Butterfly pea flower extract as an alternative dye in cytological canine mast cell tumor staining

Abstract

Mast cell tumors (MCTs) are the most common skin tumors in dogs. Laboratory diagnosis of MCTs has been
established by many techniques. The dyes generally used for tissue staining, including MCTs, are synthetic dyes,
however, the global interest in using eco-friendly natural dye has become a significant matter. We focused on the
butterfly pea (Clitoria ternatea L.) which is locally available. Limited studies are known about their efficacy as a tissue
stain. The aim of this study was to investigate the staining ability of butterfly pea flower extract in cytological canine
MCTs. The dried petals were ground into powder and dissolved in acidified ethanol to obtain the crude extraction.
After filtration, this was added with mordant, 2.5% aluminium chloride, before staining. The methanol-fixed cytological
samples of diagnosed MCTs were stained with the dye extract for 30 minutes, washed with distilled water and observed
under light microscope initially, and after the counterstaining, with hematoxylin. The slides were stained with Giemsa,
Toluidine blue and Diff-Quick stains to compare with the dye extract. The other round cell tumors including canine
transmissible venereal tumor (TVT) and melanoma were also stained with the dye extract to compare with the MCTs.
The results revealed a bright pink color specifically stained in the MCT granules. However, Giemsa, Toluidine blue and
Diff-Quick staining shown their specific stain colors in the granules in larger numbers. The dye extract staining was not
detected in either TVT or melanoma. Improvement of the dye extraction and staining conditions may provide
additional staining outcomes, thus it may be possible to serve as an alternative natural dye for future application in
cytological canine MCT diag