Role Of Transferrin In Recovery From Phlebotomy In Iron Replete Or Iron Deficient Mice
Authors: Li Huihui , Shah Jayesh , Hall Charles B. , Ginzburg Yelena 
Published: October 2019

Abstract
The most common reason for deferral of blood donation is low hemoglobin concentration and anemia. Furthermore, repeated donations, especially in young females, may lead to iron deficiency, drop of hemoglobin, and consequent iron deficiency anemia. Recent data suggests that iron deficiency, in the absence of anemia, may be associated with symptoms in otherwise healthy individuals. Our previous findings suggested that additional transferrin ameliorates anemia in mouse models of β-thalassemia; this effect appears driven by an increase in the number of circulating red blood cell (RBC) by extending RBC survival (Li Nat Med 2010). We hypothesized that exogenous transferrin increases RBC count and enables a more rapid recovery after phlebotomy. To test this hypothesis, C57 mice were fed an iron replete (35ppm iron) and iron deficient (2.5ppm iron) diet for 3 months to simulate the human condition. Mice were phlebotomized an average of 220ul of blood weekly for four weeks. Additionally, all mice received intraperitoneal (IP) injection of either of 10mg of human apo-transferrin (apo-Tf) (200 ul volume) or 200ul PBS daily Monday – Friday for 4 weeks (in total 20 days of injection). Mice (n=4/group) were sacrificed and samples analyzed at the end of the 4 weeks. RBC survival was evaluated using an in vivo biotin avidin assay previously described (Li Nat Med 2010). We analyzed all RBC indices using ADVIA 120 (Bayer). MCV decreased in phlebotomized mice on 2.5ppm compared to 35ppm diet (p=0.012) and after apo-Tf injection (p<0.05 in 2.5ppm diet, and p<0.001 in 35ppm diet). Reticulocyte counts increased by 50% in apo-Tf treated compared to PBS treated phlebotomized mice on 2.5ppm diet (p=0.006). Apo-Tf injections also resulted in an increase in RBC counts in phlebotomized mice on 35ppm diet (p=0.056). Furthermore, using antibodies to CD44 and TER119 in flow cytometry (Chen PNAS 2010), we assessed the proportion of erythroid precursors at all stages of terminal erythroid differentiation. Apo-Tf injections result in more erythroid precursors in spleen and bone marrow (p<0.05) in phlebotomized mice on 2.5ppm or 35ppm diets without changes in the degree of apoptosis or quantity of reactive oxygen species. Mice on 2.5ppm diet had smaller spleens relative to 35ppm diet (0.002 vs. 0.004 spleen / body weight, p=0.05) that increased as a consequence of apo-Tf injection (0.003 vs. 0.002 spleen / body weight, p=0.02). No differences in spleen size or spleen / body weight is observed in apo-Tf treated mice on 35ppm diet. Immunohistochemistry of spleen samples using anti-TER119 antibodies revealed more extramedullary erythropoiesis in all apo-Tf treated mice. Lastly, no difference in RBC survival was observed in mice as a consequence of diet or injection. Taken together, our data suggest that exogenous apo-Tf in recurrently phlebotomized mice on iron deficient diet stimulates erythropoiesis without a direct effect on RBC survival. This data provides initial evidence that exogenous apo-Tf accelerates recovery from phlebotomy especially in iron replete conditions, without evidence of harm in iron deficiency. The molecular mechanisms underlying the effect of iron availability and iron delivery on different stages of erythroid differentiation requires further analysis.

Disclosures:
No relevant conflicts of interest to declare.