title: Extensive early apoptosis in frozen-thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation.
abstract: Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze-thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen-thawed grafts, threshold numbers for adequate haematological recovery of 2.8-3.0 x 10(6) CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2-1.3 x 10(6) CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3-0.4 x 10(6)CD34+ cells/kg). In contrast to freeze-thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4 degrees C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen-thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.
authors.affiliation: Department of Hematology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: de Boer
authors.forename: A M
authors.initials: AM
authors.lastname: Dräger
authors.forename: H M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: F L
authors.initials: FL
authors.lastname: Kessler
authors.forename: E
authors.initials: E
authors.lastname: van der Wall
authors.forename: A R
authors.initials: AR
authors.lastname: Jonkhoff
authors.forename: J
authors.initials: J
authors.lastname: van der Lelie
authors.forename: P C
authors.initials: PC
authors.lastname: Huijgens
authors.forename: G J
authors.initials: GJ
authors.lastname: Ossenkoppele
authors.forename: G J
authors.initials: GJ
authors.lastname: Schuurhuis
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis; D002452:Cell Count; D002470:Cell Survival; D015925:Cryopreservation; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods / Q000592:standards*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007937:Leukapheresis / Q000379:methods / Q000592:standards; D008228:Lymphoma, Non-Hodgkin / Q000628:therapy; D011933:Reagent Kits, Diagnostic; D019172:Transplantation Conditioning; D014182:Transplantation, Autologous / Q000379:methods / Q000592:standards
chemical_list: D018952:Antigens, CD34; D011933:Reagent Kits, Diagnostic