Basophil counting with a new staining method using alcian blue

Abstract

Difficulties in obtaining reproducible and accurate enumeration of circulating basophils with existing techniques have hampered investigation of this infrequent cell population. A new basophil staining method is described that employs alcian blue dye for staining of heparin within basophils at low pH and in the presence of lanthanum ions. Basophil recognition is facilitated by reducing nonspecific nuclear staining. This objective is achieved because of the differences in stability of alcian blue heparin, alcian blue nucleic acid, lanthanum heparin, and lanthanum nucleic acid complexes. Reduction of pH after staining also favors solubilization of leukocyte cytoplasmic proteins, providing greater contrast between stained and unstained cells by reducing light scattering of the unstained leukocytes. The alcian blue staining method is suitable both for chamber basophil counting and automated basophil counting using continuous flow sampling and electro optical detection. The new staining method was evaluated by comparing it with the chamber counting method using toluidine blue in a triple blind study in which the results of basophil counting by the alcian blue chamber method, alcian blue automated instrument method, and the toluidine blue chamber method were analyzed for reproducibility and compared with an indirect basophil count obtained from a 1000 cell leukocyte differential and a total leukocyte count. Both alcian blue staining methods gave greater reproducibility than toluidine blue and were more accurate, as evidenced by a significantly higher correlation with the indirect basophil count. The improved reproducibility, accuracy, and convenience of this method over existing methods should facilitate the collection of more meaningful information about circulating basophil levels in health and disease.