Abstract
Apoptosis plays a critical role in maintaining tissue homeostasis and preventing pathological conditions. The accurate detection of apoptosis is crucial for both research and diagnostic pathology, yet conventional histological staining methods often lack the sensitivity to identify early apoptotic changes. This study aimed to evaluate and compare the effectiveness of classical and emerging mitochondria-targeting histological techniques in detecting apoptotic hepatocytes. Paraffinated hepatic tissue from five rabbits was analysed using haematoxylin-eosin (H&E), Goldner's trichrome (GT), immunohistochemistry for caspase-3 (IHC-Casp3) and Heidenhain's iron haematoxylin (HIH) staining. The percentage of apoptotic hepatocytes was quantified using two assessment methods, and statistical analyses determined the sensitivity of staining protocols. Histologically, the main apoptotic features identified in HIH-stained hepatic specimens were fading 'ghost' mitochondria throughout the cytoplasm, chromatin condensation, fragmentation and dissolution and nucleolar margination. A similar proportion of apoptotic hepatocytes on the HIH staining compared to IHC-Casp3 (19.00% ± 0.7 for HIH vs. 18.50% ± 0.7 for IHC-Casp3) was observed. In comparison, significantly lower values were obtained in H&E (5.75%) and GT (7.5%) stains. The lack of statistical significance of HIH vs. IHC-Casp3 demonstrates similar sensitivities. The additional quantitative analysis methods confirmed IHC as the most sensitive method (3.36 ± 3.82 apoptotic cells/field), followed by HIH, H&E, and GT. In situ histological evaluation of apoptosis remains challenging in standard H&E and GT-stained sections. HIH stain, as a cost-effective alternative to IHC, highlights early stages of apoptotic cells, providing a significant advantage over classical staining methods, emphasising their importance in diagnostic histopathology.