Assessing basophil activation by flow cytometry and mass cytometry in blood stored 24 hours before analysis

A new fluorescent-avidin–based method for quantifying basophil activation in whole blood

“…Finally, we tested whether exposure to IL-3 altered the reactivity of basophils with anti-CD63 antibody vs. Av.A488. While FITC-labeled anti-CD63 antibody did not label IL-3-stimulated basophils (a finding in accord with previous reports 8 ), we detected a modest but significant increase (vs. RPMI stimulation) in the percentage of Av.A488 + basophils after IL-3 challenge (at 20 ng/mL) (Fig. E1, C–D ); however, no histamine release above background was detected after such IL-3 challenge (data not shown).…”

“…However, the percentage of Av.A488 + basophils at baseline was significantly higher in the blood of allergic subjects (global mean ~11.4%) vs. anonymous blood donors (global mean ~3.8%) (Fig 2, E , far left panel). In some of the same blood specimens from peanut allergic patients shown in Fig 2, E , we also analyzed basophil CD203c (a surface marker reported to be associated with basophil activation) 3, 8 and found a positive correlation ( P =0.01) between the percentage of detected Av.A488 + basophils and the intensity of CD203c staining at baseline (i.e., after exposure to RPMI) but this correlation did not achieve statistical significance ( P =0.07) after activation by anti-IgE (Fig. E1, E ).…”

“…8 We used those conditions of blood storage to determine whether Av.A488 staining could be used to monitor basophil activation in whole blood from a group of 19 anonymous blood donors. We mixed 100 μL of whole blood with 1 μg/mL anti-IgE (in 100 μL of RPMI) or with 100 μL RPMI (control) alone, then gated on basophils as CD123-positive and HLA-DR-negative cells (two stable markers that, compared to FcεRI or CD203c, do not vary upon basophil activation, Fig 1, B ), 8 and compared detection of activated basophils by flow cytometry using either FITC-labeled anti-CD63 antibody or Av.A488 (both using the same detection channel; we defined “activated basophils” as those with a positive fluorescence signal for FITC-labeled anti-CD63 or for Av.488 as compared to the unstained and non-stimulated conditions) (Fig 1, C–D ). As illustrated in Fig 1, C–D , we found that both Av.A488 and FITC-labeled anti-CD63 antibody readily detected anti-IgE-induced basophil activation in the whole blood of such subjects, whose allergy status is unknown.…”

“…Peanut allergy in both trials was defined as having both a clinical reaction to ≤500 mg total of peanut protein in a double-blind, placebo-controlled food challenge (DBPCFC) to peanut and a positive skin prick test to peanut (≥to 5 mm). 8 …”

“…2, A–B (and in ref 8 and the Online Repository), we used flow cytometry to compare the ability of FITC-labeled anti-CD63 antibody vs. Av.A488 to monitor changes in whole blood basophils after their exposure ex vivo to either RPMI (control), 1 μg/mL anti-IgE (for 41 patients) or 100 ng/mL peanut extract (for 19 of the 41 patients). Compared to CD63 + basophils, we detected a higher percentage of Av.A488 + basophils in 38 of 41 patients tested, both at baseline (i.e., after RPMI incubation) and after basophil stimulation with anti-IgE, and in 17 of the 19 patients tested after basophil stimulation with peanut extract (Fig 2, B–D ).…”