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The absolute basophil count (cells/L) can be determined by manual counting of peripheral blood smears or using cell counting chambers as well as by automated hematology analyzers and fluorescence flow cytometry. Manual basophil counting of peripheral blood smears is currently regarded as the reference method, although the limitations of this method (distribution, observer, and statistical errors) are widely recognized. Automated hematology analyzers offer an advantage of larger numbers of counted cells and high throughput but are characterized by inconsistent analytical performance for basophil enumeration. Flow cytometric enumeration of circulating basophils using panels of monoclonal antibodies is being developed as novel candidate reference method for the absolute basophil count in peripheral blood. Basophil counting using fluorescence flow cytometry is characterized by high precision and statistical superiority. Emerging innovative technologies for absolute cell counts include imaging flow cytometry, mass cytometry, and on-chip blood counting, but their analytical performance for absolute basophil counts is yet to be established. Here, we describe various techniques for absolute basophil counting in peripheral blood including manual basophil counts in smears and hemocytometers and flow cytometric methodologies using double-platform, bead-based, and volumetric approaches.

The purification of basophils from peripheral blood has represented a formidable challenge for researchers since they were discovered by Paul Ehrlich in 1879. From the first published attempts in the late 1960s, it took half a century to develop robust protocols able to give sufficient numbers of pure, functionally unimpaired basophils. The existing protocols for basophil purification exploit those properties of basophils which distinguish them from other cell types such as their localization in blood, density, and the presence or absence of surface markers. Purification techniques have been used in various combinations and variations to achieve a common goal in mind: to obtain a pure population of human basophils in sufficient numbers for downstream studies. The arduous way leading up to the modern protocols is summarized in this historical retrospective. A fast protocol for purification of basophils to near homogeneity is also described.