Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.
We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.
Dobo
Irène
I
Laboratoire d'Hématologie du Centre Hospitalier Universitaire d'Angers, France. IrDobo@chu-angers.fr
Pineau
Danielle
D
Robillard
Nelly
N
Geneviève
Frank
F
Piard
Nicole
N
Zandecki
Marc
M
Hermouet
Sylvie
S
D018952:Antigens, CD34 / Q000032:analysis*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D003094:Collagen / Q000737:chemistry / Q000494:pharmacology; D003114:Colony-Forming Units Assay / Q000379:methods / Q000592:standards*; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D042381:Granulocyte Precursor Cells / Q000378:metabolism; D006098:Granulocytes / Q000166:cytology / Q000276:immunology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006689:Hodgkin Disease / Q000097:blood / Q000628:therapy; D006801:Humans; D007937:Leukapheresis; D017493:Leukocyte Common Antigens / Q000032:analysis; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008228:Lymphoma, Non-Hodgkin / Q000097:blood / Q000628:therapy; D008264:Macrophages / Q000166:cytology / Q000276:immunology; D009101:Multiple Myeloma / Q000097:blood / Q000628:therapy; D009369:Neoplasms / Q000097:blood / Q000628:therapy
D018952:Antigens, CD34; D016298:Hematopoietic Cell Growth Factors; D016179:Granulocyte Colony-Stimulating Factor; D003094:Collagen; D017493:Leukocyte Common Antigens