Stain normalization using score-based diffusion model through stain separation and overlapped moving window patch strategies

Hematoxylin and eosin (H&E) staining is the gold standard modality for diagnosis in medicine. However, the dosage ratio of hematoxylin to eosin in H&E staining has not been standardized yet. Additionally, H&E stains fade out at various speeds. Therefore, the staining quality could differ among each image, and stain normalization is a critical preprocessing approach for training deep learning (DL) models, especially in long-term and/or multicenter digital pathology studies. However, conventional methods for stain normalization have some significant drawbacks, such as collapsing in the structure and/or texture of tissue. In addition, conventional methods must require a reference patch or slide. Meanwhile, DL-based methods have a risk of overfitting and/or grid artifacts. We developed a score-based diffusion model of colorization for stain normalization. However, mistransfer, in which the model confuses hematoxylin with eosin, can occur using a score-based diffusion model due to its high diversity nature. To overcome this mistransfer, we propose a stain separation method using sparse non-negative matrix factorization (SNMF), which can decompose pathology slide into Hematoxylin and Eosin to normalize each stain component. Furthermore, inpainting with overlapped moving window patches was used to prevent grid artifacts of whole slide image normalization. Our method can normalize the whole slide pathology images through this stain normalization pipeline with decent performance. •We developed a high-performance stain normalization method using a score-based diffusion model.•We adopted a clinically plausible stain separation protocol to separate hematoxylin and eosin.•We overcome grid artifacts of normalized WSI by adopting an overlapped moving window.