Increased Reticulocytosis in Infants with Sickle Cell Disease May Be a Marker for Future Disease Severity

Abstract ²¹²⁸

The first erythropoietic stress in term neonates occurs at the time of the erythrocyte nadir (around three months of age) and is associated with increased reticulocytosis. In infants with sickle cell disease (HbSS, SCD), this nadir generally precedes the onset of clinical signs and symptoms. The objective of this study was to determine if reticulocyte levels and properties during early infancy in SCD patients are useful in disease severity prediction. Peripheral blood from 111 children with SCD who were enrolled in this observational study was analyzed within 48 hours of collection and storage at 4°C. Reticulocytes were quantified by absolute reticulocyte count (ARC) as well as reticulocyte flow cytometry phenotyping and sorting using CD71 (CD71 Hi, CD71 Lo and CD71 Neg) and CD36 markers. Among the entire group, ARC is negatively correlated with HbF (r=−0.72, p<0.0001) and F-cells (r=−0.68, p<0.0001). Analysis of lysates from sorted reticulocytes and red blood cells revealed that the least mature reticulocytes (CD71 Hi) had the lowest HbF levels. CD71 Hi sickle reticulocytes also expressed the adhesion molecule CD36 on the cell surface. CD36 was barely detectable in reticulocytes from healthy infants and older children. The proportion of sickle red cells that expressed CD71 at Hi and Lo levels was compared to age-matched healthy controls. Although red blood cells with similar levels of CD71 Lo (mean healthy infant controls 0.71±0.36% vs. 0.92±0.30% in SCD infants, p=0.43) were present in both healthy infants and infants with SCD less than five months of age, CD71 Hi expression was significantly higher in infants with SCD (mean healthy infant controls 0.19±0.08% vs. 0.58±0.25% in SCD infants, p=0.02). Examination of the reticulocyte profiles of SCD patients older than 5 months of age revealed markedly increased CD71 Lo and Hi levels when compared to similarly aged healthy controls (mean CD71 Lo healthy controls 0.31±0.30% vs SCD patients 4.06±3.45%, p=0.0001; mean CD71 Hi healthy controls 0.01±0.01% vs. SCD patients 1.55±1.42%, p=0.0001). Clinically, increased reticulocytosis during this nadir period (2–5 months of age) was associated with a worse clinical course during infancy and childhood. The ARC at physiologic nadir was identified by retrospective chart review for 37 of the 111 enrolled subjects. Significantly higher ARCs were associated with hospitalization during the first three years of life. Of the twelve children who had not been hospitalized by 3 years of age, only one had an ARC greater than 200 K/uL during the nadir period (mean ARC for the no hospitalization group 151±54 K/uL). Among subjects who were first hospitalized before age 3 years for acute chest syndrome (ACS, n=6), there was no significant increase in ARC (mean ARC 174±95, p=0.53 vs. no hospitalization group). However, patients who were first hospitalized for splenic sequestration (n=8) and vaso-occlusive crisis (VOC, n=11) had significantly higher ARCs [splenic sequestration 223±66 K/uL (p=0.017), VOC 262±102 K/uL, (p=0.004) vs. the no hospitalization group]. Twelve of 37 (32%) patients received chronic therapy with either monthly transfusions or hydroxyurea before the age of 12 years (ARC range 123–384 K/uL, mean 250±82 K/uL). Nine of the twelve (75%) had a steady state ARC of greater than 200 K/uL between ages 2 and 5 months. These data support the hypothesis that completion of the fetal-to-adult hemoglobin switching phenomenon during infancy triggers the beginning of a pathologic cascade of increased sickle hemoglobin polymerization and hemolysis followed by increased release of immature reticulocytes that express adhesion molecules including CD36. Among SCD infants with lower expression of fetal hemoglobin, the magnitude of the reticulocyte response is predicted to be further driven by uncompensated tissue hypoxia. Overall, the data suggest that increased reticulocytosis is one of the earliest clinical features of SCD, and the level of reticulocytosis during the first five months of postnatal life may help predict disease severity later in life.