Collagen and Picrosirius Red Staining: a Polarized Light Assessment of Fibrillar Hue and Spatial Distribution

Collagen plays a vital role in maintaining structural integrity and in determining tissue function. Therefore, methods to detect, quantify, and analyze collagen are valuable. Nevertheless, stains historically employed to detect collagen have disadvantages, principally a poor specificity for thin fibers. Conversely, picrosirius red, which has the capability to detect thin fibers, although frequently used, is seldom exploited to the fullest extent. Our goal was, using picrosirius red staining, circularly polarized light, and image-analysis software, not only to identify fibers and quantify collagen content, but also to assess fiber hue and the spatial distribution of the different colors. To assess collagen content, we used a subtraction technique to remove interstitial space and non-collagen elements from images of skin wounds, myocardial scars, and arterial tissue. The hue component of the resulting image was obtained, and the number of red, orange, yellow, and green (the colors of collagen fibers in order of decreasing thickness) pixels calculated. Finally, we assessed the spatial distribution of individual colors by the application of color threshold filters. Skin wound analysis demonstrated good inter-observer agreement for collagen content and fiber color. In myocardial scars, collagen content increased from 1 (61%) to 5 weeks (95%) after injury. The proportion of green (thin) fibers decreased (43 to 4%), while the proportion of orange (thick) fibers increased (13 to 65%). Color threshold application revealed regional variation in fiber color within subintimal arterial lesions. These methods increase the amount of structural information obtained from picrosirius red-stained sections.