Reversing the understanding of irreversibly sickled cells

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The incidence of sickle cell disease (SCD) has been increasing globally, with over seven million living with the disease globally (1). SCD is caused by a single point mutation (glu → val) at the sixth position of the β subunit of hemoglobin (Hb).(2) When the mutant Hb is deoxygenated, it polymerizes, which increases the rigidity and fragility of the red blood cells (RBCs) while also distorting their shape (sickling), which contributes to vaso-occlusion and other downstream events (3). Polymerization occurs when the concentration of Hb exceeds the solubility, c₀ > c_s (4). The solubility increases as the Hb oxygen saturation increases. Thus, higher oxygen pressure and lower intracellular Hb concentration are associated with less polymerization. The densities of sickle RBCs are much more heterogeneous than those of RBCs from healthy nonpatients and include abnormally dense cells with high Hb concentrations (5). These highly dense RBCs are thought to arise from dehydration secondary to disruption of cation homeostasis resulting from repeated cycles of polymerization and depolymerization (6). Among these very dense sickle cells, investigators have reported a subset that are called irreversibly sickle cells (ISCs) (7). Unlike other sickle cells that regain their normal bi-concave, discoid shape upon oxygenation (which melts the Hb polymers), these ISCs retain their distorted shape (7). Since they remain in the distorted form even under the highest oxygen pressure, these cells have been proposed to contribute significantly to the pathology in SCD (7). In this issue of Biophysical Journal, Reese et al. have employed a clever, novel microscopy-based technique to re-evaluate the nature of ISCs and, surprisingly, found that they are neither dense nor dehydrated (see Fig. 1) (8).

Figure 1.

Views on the nature of irreversibly sickled cells. The left-hand side represents commonly held views, where reversibly sickled cells can regain their normal morphology through oxygenation, but cycles of sickling and unsickling lead to cellular dehydration that can produce irreversibly sickled cells that are dense and dehydrated. The right-hand side depicts the situation implied by Reece et al., where dense distorted cells are those that contain polymers even at relatively high oxygen pressures, while distorted cells with no polymers (ISCs) are neither dense nor dehydrated, yet the mechanism of their formation is unknown.

Using microscopy, Reese et al. were able to visually identify distorted cells. Cells were placed between a coverslip and top plate, and the path length was measured using an interference technique. Absorption spectra were then collected in the Soret region, and the concentration was obtained from Beer’s law and Hb oxygen saturation obtained by fitting the measured spectra to standard ones. The volume of the cells was also calculated, from which dehydration was inferred. The measured oxygen saturation was also used to calculate the solubility (c_s) based on an empirical equation (4) so that, together with the measured Hb concentration (c₀), whether cells contained polymers or not could be deduced. A major finding was that the dense cells virtually all contained polymers, so they were not actual ISCs (cells that retain their distorted shape even in the absence of polymers). Actual ISCs (cells that were distorted yet did not have polymers) were found to have similar Hb concentrations to discoid cells, and the ISCs had larger volumes.

Previous work often separated cells using density gradient centrifugation or a similar technique and identified ISCs as those in the dense fraction that were distorted at ambient oxygen pressure. An implication of the work by Reese et al. is that most or all of those dense, distorted cells were not ISCs but rather distorted cells that actually contained polymer (that is, reversible sickle cells). That many RBCs are misidentified as ISCs was also suggested based on a comparison of the apparent abundance of ISCs prepared in room air to their abundance when they are saturated with carbon monoxide (CO) (9). The affinity of Hb for CO is about 200 times greater than that for oxygen, so it is much easier to achieve 100% saturated Hb using CO than oxygen. The measured percentage of ISCs under room air was 15%, while when CO was used, only 7% of the cells were still distorted (and would thus represent a true measure of ISCs) (9). Did the CO-saturated cells assigned as ISCs have polymer in them or were they similar to the ISCs identified by Reese et al. that are neither dense nor dehydrated? When identifying ISCs, it is important to distinguish between true ISCs and polymer-containing cells. This may be accomplished using CO, as there would likely be no polymer even in the densest cells (especially given the fraction of fetal Hb that can be present) (4). To further insure that no polymer is present, the temperature could be lowered, as solubility increases as the temperature is lowered from 37°C to 0°C (4). These considerations suggest further investigations using CO (and possibly lower temperatures) to confirm that ISCs are actually not dense or dehydrated using the novel methods developed by Reese et al. as well as more traditional methods (including density gradient centrifugation) and possibly combinations of the two sets of methods. Another interesting question raised by the study by Reese et al. concerns the nature of the nondense cells identified as ISCs. How do these cells become ISCs, what are their mechanical properties, and what role do they play in pathology of the disease? Are they poorly deformable like cells traditionally identified as ISCs (10)?

Hydroxyurea was US Food and Drug Administration approved to treat SCD in 1998, while more recently, five more therapies have been approved: voxelotor, L-glutamine, crizanlizumab-tmca, and two gene therapies (Casgevy and Lyfgenia). These approvals are a testament to the value of biomedical research, and biophysical studies (many by the investigators cited in the article) have made important contributions to understanding the mechanism of the disease that underlies current therapies. However, SCD is still responsible for substantial morbidity and mortality. Despite advances in knowledge, more work is needed, and the paper by Reese et al. is a good example.

Author contributions

D.B.K.-S. wrote this commentary and did the artwork.

Declaration of interests

The author declares no competing interests.

Editor: Guy Genin.