Distinguishing BCR signaling events and FL biopsy cell subsets by flow cytometry. (A) Phosphoproteins detected by flow cytometry (dark blue rings) are highlighted on a model of BCR signaling that includes regulation by protein tyrosine phosphatases. The BCR can instruct B cells to proliferate, alter innate immune signaling thresholds, induce B-cell anergy, or initiate cell death in a manner thought to depend on the strength, duration, and path of signaling. We detected BCR-mediated phosphorylation of Syk, Btk, Erk1/2, and p38 in subsets of primary human B cells. (B) Flow cytometry analysis of light chain isotype of CD20⁺ cells from a peripheral blood mononuclear cell (PBMC) sample (healthy donor), an FL tumor biopsy specimen from a different patient (FL-P12), and the Ramos lymphoma cell line. Ramos cells are clonal in origin and were of λ isotype. FL tumor cells in FL-P12 were κ isotype and vastly outnumbered the TIL B cells, as is commonly observed in FL tumor biopsy specimens. (C) Flow cytometry analysis of an FL biopsy specimen with an unusually large number of infiltrating nonmalignant B cells (FL-P10). Analysis of light chain isotypes present in the CD20⁺ subset of cells suggested that the tumor B cells were κ isotype and indicated that all B cells in the sample were exclusively κ or λ isotype. Expression of λ isotype was compared with Bcl-2 expression to identify FL tumor and nonmalignant B-cell populations. FL tumor B cells were κ isotype and overexpressed Bcl-2. Nonmalignant tumor-infiltrating host B cells did not overexpress Bcl-2 and were either κ or λ isotype. CD20 expression was also compared with Bcl-2 and with λ light chain expression in the total population of cells in the sample.