Histopathologic staining of low temperature cutaneous burns: Comparing biomarkers of epithelial and vascular injury reveals utility of HMGB1 and hematoxylin phloxine saffron

Histopathology remains the gold standard for evaluation of burn depth, progression, and healing, but burn literature offers little guidance on the best stains for analysis of these complex and evolving injuries. A battery of histochemical and immunohistochemical stains was compared on adjacent sections to determine the best stains for histopathologic study and imaging of burns. Using a validated porcine model of vertical burn progression, full-thickness cutaneous biopsies were stained using hematoxylin and eosin, Hematoxylin phloxine saffron (HPS), Masson Trichrome, Elastin Von Gieson, Movatt's Pentachrome, vimentin, CD31, KI-67, caspase 3a, and high mobility group box 1. Depth of collagen degeneration, cellular necrosis, apoptosis, and vascular occlusion; and reparative processes of cellular hyperplasia, reepithelialization, and new collagen deposition were measured by ocular microscopy. High mobility group box 1 was superior for necrosis between 1 and 24 hours postburn. Vimentin underestimated necrosis until 48 hours postburn. For overall assessment, hematoxylin and eosin and HPS were comparable, except for analysis of thermally injured collagen, vessel occlusion, erythrocyte extravasation, and polariscopic study of collagen deposition, where HPS was superior. HPS stain offers specific advantages in histopathologic burn analysis. Inexpensive and rapid to produce, HPS allows users to analyze eosinophilic components more precisely than standard hematoxylin and eosin.