Sequential gating strategy for identification of plasma cells.

The gating strategy is demonstrated in a patient with MGUS, with approximately even numbers of normal and myeloma plasma cells. (A) Plot showing all cells, with CD138 (Syndecan-1, B-B4) expression against CD38 expression. High CD138 and CD38 expression (region 1) usually provides the best separation of bone marrow plasma cells from other leukocytes, but also contains a significant proportion of cells binding antibodies nonspecifically (the diagonal line of dots). Contaminating cells can be removed in most cases using a region set on forward- and side-scatter characteristics. (B) Scatter characteristics of cells identified in region 1 (R1). Plasma cell scatter characteristics vary from patient to patient, in keeping with their morphologic heterogeneity. However, by excluding cells with very low or very high forward- and light-scatter characteristics (apoptotic cells or debris and doublets or electronic noise, respectively), it is possible in most cases to exclude the majority of contaminating events. (C) Plot showing all cells, with CD38 against CD45 expression. The neoplastic plasma cells are clearly visible as a distinct population in the upper left area of the plot; however, the normal plasma cells in the upper right are not a discrete population and overlap with other leukocytes as well as nonspecific binding events. The contaminating leukocyte population is, in this case, normal B-cell progenitor cells, which are often more numerous in MGUS patients than plasma cells. Activated T cells expressing the same level of CD38 as plasma cells are also common in myeloma. (D) The same plot as (C), but the only cells displayed are those satisfying the scatter region (R2), indicating that 2 clear populations are present with no obvious contamination. The rationale for using CD38 versus CD45, as opposed to CD138 versus CD38, as the primary phenotypic gate has been reported previously,29but is, briefly, because of the following: (1) an immunophenotypic gate set on dual positivity is more likely to contain contamination than a gate set on the basis of one positive and one negative marker because a cell that binds one antibody nonspecifically also binds others, and hence is more likely to contaminate a dual-positive region; and (2) CD138 is expressed at a significantly lower level by circulating plasma cells than by paired marrow counterparts, whereas CD38/CD45 expression does not vary and therefore provides a more consistent approach for all samples. Internal controls to ensure that plasma cells have been accurately identified are shown in Figure 2.