Robust expression of CCR3 as a single basophil selection marker in flow cytometry.

BACKGROUND: Basophil activation tests (BAT) rely on different combinations of basophil selection and activation markers. Whereas activation markers, especially CD63, are widely validated, the most suitable and robust marker for basophil selection is still a matter of debate. AIMS: Comparison of cell surface expression of two commonly used basophil selection markers (IgE, CD123/HLA-DR) with CCR3 in an unselected group of atopic and nonatopic donors in resting and activated basophils. METHODS: EDTA blood of 94 healthy adults, about half of them atopic by history, was analyzed using two different staining strategies: anti-CD123-PE/anti-HLA-DR-PerCP/anti-lin1-FITC and anti-IgE-FITC/anti-CD3-PerCP/anti-CCR3-PE. Additionally 40 pollen-allergic patients were recruited for the assessment of CCR3 expression after basophil activation. RESULTS: In resting basophils, cell surface expression of the three basophil selection markers was most constant for CCR3. IgE gating strategy showed the highest variation and up to 80% of nonbasophils in the selected gate in certain donors. During basophil activation, a shift of the mean fluorescence intensity for CCR3 toward the lower third of the CCR3-positive population could be demonstrated, but neither were CCR3-positive cells significantly lost for further analysis nor was differentiation between CCR3-positive and CCR3-negative cell populations hampered by this shift. CONCLUSIONS: CCR3 is a stable and highly expressed basophil selection marker, independent of the atopic background or basophil activation state and allows an accurate identification of basophils without need of a second marker. The basophil markers CD123/HLA-DR and IgE showed significantly higher interindividual variability in cell surface expression and are therefore less suited as selection markers.

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.