Introduction
Sickle cell disease is a group of inherited blood disorders caused by a single point mutation in the hemoglobin gene. This gene mutation produces hemoglobin S (HbS), an abnormal hemoglobin variant that alters the shape and flexibility of red blood cells (RBCs). In 2021, over 500,000 newborns globally were diagnosed with sickle cell disease, the majority born in sub-Saharan Africa, and over 8 million people were living with this condition globally.
Under normal conditions, RBCs are biconcave (disk-shaped) and flexible, allowing them to navigate narrow capillaries smoothly. In sickle cell disease, however, low-oxygen conditions trigger HbS to polymerize, which increases RBC rigidity and causes cells to adopt a crescent or “sickle” shape. Although some cells can recover their normal shape upon reoxygenation, other sickled cells, referred to as “irreversibly sickled cells” (ISCs), remain permanently distorted.
For decades now, researchers have attributed the pathophysiology of sickle cell disease to ISCs. This longstanding hypothesis holds that ISCs are denser, highly dehydrated, and rigid, such that they are more likely to cause vaso-occlusion and thus contribute to several acute health complications in sickle cell patients. However, ISCs remain poorly characterized, largely because of limitations in traditional methods used to study them. Until now, techniques used to study ISCs either have been technically unsuitable or have relied on inferring, rather than directly measuring, the biophysical properties of ISCs. (see Mohandas, N., et al. and Kaul D.K., et al.)
In their study, “Novel single-cell measurements suggest irreversibly sickled cells are neither dense nor dehydrated,” Reese et al. reexamine the biophysical properties of ISCs by using a novel, single-cell optical method. This approach enables them to measure ISC properties, including intracellular hemoglobin concentration, oxygen saturation, and cell volume, at a single-cell level. The authors also examine oxygenated sickle cells with normal biconcave shapes and those distorted by internal sickle fibers, allowing for a comprehensive assessment of the sickle cell population.
Key components of the optical approach
The optical technique used by Reese et al. involves placing a small (1 µL) drop of blood between two glass coverslips to compress cells into a uniform and measurable thickness of ~2 µm. Using a commercial device to measure the interference of reflected light beams across a range of wavelengths as it passes through the sample, the authors confirmed thickness uniformity, enabling precise measurements of oxygenation levels and intracellular concentration in each cell.
A microspectrophotometer that captures light absorbance between 400 and 450 nm allowed the authors to scan single RBCs and quantify oxygenated and deoxygenated hemoglobin concentrations. By combining cell area measurements from images with the known thickness, the authors also estimated cell volume, from which dehydration was inferred, and categorized cells as either discocytes (normal, disk-shaped cells) or distorted (sickled) cells. Their approach thus provides precise data on hemoglobin concentration and oxygenation levels at the single-cell level.
New insights: ISC cell density, oxygen saturation, and shape
In their study, Reese and colleagues challenge the prevalent notion that ISCs are rigid and permanently deformed. Using their novel optical approach, they measure single-cell shape, density, and oxygenation levels in single ISCs collected from three different sickle patients.
When categorizing cells as either discocytes (regular-shaped cells) or “distorted” sickled cells, Reese and colleagues found that sickled cells were not always highly concentrated or more dehydrated than normal discocytes, as previously assumed. Instead, many distorted cells exhibited varying degrees of oxygenation, with some exhibiting oxygen saturation levels higher than expected.
To delve further, Reese and colleagues conducted a follow-up experiment on blood samples from two additional patients in which, two and three days after blood collection, cells were exposed to pure oxygen. Interestingly, many of the previously sickled (distorted) cells in these samples restored a concentration distribution and shape like those of normal discocytes, with oxygen saturation levels reaching 96–98%. Considering these findings, Reese and colleagues propose that there are three, rather than two, categories of sickled cells: (1) distorted cells containing HbS polymers and a hemoglobin concentration that exceed solubility at their O2 saturation, (2) discocytes (non-distorted cells) devoid of HbS polymers, and (3) ISCs, which are distorted cells with oxygen saturation below solubility but without polymers. In other words, not every ISC is truly “irreversible.” Even distorted cells can regain near-normal oxygenation and shape under certain conditions.
Irreversible sickled cells may thus play a lesser role in obstructing blood flow than previously thought. Because not all distorted cells are equal, exhibiting varying densities and rigidities, some may move more easily through capillaries without contributing equally to the pathophysiology of sickle cell disease. This evidence reframes our understanding of ISCs, suggesting that the “polymer-laden cells”—those that are deformed, have higher hemoglobin concentrations, and are more rigid due to polymerized HbS—are those most problematic for blood flow. If there were higher hemoglobin concentrations within the blood, ISCs would be more likely to cluster together and impede microcirculation, but this is not what Reese and colleagues observed.
Conclusions and potential implications
This study by Reese and colleagues provides valuable insights, especially regarding the significance (and consequent necessity) for biophysical approaches that enable single-cell analysis. By achieving accurate and quantifiable measurements at the single-cell level, their approach challenges long-held assumptions about this disease and offers a fresh perspective on its cellular dynamics. These findings have significant implications for both diagnosis and treatment. For instance, measuring ISCs not based solely on shape or density but rather on polymer presence could help provide a better picture of individual patient states, and novel treatments could target ISC flexibility.