Valid statistical approaches for clustered data: A Monte Carlo simulation study

The translation of preclinical studies to human applications is associated with a high failure rate, which may be exacerbated by limited training in experimental design and statistical analysis. Nested experimental designs, which occur when data have a multilevel structure (e.g., in vitro: cells within a culture dish; in vivo: rats within a litter), often violate the independent observation assumption underlying many traditional statistical techniques. Although previous studies have empirically evaluated the analytic challenges associated with multilevel data, existing work has not focused on key parameters and design components typically observed in preclinical research. To address this knowledge gap, a Monte Carlo simulation study was conducted to systematically assess the effects of inappropriately modeling multilevel data via a fixed effects ANOVA in studies with sparse observations, no between group comparison within a single cluster, and interactive effects. Simulation results revealed a dramatic increase in the probability of type 1 error and relative bias of the standard error as the number of level-1 (e.g., cells; rats) units per cell increased in the fixed effects ANOVA; these effects were largely attenuated when the nesting was appropriately accounted for via a random effects ANOVA. Thus, failure to account for a nested experimental design may lead to reproducibility challenges and inaccurate conclusions. Appropriately accounting for multilevel data, however, may enhance statistical reliability, thereby leading to improvements in translatability. Valid analytic strategies are provided for a variety of design scenarios.