CLL cells that migrate have a strikingly similar phenotype to those derived from the LN. (A) CLL PBMCs were introduced into the circulating model system coated with human endothelial cells (human umbilical vein endothelial cell²³  or human microvascular endothelial cell-1^9,10 ), and samples were collected from port C (migrated) and port D (circulating) after 48 hours. Matched CD5⁺/CD19⁺ CLL cells from each compartment were analyzed using multicolor flow cytometry. (B) Compared with CLL cells that remained circulating, migrated CLL cells had a phenotypic pattern strikingly similar to LN-CLL cells: higher HLA-DR (n = 7, Ai), CD5 (n = 12, Aii), CD80 (n = 19, Aiii), CD86 (n = 7, Aiv), CD69 (n = 26, Av), CD49d (n = 36, Avi), and CD38 (n = 32, Avii), and reduced CXCR4 (n = 34, Aviii). CLL cells that remained circulating had a pattern reflective of PB-CLL cells. (C) PBMCs from 11 patients were introduced into the circulating model system in the absence of endothelial cell coating. The numbers of CLL cells migrating was much lower than in the presence of endothelial cells and only sufficient for analysis migrated from 8 patients. In these 8 cases, the migrated CLL cells had increased CXCR4 expression compared with those that remained circulating. HMEC, human microvascular endothelial cell.