The granulocytic lineage consists of three mature cell types: neutrophils, basophils, and eosinophils. The only way to truly identify the frequency of these in peripheral blood is based on histological staining with hematoxylin and eosin (H&E) and evaluation of relevant morphological features that distinguishes these three types based on their size, color, and shape of the nucleus.
In a CFU assay, one only has the size of the mature cells to help identify the progenitor type and cannot visualize the shape of the nuclei. The shape of the nuclei is key in determining the difference between, for example, granulocytes and monocytes. The only way to identify the composition of the cells within a CFU-GM colony is to pluck the colony and evaluate the mature cells within the colony following a cytospin and subsequent H&E staining. However, most colonies are a heterogeneous mix of immature and mature cells that represents the lineage potential of the progenitor.
Alternatively, cells plucked from individual colonies can be stained with cell surface markers that are characteristic for the various mature cell types. However, this approach often involves staining of multiple surface markers to be definitive. Historically, histological staining, cell-surface expression by flow cytometry, and cytokine requirements have all been used to confirm lineage specific progenitor types.
Recommended Reading: Görgens A et al. (2013) Revision of the human hematopoietic tree: Granulocyte subtypes derive from distinct hematopoietic lineages. Cell Rep 3(5): 1539–52.