Table 2 Summary points and recommendations for the conduct of a confirmatory multicenter study including open questions that require further discussion and will be subject matter of future research

Minimum validity and reliability criteria need to be fulfilled before engaging in a confirmatory multicenter study (Table 1)

Are dose–response effects a prerequisite for the confirmation?

If uncertainty is still high, optimization of evidence via (within-lab, in-house) replication studies to (i) increase sample size, (ii) improve internal validity, (iii) introduce systematic homogenization and/or (iv) flanking experiments

What if evidence from pilot, exploration and within-lab replication are contradictory (positive and negative results)?

(Standardized) protocols should be in place before starting a confirmatory study

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(Animal) Model(s) should be disease relevant and limitations be acknowledged

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Depending on the experimental objective control groups should include positive, negative controls and/or in case available a comparator from standard clinical care

What requirements need to be fulfilled to use historical control groups?

For planning a confirmatory study, sample size calculation should be based on smallest effect (size) of interest (clinical/biological relevant) or a shrinkage of the effect size(s) from exploratory studies should be considered

Field specific effect sizes distributions are scarce, how can the situation be improved?

What is the optimal approach to calculate the sample size?

Flanking experiments (triangulation) might be performed early on and are highly recommended for confirmatory studies

How can in vitro studies be integrated in the confirmatory study design and sample size calculation?

Introduction of sources of variation like sex or strain (systematic heterogenization)

How to best balance standardization and systematic heterogenization?

Multicenter considerations include (i) harmonization of protocols, (ii) skills and expertise of partner lab(s), (iii) balanced design and (iv) block randomization across centers

Which experiments should be confirmed in several laboratories?