Evaluation of eluents from separations of CD34+ cells from human cord blood using a commercial, immunomagnetic cell separation system.
Human CD34+ cells from cord blood were separated in a two-step process using a commercial, immunomagnetic cell retention system. The performance of the system was evaluated by analyzing a number of eluents from the separations with a number of analytical techniques. In addition to cell counts and flow cytometry analysis, a new experimental technique that is undergoing development, cell tracking velocimetry (CTV), was used. CTV measures the degree to which a cell is immunomagnetically labeled, known as the magnetophoretic mobility, of a population of cells on a cell-by-cell basis and presents the results in the form of a histogram similar to flow cytometry data. The average recovery and purity of CD34+ cells from 10 separations was 52% and 60%, respectively. CTV analysis indicated that the mean magnetophoretic mobility of the positively enriched CD34 cells was 9.64 x 10(-5) mm3/T-A-s, while the mean mobility from negative eluents was -2.02 x 10(-6) mm3/T-A-s, very similar to the mobility of unlabeled cells. Within the positive eluents, the range of magnetophoretic mobility was approximately 50-fold, representing a plausible 50-fold range in surface CD34 antigen expression. CTV analysis also indicated that in some separations, positive cells were not retained by the immunomagnetic cell retention system. Finally, preliminary studies indicate that monocytes might be a primary cause in the lower purities and recoveries seen in this study. It is suggested that the monocytes phagocytose the magnetic nanobeads and become sufficiently magnetized to be retained within the Miltenyi column, reducing the purity of the positive eluent.
Melnik
K
K
Department of Pathology, Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43220, USA.
Nakamura
M
M
Comella
K
K
Lasky
L C
LC
Zborowski
M
M
Chalmers
J J
JJ
D018952:Antigens, CD34 / Q000032:analysis* / Q000276:immunology; D002452:Cell Count; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D008280:Magnetics; D036103:Nanotechnology
D018952:Antigens, CD34