Abstract
Hematoxylin and eosin (H&E) staining, which has been considered the gold standard in histopathology practice for over a century, continues to form the basis of diagnostic processes. Hematoxylin, a natural flavonoid derived from the
Haematoxylum campechianum
tree, requires oxidation to convert to hematein to obtain staining ability and metallic mordants to bind with tissue. This review comprehensively analyzes the chemical mechanisms, formulation diversity, application protocols, and technological integration of hematoxylin in light of the current literature. This review details modern safety paradigms that necessitate the transition from toxic mercury oxide to safe sodium iodate-based formulations, as well as semi-oxidation strategies that extend dye life. Alongside traditional aluminum and iron mordant variants (Mayer, Harris, Gill, Weigert), the new generation chromium mordant hematoxylin X innovation, which stands out for its stability and staining capacity, is technically evaluated. Furthermore, progressive and regressive methods that directly affect staining quality, artifact management, and quality control standards (stain verification, duty cycle tracking) are examined. In conclusion, this review focuses on environmentally-friendly chemistry practices that prioritize laboratory safety and the environment, as well as the transformation brought about by artificial intelligence (AI)-powered Virtual H&E Staining technologies. Regardless of how rapidly the digital pathology revolution progresses, it has been concluded that the familiar morphological signature provided by hematoxylin will remain an essential guide for pathologists, particularly in unlocking the secrets of nuclear chromatin and preparing the groundwork for molecular analyses.