Counterflow centrifugal elutriation as a method of T cell depletion may cause loss of immature CD34⁺ cells

Counterflow centrifugal elutriation (CCE) is capable of separating cells on the basis of size. CCE has been used successfully to deplete allogeneic bone marrow (BM) grafts of T lymphocytes to decrease the risk of acute graft-versus-host disease. Previous studies have shown that more immature CD34⁺ cells in human BM tend to be smaller than more mature CD34⁺ cells. Human BM was subjected to CCE with the 4 ml standard chamber at constant rotor speed (2300 r.p.m.) and increasing flow-rate (14-23 ml/min, rotor-off). The eleven fractions collected were assayed for CD34⁺ and CD3⁺ cells, and for CFU-GM, HPP-CFC and long-term culture initiating cells (LTC-IC). The CD3⁺ T cells were enriched in the early (small-cell) fractions 14-17 ml/min. CD34⁺ cells were enriched in fractions 17-21 ml/min, and CFU-GM were concentrated in the same fractions. HPP-CFC and LTC-IC showed nearly identical CCE profiles, with enrichment in fractions 16-18 ml/min. When fraction ≤17 ml/min was chosen as cut-off, the small-cell fraction contained 94.0% of all CD3⁺ cells, 44.4% of total cells, 33.2% of CD34⁺ cells and 34.7% of CFU-GM; however, 67.6% of HPP-CFC and 72.4% of LTC-IC were recovered in this small-cell fraction. These data suggest that T cell depletion through CCE as used by us, while losing only minor proportions of CD34⁺ cells and CFU-GM, carries the risk of losing the majority of more immature progenitor cells. This may lead to an increased risk of graft failure, in particular in HLA-mismatched transplants.