Reference values of diaphragmatic dimensions in healthy children aged 0–8 years

Diaphragmatic thickness (Tdi) and diaphragm thickening fraction (dTF) are widely used parameters in ultrasound studies of the diaphragm in mechanically ventilated children, but normal values for healthy children are scarce. We determined reference values of Tdi and dTF using ultrasound in healthy children aged 0–8 years old and assessed their reproducibility. In a prospective, observational cohort, Tdi and dTF were measured on ultrasound images across four age groups comprising at least 30 children per group: group 1 (0–6 months), group 2 (7 months-1 year), group 3 (2–4 years) and group 4 (5–8 years). Ultrasound images of 137 healthy children were included. Mean Tdi at inspiration was 2.07 (SD 0.40), 2.09 (SD 0.40), 1.69 (SD 0.30) and 1.72 (SD 0.30) mm for groups 1, 2, 3 and 4, respectively. Mean Tdi at expiration was 1.64 (SD 0.30), 1.67 (SD 0.30), 1.38 (SD 0.20) and 1.42 (SD 0.20) mm for groups 1, 2, 3 and 4, respectively. Mean Tdi at inspiration and mean Tdi at expiration for groups 1 and 2 were significantly greater than those for groups 3 and 4 (both p < 0.001). Mean dTF was 25.4% (SD 10.4), 25.2% (SD 8.3), 22.8% (SD 10.9) and 21.3% (SD 7.1) for group 1, 2, 3 and 4, respectively. The intraclass correlation coefficients (ICC) representing the level of inter-rater reliability between two examiners performing the ultrasounds was 0.996 (95% CI 0.982–0.999). ICC of the inter-rater reliability between the raters in 11 paired assessments was 0.989 (95% CI 0.973–0.995). Conclusion: Ultrasound measurements of Tdi and dTF were highly reproducible in healthy children aged 0–8 years. Trial registration: ClinicalTrials.gov identifier (NCT number): NCT04589910. What is Known: • Diaphragmatic thickness and diaphragm thickening fraction are widely used parameters in ultrasound studies of the diaphragm in mechanically ventilated children, but normal values for healthy children to compare these with are scarce. What is New: • We determined normal values of diaphragmatic thickness and diaphragm thickening fraction using ultrasound in 137 healthy children aged 0–8 years old. The diaphragmatic thickness of infants up to 1 year old was significantly greater than that of children from 2 to 8 years old. Diaphragmatic thickness decreased with an increase in body surface area. These normal values in healthy children can be used to assess changes in respiratory muscle thickness in mechanically ventilated children. What is Known: • Diaphragmatic thickness and diaphragm thickening fraction are widely used parameters in ultrasound studies of the diaphragm in mechanically ventilated children, but normal values for healthy children to compare these with are scarce. What is New: • We determined normal values of diaphragmatic thickness and diaphragm thickening fraction using ultrasound in 137 healthy children aged 0–8 years old. The diaphragmatic thickness of infants up to 1 year old was significantly greater than that of children from 2 to 8 years old. Diaphragmatic thickness decreased with an increase in body surface area. These normal values in healthy children can be used to assess changes in respiratory muscle thickness in mechanically ventilated children.


Introduction
The main driver of inspiration, the diaphragm muscle, is a musculo-fibrous membrane separating the thoracic and abdominal cavities. It consists of a non-contractile central fibrous portion (centrum tendineum diaphragmatis) and a peripheral muscular section that is partitioned into the sternal, costal and lumbar muscular groups. The diaphragm has a role in both the low-intensity, perpetual cycle of breathing and in more rapid and strenuous settings, such as talking, singing, sneezing and in situations of acutely increased ventilation [1,2]. Studies in mechanically ventilated adults have demonstrated rapid development of diaphragm weakness or critical illness-associated diaphragmatic weakness [3][4][5][6][7]. This condition reflects the inability of the diaphragm to generate normal levels of maximal force [8][9][10][11]. Diaphragmatic thickness (Tdi) and diaphragm thickening fraction (dTF) measured on ultrasound images are widely used parameters to evaluate diaphragm function in patients with suspected diaphragm weakness [4,[11][12][13][14][15], including critically ill children [16][17][18][19][20]. For the sake of comparison, however, limited data is available about normal Tdi and dTF values in healthy children [21][22][23].
In children, the chest wall musculature is still immature, with fewer type-1 muscle fibres (slow fibres for quiet breathing) than in adults. Although the proportion of type-1 muscle fibres increases from 9% at 27 weeks gestational age to 25% at term age and already reaches the adult level during the second postnatal year, a child has to develop muscle mass and greater contraction strength during childhood and puberty [24][25][26]. Studies in ventilated children with a median age between 3 months and 5 years old found evidence of decreases in Tdi during the course of mechanical ventilation compared to baseline values [17,18,20,27,28]. Decrease in thickness is associated with atrophy, which in turn is associated with diaphragm weakness and worse outcome in adults [29]. Smaller diaphragm thickness is also found at initiation of mechanical ventilation in sick adult patients [3,30]. The lack of normal values in children makes it hard to establish whether even critical illness already influences changes in respiratory muscle thickness in children.
We performed a study to determine Tdi at end-inspiration, Tdi at end-expiration and dTF with the use of ultrasound in children aged 0-8 years and assessed the intra-rater and inter-rater reproducibility. This age group represents the largest patient group in paediatric intensive care units around the world.

