Improved Randolph stain for direct leukocyte differentiation and determination of total eosinophil count in a hemocytometer.

Identification and quantification of eosinophilic granulocytes are commonly performed indirectly by total leukocyte count and white cell differentiation in smears or cytocentrifuge preparations. Using a combination of four dyes, phloxine, Biebrich scarlet, methylene blue, and crystal violet, at 50-800 micrograms/ml, we have substantially improved an earlier method for differentiating leukocytes in a hemocytometer. This direct method allowed a rapid and reliable enumeration of eosinophils and their differentiation from neutrophils, lymphocytes and monocytes in peripheral blood and leukocyte fractions. The results obtained using this stain correlated with the leukocyte counts calculated from May-Grünwald-Giemsa stained smears in 100 patients with eosinophilia of various etiologies (r = 0.95; p < 0.01). This simple method is a useful improvement for eosinophil enumeration in field studies and biological experiments where the purity of cell suspensions must be evaluated without delay. The method cannot be substituted for the commonly used indirect technique which also allows the identification of other leukocyte lineages and their precursors.