The dielectrophoresis enrichment of CD34+ cells from peripheral blood stem cell harvests.
There is considerable interest in isolating the CD34+ cell population from leukaemic patients undergoing peripheral blood stem cell harvests. The techniques currently available make use of antibodies specific to the CD34+ surface markers. However, all of these techniques involve disturbance of the cell surface, are time-consuming and relatively expensive. In this study, we have used dielectrophoresis, which does not rely on the presence of cell-specific markers, to separate CD34+ cells from peripheral blood stem cell harvest samples containing an untreated natural mixed cell population. The separation is achieved by exploiting differences in the inherent dielectric properties of the various cell types. Samples obtained from peripheral blood stem cell harvests were resuspended in medium with a conductivity of less than 50 microS/cm and introduced into the dielectrophoretic separation chamber. Alternating field frequencies, from 500 kHz to 5 kHz, were used to collect cell fractions which were analysed by FACS, using a CD34-specific antibody, to quantify the CD34+ population within the fractions. On average a nearly five-fold increase in the frequency of the CD34+ cell population was observed in the fractions collected within the 50-10 kHz range. For this dielectrophoretic separation technique to be suitable in harvesting CD34+ cells for transplantation, it is important to demonstrate that the cells remain viable after the separation process. Cells obtained from each fraction grew when plated in colony assay cultures, GM-CFU and BFU-E, demonstrating that the cells remain normal, viable and capable of colony formation when cultured for 2 weeks. The number of colonies formed correlated with the percentage of CD34+ cells in each fraction. The dielectrophoretic separation technique is simple to operate, the separation is fast, the procedure non-invasive and although not tested has the potential to be incorporated as a batch-wise online facility with the standard harvesting equipment to increase the yield and speed of CD34+ cells in the PBSC harvest.
Department of Haematology, University of Wales College of Medicine, Cardiff, UK.
M
M
Stephens
M S
MS
Talary
R
R
Pethig
A K
AK
Burnett
K I
KI
Mills
D018952:Antigens, CD34 / Q000097:blood*; D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage; D001781:Blood Component Removal / Q000295:instrumentation / Q000379:methods*; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D003520:Cyclophosphamide / Q000008:administration & dosage; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000008:administration & dosage; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D006801:Humans
D018952:Antigens, CD34; D018906:Antineoplastic Agents, Alkylating; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003520:Cyclophosphamide