2.1. Study design and population
This cross-sectional controlled study was carried out from November 2021 to November 2022 at the Department of Pediatrics of Montpellier University Hospital, a tertiary care academic institution in southern France.
Children and adolescents aged 6 to 17 years with a confirmed diagnosis of sickle cell disease (i.e., homozygous HbS/S or heterozygous HbS/C mutations) were consecutively screened during their routine follow-up, which included a hematology consultation (physical examination, blood test), a cardiology consultation (electrocardiogram, transthoracic echocardiography), a respiratory plethysmography and a CPET. Patients enrolled in the study were offered to fill in the study questionnaires.
Healthy age and sex-matched controls were recruited in our pediatric CPET laboratory during the same study period. The control group consisted of children referred for a non-severe functional symptom linked to exercise (murmur, palpitation, or dyspnea) or for a medical sports certificate. As in similar previous controlled CPET studies, these subjects were classified in the control group only after a completely normal check-up, including physical examination, electrocardiogram, echocardiography, and spirometry.[22, 23, 30]
2.2. Collected data
In the sickle cell group, the following patient characteristics were collected from electronic health records: sickle cell related-treatment, number of vaso-occlusive crises, and acute chest syndrome. Collected biological data were blood and reticulocyte count and hemoglobin electrophoresis (HbA, HbA2, HbF, HbS ± HbC). Anemia was defined as a hemoglobin level lower than − 2 standard deviation (SD) above the mean for age.[31]
The following clinical data were systematically collected when the CPET was performed: sex, age, weight, height, body mass index (BMI), heart rate, blood pressure, and cardiac functional status (NYHA). Echocardiographic parameters included the left ventricle and right ventricle conventional measures, normalized in Z-scores.[32] Z-scores > 1.64 indicated ventricular hypertrophy or dilatation.
In the control group, we collected anthropometric and CPET data.[30]
2.3. CPET procedure
The CPET laboratory used the following equipment: pediatric face masks (Hans Rudolph, Shawnee, KS), a calibrated gas analyzer (Quark CPET, Cosmed Srl, Pavonna di Albano, Italy), breath-to-breath measurement software (Windows 7–10, Omnia, Cosmed), 12-lead ECG equipment (Norav, Medical, Mainz-Kastel, Germany) and pulse oximeter (Masimo, Neuchâtel Switzerland), and a manual sphygmomanometer with adapted pediatric cuffs. For the plethysmography the following equipment was used: a cabin (MasterScreen Body, Jaeger, Carefusion, Germany), and a breath-by-breath software (Windows 7–10, SentrySuite 2.7, Carefusion, Germany).
The following lung function parameters at rest were collected: forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC, total lung capacity (TLC), functional residual capacity (FRC). Measurements were normalized using pediatric Z-score reference values. Z-scores < -1.64 indicated reduced values.[33, 34] Reduced FVC and TLC with normal FEV1/FVC indicated restrictive ventilatory defect, and reduced FEV1/FVC and TLC indicated mixed ventilatory defect.
We used a CPET pediatric cycle ergometer standardized protocol adapted to the patient's age, with a homogeneous incremental overall duration between 10 and 12 minutes: a 1-min rest; a 3-min warm-up (10–20 watts) in increments of 10, 15, or 20 watts each minute; a pedaling rate of 60–80 revolutions per minute; a 3-min active recovery (20 watts); and a 2-min rest.[35]
The exercise test was considered maximal when the patient experienced exhaustion despite active encouragement. The main cardiopulmonary fitness parameter used in this study was the VO2max. When the VO2max did not reach a plateau, the peak VO2 (VO2peak) was collected, as usual in pediatrics.[36] VO2max was expressed in raw values and Z-score pediatric reference values.[30] Impaired aerobic capacity was defined as a VO2max Z-score < -1.64 (from a statistical approach to define abnormality from normality based on 5th percentile values).
We also collected other maximal parameters (maximal heart rate), ventilatory parameters (maximal respiratory rate (RRmax), maximal tidal volume (VTmax)), and submaximal parameters (ventilatory anaerobic threshold (VAT), VE/VCO2 slope, oxygen uptake efficiency slope (OUES)), as previously detailed.[24] Impaired VAT, suggesting the existence of muscular deconditioning, was defined as a VAT < 55% of predicted VO2max, according to the pediatric reference values (i.e., percent-predicted VAT).[37]
2.4. Study questionnaires
Children with sickle cell disease (self version) and their parents (proxy version) filled in the PedsQL™ 4.0. questionnaire, a generic HRQoL questionnaire with four multidimensional scales (physical functioning, emotional functioning, social functioning, and school functioning) and three summary scores ranging from 0 to 100 (total scale score, physical health summary, and psychosocial health summary, including emotional, social, and school functioning).[38–40] Higher scores indicate better HRQoL.[38] Psychometric properties showed reliability, validity, and responsiveness to clinical change over time, including for the French version of the PedsQL.[39, 40]
The self-reported level of physical activity was assessed by the Ricci and Gagnon questionnaire, composed of 9 items, with a total score ranging from 9 to 45 ([9–18] = physical inactivity; [18–35] = moderate physical activity; [36–45] = intensive physical activity).[41, 42]
We also used a sickle cell disease knowledge questionnaire, a non-validated instrument used in patient education in our institution (47 questions, 5 domains, 7 scores, sickle cell knowledge overview score of 70, Appendix 1).
2.5. Sample size and statistical analysis
The study was designed to have 90% power to detect an absolute VO2max group difference of 9 ± 9.2 mL/kg/min, based on previous pediatric CPET studies,[10] in a two-sided test with a 5% alpha level. A total of 72 participants were required to be conclusive, considering a 1:2 case-control ratio and 30% uncomplete or missing CPET data (24 subjects with sickle cell disease and 48 controls).
The study populations were described with medians and inter-quartile ranges for quantitative variables and with numbers and percentages for qualitative ones. Each case was matched with a control of the same sex and ± 0.5 years around its age, in a 1:2 ratio, using a greedy algorithm. Quantitative variables were compared using the Student’s t-test when the distribution was Gaussian and with the Mann-Whitney test, otherwise. For qualitative variables, groups were compared using the χ2 test or Fisher’s exact test. Pearson's coefficient was used to assess the correlation between two quantitative variables, following the verification of their normal distributions.
A multiple linear regression was used to identify the parameters associated with VO2max in the sickle cell disease group. All clinically relevant variables were proposed, parameters with a P-value < 0.157 in univariate analysis were selected by clinicians, and a top-down elimination procedure was applied to select the model, retaining only variables with a P-value < 0.1. Linear regression coefficient ß were provided with 95% confidence intervals (95%CI).
The statistical significance was set at 0.05 and analyses were performed using software Easy Med Stat, version 3.21.5 (Easymadestat, Levallois-Perret, France) and SAS® 9.04 (SAS Institute, Cary, NC, USA).