Extended Data Fig. 10: The VirtualMultiplexer can greatly accelerate histopathology workflows.

We performed a runtime estimation of all components of the VirtualMultiplexer framework across imaging datasets of different scales: an in-domain TMA from the EMPaCT dataset (A), an out-of-domain TMA from the PDAC dataset (B), an out-of-domain needle biopsy from the SICAP dataset (C), and an out-of-domain WSI from the in-house dataset (D). We calculated that applying the trained VirtualMultiplexer on a single EMPaCT TMA core (6000 × 6000 pixels at 20X magnification-0.24μm/pixel) for one marker resulted in a total runtime of 2.81 seconds, and the same process for an out-of-distribution TMA core resulted in a runtime of 10.88 seconds, with the increase attributed to stain normalization. However, the stain normalization step is crucial as it alleviates the appearance disparity between the training and the out-of-distribution samples (Supplementary Fig. 1), and allows for a faithful application of the VirtualMultiplexer to unseen datasets. The above result implies that virtual staining of a hypothetical TMA slide containing 250 out-of-distribution TMA cores for 6 markers would be feasible in ≈ 65.8 minutes (preprocessing: ≈ 9.9 seconds per core, virtual staining and post-processing: ≈ 0.98 seconds per core and marker). Conversely, performing the IHC staining for the same hypothetical TMA for 6 IHC markers could take an estimated time of approximately 1 day, when applied in a cutting-edge pathology laboratory using the latest protocols⁸⁵. When applied in a biology lab that does not specialize in pathology, however, IHC staining could take up to 5 days per marker (sectioning: 1 day, staining: 2 days, slide drying: 1 day, imaging: 1 day), leading to a minimum of 5 days, if done simultaneously for all 6 markers, and more than 10 days, if performed mostly sequentially. Importantly, as our method scales linearly with the size of the tissue (TMA to WSI) and with the number of markers, similar time gains would be feasible for virtually staining needle biopsies and WSIs.