Surface area-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries

Background

The mature human red blood cell (RBC) exhibits remarkable deformability and durability. Throughout their lifespan, RBCs circulate the body a vast number of times without repair, squeezing through capillaries and splenic inter-endothelial slits as small as 1-3µm. Therefore, the ability to undergo large deformations is key for RBC function. RBC deformability is determined by factors such as plasma membrane viscoelasticity, cytoplasmic viscosity, and cellular geometry- such as the surface area to volume (SA:V) ratio.Altogether, the individual effects of cell geometry and cellular rigidity on capillary traversal are not well understood.

The surface area of a healthy RBC is kept constant, and any increases in volume decrease the SA:V ratio, which adversely impacts RBC rheology. Upon infection with a malaria parasite, key biomechanical properties of the RBC are subverted. However, it remains unclear to what extent the higher rigidity of infected RBCs contributes directly to trapping of infected RBCs in small capillaries.

In their work, Namvar et al (1) monitored RBC traversal in an array of microchannels, with diameters such as those found in the smallest vessels, and examined the effects of independently manipulating cellular rigidity and SA:V ratio.

 

 

Key findings and developments

The authors began by calibrating measurements of RBC geometry. For this, they used a modified version of the microfluidic device called a Human Erythrocyte Microchannel Analyser (HEMA) to assess the ability of RBCs to traverse into wedge-shaped microchannels with a 5 µm diameter entry, and a 1.4 µm diameter exit. They then determined the minimum cylindrical diameter (MCD), which is the position where the RBC becomes lodged. Conforming the RBCs into the microchannels allows estimation of the cell surface area and volume. This analysis showed an average volume of 99 fL, an average surface area of 149 µm², and a SA:V ratio of 1.50, with an average MCD of 3.03 µm.
The authors then examined the behaviour of RBCs under conditions in which the cellular rigidity was modified by chemical treatment with various concentrations of glutaraldehyde, but the SA:V ratio was held constant. They then used an ektacytometer to measure the elongation index of RBCs at shear stresses ranging from 0-20 Pa. At the physiologically relevant shear stress of 3 Pa, the ability of the RBCs treated with 0.004% glutaraldehyde to elongate was largely abrogated. No significant changes in SA:V ratio were observed between fixed and unfixed RBCs at glutaraldehyde concentrations of 0.001 and 0.004%, and the cells reach a similar MCD as unfixed RBCs. Altogether, these data show that cellular rigidity has little impact on RBC traversal into small capillaries.

As a next step, they examined the impact of altered cell volume on microchannel traversal and RBC elongation. Cell volume was altered by varying the osmolarity of the buffer solution. The authors found that at osmolarities close to the physiological range, the EI showed little dependence on osmolarity. Using the HEMA device, they showed that as cells over-hydrate at lower buffer osmolarities, there is a gradual increase in cell volume and they become lodged closer to the capillary entrance. Altogether, the work shows that moderate swelling of RBCs limits their traversal into microchannels but has less effect on their ability to undergo elongation.

The authors next explored the relevance of SA:V ratio changes in the context of Plasmodium infections. P. falciparum-infected RBCs(trophozoites) exhibited a rigidity profile similar to that of RBCs treated with 0.005% glutaraldehyde. In these cells, there was no significant difference in the mean SA:V ratio or MCD value compared to uninfected RBCs. Conversely, P. knowlesi-infected RBCs showed a decreased SA:V ratio and a higher MCD value compared to uninfected RBCs. Moreover, P. knowlesi-infected RBCs had reduced ability to elongate in flow, but appeared less rigid than P. falciparum-infected cells. These data confirm that SA:V ratio is a more important determinant than cell stiffness of the ability of RBCs to traverse small microchannels, with implications for the pathology of P. knowlesi infections.

Next, the authors compared the SA:V ratio of reticulocytes to mature RBCs, and their ability to traverse microchannels in the HEMA device. They found that two separate populations of reticulocytes (CD71+ and CD71-) exhibit the same SA:V ratio as mature RBCs and were found to reach the same MCD.

At any given point in the HEMA device, an RBC undergoes a complex distribution of stresses that cannot be measured experimentally. The authors developed a 3D computational simulation model of RBC traversal into the HEMA chip, to estimate the forces that RBCs would experience, and to probe the physical basis for the importance of SA:V ratio on RBC traversal through microchannels. This allowed them to find differences in pressure as RBCs traverse the microchannels, that best fit the experimental measurements. Using their 3D simulation model, the authors carried out two analyses of RBCs within the HEMA device, considering a) only membrane stiffness variation and b) only SA:V ratio variation. Simulations were performed on RBCs with different SA:V ratios. The authors concluded that passage of RBCs into the microchannels is highly dependent on the SA:V ratio.

The authors then performed simulations to predict whether high cell stiffness or low SA:V ratio would restrict the passage of a population of RBCs through the smallest capillaries encountered under physiological conditions, and found that the average MCD for healthy RBCs was between 2.73 and 3.29. Equally, they performed simulations to determine the minimum required pressure for cells with different rigidities and SA:V ratios to squeeze into small capillaries. They found that while shear modulus was not important, the minimum required pressure steeply increased as the SA:V ratio decreased. Altogether, the simulations showed that RBCs with a low SA:V ratio are more prone to trapping in small capillaries than cells with high cellular rigidity.

 

What I like about this preprint

I chose this preprint because I think while the focus in parasitology has been largely on the cell biology and molecular level, there is still a lot we don’t know from a biophysics point of view. I think this work addresses one important topic to better understand sequestration. I like the range of techniques approached, and I think this opens further questions for future exciting research.

 

References

1. Namvar A., et al, Surface-to-volume ratio, not cellular rigidity, determines red blood cell traversal through small capillaries, bioRxiv, 2020.

 

doi: https://doi.org/10.1242/prelights.23277

Read preprint (No Ratings Yet)