Stressed Erythropoiesis In Children with Sickle Cell Disease Is An Indicator of Low Fetal Hemoglobin Production and Increased Disease Severity

Abstract 2666

Acute erythroid stress causes elevated levels of fetal hemoglobin (HbF) in primates. However, relationships between the chronic erythroid stress in children with Sickle Cell Disease (HbSS) and HbF production are less well understood. For this study, peripheral blood from children with HbSS was analyzed within 72 hours of collection and storage at 4°C. The level of erythroid stress was determined by absolute reticulocyte counts (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (HiCD71, LoCD71 and NegCD71) and CD36. Immature reticulocytes were identified by HiCD71 expression and included the CD36+ population. Sorted populations from at least three subjects were utilized to assess fetal globin expression during reticulocyte maturation according to quantitative polymerase chain reaction (qPCR), and high pressure liquid chromatography (HPLC). While the number of gamma-globin mRNA copies/cell decreased with reticulocyte maturation, the ratio of gamma/gamma+beta mRNA remained stable. HbF protein levels in the HiCD71 and LoCD71 populations were equivalent for each patient with average values of 4.3±4.2% HiCD71 vs. 4.4±4.0% LoCD71, p=0.96. By comparison, HbF levels in the NegCD71 were increased in all subjects (14.2±9.5% NegCD71, p<0.05) consistent with prolonged survival of the HbF expressing cells. Unexpectedly, the percentage of immature reticulocytes neither correlated with the total ARC nor total HbF expression in the peripheral blood. Further analysis of 82 HbSS patients 4–21 years of age was performed to determine if ARC, by itself, might provide a simple clinical indicator of HbF expression or disease severity in this population. Thirteen patients had been treated with HU, 17 had received regular RBC transfusions (Tx), and the remainder (n=52) received neither HU nor monthly Tx within 4 months of analysis (Ctrl). A negative correlation between ARC (K/uL) and HbF levels was present in both the HU and Ctrl groups (HU r=-0.76, p=0.0016; Ctrl r=-0.34, p=0.013), but the correlation was lost in the Tx group (r=-0.21, p=0.43). Clinical events were defined as acute chest syndrome, splenic sequestration, conditional and abnormal transcranial Doppler (TCD),silent cerebral infarct, overt stroke, laparascopic cholecystectomy and bacteremia. There was no significant correlation between ARC and clinical events in the HU and Tx groups. Within the Ctrl group, those with an ARC below 300 (21 of 52, 40.4%) had a statistically lower event rate (ARC<300 mean event rate=0.23±0.18 events/yr vs. ARC>300 mean event rate=0.59±0.60 events/year, p=0.0028). These data suggest that increased levels of erythroid stress as measured by reticulocyte production and maturation in pediatric HbSS subjects is not associated with elevated gamma-globin mRNA or HbF levels. Instead, higher levels of reticulocytosis were associated with lower HbF levels and increased disease severity.