title: Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.
abstract: 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.
  
  
  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 degrees 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 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) 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 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) 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 recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.
authors.affiliation: Abteilung Hämatologie und Onkologie, Medizinische Hochschule Hannover, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Arseniev
authors.forename: L
authors.initials: L
authors.lastname: Goudeva
authors.forename: J G
authors.initials: JG
authors.lastname: Kadar
authors.forename: I
authors.initials: I
authors.lastname: Südmeier
authors.forename: K
authors.initials: K
authors.lastname: Battmer
authors.forename: S
authors.initials: S
authors.lastname: Matheja
authors.forename: H
authors.initials: H
authors.lastname: Mitschulat
authors.forename: W
authors.initials: W
authors.lastname: Stangel
authors.forename: H
authors.initials: H
authors.lastname: Link
mesh_terms: D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D001793:Blood Preservation / Q000295:instrumentation*; D015925:Cryopreservation / Q000295:instrumentation*; D005434:Flow Cytometry / Q000295:instrumentation*; D018380:Hematopoietic Stem Cell Transplantation / Q000295:instrumentation*; D006801:Humans; D007937:Leukapheresis / Q000295:instrumentation; D007958:Leukocyte Count; D011138:Polytetrafluoroethylene; D011786:Quality Control
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D011138:Polytetrafluoroethylene