Primitive erythroblasts may associate with fetal liver macrophage cells in vivo. (A) ImageStream images of liver cells viewed in brightfield (BF) or stained with anti–ϵγ-globin (ϵγ) and Ter119 antibodies and Draq5. The right panel is a combination of images of the 3 fluorescent stains. ImageStream analysis software facilitates the identification of different erythroid populations in the liver by quantifying brightfield characteristics (contrast and gradient RMS, ie, irregular surface) as well as fluorescent stain intensities. Primitive orthochromatic erythroblasts (row 1) were contrast^hi, gradient RMS^hi, ϵγ-globin^hi, Ter119^{lo, large}, Draq5^{+, small}. Primitive enucleated erythrocytes (row 2) were contrast^hi, gradient RMS^hi, ϵγ-globin^hi, Ter119^{lo, large}, Draq5⁻. Immature and mature definitive erythroblasts (rows 3 and 4, respectively) were distinguished from primitive erythroid cells by their nuclear size as well as by being contrast^lo, gradient RMS^lo, ϵγ-globin^lo, and Ter119^hi. (B) Cytospin preparations of E14.5 liver erythroblast islands stained with Wright-Giemsa reveal the presence of small numbers of primitive erythroid cells (*) attached to macrophage cells (m). (C) Immunohistochemistry of E15.5 liver reveals the very common spatial association (arrowheads) of ϵγ-globin–positive primitive erythroid cells (red) with F4/80-positive macrophage cells (blue). Scale bar = 20 μm.