Patients selection
In this retrospective study a total of 388 patients, with a first episode of bronchiolitis, admitted to the Bambino Gesù Pediatric Hospital of Rome (Italy) in the period from January to December 2017, were enrolled. Of all the hospitalized children, we selected only those aged 12 months or less (age with higher incidence of bronchiolitis). We excluded from the study patients with pathologies or infections that could modify the clinical course of bronchiolitis worsening the prognosis. In particular, we excluded: 80 patients (21%) infected by unknown viruses; 45 patients (12%) with history of prematurity (less than 37 weeks); 40 patients (10%) with immunodeficiency; 20 patients (5%) with congenital heart diseases. The analysis was conducted on only 203 patients (52% of enrolled patients): 112 boys and 91 girls. For all patients, disease severity score was constructed based on the following variables: dyspnea, tachypnea, hypoxia (oxygen saturation < 92), cough, fever and length of hospitalization. For each symptom, the patient was “accredited” one point. Patients with the median score or above were classified as having a severe illness.
Based on the degree of bronchiolitis (moderate or severe) oxygen was administered with low or high flow through nasal tubes.
Virus infections were detected in nasopharyngeal secretions by real-time PCR (RT-PCR). The characteristics of all the patients included in the study are shown in Table
1. Based on the clinical differences highlighted on patients admitted from January to December 2017, we selected a group of patients admitted from January to March 2018 [
7] to conduct a pilot study on OxS and platelet characteristics. In particular, a group of patients with diagnosis of moderate bronchiolitis (15 boys and 12 girls) was selected. The diagnosis of moderate bronchiolitis was performed according to Berardi et al., [
8] (see Table
2). We chose patients with moderate bronchiolitis because in this form of bronchiolitis the oxygen support is delivered with low flow rates, while in patients with severe forms of bronchiolitis the oxygen support is delivered with high flows. Moreover, severe forms of bronchiolitis could alter the pro-oxidant state of the blood and the activation of the platelets in the two different sexes.
Table 1Mean characteristics of patients admitted from January to December 2017
Age, months | 2.4 (1–10) | 3.3 (1–12) | ns |
Hospitalization (days) | 5.13 (6–19) | 5.8 (1–19) | ns |
Illness duration (days) | 10.5 (6–20) | 8.7 (3–19) | ns |
CRP, mg/dL | 0.92 (range 0.46–6.28) | 1.11 (range 0.36–14.32) | P < 0.05 |
RSVB (% of patients) | 58% | 47% | P = 0.030 |
RSVA (% of patients) | 11.6% | 16% | ns |
Rhinovirus (% of patients) | 6.25% | 4% | ns |
Metavirus | 9.8% | 7% | ns |
Other viruses | 7.1% | 19% | P = 0.05 |
Coinfections (% of patients) | 4.46% | 7% | ns |
Complications of the disease | 12.5% | 11% | ns |
Mild thrombocytosis | 78.3% | 90% | P = 0.01 |
Severe thrombocytosis | 21.7% | 10% | P = 0.05 |
Oxygen therapy (% of patients) | 42.86.% | 48% | ns |
Cortisone therapy (% of patients) | 46.4% | 60% | P = 0.05 |
Antibiotic therapy (% of patients) | 35.7% | 35% | ns |
Aerosol therapy with 2 ml of 3% hypertonic solution (% of patients) | 62% | 54% | ns |
Table 2Characteristic of patients with moderate bronchiolitis
Respiratory rate | Increased |
Respiratory effort | Tracheal tug Nasal Flare Moderate chest wall retraction |
Oxygen saturations | Saturation 90–95% |
Feeding | 50–75% of normal feeds |
Apnoea | Brief episodes |
This selected group of patients with moderate bronchiolitis was limited to patients infected by RSVB, that is the most common cause of bronchiolitis in both male and female infants and that frequently causes severe morbidity and mortality [
1].
