A three-step procedure for the purification of human basophils from buffy coat blood

Objective and Design: We report a method for basophil purification from buffy coats, which avoids positive selection of the cells and gives rise to good purity, yield and functional integrity of the cells. Subjects: Buffy coat blood (concentrated leukocyte fraction derived from 450 ml venipuncture donations) obtained from healthy blood donors (n=51). Methods: Basophils were enriched by a three-step process starting with Ficoll density centrifugation (1.6+/-0.1% basophil purity) followed by counter current centrifugal elutriation (17.7+/-1.4% basophil purity). The final stage involved negative selection using Dynal immunomagnetic beads directed against CD2, CD14, CD16 and CD19 positive cell contaminants. Functional integrity of which was assessed by comparing the anti-IgE or calcium ionophore A23187 induced histamine release from basophils obtained from each enrichment step. Furthermore, basophil morphology was investigated using light and electron microscopy. Results: The final mean basophil purity of 67.3+/-1.4% with a yield of 3.5+/-0.5 x 10(6) basophils and a recovery of 21.8+/-2.4% was achieved. Net histamine release from basophils stimulated with optimal concentrations of antihuman IgE was 39.1+/-6.5% after Ficoll centrifugation, 41.6+/-7.7% following elutriation and 35.7+/-6.8% from the final purified fraction. Additionally, basophils enriched with our method showed intact morphology by electron microscopy and were functionally active to non-immunological stimulation. Conclusions: These results compare favourably with previous studies, which have often required the use of positive selection via the Fc(epsilon)RT receptor, which may result in cell degranulation, or cell sorting, which cannot be applied to large cell numbers. Our method provides a reproducible technique for basophil enrichment when large numbers of functionally intact basophils are required.