Method
We performed a prospective cohort study from September 1, 2020, until March 1, 2021.

Study population
Healthy children aged 0-8 years old were eligible for inclusion. We created four age subgroups for analysis: 0-6 months, 7 months to 1 year, 2-4 years and 5-8 years. Participants were recruited through healthcare professionals working at the Erasmus MC-Sophia Children's Hospital as well as through the research team members' families, friends and neighbours. In addition, brochures were placed in waiting rooms and elevators in the Erasmus MC Sophia Children's hospital in which parents were invited to have their healthy children participate in this study.
Children who met any of the following criteria could not participate: neuromuscular disease, chronic lung disease, abdominal or thoracic surgery less than 3 months ago, known abnormalities of the diaphragm, and having been mechanically ventilated in the past 6 months.
Informed consent from the parents of each participant was obtained.

Diaphragm measurement
After having received thorough training from an experienced ultrasound technician (JM), two researchers (JS and AD) performed ultrasound measurements at the children's homes, supported by a written protocol. Parents were present to keep the child as calm as possible. If necessary, the child was distracted with a video on the phone, a picture book or other toy and a pacifier.
The procedure did not start until the child lay as much as possible in a 30-degree position with head and shoulders on a pillow either on a sofa, a bed or on the lap of a parent and was breathing quietly.
Ultrasound measurement of the diaphragm was performed with a Lumify L 12-4 linear array transducer (Philips Medical Systems B.V., Best, Netherlands) in B mode with the child in a 30-degree supine position, with the head on a pillow. The diaphragm thickness was measured on the right side, with the transducer placed perpendicular to the ribs in the ninth or tenth intercostal space between the anterior and mid axillary lines, in the zone of apposition between the lung and liver. In accordance with the adult literature, we measured only the right hemidiaphragm since the feasibility and repeatability of right hemidiaphragm measurements are superior to those of the left hemidiaphragm. Studies in adults did not show significant differences between Dtf and Tdi between the left and right hemidiaphragms [13,31]. In the zone of apposition, the diaphragm is observed as a three-layered structure, a non-echogenic central layer bordered by two echogenic layers: the peritoneum and the diaphragmatic pleurae ( Fig. 1) [12,13,15,32,33].
Tdi in this position was defined as the vertical distance in millimetres between the midpoint of the diaphragmatic pleura and the midpoint of the parietal peritoneum. dTF was quantified by the percentage change in the right hemidiaphragm thickness from end-expiration to end-inspiration during tidal breathing. dTF was calculated with the formula: end-inspiratory thickness of diaphragm (Tdi-insp) minus end-expiratory thickness of diaphragm (Tdi-exp) divided by end-expiratory thickness × 100. This index is widely used in various studies and is well applicable (Fig. 1) [15,33,34].
Per ultrasound moment, three measurements were made when the optimum position was reached with the clearest view. The mean of the three values was used in the head analysis. For participants where it was not possible to do three measurement but only two, the mean of the two values was used, and this was used in a sensitivity analysis. Short axis Digital Imaging and Communications in Medicine (Dicom) videos were recorded for offline B-mode analysis, and calculations were made with dedicated software (Radi-Ant DICOM viewer from Medixant).
We calculated the inter-rater reliability in terms of [1] performing the ultrasounds and [2] in terms of analysing the recorded images. The procedure of calculating the inter-rater reliability in performing the ultrasounds was as follows: each examiner independently made ultrasounds of 5 children who came to the hospital with their parents in the same position 5 min after each other on the same day. The inter-rater reliability expressed as variability between the two examiners was calculated by analysing the thickness of the diaphragm of the recorded images of these 5 subjects performed simultaneously. AD analysed these recorded images. The inter-rater reliability indicates the level of agreement between independent raters who assessed the images. This was calculated from measurements of Tdi at end-inspiration and at end-expiration of eleven randomly selected ultrasound images. These images were assessed at two different times by two raters independently (AD and JS), without knowing each other's results.

