title: CD133 immunomagnetic separation: effectiveness of the method for CD133(+) isolation from umbilical cord blood.
abstract: OBJECTIVE
  Umbilical cord blood (UCB) is a rich source of stem cells, the characterization and isolation of which requires specific stem cell markers and reliable and reproducible protocols.
  
  
  METHODS
  We assessed CD133 isolation in 39 UCB samples, using a commercial immunomagnetic cell-sorting protocol, and, because of its non-reproducibility, we applied optimized protocols in an effort to improve it. These included extra-labeling of the selected CD133(+) subpopulation and indirect labeling using anti-phycoerythrin (PE) microbeads, goat anti-mouse IgG microbeads or a combination of both. The CD34 isolation was used as a control.
  
  
  RESULTS
  The mononuclear cell fraction expressed 0.53±0.06% CD133. The corresponding value for CD34 was 1.64±0.15%. Following the manufacturer's instructions, the CD34 isolation resulted in a population expressing 93±1.25% CD34 while, after the corresponding process, CD133(+) expression ranged from 10% to 85% (median 60%). The optimized isolation protocols did not result in improved CD133(+) yield. The variation in the purity of the CD133 population cannot be attributed to the different clones of CD133 used, because they do not cross-block, while other factors such as glycosylation, which could possibly interfere, do not apply in normal hematopoietic stem cells (HSC).
  
  
  CONCLUSIONS
  CD34 isolation by the immunomagnetic method results in highly pure CD34(+) population, while the efficient and reproducible yield of a pure CD133(+) population is not feasible. Therefore quantification of the positive cells should follow each isolation procedure in order to confirm the number of CD133(+) cells.
authors.affiliation: Department of Pediatric Hematology-Oncology, University Hospital of Heraklion, University of Crete Medical School, Heraklion, Crete, Greece.
authors.forename: Iordanis
authors.initials: I
authors.lastname: Pelagiadis
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Relakis
authors.forename: Lida
authors.initials: L
authors.lastname: Kalmanti
authors.forename: Helen
authors.initials: H
authors.lastname: Dimitriou
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D018952:Antigens, CD34 / Q000378:metabolism; D002469:Cell Separation; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006023:Glycoproteins / Q000378:metabolism*; D018189:Immunomagnetic Separation / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D051379:Mice; D008863:Microspheres; D010455:Peptides / Q000378:metabolism*
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; D010455:Peptides; C000606024:Prom1 protein, mouse