Development of AIHA in mice treated on a weekly basis with rat RBCs. Female C57/Bl6 mice age 8 to 10 weeks were immunized on a weekly basis with rat RBCs for 10 weeks. (A) Levels of IgG-specific autoantibodies on mouse RBCs were measured by flow cytometry and are expressed as percentage of background unstained cells. The bars in each panel represent the mean values and n is the number of mice in each group. The autoantibody levels in rat immunized compared with control uninjected mice were significantly higher (P < .05). (B) Numbers of circulating reticulocytes in mice expressed as a percentage. There was a significant difference in the reticulocyte counts between the rat-immunized group and control uninjected mice (P < .05). (C) RBCs obtained from mice immunized with rat RBCs (n = 4) or control mice (n = 5) were fluorescently labeled with PKH-26 and injected intravenously into an equivalent number of C57Bl/6-naive mice. At times indicated, venous blood was sampled and analyzed by flow cytometry for the fraction of fluorescent RBCs. To show the clearance kinetics, injected RBCs at 1 minute after injection were taken as 100%, and the remaining RBCs were calculated at different time points as the average for each group of mice (error bars depict the standard error of the mean SEM). The difference at all points between the mice receiving RBCs from rat-immunized mice and those receiving control RBCs was calculated as P = .04. (D) Levels of IgG-specific anti–rat xenoantibodies in plasma were measured by first incubating rat erythrocytes with diluted mouse plasma (1 in 50 000) followed by staining with FITC-conjugated anti–mouse IgG. The analysis was performed by flow cytometry and is presented as relative fluorescent units on the y axis.