A single step density gradient separation for large scale enrichment of mobilized peripheral blood progenitor cells collected for autotransplantation

Abstract

Peripheral blood leukocytes are becoming the preferred source of hematopoietic progenitor/stem cells for autologous transplantation. However, in vitro purging procedures are complex and expensive when applied to peripheral blood progenitor cells harvests. This is mainly due to the large quantities of nucleated cells present in leukapheresis collections. Aiming to reduce total cellularity without significant loss of CD34⁺ cells, we developed an in vitro cell separation procedure based on ficoll/metrizoate gradient used at a final density of 1.067 g/ml. To obtain this density, standard Lymphoprep (1.077 g/ml) was diluted with normal saline solution (NaCl 9 g/l). Twenty-six leukapheresis collections (median cellularity 21.1 × 10⁹, range 2.8–60) from 14 patients with non-Hodgkin’s lymphoma, multiple myeloma or plasma cell leukemia were processed (median two leukaphereses per patient). Mean (± s.d.) recovery of total nucleated cells, CD34⁺ cells and CFU-GM was 20.9 ± 10%, 74.7 ± 22% and 70.5 ± 19%, respectively. Cumulative per patient progenitor cell recovery was always above 50%, and as high as 80% in 10/14 patients, while total cellularity was reduced to a median 21.5% (10–33%) of pre-separation values. Contaminating neoplastic cells, identified by immunofluorescence in five collections, were reduced by 1–2 logs. The results indicate that our density gradient separation is an effective method to reduce total cellularity prior to immunological purging, without significant loss of progenitor cells.