Absolute Reticulocyte Count Acts as a Surrogate for Fetal Hemoglobin in Infants and Children with Sickle Cell Anemia

Abstract

Hemoglobin switching is largely complete in humans by six months of age. Among infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). The objective of this study was to determine if absolute reticulocyte count (ARC) is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study. Hematologic data, including ARC and HbF levels, were measured at steady state. F-cells were enumerated by flow cytometry. Initial studies compared infants with ARC greater than or equal to 200 K/μL (ARC ≥ 200) based upon the previously reported utility of this threshold as a predictive marker for SCA severity. Mean HbF and F-cell levels were significantly lower in the ARC ≥ 200 group when compared to the ARC < 200 group. Both HbF and F-cell percentages were negatively correlated to ARC in infants and in children between the ages of 1 and 9 years. However, the inverse relationship was lost after the age of 10 years. Overall, decreased expression and distribution of HbF during childhood SCA is well-correlated with increased reticulocyte production and release into the peripheral blood. As such, these data further support the clinical use of reticulocyte enumeration as a disease severity biomarker for childhood sickle cell anemia.

Fig 1. Relationship of ARC or Hemoglobin with HbF According to Age Group.

A-C: Correlation of HbF and ARC in patients with SCA who are less than 1 year of age, 1–9 years of age and 10–20 years old, respectively. D-F: Correlation of HbF and hemoglobin in the same age groups, respectively. HbF% is on the x-axis in each panel. Correlation coefficients (r) and p- values are shown in each panel. ARC, Absolute Reticulocyte Count; Hb, hemoglobin. Each dot represents a steady state value for a separate patient.

Fig 2. Relationship of ARC and F-cells by Age Group.

A. Correlation of F-cell % and ARC in infants with SCA (less than 1 year of age) at steady state. B. Same correlation with children with SCA who are between 1 and 9 years of age and C. between 10 and 20 years of age. ARC, Absolute Reticulocyte Count. Each dot represents a steady state value for a separate patient.

Fig 3. Representative Flow Dot Plots for Pancellular vs. Heterocellular HbF Distribution.

A. Erythrocytes from a 3 month old infant with SCA stained with fluorescent antibodies against HbF, revealing a pancellular HbF distribution. B. Representative F-cell staining from an older child with SCA, representative of heterocellular HbF distribution.

Fig 4. Comparison of F-cell and HbF levels in Infants and Children with ARC<200 K/μL and ARC ≥ 200K/μL.

A. F-cell level comparisons in infants less than 1 year of age and children between ages 1–9 years with an ARC < 200 K/μL (open bars) and ARC ≥ 200 K/μL (shaded bars) (*p = 6.2 E-5, **p = 1.7E-6). B. Comparison of HbF levels in infants and children ages 1–9 years and ARC < 200 K/μL (open bars) and ARC ≥ 200 K/μL (shaded bars) (*p = 2.2E-5,**p = 7.4E-7). Standard deviation bars are included.