Primitive pyrenocytes constitute a distinct cellular population in the fetal bloodstream. (A) Relative size of nucleated primitive erythroid cells as determined by ImageStream analysis reveals that pyrenocytes account for approximately 2% of nucleated blood cells in E15.5 blood (red line), less than 1% at E14.5 (blue line), and are not detected at E12.5 (green line). (B) Example of images of fetal blood cells visualized using the ImagesStream. Concurrent brightfield (BF) images and fluorescent images (anti–ϵγ-globin antibodies [ϵγ], Ter119, and the DNA stain [Draq5]) of larger primitive orthochromatic erythroblasts (rows 1 and 2) and of smaller primitive pyrenocytes (rows 3 and 4). (C) Primitive pyrenocytes and primitive orthochromatic erythroblasts (EryP) in the E15.5 circulation have similar nuclear sizes. Error bars represent SD. (D) E15.5 blood stained with annexin V (AnV), Ter119, and Draq5 (shown as merged images), and 7AAD analyzed by the ImageStream. Row 1 is a primitive pyrenocyte that is annexin V–positive and 7AAD-negative. Row 2 is a primitive orthochromatic erythroblasts that is negative for annexin V and 7AAD.