Flowcytometric analysis of basophil counts in human blood and inaccuracy of hematology analyzers

Background: Differential leukocyte counts are of proven clinical value, but information about basophil counts in normal and disease conditions is scarce although basophils are regarded as key effector cells in allergy. Aims: To establish and validate flowcytometric methods for counting basophils in peripheral human blood, to determine reference values, and to examine the accuracy of two widely used hematology analyzers. Methods: Basophils were measured in whole blood by flowcytometry after staining with antibodies against the IL-3-receptor (CD123) or the eotaxin-receptor (CCR3) combined with other markers used for gating or validation purposes. Results: The basophil percentages in 95 healthy adults showed an excellent correlation between the two independent flowcytometric methods, demonstrating that both are accurate and precise. The most robust maker is CCR3, which seems to be sufficient to specifically identify basophils. Normal values of relative and absolute blood basophils counts were 0.22-1.28% and 0.014-0.087 G/L (95% reference intervals), respectively. Basophil counts measured with two hematology analyzers Coulter GEN-S and ADVIA-120 showed no correlation between these instruments. Comparing the data obtained by flowcytometry and the analyzers demonstrate that basophil counts of the GEN-S are erratic, while the ADVIA-120 gives at least an estimation of true basophil numbers. Conclusions: We provide a solid description and validation of a novel and rapid method for the flowcytometric enumeration of basophil in whole blood. The fact that the most heavily used Hematology autoanalyzer gives completely erroneous results could explain why basophils counts have not yet received recognition as a clinically useful diagnostic marker.