Cyclical Changes in Erythrocyte Characteristics during Chronic Transfusion Therapy in Pediatric Sickle Cell Patients

Chronic Transfusion Therapy (CTT) is increasingly important in the treatment of sickle cell disease (SCD), and is now the standard of care for primary and secondary stroke prevention in pediatric patients. However, little is known about its effects on sickle RBC properties, including endogenous HbF synthesis and sickle red blood cell (RBC) lifespan. A better understanding of the unique transfusion biology of SCD would improve our ability to design optimal clinical protocols, especially those that address the critical question of how to transition patients from CTT to other therapies (e.g., hydroxyurea) while minimizing risk. We studied cellular changes during one transfusion cycle in five pediatric patients currently on stable CTT for various clinical indications. Flow cytometry was used to identify TfR+ reticulocytes, HbA+ RBC to quantitate the number of donor RBC, and F+ cells to identify endogenous and donor RBC that contain HbF. Three color flow cytometric assays of these cell types, performed immediately before and after transfusion, and then weekly until the next transfusion, enabled us to evaluate cellular information during a complete transfusion cycle, including:

1. endogenous TfR+ reticulocytes;

2. endogenous HbS+F+ cells;

3. donor and endogenous mature RBC; and

4. endogenous TfR+F+ and TfR−F+ cells.

These data were used to evaluate transfusion-dependent changes in the amount of erythropoietic stress, the effect of transfusion therapy on HbF synthesis, and the survival of donor RBC. As expected, all variables returned to approximately the same values at the beginning of each transfusion cycle. In all five patients, the %TfR+ cells in the HbS+ subpopulation was high prior to transfusion, decreased during the first week of the cycle due to suppression of erythropoiesis, and then returned to a high value in subsequent weeks. The % TfR+ cells in the HbS+ subpopulation was remarkably variable among the five subjects, with pretransfusion values ranging from 7.4 to 41.1%. The presence of such high percentages of immature RBC in the endogenous population for some patients was an unexpected finding during CTT. A possible cause for this variability is a shorter survival of endogenous RBC in some patients during CTT, which would increase the percent TfR+ cells, but have less effect on the absolute number of TfR+ cells. Variability in sickle cell survival would have a direct impact on the required period between transfusions, and may influence the type of transfusion program that would be selected for the patient. The percentage of F+ cells within the HbS+TfR+ population is a real time measure of HbF production. In four of five patients there was a modest decrease in HbS+TfR+F+ cells during the first one or two weeks after transfusion, followed by an increase toward the original pre-transfusion value, consistent with decreased production of HbF due to decreased erythropoietic stress just after the transfusion. The number of HbA+ donor RBC was calculated from the % HbA+ cells and the red cell count, and normalized to the immediate post-transfusion point. The slope of the resultant curve (%/day) is a measure of the survival of the donor RBC. In six normal subjects we observed a range of 0.95–1.42 %/day during the first month after reinfusion of biotin-labeled autologous RBC. For 4/5 transfused sickle cell patients the removal rate of HbA+ cells ranged from 1.00–1.28 %/day. In one patient with splenomegaly, this slope was 2.38 %/day, suggesting an increased rate of removal of transfused RBC in this subject. The techniques described here allow a detailed evaluation of cellular changes that take place during CTT. Patients maintain a surprisingly high percentage of TfR+ cells in the endogenous population and suppress HbF as expected. The survival of donor RBC appears to be normal in the absence of splenomegaly.