CML leukapheresis products can be enriched for CD34+ cells and simultaneously depleted of CD15+ cells using a simple Ab cocktail.
BACKGROUND
CML progenitor-cell studies would be greatly facilitated if samples could be repeatedly accessed from a source of well-characterized cells. The present study was designed to develop a simple, inexpensive Ab cocktail that would provide subpopulations of cells enriched for CD34+ cells and simultaneously depleted of CD15+ mature myeloid cells.


METHODS
Cells from leukapheresis products from CML patients at diagnosis were incubated with each of two Ab cocktails. The standard cocktail (debulking, DB), containing 11 Abs, is recommended for obtaining a highly enriched population of CD34+ cells. The efficacy of an alternative, simpler cocktail (CML custom, CC), containing only four Abs was tested. The recoveries of CD34+ cells, CD15+ cells, colony-forming unit granulocyte-macrophage, and LTCIC were monitored. The samples were then cryopreserved, thawed, and the recoveries remeasured.


RESULTS
The purity of CD34+ cells was significantly superior using the DB cocktail than with the CC cocktail. Conversely, using the CC cocktail, the yield of CD34+ cells was significantly higher compared to the DB cocktail. These results were maintained even when the amount of Ab was reduced 10-fold. Both Ab cocktails consistently removed > 99% of the CD15+ cells. Consistent with the CD34+ cell-enrichment data, higher colony-forming cell (CFC) frequencies were obtained with the DB cocktail, although superior yields of CFC were obtained with the CC cocktail. After cryopreservation and thawing the yield of CD34+ cells remained high, and a further reduction in the number of CD15+ cells was obtained.


CONCLUSIONS
A method is described that allows the rapid and efficient debulking of large CML samples. This strategy will provide a source of well-characterized CML stem/progenitor cells that can be repeatedly accessed.
Richmond
L J
LJ
Department of Haematology, Glasgow Royal Infirmary, UK.
Alcorn
M J
MJ
Pearson
C
C
Cameron
G
G
Thomas
T
T
Eaves
C J
CJ
Eaves
A C
AC
Holyoake
T L
TL
D018952:Antigens, CD34 / Q000096:biosynthesis*; D002469:Cell Separation / Q000379:methods*; D015925:Cryopreservation; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood* / Q000628:therapy*; D016256:Lewis X Antigen / Q000096:biosynthesis*; D022423:Myeloid Cells / Q000276:immunology; D033581:Stem Cell Transplantation; D013601:T-Lymphocytes / Q000276:immunology*
D018952:Antigens, CD34; D016256:Lewis X Antigen; D016178:Granulocyte-Macrophage Colony-Stimulating Factor