Properties of human basophils isolated by fluorescence-activated cell sorting

Abstract

The aim of the current study was twofold: (1) to consider the applicability of fluorescence-activated cell sorting (FACS) to basophil isolation from small blood volumes and (2) to compare basophils obtained from children with asthma to basophils from healthy children. With FACS, basophil suspensions were prepared with a purity of 84% (range, 75% to 95%) and a recovery of 20% (range, 15% to 30%). The purified basophils had a total histamine content of 1.6 ± 0.12 pg per cell, not differing significantly from total histamine content observed in “total” leukocyte suspensions (1.4 ± 0.07 pg per basophil). The same was true for IgE receptor-mediated histamine release (29 ± 4% versus 27 ± 4%) and for ionophore A23187-induced histamine release (41 ± 6% versus 51 ± 9%). Sorted basophils from subjects with asthma released more histamine after IgE receptor activation (0.67 ± 0.09 pg per cell) than basophils from healthy children (0.40 ± 0.04 pg per cell; p < 0.02). Expressed as percent release, no significant difference was observed (37 ± 3.2% versus 30 ± 2.7%). Ionophore A23187-induced histamine release did not differ significantly between subjects with asthma and control subjects, neither expressed as picograms per cell (1.21 ± 0.17 pg per cell versus 1.02 ± 0.11 pg per cell) nor expressed as percent release (66 ± 4.4% versus 74 ± 3.2%). The IgE receptor-mediated change in membrane potential induced by Ca ++ exposure and by ionophore A23187 exposure demonstrated no significant differences between subjects with asthma and healthy children: 12 ± 1.6 mV versus 14 ± 1.0 mV after Ca ++ exposure and 11 ± 1.8 mV versus 12 ± 1.4 mV in the presence of ionophore A23187. FACS is well suited for the preparation of high-purity basophil suspensions without significant selection bias or cell damage. In response to IgE stimulation, purified basophils from subjects with asthma release more histamine than basophils from healthy children. No such difference could be demonstrated in ionophore A23187-induced histamine release.