Flow cytometric analysis and gating of blood B cells showing the relative lack of IgD⁻ B cells in a BMT recipient 1 year after grafting.

Both specimens, normal and patient, were processed concurrently. Ficoll-isolated mononuclear cells (MNCs) were stained with anti-IgD–FITC (goat F(ab′)₂ antihuman delta chain), anti-(CD4, CD8, CD14, CD16)–PE, and anti-(CD19, CD20)–peridinin chlorophyll protein. Data were acquired on FACSCAN cytometer. For analysis, first the MNC gate (R1, R4) was drawn on the forward- versus side-scatter dot plots (left). Then, B cells were gated on the CD19/CD20 versus CD4/CD8/CD14/CD16 dot plots (R2, R5), excluding non-B cells binding anti-CD19/CD20 nonspecifically, “B cell + non-B cell” doublets, and CD20^low T cells (middle). Finally, CDl9/CD20 versus IgD dot plots were created exclusively of the cells falling within the MNC gate and the B-cell gate, ie, R1 and R2 in the normal and R4 and R5 in the patient (right). To calculate the percentage of IgD⁻ B cells, regions R3 and R6 were set so as to encompass the B cells showing only background FITC fluorescence. In this example, 18% of B cells in the normal were IgD⁻ versus 4% of B cells in the patient.