Standardization of the CFU-GM Assay Using Hematopoietic Growth Factors

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Abstract
The colony-forming unit-granulocyte-macrophage (CFU-GM) assay is used commonly to assess adequacy of progenitor number in bone marrow transplantation. The assay is poorly standardized, resulting in variability of results between and within laboratories. We assessed three variables that contribute to the lack of standardization. The colony-stimulating activity of human placental-conditioned medium (HPCM) was compared with combinations of recombinant hematopoietic growth factors (HGF) in 5 normal bone marrow donors. A protocol for batch testing of fetal calf serum (FCS) is described. In addition, a rigid training program has been introduced to minimize interstaff and intrastaff variability in the counting of colonies. We show that a five-factor combination of interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and stem cell factor (SCF) produces a mean increase of 85% in colony number. Some combinations of three HGF produce similar growth to HPCM, and all four HGF combinations are equivalent or superior to HPCM. Batch testing of FCS shows variability between batches. We show significant interstaff and intrastaff variability between a new and experienced staff member that improves following a period of training. In summary, the use of recombinant HGF in association with a rigorous program of batch testing of FCS and staff training results in a CFU-GM assay that can be standardized between laboratories.