Background

Major human B cell subsets are currently defined by pauci-color flow cytometry protocols that typically include IgD, CD27, CD38, and CD24 to classify the major accepted peripheral blood populations (transitional, naïve, memory and plasmablast subsets). By and large, the expression of other informative markers (including IgM, CD23, CD10, CD21 and CD95, as well as chemokine receptor expression) is assessed by parallel staining of several sample aliquots with different combinations of the aforementioned markers in 4–5 color schemes. These approaches suffer from multiple shortcomings including: 1) the limited ability of the “defining” markers such as IgD, CD27 and CD38 to properly discriminate major populations; 2) the inability to ascertain the actual co-expression of multiple markers in a single population possibly leading to faulty assumptions of extended phenotypes and, by extension, preventing the discovery of new sub-populations; and 3) the need for larger number of cells to perform multiple stainings, a major practical limitation when dealing with rare samples. Combined, it seems obvious that limited use of available markers not only fails to differentiate multiple populations within the conventional core subsets, but could potentially lead to erroneous attribution of functional properties. To address these limitations, we have developed several multicolor panels to fully characterize human B cells. These multicolor panels share seven so-called anchor markers. Antibodies against CD19 and CD3, along with the Fixable Aqua Dead Cell Stain, allow the unambiguous identification of live CD19+ CD3− B cells. The inclusion of four developmental markers (IgD, CD24, CD27 and CD38) in the same panel makes it feasible to compare and integrate these different classification schemes and provide precise identifications of the core human B cell subpopulations (2).