Basophilic macrocytes, splenomegaly, and extramedullary hematopoiesis in Pen KO mice. (A-B) Wright-Giemsa–stained blood smears of 2 representative Pen^−/− mice. Note the basophilic macrocytes, target cells, overall larger RBCs, and the absence of microspherocytes. (C) A Wright-Giemsa–stained blood smear of a Pen WT mouse. Note the smaller RBC size, the absence of basophilic macrocytes, and a pale blue “shift cell” in the right lower corner. (D) The spleen of the Pen^−/− mouse seen in panel A. A WT spleen is included for comparison. Bar = 1 cm. (E) A Wright-Giemsa–stained cytospin preparation of the spleen cells of the Pen^−/− mouse seen in panel D. Most cells are intensely basophilic proerythroblasts or erythroblasts (arrowheads) or enucleated, basophilic RBCs (arrows). (F) A Wright-Giemsa–stained cytospin preparation of the spleen cells of a Pen WT mouse. Most cells are lymphocytes. Panels A, B, C, E, and F have the same magnification (300×). (G) The spleen of a Pen^−/− mouse with multiple infarcts (pale scars). Bar = 1 cm. (H) The hepatic tumor of the same Pen^−/− mouse seen in panel G. Bar = 1 cm. (I) A hematoxylin and eosin–stained thin section of panel H. The asterisk denotes extramedullary hematopoiesis; arrowheads, hepatocytes. (J) Flow cytometric analyses of the spleen cells seen in panels D-F. RBCs were removed by hypotonic lysis before staining with MAb. The percentage of cells in each quadrant/gate is shown. CD3 is a marker for T cells while CD19 is a marker for B cells. The frequency of TER119⁺ erythroblasts in Pen^−/− spleen is increased by 150-fold.