Allosteric Hemoglobin Effector Loaded Erythrocytes Improve the Efficiency of Transfusion to Prevent Sickling.

Sickle cell disease (SCD) is characterised by abnormal haemoglobin S (HbS). Under hypoxic conditions, HbS crystallizes, inducing sickling of red blood cells. Consequently, patients have a high risk of vaso-occlusive painful crisis. Red cell exchange transfusions remain an effective therapy in the acute and chronic treatment of SCD: the patient’s red blood cells (RBC) are removed and replaced by homologous normal red cells. Red cell exchange can provide needed oxygen carrying capacity while reducing the overall viscosity of blood (P.S. Swerdlow, 2006). We propose a novel preventive and therapeutic approach for SCD based on red blood cell transfusion. We hypothesise that loading RBC with an allosteric effector of hemoglobin can reduce RBC sickling. Indeed, the entrapment of Inositol Hexaphosphate (IHP) inside RBC reduces the oxygen-hemoglobin affinity, which is measured by a right shift of the oxygen dissociation curve. Thus, RBC-IHP have an increased capacity to deliver oxygen to tissues. It is also expected that the deoxygenation of SS RBC is reduced and sickling is avoided. IHP was entrapped into human RBC by hypotonic reversible lysis followed by a resealing step. RBC-IHP were characterised by the amount of IHP entrapped into RBCs and the P50 measurement. Unprocessed human RBC were used as control.

The potential anti-sickling effect of RBC-IHP was investigated using an in vitro model. Firstly, an experimental model to observe the relationship between sickling and oxygen concentration was set up : patients cells were submitted to deoxygenation by nitrogen bubbling for 30 min, and then re-oxygenated with different concentrations of oxygen (2, 5, 8, 15, 22%) for 30 min. The percentage of sickled cells was assessed by microscopy (about 500 cells checked). We observed that sickled cells recovered a normal shape upon reoxygenation (>15%O2), and a steady state between 5 and 8 % of oxygen, allowing the development of a reliable experimental model. Next, patient blood samples (n=6), harvested just prior to red cell exchange, were studied. RBC were washed 3 times with phoshate buffer before use. Different proportions of RBC-IHP (10%, 30% or 50%) were mixed with patients red cells and submitted to deoxygenation (0% O2) for 30 min and reoxygenation (5% O2) for 30 min. The final hematocrit of the suspensions was approximately 15%. The percentage of sickled cells in the suspensions was evaluated by microscopy and corrected according to the appropriate dilution factor. After full deoxygenation, 10% to 50% of cells were sickled, which appeared to be dependent on the HbS level in the blood samples. For all patients, RBC-IHP exhibited an enhanced anti-sickling effect: sickling was reduced by 19, 34, and 67% according to the RBC-IHP proportions 10%, 30% and 50%, respectively. Indeed, for equivalent RBC proportions RBC-IHP (50%) was 1.4 to 9 times more efficient compared to the unprocessed control RBC.

Thus, RBC-IHP has the capacity to prevent sickling in a dose-dependent manner and is efficient at low proportions (10%). Consequently, RBC-IHP can improve classical transfusion therapy in terms of transfused volume, frequency and preventive sickling effect.