Xylene-Free Papanicolaou Staining

Xylene (XL) is the most commonly used clearing agent in Papanicolaou staining. XL is hazardous and toxic chemical and prolonged exposure to XL can cause many ill-health effects. The health risk due to XL can be minimized by substituting XL with less hazardous clearing reagents such as Pine Oil (PO), Eucalyptus oil (EO), or Limonene (LM). The objective of this study was to compare the clearing ability, staining quality, preservation of morphology, physical properties, and cost of XL, PO, EO, and LM.

Four smears were prepared from each of 50 serous effusions and were subjected to Papanicolaou stain. Out of four smears, one each was exposed to clearing specifically with XL (control), PO (test), EO (test), and LM (test). Test smears were compared with control for clearing, staining and morphology; graded as excellent, good or fair and further scored as 3, 2, 1, and the quality index (QI) was calculated. Statistical analysis was performed and the p value was calculated. In addition, the physical properties and cost of all the reagents were compared.

QI was 0.96 for both XL and PO, whereas 0.92 and 0.54 for EO and LM, respectively. Compared to XL, the quality of staining, clearing and morphology of PO and EO were statistically not significant, whereas the difference was statistically significant with LM (p = 0.005). Physical properties such as volatility, flammability, miscibility with alcohol and DPX and the refractive indices of all the reagents were almost similar and all were recyclable. Odor was pungent for XL and EO but was pleasant for PO and LM. The cost was less for PO as compared to others.

PO was a natural, less hazardous, less toxic, and economical clearing agent and can be considered as a substitute for XL.