intensive care unit in infants with transient neonatal tachypnoea or respiratory 
distress syndrome born by caesarean section. Eur J Pediatr 180, 869–876 (2021). 
11. 
Gautam, N. K., Venkatesh, H. A., Pejaver, R. & Nagesh, K. Diagnostic Utility of 
Bedside “Point of Care Lung Ultrasound” in Predicting the Need For NICU Admission 
in Late Preterm and Term Newborns Having Respiratory Distress Soon After Birth in 
the Transition Period: A Prospective Observational Study. Pediatr Pulmonol 60, 
(2025). 
12. 
Aichhorn, L., Küng, E. & Schwaberger, B. Neonatologist performed lung ultrasound: 
NPLUS—proposal for a consistent ultrasound terminology. Frontiers in Pediatrics 
vol. 10 Preprint at https://doi.org/10.3389/fped.2022.1007672 (2023). 
13. 
Renz, D. M. et al. Chest X-rays in children and adolescents: Indications and 
limitations. Radiologe 62, 140–148 (2022). 
14. 
Ollier, V. et al. Semi-quantitative lung ultrasound score during ground 
transportation of outborn neonates with respiratory failure. Eur J Pediatr 181, 
3085–3092 (2022). 
15. 
Raimondi, F. et al. Neonatal Lung Ultrasound and Surfactant Administration: A 
Pragmatic, Multicenter Study. Chest 160, 2178–2186 (2021). 
16. 
Rodriguez-Fanjul, J. et al. Early surfactant replacement guided by lung ultrasound in 
preterm newborns with RDS: the ULTRASURF randomised controlled trial. Eur J 
Pediatr 179, 1913–1920 (2020). 

Heterogeneity Index in Neonatologist-performed Lung Ultrasound in neonates receiving respiratory 
support 
Version 1.0, 17.03.2025 
Page 7/ 7 
17. 
Schmidt, A. R. & Ramamoorthy, C. Bronchopulmonary dysplasia. Paediatr Anaesth 
32, 174–180 (2022). 
18. 
Alonso-Ojembarrena, A. et al. The Predictive Value of Lung Ultrasound Scores in 
Developing Bronchopulmonary Dysplasia: A Prospective Multicenter Diagnostic 
Accuracy Study. in Chest vol. 160 1006–1016 (Elsevier Inc., 2021). 
19. 
Mongodi, S. et al. Quantitative Lung Ultrasound: Technical Aspects and Clinical 
Applications. Anesthesiology 134, 949–965 (2021). 
20. 
Rodriguez-Gonzalez, M., Rodriguez-Campoy, P., Estalella-Mendoza, A., Castellano-
Martinez, A. & Flores-Gonzalez, J. C. Characterization of Cardiopulmonary 
Interactions and Exploring Their Prognostic Value in Acute Bronchiolitis: A 
Prospective Cardiopulmonary Ultrasound Study. Tomography 8, 142–157 (2022). 
21. 
Brat, R. et al. Lung ultrasonography score to evaluate oxygenation and surfactant 
need in neonates treated with continuous positive airway pressure. JAMA Pediatr 
169, 1–8 (2015). 
22. 
Ghorayeb, S. R., Bracero, L. A., Blitz, M. J., Rahman, Z. & Lesser, M. L. Quantitative 
ultrasound texture analysis for differentiating preterm from term fetal lungs. in 
Journal of Ultrasound in Medicine vol. 36 1437–1443 (Wiley Blackwell, 2017). 
23. 
Ahmed, B. & Konje, J. C. Fetal lung maturity assessment: A historic perspective and 
Non – invasive assessment using an automatic quantitative ultrasound analysis (a 
potentially useful clinical tool). European Journal of Obstetrics and Gynecology and 
Reproductive Biology 258, 343–347 (2021). 
24. 
Cobo, T. et al. Feasibility and reproducibility of fetal lung texture analysis by 
automatic quantitative ultrasound analysis and correlation with gestational age. 
Fetal Diagn Ther 31, 230–236 (2012). 
25. 
Burgos-Artizzu, X. P., Perez-Moreno, Á., Coronado-Gutierrez, D., Gratacos, E. & 
Palacio, M. Evaluation of an improved tool for non-invasive prediction of neonatal 
respiratory morbidity based on fully automated fetal lung ultrasound analysis. Sci 
Rep 9, (2019). 
26. 
Du, Y. et al. Ultrasound-based radiomics technology in fetal lung texture analysis 
prediction of neonatal respiratory morbidity. Sci Rep 12, (2022). 
27. 
Du, Y. et al. Application of ultrasound-based radiomics technology in fetal-lung-
texture analysis in pregnancies complicated by gestational diabetes and/or pre-
eclampsia. Ultrasound in Obstetrics and Gynecology 57, 804–812 (2021). 
28. 
Palacio, M. et al. Prediction of neonatal respiratory morbidity by quantitative 
ultrasound lung texture analysis: a multicenter study. Am J Obstet Gynecol 217, 
196.e1-196.e14 (2017). 
29. 
Perez-Moreno, A. et al. Clinical Feasibility of Quantitative Ultrasound Texture 
Analysis: A Robustness Study Using Fetal Lung Ultrasound Images. Journal of 
Ultrasound in Medicine 38, 1459–1476 (2019). 
30. 
Thabane, L. et al. A tutorial on pilot studies: the what, why and how. BMC Research 
Methodology 10, 37–58 (2010).