SignificanceAlthough the registration of restained sections allows nucleus-level alignment that enables a direct analysis of interacting biomarkers, consecutive sections only allow the transfer of region-level annotations. The latter can be achieved at low computational cost using coarser image resolutions.PurposeIn digital histopathology, virtual multistaining is important for diagnosis and biomarker research. Additionally, it provides accurate ground truth for various deep-learning tasks. Virtual multistaining can be obtained using different stains for consecutive sections or by restaining the same section. Both approaches require image registration to compensate for tissue deformations, but little attention has been devoted to comparing their accuracy.ApproachWe compared affine and deformable variational image registration of consecutive and restained sections and analyzed the effect of the image resolution that influences accuracy and required computational resources. The registration was applied to the automatic nonrigid histological image registration (ANHIR) challenge data (230 consecutive slide pairs) and the hyperparameters were determined. Then without changing the parameters, the registration was applied to a newly published hybrid dataset of restained and consecutive sections (HyReCo, 86 slide pairs, 5404 landmarks).ResultsWe obtain a median landmark error after registration of 6.5 mm (HyReCo) and 24.1 mm (ANHIR) between consecutive sections. Between restained sections, the median registration error is 2.2 and 0.9 mm in the two subsets of the HyReCo dataset. We observe that deformable registration leads to lower landmark errors than affine registration in both cases (p < 0.001), though the effect is smaller in restained sections.ConclusionDeformable registration of consecutive and restained sections is a valuable tool for the joint analysis of different stains.