Quantification of Solution-Free Blood Cell Staining by Sorption Kinetics of Romanowsky Stains to Agarose Gels

Abstract

Imaging and quantification of stained blood cells are important for identifying the cells in hematology and for diagnosing diseased cells or parasites in cytopathology. Romanowsky staining have been used traditionally to produce hues in blood cells using anionic eosin Y and cationic methylene blue. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to premature precipitation or evaporation of methanol, leading to the inherent inconsistency of solution-based Romanowsky staining. Here, we demonstrate that staining and destaining of blood smear are controllable by the contact time of agarose gel stamps. While the extent of staining and destaining are discernable by hue values of stamped red blood cells in micrographs, quantification of adsorbed and desorbed Romanowsky dye molecules (in particular, eosin Y, methylene blue, and azure B) from and to the agarose gel stamps needs a model that can explain the sorption process. We find predictable sorption of the Romanowsky dye molecules from the pseudo-second-order kinetics models for adsorption and the one phase decay model for desorption. Thus, the method of agarose gel stamping demonstrated here could be an alternative to solution-based Romanowsky staining with predictable quantity of sorption and timing of contact.