Characterization and isolation of peripheral blood B-cell subsets. (A) Total PBMCs were stained with PE-Cy5-conjugated anti-CD20 mAb to enumerate the percentage of B cells (●) in blood from chronic virus carriers (CC) and IM patients. Purified B cells were subsequently double-stained with FITC-conjugated anti-IgD and PE-conjugated anti-CD27 Abs to enumerate the percentages of IgD⁺CD27⁻ naive, IgD⁻CD27⁺ isotype-switched memory and IgD⁺CD27⁺ nonswitched memory B cells. Median values for each group are shown by the horizontal bars; from P values, the only significant difference was for total percentage B cells in CC versus IM blood (P < .001). (B) Purified B cells double-stained as in panel A to identify naive, switched memory and nonswitched memory subsets were separated using the indicated sort gates and the isolated B-cell populations subsequently reanalyzed to determine the sort purities. Shown are representative data from donor CC17.