New Insights into F-Cells, F-Reticulocytes and the Role of Fetal Hemoglobin in Protecting Sickle Red Cells from Hemolysis upon Mechanical Stress

Sickle cell disease (SCD) is an autosomal hereditary recessive disorder caused by a single point mutation in the first exon of the β-globin gene generating abnormal hemoglobin (HbS). Hypoxia drives HbS polymerization and fiber formation that are responsible for red blood cell (RBC) sickling. In SCD patients expression of fetal hemoglobin (HbF) is higher than in healthy individuals and varies considerably. Although clinical studies have shown that HbF levels were inversely correlated with disease severity, studies addressing the relationship between the expression level of HbF at the cellular level, i.e. in F-cells, and the impact on RBC morphology and survival are scarce.

In this study, we investigated the distribution and the expression level of HbF at the cellular level in a large cohort of SCD patients (SS or Sβ°) using high throughput imaging techniques, functional sickling assays and a novel microfluidic device.

We determined the percentage of HbF-expressing cells (F⁺RBCs) in 46 patient blood samples and found that it was highly variable (mean: 44% +/- 24%) and correlated with the percentage of HbF measured by HPLC (R² = 0.86). To estimate the content of HbF in F⁺RBCs at the single cell level we developed a method based on imaging flow cytometry and defined three different populations according to the expression level of HbF: low, medium and high F⁺RBCs (Figure 1). We determined the percentage of each subpopulation in 30 SCD blood samples and found that the low F⁺RBCs were the most prevalent, with a median of 17.85% of total circulating RBCs. The high F⁺RBCs were the least abundant, with a median of 0.63%, while medium F⁺RBCs accounted for 4.03% of RBCs.

Next, we investigated the relationship between F⁺RBCs and irreversibly sickled cells (ISCs) using imaging flow cytometry. The %ISCs was significantly higher in the low than in the medium subpopulation and no ISCs were found in the high F⁺RBC subpopulation. Applying Percoll density fractionation we found that F⁺RBCs were predominant in the low dense (LD) fraction and poorly present in the high dense (HD) fraction; this was associated with low and high %ISCs in the LD and HD fractions, respectively.

To investigate the potential role of HbF in the survival of circulating sickle RBCs we determined the percentage of F-cells within the reticulocyte and mature RBC populations using flow cytometry. Analyzing 46 blood samples, we found that the percentage of F⁺ reticulocytes (%F⁺Retics) was significantly lower than the percentage of F⁺ mature RBCs (%F⁺RBCs) within the same blood sample of all patients, indicating selective advantage of the F⁺Retics during maturation. We hypothesized that HbF might exert a protective effect by preserving the deformability of RBCs, protecting them from mechanical destruction. To investigate our hypothesis, we designed a microfluidic biochip to evaluate the resistance of F⁺RBCs undergoing repetitive mechanical stress, flowing through 5 x 5 μm channels (Figure 2). We measured the %F⁺RBCs in cell suspensions before (input) and after (output) flowing in the chip. We observed a significant increase of %F⁺RBC population in the output suspensions, indicating that F⁺RBCs withstand mechanical stress better than non-F-cells. Moreover, we found that high but not low F⁺RBCs were protected against lysis, indicating that a minimal threshold is needed for HbF to exert its protective role. In line with this finding, we measured the HbF/HbS ratio in the plasma (free hemoglobin) and in RBCs of SCD patients and found that it was much lower in the plasma, providing direct evidence that intravascular hemolysis targets RBCs with no or low HbF.

Finally, there was no correlation between the percentage of reticulocytes and the %HbF or the %F⁺RBCs. However, a negative correlation was found between the percentage of reticulocytes and the %F⁺Retics, indicating that this latter is a more relevant marker of hemolysis and medullar regeneration than %HbF or %F⁺RBCs.

Our study gives new insights into the role of the mechanical dimension as a critical contributing factor to anemia in SCD. It reveals a cellular mechanism through which HbF exerts its protective role in vivo, showing evidence that high HbF levels protect RBCs from increased density and from hemolysis upon mechanical stress. Our findings pave the way to future clinical studies in which the relationship between HbF expression at the early reticulocyte stage and clinical manifestations could be finely addressed.

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