title: Flow cytometry assessment of apoptotic CD34+ cells by annexin V labeling may improve prediction of cord blood potency for engraftment.
abstract: BACKGROUND
  Nonviable CD34+ cells are commonly assessed by standard flow cytometry using the nuclear stain 7-aminoactinomycin D (7AAD). 7AAD, however, only detects necrotic and late apoptotic cells, not earlier apoptosis, which engraft poorly in animal models of cord blood (cord) transplantation. The standard method, therefore, may overestimate engraftment potency of cord units under certain conditions.
  
  
  METHODS
  To detect apoptotic events, costaining with 7AAD and annexin V (AnnV), in parallel with the quantitative, standard enumeration, was used. Cord units were assessed before and after cryopreservation using both staining methods and colony-forming units (CFU) to determine if graft potency can be predicted using a "functional flow cytometry" approach.
  
  
  RESULTS
  Significant numbers of CD34+ AnnV+ events were found within the 7AAD-gated population. Nonapoptotic cell dose (CD34+ AnnV-) correlated well with CFUs in both a small-scale (n = 10) and a large-scale banking study (n = 107). Finally, following samples postthaw with time showed increasing numbers of apoptotic CD34+ cells and consequently the AnnV assessed dose was better at predicting the CFU compared with just the standard enumeration.
  
  
  CONCLUSIONS
  Defining the apoptotic population of CD34+ cells improved the prediction of CFU, making this method a rapid test of potency for assessment of cord units for clinical use.
authors: Anthony Nolan Research Institute, Royal Free Hospital, London, UK. richard.duggleby@yahoo.co.uk
authors: Richard C
authors: RC
authors: Duggleby
authors: Sergio
authors: S
authors: Querol
authors: Robert C
authors: RC
authors: Davy
authors: Laura J
authors: LJ
authors: Fry
authors: Daniel A
authors: DA
authors: Gibson
authors: Roger B V
authors: RB
authors: Horton
authors: Salmah N
authors: SN
authors: Mahmood
authors: Susana G
authors: SG
authors: Gomez
authors: J Alejandro
authors: JA
authors: Madrigal
mesh_terms: D017304:Annexin A5 / Q000378:metabolism*; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods* / Q000592:standards; D003609:Dactinomycin / Q000031:analogs & derivatives; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D011237:Predictive Value of Tests
chemical_list: D017304:Annexin A5; D018952:Antigens, CD34; D015415:Biomarkers; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D