Abstract Title Virtual PAS Stain from Frozen Section H&E Kidney Biopsy Slides Background Chronic Kidney Disease (CKD) affects approximately 14% of the US adult population and is a leading cause of End-Stage Renal Disease (ESRD), requiring dialysis or kidney transplantation. Retrospective analysis shows that about 20% of viable kidneys are discarded due to the on-call pathologists' evaluations of frozen section biopsies. Frozen section Hematoxylin & Eosin (H&E) biopsies are the current standard for pre-transplant assessment but are often reviewed by pathologists with limited renal specialization and are difficult to interpret due to freezing artifacts. To address these limitations, we have developed DUal-mode Emission and Transmission (DUET), a novel microscopy technique that enables the creation of virtual Periodic Acid-Schiff (vPAS) images without additional slides or advanced optical methods. Methods DUET images are generated by scanning pixel-registered brightfield and fluorescence images from 29 frozen H&E-stained kidney biopsy slides. Collagen masks were extracted using a NeuralNet training model and were digitally overlaid to generate a vPAS stain. Four renal pathologists and three general pathologists scored the following features: percent sclerotic glomeruli, interstitial fibrosis, tubular atrophy (IF/TA), inflammation, arteriosclerosis, and arteriolar hyalinosis. The pathologists first assessed only the H&E WSIs; following a two-week washout period, reviewed the cases with both H&E and vPAS WSIs. Inter-pathologist agreement was analyzed separately for renal and general pathologists by two-way intraclass correlation coefficient (ICC) to identify any group differences. Results Among general pathologists’ evaluations, ICC scores from the H&E to virtual PAS stain increased for IF/TA percentage (0.103 to 0.722), inflammation percentage (0.208 to 0.310), and arteriosclerosis number (0.033 to 0.428). For renal pathologists’ evaluations, ICC scores from the H&E to virtual PAS stain increased for percent sclerotic glomeruli (0.794 to 0.877), IF/TA percentage (0.201 to 0.435), inflammation percentage (0.031 to 0.343), and arteriosclerosis number (0.581 to 0.615). No improvement in ICC score was observed for arteriolar hyalinosis in either pathologists’ group. Conclusion We have demonstrated that a frozen virtual PAS stain shows higher consistency among pathologist evaluations when compared to a frozen H&E stain for various metrics used to assess donor kidney viability. This virtual stain allows for easier visualization of IF/TA, inflammation, and arteriosclerosis, as ICC scores for general and renal pathologists increased when comparing the virtual stain to a standard H&E stain for frozen kidney biopsies. DUET-generated vPAS stains offer a rapid, cost-effective solution for enhancing renal pathology assessments from frozen section H&E biopsies. This approach has the potential to reduce diagnostic variability, improve transplant decision-making, and optimize donor kidney utilization.