Bias in group-level EEG microstate analysis

Microstate analysis is a promising technique for analyzing high-density electroencephalographic data, but there are multiple questions about methodological best practices. Between and within individuals, microstates can differ both in terms of characteristic topographies and temporal dynamics, which leads to analytic challenges as the measurement of microstate dynamics is dependent on assumptions about their topographies. Here we focus on the analysis of group differences, using simulations seeded on real data from healthy control subjects to compare approaches that derive separate sets of maps within subgroups versus a single set of maps applied uniformly to the entire dataset. In the absence of true group differences in either microstate maps or temporal metrics, we found that using separate subgroup maps resulted in substantially inflated type I error rates. On the other hand, when groups truly differed in their microstate maps, analyses based on a single set of maps confounded topographic effects with differences in other derived metrics. We propose an approach to alleviate both classes of bias, based on a paired analysis of all subgroup maps. We illustrate the qualitative and quantitative impact of these issues in real data by comparing waking versus non-rapid eye movement sleep microstates. Overall, our results suggest that even subtle chance differences in microstate topography can have profound effects on derived microstate metrics and that future studies using microstate analysis should take steps to mitigate this large source of error.