Transferrin, the major plasma iron binding molecule, interacts with cell surface receptors to deliver iron, modulate hepcidin expression, and regulate erythropoiesis. Transferrin binds and releases iron via either or both of two homologous lobes (N and C). To test the hypothesis that the specificity of iron occupancy in N C lobe influences transferrin function, we generated mice with mutations to abrogate iron-binding in either lobe ( or ). Mice homozygous for either mutation have hepatocellular iron loading and decreased liver hepcidin expression (relative to iron concentration), although to different magnitudes. Both mouse models demonstrate some aspects of iron-restricted erythropoiesis, including increased zinc protoporphyrin levels, decreased hemoglobin levels, and microcytosis. The mice moreover demonstrate the anticipated effect of iron restriction on red cell production (i.e., no increase in RBC count despite elevated erythropoietin levels) along with a poor response to exogenous erythropoietin. In contrast, the mice have elevated RBC counts and an exaggerated response to exogenous erythropoietin sufficient to ameliorate the anemia. Observations in heterozygous mice further support a role for relative N C lobe iron occupancy in transferrin-mediated regulation of iron homeostasis and erythropoiesis.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Decreased erythrocyte deformability, as measured by ektacytometry, may be associated with disease severity in sickle cell anemia (SCA). Heterogeneous populations of rigid and deformable cells in SCA blood result in distortions of diffraction pattern measurements that correlate with the concentration of hemoglobin S (HbS) and the percentage of irreversibly sickled cells. We hypothesize that red cell heterogeneity, as well as deformability, will also be influenced by the concentration of alternative hemoglobins such as fetal hemoglobin (HbF) and the adult variant, HbA. To test this hypothesis, we investigate the relationship between diffraction pattern distortion, osmotic gradient ektacytometry parameters, and the hemoglobin composition of SCA blood. We observe a correlation between the extent of diffraction pattern distortions and percentage of HbF and HbA. Osmotic gradient ektacytometry data indicate that minimum elongation in the hypotonic region is positively correlated with HbF, as is the osmolality at which it occurs. The osmolality at both minimum and maximum elongation is inversely correlated with HbS and HbA. These data suggest that HbF may effectively improve surface-to-volume ratio and osmotic fragility in SCA erythrocytes. HbA may be relatively ineffective in improving these characteristics or cellular hydration at the levels found in this patient cohort.