The analyses were carried out after the diagnosis of bronchiolitis (about 2 or 3 days after admission).
Age-matched healthy subjects (10 boys and 10 girls) were used as control group (HC). This group consisted of healthy children who have no clinical symptoms and that came to the hospital for a check-up after diarrhea, poor growth or dehydration.
In this study the use of healthy subjects excludes that data obtained in patients are not due to artifacts, but to the disease. Informed consent by parents of both patients and HCs was obtained. The study has been approved by the Institutional Review Board of the Bambino Gesù (approval number: 1489OPBG2017). The investigation conforms the principles outlined in the Declaration of Helsinki.
Platelet isolation
Fresh whole blood samples were collected in acid-citrate- dextrose tubes (ACD; NIH formula A), and immediately centrifuged at 200 g for 12 min at room temperature to separate platelet-rich plasma (PRP). Additional ACD was added (one part ACD per three parts PRP) and platelets were pelleted at 800 g for 15 min as previously reported [
9] for ROS measurement.
ROS released in fresh whole blood were measured by electron para-magnetic resonance (EPR) spin trapping technique as previously reported [
10]. Briefly, the spin trap 1-Hydroxy-3-Carboxy-Pyrrolidine (CPH; ENZO Life Sciences, Farmingdale, NY) was incubated (1 mM) with fresh whole blood for 20 min at 37 °C. Samples were then drawn up into a gas-permeable Teflon tube and inserted into a quartz tube. EPR spectra were measured in air at 37 °C on a Bruker e-Scan (Bruker, Rheinstetten, Germany). The intensity of the CPH-derived characteristic 3-line spectrum attributable to the nitroxide 3-carboxyproxyl radical (CP
●) (hyperfine coupling constant 1.63 ± 0.04 mT) was measured as peak-to-peak linewidth and taken as the measure of the totality of oxidants formed. Spectrometer conditions: modulation frequency, 100 kHz; microwave frequency, 9.4 GHz; microwave power, 20 mW; gain 1 × 10
4; modulation amplitude, 0.1 mT; conversion time, 20.5 ms; time constant, 82 ms; sweep time, 21 s; number of scans, 1 [
10].
Platelet activation and platelet apoptosis
Platelet activation was assessed by flow cytometry using fluorescein Isothiocyanate-conjugated annexin V. Evaluation of apoptosis was performed by flow cytometry after double staining using fluorescein Isothiocyanate-conjugated annexin V (1: 200) and 0.05% trypan blue for 10 min at room temperature. All samples were analyzed by FACSCalibur cytometer (Becton Dickinson, Mountain View, CA, USA) in the FL1 and FL3 channels to determine the percentage of dead cells.
Platelet adhesion
Heterotypic adhesion (platelets-lymphocytes) was evaluated by using monoclonal anti-CD62 IgG-Phycoerythrin-conjugated (1: 20; Becton Dickinson), while homotypic adhesion (platelets-platelets) was evaluated by using monoclonal anti PAC-1 IgG-FITC-conjugated (1: 20; Becton Dickinson). All samples were stained at 4 °C for 30 min with monoclonal antibodies and analyzed on a FACSCalibur cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a 488 nm argon laser. At least 20,000 events have been acquired.
Statistical analyses
To compare average values of a continuous variable between two groups we used the Student’ T test, and to analyze relationship between two categorical variables we used Chi Squared test.
Cytofluorimetric results were analyzed by using the Kolmogorov–Smirnov test using Cell Quest Software. At least 20,000 events were acquired. The percentage of platelet positives to annexin V, CD62 and PAC-1 were used to provide a semi-quantitative analysis. Results are displayed as average value ± standard deviation, unless otherwise specified. Statistical analysis was performed by one-way analysis of variance (ANOVA). When a significant interaction was detected, we also performed a Bonferroni post-hoc test. p < 0.05 was considered as a threshold for a significant difference.