Parameters
Each child's age, sex, height, and weight were noted, and Tdi-insp, Tdi-exp (in millimetres) and dTF (as a percentage) were measured during tidal breathing.

Sample size
The desired sample size was based on the available literature, and the choice of sample size was not made using a formal sample size calculation. All literature about reference values of the diaphragm were described as mean values. The study of van Doorn et al. described a sample size estimation for reference values of the diaphragm in a population of 0-80 years old. They decided to enrol 10 healthy subjects for regressionbased reference limits for each 10 years category. Boon et al. also chose 10 subjects per age category. Vishwanath et al. chose 14-29 subjects per age category. We chose at least the same number of subjects per age group as in these earlier studies and aimed for 30 subjects per age group (120 children in total) [21,22,31,35].

Data analysis
Data are presented as mean (standard deviation, SD) for normally distributed data and median (Interquartile range, IQR) for non-parametric data. Differences in patients' characteristics between the four age groups were tested with the one-way ANOVA test for normally distributed variables and the Kruskal-Wallis test for continuous variables that were not normally distributed.
Inter-rater reliability of the results was assessed using the intraclass correlation coefficient (ICC) for average measures with a two-way random effects model for continuous data. An ICC value below 0.5 is considered to indicate poor reliability, between 0.5 and 0.75 moderate reliability, between 0.75 and 0.9 good reliability and any value above 0.9 excellent reliability [36].
Correlations between body surface area, age, weight and length with Tdi and dTF were assessed using Pearson correlation coefficients (r p ) with 95% confidence interval.

Ethical considerations
The Medical Ethics Review board at Erasmus MC Rotterdam approved the study (METC No. NL70476.078.19). The study was performed in accordance with the ethical standards of the Declaration of Helsinki.

Results
From 1 September 2020 to 1 March 2021, 137 healthy children were enrolled. In 13 of them, only two measurements instead of three could be performed because of movement artefacts. The two measurements in these 13 children are included in the sensitivity analysis. Age groups 1 and 2 included 30 subjects in each; age group 3 included 29 subjects because we lost the ultrasound image of one. Age group 4 was the largest group, with 48 inclusions. Baseline characteristics are shown in Table 1.

Primary outcome
Mean Tdi-insp ranged from 2.09 (SD 0.40) to 1.69 (SD 0.30) mm; the mean Tdi-exp ranged from 1.64 (SD 0.30) to 1.38 (SD 0.20) across groups. Median dTF ranged from 25.4% (SD 10.4) in the two youngest age groups to 21.3% (SD = 7.1) in the oldest age group. Tdi-insp and Tdi-exp differed significantly between groups, with higher values observed in groups 1 and 2 than in groups 3 and 4. dTF did not significantly differ between age groups. (Tables 2 and 3, Fig. 2a and b, Fig. 3). Weak to moderate negative correlations between age, body surface area, weight and height on the one hand and dTF, Tdi-insp and Tdi-exp on the other hand were demonstrated (Table 4 and Fig. 4). For example, the correlation coefficient between Tdi-insp and body surface was -0.400 (-0.532, -0.246), and the correlation coefficient between Tdi-exp and body surface area was -0.351 (-0.490, -0.193).
The ICC representing the level of inter-rater reliability between the two examiners performing the ultrasounds was 0.996 (95% CI 0.982-0.999). The ICC of the inter-rater reliability between the raters in 11 paired assessments was 0.989 (95% CI 0.973-0.995).

