NSH Poster Podcast (2025): Enhanced Collagen Detection in Liver Fibrosis: A Comparative Study of Picrosirius Red Staining With and Without Bouin’s Pretreatment

Title: Enhanced Collagen Detection in Liver Fibrosis: A Comparative Study of Picrosirius Red Staining With and Without Bouin’s Pretreatment

Authors: Nate Rampy, BS, Amber Moser, BS, HTL(ASCP)cm, Hannah Benton, BS, Brad Bolon, DVM, MS, PhD, DACVP, DABT and Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC, Premier Laboratory, LLC, Longmont, Colorado; GEMpath, Inc., Longmont, Colorado

Abstract: The use of Bouin’s solution as a post-fixation treatment, rather than a primary fixative, remains largely unexplored in Picrosirius Red (PSR) procedures for collagen detection. In this study, we compared the effectiveness of PSR staining in liver samples from mouse, rat, and human with and without Bouin’s solution as a pretreatment step. Liver sections were fixed in 10% neutral buffered formalin, processed and embedded in paraffin before being sectioned at 4 microns and stained with PSR. Bouin’s was applied prior to staining for 60 minutes at 70º C, not as a fixative, but as a mordant to enhance dye-tissue interactions. Stained slides were scanned at 20x with an Aperio AT2. Visual assessment and image analysis in bright field microscopy demonstrated that the slides pretreated with Bouin’s had significantly improved collagen differentiation, with enhanced contrast. By comparison, slides stained without the Bouin’s pretreatment showed weaker and less distinct collagen staining. Our findings suggest that Bouin’s pretreatment significantly improves collagen staining contrast and differentiation. The use of Bouin’s pretreatment may serve as a valuable revision to the standard histology protocol for PSR fibrosis evaluation as well as general collagen visualization.