Early Pathogenesis of Sickle Cell Anemia: Absolute Reticulocyte Counts Are Correlated with Increased Detection of CD36+ Reticulocytes during the First Two Years of Postnatal Life
Authors: Meier Emily Riehm , Byrnes Colleen , Martin Brenda , Tumburu Laxminath , Lee Y. Terry , Luban Naomi L.C. , Miller Jeffery L. 
Published: October 2019

Abstract
Reticulocytosis begins early in life in infants with sickle cell anemia (HbSS, SCA) as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Almost one quarter of children with SCA will have at least one episode of splenic sequestration before the age of 2 years, and immature (CD36+) reticulocytes released into circulating blood may contribute to this early pathology because of splenic trapping of those cells. Additionally, clinical studies have identified that absolute reticulocyte counts (ARC) greater than 200 K/µL during early infancy (60-196 days of age) may be a useful marker to identify SCA infants who are at the highest risk for SCA-associated events during childhood. The objective of this study was to determine if expression of the adhesion marker named CD36 changes with ARC levels during early childhood in pediatric SCA patients.
Infants between the ages of 6 and 12 months were enrolled in a prospective, longitudinal, observational study after written consent was obtained from a parent or legal guardian. Participants were not receiving hydroxyurea or monthly blood transfusions. After consent was provided, peripheral blood was obtained during study visits at steady state and analyzed within 48 hours of collection and storage at 40 C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated with flow cytometry following intracellular staining with a HbF fluorescent antibody. Reticulocytes were identified by flow cytometry after thiazole orange staining and were further quantitated with CD36, CD45, CD71 and CD235a staining. CD36+ reticulocytes were defined here by surface CD36 detected at levels greater than two standard deviations above unstained controls. Calculations were made to determine the proportion of total circulating reticulocytes that expressed surface CD36+ as well as the absolute number of circulating CD36+ reticulocytes/microliter of whole blood. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t-tests were used to compare means.
Mean age at enrollment was 278±70 days and 62.5% of the participants were male. None of the eight enrolled patients had SCA complications or hospitalizations prior to enrollment. Mean HbF and F-cell levels were 40.8±11.1% and 92.8±8.8%, respectively. Mean hemoglobin was 10±0.6 g/dL and ARC 189.1±69.4 K/µL and mean number of absolute CD36+ red blood cells was 29.3±19.1 K/µL. ARC increased over time in 6 of the 8 (75%) participants for the study observation period.
ARC was positively correlated with the proportion of CD36+ reticulocytes (r=0.78, p<0.0001) as well as the absolute number of circulating CD36+ reticulocytes in peripheral blood (r=0.76, p<0.001). Similar correlations in HbF and F-cells were not found (HbF vs. CD36+ reticulocytes, r=-0.23, p=0.22; HbF vs. absolute CD36+ reticulocytes, r=-0.23, p=0.23; F-cells vs. CD36+ reticulocytes, r=-0.22, p=0.24; F-cells vs. absolute CD36+ reticulocytes, r=0.23, p=0.20). As expected, increased ARC was positively correlated with age (r=0.5854, p=0.0013) as was the absolute number of CD36+ reticulocytes (r=0.5736, p=0.0017). HbF, F-cell, and hemoglobin levels were all negatively correlated with age, though the relationship was not as robust (HbF vs. age, r=-0.47, p=0.01; F-cells=-0.39, p=0.05; hemoglobin -0.31, p=0.12).
The incidence of splenic sequestration is highest in the first two years of life. Results from this cohort of SCA infants reveal that both ARC levels and CD36+ reticulocytes increase during the first 2 years of life, and the increases in ARC are positively correlated with the proportion and absolute number of circulating CD36+ reticulocytes in peripheral blood. This relationship provides a potential mechanism for the previously reported relationship of early reticulocytosis with increased risk of future SCA complications and events. As such, longitudinal studies should be pursued to determine if increasing CD36+ reticulocytes during early childhood serves as a marker or contributes to early SCA-related pathologies including splenic sequestration.

Disclosures
No relevant conflicts of interest to declare.