Analysis of peripheral blood granulocyte-macrophage colony growth by limiting dilution assay.

Analysis of myeloid progenitor cells in the peripheral blood (peripheral blood colony-forming unit granulocyte-macrophage; PBCFU-GM) is limited by their low frequency and by the presence of inhibitory cell populations. These factors limit the study of cytokines and cellular influences on PBCFU-GM in semisolid media assays and complicate the interpretation of data. We have developed a limiting dilution assay (LDA) in liquid culture for PBCFU-GM that allows evaluation of inhibitory or accessory effects of other cell populations and estimation of progenitor cell frequency. Using this system we have examined the inhibitory effect of autologous monocytes on in vitro colony growth. After monocyte depletion by counterflow centrifugal elutriation and adherence, colony growth with recombinant human granulocyte-macrophage colony-stimulating factor was linear over a wide range of cell densities, indicating a direct proliferative effect on circulating myeloid progenitor cells. Simultaneous PBCFU-GM assays in agar demonstrated monocyte inhibition but did not afford reliable interpretation of either progenitor frequency or linear growth kinetics in a statistically verifiable fashion. LDA in liquid culture may be a useful tool to study the effects of various cytokines and cell populations on PBCFU-GM in vitro and in vivo.