title: Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts.
abstract: Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.
authors.affiliation: Department of Hematology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands. GJ.Schuurhuis@vumc.nl
authors.forename: Fransien
authors.initials: F
authors.lastname: de Boer
authors.forename: Angelika M
authors.initials: AM
authors.lastname: Dräger
authors.forename: Herbert M
authors.initials: HM
authors.lastname: Pinedo
authors.forename: Floortje L
authors.initials: FL
authors.lastname: Kessler
authors.forename: M
authors.initials: M
authors.lastname: Monnee-van Muijen
authors.forename: Geert
authors.initials: G
authors.lastname: Weijers
authors.forename: Guus
authors.initials: G
authors.lastname: Westra
authors.forename: Elsken
authors.initials: E
authors.lastname: van der Wall
authors.forename: Tanja
authors.initials: T
authors.lastname: Netelenbos
authors.forename: Jan W
authors.initials: JW
authors.lastname: Oberink
authors.forename: Peter C
authors.initials: PC
authors.lastname: Huijgens
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Schuurhuis
mesh_terms: D018952:Antigens, CD34; D017209:Apoptosis; D002465:Cell Movement; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000592:standards*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D007937:Leukapheresis / Q000592:standards*; D008223:Lymphoma / Q000628:therapy; D009101:Multiple Myeloma / Q000628:therapy; D013194:Staining and Labeling; D013997:Time Factors; D014182:Transplantation, Autologous
chemical_list: D018952:Antigens, CD34; D005456:Fluorescent Dyes