Discussion
To determine normal values of diaphragm thickness and thickening fraction in young children, we performed measurements in healthy children aged 0 to 8 years old. To our knowledge, this is the largest study in this age group collecting normal values during spontaneous breathing for thickness of the diaphragm at end-inspiration and at end-expiration in combination with thickening fraction. Establishing normal values is important, for example, to be able to judge whether critical illness induces changes in respiratory muscle thickness.
Both the Tdi at end-inspiration and at end-expiration in infants from 0 to 1 year old were thicker than those of the children from 2 to 8 years old, a finding we had not expected. Children with the average of 3 ultrasound measurements and children with the average of 2 measurements included showed small differences. This indicates that even averaging two ultrasounds of the diaphragm results can be fairly accurate.
To relate our findings to those of other studies, it is important to know how the diaphragm muscle measurements were made. It must be taken into account that the membranes are biologically active and that mechanical forces related to ventilation can lead to inflammation with remodelling and thickening of the tissue [37]. We measured the diaphragm thickness as the vertical distance in millimetres between the midpoint of the diaphragmatic pleura and midpoint of the peritoneal membrane, but other studies measured between the inner lines or outer lines of both membranes. Therefore, thicknesses may differ slightly from each other (see Table 5) [37,38].

Normal diaphragm values in relation to other publications in healthy children and adults
The diaphragm thickness for children aged 2 to 8 years established in the present study compares well with that found by Vishwanath et al. in healthy children aged 8 to 10 years, while the mean dTF was smaller (Table 5) [35]. Of note, however, Vishwanath and colleagues did not describe how dTF and Tdi were exactly measured concerning the pleura and peritoneal membrane.
Rehan et al. measured Tdi at end-expiration in 15 newborns (appropriate for gestational age, and one or 2 days old) and found greater thickness than we found in our youngest age group, which had a much higher mean age of 5 months  [40]. In contrast to our finding, they found that the thickness of the diaphragm at end-expiration were much higher for all age groups with means of 3.4 mm (SD = 0.7) for the  , respectively, in our study. These differences could be explained by their measurement of the whole peritoneal and pleura membrane instead of the vertical distance between the midpoint of both membranes like we did and more importantly, their recording on a different place than the point of apposition. They placed their probe between the anterior and mid-axillary lines, in the subcostal or lower intercostal area, and directed medially, cranially and dorsally. This is better for measurement of excursion but not for the measurement of thickness of the diaphragm [38,41].
The values of Tdi-exp in spontaneously breathing adults, varying from 1.4 to 1.9 mm, are comparable of those of spontaneously breathing children, varying from 1.4 to 1.7 mm across all age groups. Only Boon et al. find higher values in adults, but the lower limit of normal was 1.7 mm. The values of Tdi-insp in spontaneously breathing adults are higher than those in children from 2 to 8 years old, but comparable to those of children from 0 to 1 year old in our study and in the study of Alonso-Ojembarena et al. (Table 5) [21,23,31,35,42,43]. Older children over 10 years appear to have similar thickness of the diaphragm as adults as shown in two studies [21,35].

