Evaluation of Bixa orellana and Dianella nigra as Natural Alternatives to Hematoxylin and Eosin for Cytosmear

Abstract

Introduction: Conventional cytological staining methods such as hematoxylin and eosin (H&E) involve toxic, expensive, and environmentally hazardous chemicals. In light of growing interest in sustainable laboratory practices, this study evaluates the staining efficacy of plant-based alternatives—Dianella nigra for nuclear staining and Bixa orellana (sindoor) for cytoplasmic staining. Materials and Methods: Buccal smear samples (n = 15) were collected and fixed in 95% ethanol. A nuclear stain was prepared from freshly extracted juice of Dianella nigra, while the cytoplasmic stain was derived by dissolving Bixa orellana in ethanol. Staining quality was assessed using three parameters: nuclear clarity, cytoplasmic contrast, and overall staining quality, and compared with smears stained using standard H&E. Statistical analysis included interobserver reliability (Cohen’s κ), Shapiro-Wilk test for normality, and Pearson correlation. Results: Natural stains demonstrated high efficacy in cytoplasmic contrast (mean ± SD: 2.80 ± 0.41) and overall staining quality (2.87 ± 0.35), with slightly reduced nuclear clarity (2.07 ± 0.59) compared to H&E (3.00 ± 0.00 for all parameters). Interobserver reliability was substantial to almost perfect for both stains (κ = 0.86–0.88 for natural stains; κ = 0.91–0.95 for H&E). A strong positive correlation was observed between nuclear clarity and overall staining quality in both groups (r = 0.91 for natural stains, r = 0.93 for H&E; p < 0.001). Conclusion: Natural dyes such as Dianella nigra and Bixa orellana offer promising alternatives to conventional H&E staining, combining diagnostic clarity with eco-friendliness and cost-effectiveness. While slight variability in staining intensity persists, their high interobserver reliability and strong correlation metrics support their potential utility in routine cytological evaluations. Further standardization may enable broader application in clinical and educational settings.