Re-Assessing the Red Blood Cell Lifespan in Sickle Cell Anemia: Does Size Matter?
Authors: Mazer Norman Alan , Tomka Tomas , Winter Michael , Koerner Annette , Ballas Samir K. 
Published: October 2019

Abstract
Background: Does the size of RBCs in sickle cell anemia (SCA) influence their lifespan? According to Ballas and Marcolina's study of 26 SCA patients (Hemoglobin 2000), the half-life of 51Cr labelled RBCs (T1/2) had a statistically significant negative correlation with MCV (p = 0.009), that was described by the regression equation: T1/2 (days) = 9.3 - 0.22 x [MCV (fL) - 87]. We have used this equation to derive a quantitative relationship between the RBC lifespan (LSRBC) and RBC size (MCV) and have tested this relationship with the data of Higgs et al. (N Engl J Med 1982) in SCA patients with and without concomitant α-thalassemia. We also reassessed the RBC survival kinetics of non-F-cells in 12 SCA patients (Franco et al. Blood 2006) to explore a size effect. A mechanistic hypothesis for a causal relationship between RBC size and LSRBC is proposed.
Methods: The numerical relationship between LSRBC and T1/2 was derived using Engstedt's theoretical model (Acta Med Scand. Suppl. 1957) for the case of auto-transfusion in patients with hemolytic anemia. Combining this relationship with the experimental regression equation of T1/2 vs. MCV yielded a prediction of the dependence of LSRBC vs. MCV, which was accurately represented by a 2nd degree polynomial. This dependence was used to estimate the LSRBC values corresponding to the 3 groups of SCA patients studied by Higgs et al. (N Engl J Med 1982), whose mean MCV values were 90.1 fL (4 α-globin genes), 84.4 fL (3 α-globin genes) and 71.2 fL (2 α-globin genes). For comparison, we estimated LSRBC values for patients with 4 and 2 α-globin genes directly from the T1/2 values reported by De Ceulaer et al. (N Engl J Med 1983). An indirect estimate of LSRBC was derived from the ratio of the reticulocyte lifespan to the % reticulocytes reported in the 3 groups, with the former parameter set to 1.82 days in order to match the LSRBC prediction of the 4 α-globin gene group. A further estimate was derived from the levels of total bilirubin, Hb and MCHC using a theoretical expression that was also adjusted to match the 4 α-globin gene prediction. From the data on RBC survival kinetics in SCA patients reported by Franco et al. (Blood 2006), we estimated the T50% survival times of non-F-cells and explored their relationship to MCV.
Results: The dependence of LSRBC vs. MCV derived from Ballas and Marcolina's data (Hemoglobin 2000) shows a marked decrease from 25 days to 10 days as MCV increases from 73 to 101 fL (Figure 1). Using this relationship, a corresponding increase in the predicted LSRBC values is shown for the SCA patients of Higgs et al. (N Engl J Med 1982) with α-gene numbers corresponding to 4 (no α-thalassemia), 3 (heterozygous α-thalassemia) and 2 (homozygous α-thalassemia), respectively (Figure 2). The estimated LSRBC values derived from the data of De Ceulaer et al. (N Engl J Med 1983) in patients with 4 and 2 α-globin genes are in good agreement with our predictions (Figure 2). Indirect estimates of LSRBC, based on the % reticulocytes and total bilirubin levels reported by of Higgs et al. and adjusted to match the 4 α-globin gene group, are also in good agreement with the predicted LSRBC values for the 3 and 2 α-globin gene groups (Figure 2). Lastly the T50% survival times for non-F-cells derived from Franco et al. (Blood 2006) are seen to be inversely related to the MCV with a variation of about 3-fold (Figure 3).
Discussion: Our reassessment of literature data in SCA patients illustrates a strong inverse relationship between the RBC lifespan and RBC size (MCV). The confounding of this finding by the presence of hetero- and homozygous α-thalassemia in the lower MCV groups cannot be excluded, nor can the influence of the MCHC, which varies weakly with MCV. Nonetheless, we suggest that this relationship could result from the following causal mechanism. Smaller RBCs (initially in the oxygenated state) should have a shorter capillary transit time than larger RBCs, as seen in theoretical simulations (Secomb and Hsu, Biophysical journal. 1996) and experimental studies (Du et al. PNAS 2015). A shorter transit time should limit the time for deoxygenation and HbS polymerization; leading to less sickling, less cell damage and less hemolysis. The reduced rate of hemolysis should result in a longer RBC lifespan. Our quantitative analysis and causal hypothesis suggest that size matters in the lifespan of sickle cells. The clinical and therapeutic implications of this hypothesis require further consideration.

Disclosures
Mazer: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Tomka:Roche: Employment. Winter:Roche: Employment. Koerner:F. Hoffmann-La Roche Ltd: Employment.