Normal diaphragm values (dTF) in relation to publications in ventilated children
Six studies in ventilated children, with a median age between 3 months and 5 years old, found either a decrease or an increase in Tdi during the ventilation period (Table 5) [17-20, 28, 44]. Montoro et al. studied ventilated children between 0 and 18 years old with a median age of 3 months, comparable with that of our youngest age group [17]. The median dTF at baseline was comparable with our finding, but was higher before extubation. The effect of the supporting ventilation mode or the breathing work during spontaneous breathing through an endotracheal tube before extubation could have played a role here. Lee et al. studied 31 ventilated children between 1 month and 18 years old (median age 3 years) [19]. The dTF at baseline in Lee et al. was comparable with our findings, but during the course of ventilation became lower than the values we established, both in the success group and in the three patients who failed extubation. After extubation, dTF in the successfully extubated group was higher and comparable with values in our cohort of children from 2 to 4 years old. The dTF in the ones who failed extubation was less than 17%, lower than the 24.1% we established for this age group in healthy children. Glau et al. found a linear correlation of a decreasing dTF with less spontaneous breathing via a support ventilation NA not available, PS pressure support, PRVC pressure controlled, volume controlled ventilation, SB spontaneous breathing (through the tube), SIMV synchronized intermittent mandatory ventilation *Mean (SD) **Median(IQR); measurement Tdi/ dTF ***Between midpoint of diaphragmatic pleura and peritoneal membrane or between inner edges of both layers or between outer edges of both layers mode in 56 ventilated children between 0 and 18 years old (median age 17 months) (beta coefficient 9.4, 95% CI 4.2, 14.7, p = 0.001) [18]. The dTF was very low at baseline and still low before extubation. Notable in that study is the use of neuromuscular blockade infusion (NMB) in one-third of the patients, which may have led to this low dTF. The use of NMB in patients in the study of Montoro et al. led to a greater decrease in thickness and increased the daily atrophy rate in the ventilated patients. The dTF values established by IJland et al. at baseline and before extubation in ventilated children with a median age of 4 months were comparable with those of Glau et al. Three children in whom extubation had failed had a very low dTF under 5%. None of the studies in ventilated children had standardized the dTF before extubation for level of sedation or level of ventilation support, so it is difficult to compare all studies. Xue et al. found in 50 ventilated children between 0 and 18 years old, with a mean age of 3 years that a cut-off value for dTF greater or equal to 21% was associated with successful weaning, defined as passing a spontaneous breathing trial of pressure support 8 cmH 2 O above PEEP 5 cmH 2 O. This cut-off value of 21% is comparable with the normal dTF we found in healthy children in the same age group [28]. Abdel Rahman et al. established a cut-off value of dTF of 23.2% for predicting weaning failure in 106 ventilated children. They did not measure dTF per age group, but per failed weaning group or successful weaning group [44]. Thus, it seems plausible to aim for a normal physiological dTF to estimate the success of extubation. However, various factors may impact diaphragmatic dynamics and thus influence diaphragmatic measurement in ventilated children. They may experience respiratory distress or asynchrony with the ventilator. Moreover, sedation or NMB can influence the diaphragm dynamics, and the intra-abdominal pressure in ventilated patients will be different from that in spontaneously breathing children. More research is needed assessing the diaphragm and respiratory muscle effort from the initiation of ventilation to extubation in children [45].
We found that the diaphragm of children from newborn age up to the age of 1 year was thicker than that of older children and comparable with the diaphragm thickness in adults, which suggests that the diaphragm has already largely been constructed at birth -and becomes notably longer rather than thicker as the child grows. This seems to make sense from the "survival perspective", because the young child has to assure full alveolar ventilation from the first breath onward, but does not need to be able to walk. This scenario is different from growth of the other skeletal muscles in children [46]. The need of a strong diaphragm in newborns is confirmed by the above-mentioned study of Rehan et al. in 15 healthy infants, in which diaphragm thickness was positively associated with body size [22]. The authors stated that thickness has implications for diaphragm strength and found that predicted maximal trans-diaphragmatic pressures were independent of body size and length and were greater than those predicted for adults [22]. In addition, the authors suggested that sufficient transdiaphragmatic pressure is necessary to allow the infant to generate sufficient pressure to overcome substantial elastic and resistive loads at the first breath after birth [22].
The strength of our study is that this is the largest study that established normal values of the thickness of the diaphragm in combination with the thickness fraction of the diaphragm of healthy children aged 0-8 years. Some limitations can be identified. First, the oldest age group included more participants than did the younger age groups, mainly caused by the fact that older siblings of the younger ones wished to undergo ultrasound examination as well. We found it unethical to discard their results. Second, the method of diaphragm measurement may not have been the optimal one. The current general consensus is to measure just the muscle within the lines of the fascia. This consensus was not yet available at the beginning of our study [37,38]. On the other hand, we measured healthy children whose membranes are not expected to be affected by disease or mechanical forces related to ventilation as in ventilated children. Third, we did not verify the 30-degree supine position with a degree arc, but approximated this as much as possible by positioning the child with head and shoulders on a pillow. This could have influenced our measurements. Fourth, we only used 5 ultrasounds for calculating inter-rater reliability of performing the ultrasounds. These results were calculated by one of the two who made the ultrasounds. This may have created bias; however, the assessments of the inter-rater reliability of the 11 paired assessments were calculated by two raters.

Conclusion
This study provides normal values of diaphragm thickness and thickening fraction in healthy children aged 0 to 8 years. The diaphragm thickness of infants up to 1 year old was significantly greater than that of children from 2 to 8 years old. With an increase in body surface area, the diaphragm thickness decreased. These normal values in healthy children can be used to assess changes in respiratory muscle thickness in ventilated children.