Representative dot plot profiles and gating strategies for 3 colour (A) and 5 colour (B) flow cytometric analysis of IgM memory B cells in normal and splenectomy patients.

A. i) Lymphocytes were identified by forward (FSC) and side scatter(SSC), ii) B cells by CD19 expression (% B cells./lymph), iii) memory B cells by CD19, CD27 expression (% memory B cells/B cells), iv) IgM memory B as CD19+CD27+IgM+ (% IgM memory B cells/memory B cells) and switched memory B cells as CD19+CD27+IgM− (% switched memory B cells/memory B cells). Lymphocytes were also sequentially gated v) for CD19 CD27+ memory B cells (% memory B cells/lymph) and (vi) for CD27+ and IgM to identify IgM memory B cells [upper right quadrant] (% IgM memory B cells/B cells) and switched memory B cells [lower right quadrant] (switched memory B cells/B cells). Parameters used in subsequent quantitative analysis were B cells as a proportion of lymphocytes (ii), memory B cells as a proportion of B cells (iii) or total lymphocytes (v), and IgM memory cells as a proportion of B cells (vi). B. i) Blood leukocytes were identified by forward (FSC) and side scatter (SSC), ii) Lymphocytes selected by CD45 and log SSC, iii) B cells were identified by CD19 expression (% B cell/lymph) and iv) memory B cells were identified as CD19+CD27+IgD^lo cells (% memory B cells/lymph). v) memory B cells were identified as IgD^loIgM+ (% IgM memory B. memory B cells) and switched memory B cells as CD19+CD27+IgM−IgD− (% switched memory B cells/memory B cells). vi) lymphocytes gated as B cells and plotted for IgM and IgD are shown for comparison to identify the predominant naïve unswitched IgD^hi IgM+ B cell population.