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.