Evaluation of the Potential of Kiat-Kiat (Citrus Reticulata) Oil D-Limonene as Deparaffinization Agent in Histopathological Preparations

This study evaluates the potential of kiat-kiat (Citrus reticulata) peel–derived oil D-limonene as a potential sustainable deparaffinizing substitute to xylene. Specifically, it compared the macroscopic and microscopic performance of Solution 1 (oil D-limonene mixed with 90% ethanol and Polysorbate 80), pure oil D-limonene extract, cedarwood oil (commercial xylene alternative), and xylene (positive control). Macroscopic evaluation included deparaffinization capacity, ease of wax removal, and macroscopic artifacts, while microscopic evaluation assessed nuclear staining, cytoplasmic staining, and clarity of staining. Oil extraction was performed using absolute ethanol and rotary evaporation, and the extract was applied to pig liver tissues processed using Hematoxylin and Eosin (H cedarwood oil = 45.50) and microscopic quality (mean rank: xylene = 40.50; cedarwood oil = 40.50). Pure oil D-limonene and Solution 1 demonstrated moderate potential, with better microscopic performance for D-limonene (mean rank = 25.50) and better macroscopic performance for Solution 1 (mean rank = 25.50). However, both were inferior to xylene and cedarwood oil in wax removal and artifact reduction. The study concludes that kiat-kiat-derived D-limonene shows limited but observable potential as a natural deparaffinizing agent but requires further optimization. Future studies should focus on formulation refinement and improved extraction methods.