Group size, randomization, blinding, data and statistical analysis
No published data are currently available concerning the impact of benralizumab and mepolizumab against AHR in in human isolated bronchi. The only paper regarding the effect of a mAb on the AHR induced by histamine in passively sensitized bronchi has been performed on the anti-IgE mAb omalizumab (Berger et al., 2007). The results of that study reported that repeating experiments on n=4 different bronchi permitted to detect a small but significant reduction (-≃30%, p<0.05) of histamine-induced AHR in passively sensitized bronchi treated with omalizumab, compared to control tissue (Berger et al., 2007).
In the present study it is not possible the calcutation of the sample size due by the fact that no studies have been conducted so far on neither benralizumab nor mepolizumab in an experimental setting by using passively sensitized human isolated bronchi. However we can set n=5 as the number of repetitions required to have >95% possibility (p<0.05) to detect, with at least 80% power, a relatively small but significant alteration of bronchial contractility induced by benralizumab and mepolizumab, accordingly with the results reported by Berger and colleagues (Berger et al., 2007). This sample size satisfies the guidelines of the British Journal of Pharmacology for preclinical studies, where n refers to independent values and not replicates (available at: https://bpspubs.onlinelibrary.wiley.com/hub/journal/14765381/author-guidelines.html).
The total number of bronchial rings necessary to complete the study was n=366, including C-, C+, and time control tissues. Isolated airways were collected from at least n=5 different donors. The treatment with each specific concentration of either benralizumab or mepolizumab was performed by using specimens collected from the same patient, and experiments were repeated 5 times in samples originating from 5 different donors. In the case the amount of samples from a subject did not permit to test all the benralizumab and mepolizumab concentrations in the same experiment, the remaining concentrations were assessed in specimens collected from other patients in parallel with further C-, C+, and time controls in order to provide controlled results not affected by potential bias.
The protective effects of different concentrations benralizumab and mepolizumab against AHR were compared each other and with C- and C+ tissues. Values were reported as mean±standard error of the mean (SEM), the statistical significance was assessed by the Student’s t -test and analysis of variance (ANOVA), and the level of statistical significance was defined as P<0.05. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Detailed dataset of sectional tissues is reported in e-Table 2. Data were collected and managed in order to perform pre-specified statistical analysis; any so called “p-hacking” were avoided in order to prevent that any potential bias could affect the robustness of results, as reported by Head and colleagues (Head et al., 2015).
Each bronchial ring was randomly assigned to a specific treatment by using a computer generated sequence. All the study procedures were performed under blinded condition, in which both the operator and data analysis were blinded.
All data analysis was performed using computer software GraphPad Prism 5 (La Jolla, CA, USA) and OpenEpi (available at: https://www.openepi.com) was used for the power calculation and randomization.