Background: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. Materials and Methods: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 °C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. Results: The mean content of the divided apheresis products was 4.9 × 109 leukocytes with 86% MNC, 6.89 ×106 CD34+ cells, 2.1 ×105 granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 ×105 erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 ×106 CD34+ cells, 2.5 × 105 CFU-GM, and 8.1 × 105 BFU-E in Fresenius containers, and 6.1 × 106 CD34+ cells, 1.3 × 105 CFU-GM, and 7.7 × 105 BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. Conclusions: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be

Keywords:
Hematopoietic progenitor cells, CD34+ cell analysis, Cryopreservation, Flow cytometry, multiparameter, Frozen cells, Storage container
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© 1995 S. Karger AG, Basel
1995
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