MED-PHCVLGAug 22, 2025

Deep learning-enabled virtual multiplexed immunostaining of label-free tissue for vascular invasion assessment

arXiv:2508.16209v11 citationsh-index: 17BME Frontiers
Originality Incremental advance
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This approach improves diagnostic accuracy and efficiency for pathologists in histopathology, though it is incremental as it builds on existing virtual staining methods by extending to multiplexed applications.

The paper tackled the problem of laborious and costly traditional immunohistochemistry for vascular invasion assessment in thyroid cancers by developing a deep learning-based virtual multiplexed immunostaining framework from label-free tissue, achieving high concordance with histochemical staining results as validated by pathologists.

Immunohistochemistry (IHC) has transformed clinical pathology by enabling the visualization of specific proteins within tissue sections. However, traditional IHC requires one tissue section per stain, exhibits section-to-section variability, and incurs high costs and laborious staining procedures. While multiplexed IHC (mIHC) techniques enable simultaneous staining with multiple antibodies on a single slide, they are more tedious to perform and are currently unavailable in routine pathology laboratories. Here, we present a deep learning-based virtual multiplexed immunostaining framework to simultaneously generate ERG and PanCK, in addition to H&E virtual staining, enabling accurate localization and interpretation of vascular invasion in thyroid cancers. This virtual mIHC technique is based on the autofluorescence microscopy images of label-free tissue sections, and its output images closely match the histochemical staining counterparts (ERG, PanCK and H&E) of the same tissue sections. Blind evaluation by board-certified pathologists demonstrated that virtual mIHC staining achieved high concordance with the histochemical staining results, accurately highlighting epithelial cells and endothelial cells. Virtual mIHC conducted on the same tissue section also allowed the identification and localization of small vessel invasion. This multiplexed virtual IHC approach can significantly improve diagnostic accuracy and efficiency in the histopathological evaluation of vascular invasion, potentially eliminating the need for traditional staining protocols and mitigating issues related to tissue loss and heterogeneity.

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