ECP generation of dendritic cells. The percentage and absolute number of DCs generated in ECP-processed monocytes. (A) The percentage of monocytes induced by ECP to enter the DC differentiation pathway was assessed by coexpression of cytoplasmic CD83 and cell-surface HLA-DR. Two-color flow cytometric analysis of 10 000 monocytes from 3 CTCL and 3 GVHD patients and from 3 normal subjects is shown at the 3 time points: from the leukapheresis harvest prior to ECP (pre ECP), immediately after ECP (ECP Day 0), and 18 hours after ECP (ECP Day 1). The monocyte population (confirmed by CD11c staining) was gated using forward and side scatter. Coexpression of cytoplasmic CD83 and cell membrane HLA-DR was assessed in the gated monocyte population. The last set of bars demonstrates the mean ± SD for all 9 subjects, revealing significant enhancement in CD83 expression after 18-hour incubation (P < .001), compared with pre- and immediately post-ECP. (B) The absolute number of ECP-processed monocytes that entered the DC differentiation pathway was determined for each test subject. The absolute number of DCs was calculated as the product of 2 numbers: (1) the percentage of leukocytes cytometrically gating, by forward and side scatter, in the Monocyte/DC region and (2) the total number of cells in that region displaying the CD11c marker typical of both monocytes and DCs. The absolute number of induced CD83⁺ cells varied with white blood cell count of the subject, but exceeded 50 million in all but 1 CTCL patient and exceeded 300 million in 1 normal subject. The absolute number of DCs was then calculated by multiplying the CD83⁺ percentage by the relevant total volume.