Two-day collection and pooling of peripheral blood stem cells with semiautomated density gradient cell separation

Autologous and allogeneic PBSC collection and cryopreservation have been sh own to be feasible in the pediatric population, This technique may be assoc iated with complications, including volume overload and DMSO toxicity, To d ecrease these risks, we developed a technique by which PBSC are collected o ver a 2-day period and pooled prior to cryopreservation, PBSC are harvested on day 1 and stored with tissue culture medium on a rocker at ambient temp erature, On day 2, a second PBSC harvest ts performed, and the two harvests are pooled, separated on a Ficoll gradient in a semiautomatic closed syste m, and frozen with 10% DMSO after a soft spin for volume reduction. Cells f rom each day's harvest are tested for cell count and viability, A total of 36 collections in 26 patients were performed, This technique resulted in a 73% +/- 0.0% reduction in volume (mean +/- SEM) and an 88% +/- 0.9% depleti on of RBC, Mononuclear cell (MNC) count recovery was 88% +/- 2.6%, and the MNC dose delivered to the patient was 3.1 +/- 0.6 X 10(8) cells/kg. Cell vi ability was >98% before and after processing. Seventeen patients have been transplanted thus far, and all these patients engrafted with minimal toxici ty, These data indicate that storing PBSC for up to 24 h after harvest does not decrease PBSC viability or delay engraftment.