Towards standardizing basophil identification by flow cytometry

BACKGROUND: Basophils normally make up <2% of the white blood cells (WBC). There is no clear consensus for basophil identification by flow cytometry. The increased demand for basophil activation test (BAT) to identifying and monitoring allergic patients highlights the need for a standardized approach to identify basophils. METHODS: Using flow cytometry we analyzed whole blood stained with antibodies against: IgE, CD123, CD193, CD203c, CD3, HLADR, FcɛRI, CRTH2 and CD45. We examined unstimulated blood as well as blood stimulated with Anti-IgE and fMLP. Finally, we compared the results to a complete blood count (CBC) from an FDA approved hematological analyzer. RESULTS: Basophil identification relying on just one surface marker performed worse than approaches utilizing two identification markers. The percentage of basophils from WBC determined by flow cytometry results had a good correlation with the CBC results even though the CBC results were generally higher. Stimulating whole blood with the basophil activators did not interfere with the basophil identification markers. CONCLUSION: In flow cytometry assays, two surface markers should be used for identifying basophils and if a very pure basophil fraction is desired a third marker can be considered. In our hands the approaches that included CD123 in combination with either CD193, HLADR(negative) or FcɛRI performed the best.