Modified technique for soft tissue processing and staining

Abstract

Use of xylene as a clearing agent during tissue processing and the subsequent hematoxylin and eosin staining process have become inseparable parts of histopathology laboratories. While using these reagents, one must keep in mind the well-documented health hazards of xylene and the stability, the cost factor, and the shortage of hematoxylin, which has been faced once and may recur since it is obtained from a natural resource. Thus, this study was carried out to find suitable substitutes for xylene and hematoxylin. To compare xylene and chloroform, ten biopsy tissues fixed in formalin were cut into two; one piece of each biopsy was cleared in xylene and the other piece in chloroform. All the other reagents used in the tissue processing remained the same. The tissues were then sectioned and stained with hematoxylin and eosin. Further, two tissue sections were obtained from tissues of differing natures and stained with hematoxylin and eosin and Celestine blue and eosin, in order to compare and evaluate hematoxylin and Celestine blue. In both cases, the staining intensity of selected fields was evaluated at regular intervals. Staining intensity of sections following clearing with chloroform was more stable over a period of time as compared to those cleared with xylene. The staining of sections with Celestine blue was comparable to that of hematoxylin and was not affected by the nature of the tissue. Results of the present study suggest that chloroform has specific advantages as a clearing agent as compared with xylene. Furthermore, Celestine blue is a steadfast substitute for hematoxylin.