Erythrocyte morphological symmetry analysis to detect sublethal trauma in shear flow

Antony P Mcnamee,John F Fraser,Michael J Simmonds,Masaya Hakozaki,Jarod T Horobin,Masataka Inoue,Nobuo Watanabe

doi:10.1038/s41598-021-02936-2

Abstract

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. Their viscoelasticity is intrinsically linked to physical properties of the cytosol, cytoskeleton, and membrane—all of which are highly sensitive to supraphysiological shear exposure. Given the need to minimise blood trauma within artificial organs, we observed RBC in supraphysiological shear through direct visualisation to gain understanding of processes leading to blood damage. Using a custom-built counter-rotating shear generator fit to a microscope, healthy red blood cells (RBC) were directly visualised during exposure to different levels of shear (10–60 Pa). To investigate RBC morphology in shear flow, we developed an image analysis method to quantify (a)symmetry of deforming ellipsoidal cells—following RBC identification and centroid detection, cell radius was determined for each angle around the circumference of the cell, and the resultant bimodal distribution (and thus RBC) was symmetrically compared. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased. These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow.

Highlights

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces
While haemolysis in artificial organs has several determinants, exposure to supraphysiological shear stresses greater than tenfold the upper limits of normal circulation is considered the primary candidate to cause red blood cell (RBC) membrane rupture and d estruction[2,3,4,5,6]
While only normal ellipsoidal RBC are observed at the 300-s timepoint of exposure to 10, 30, and 40 Pa (Fig. 2A–C), the 60 Pa shear condition (Fig. 2D) induced RBC with ‘unstable’ abnormal morphology and increased the presence of cell fragments/extracellular vesicles

Summary

Introduction

The viscoelastic properties of red blood cells (RBC) facilitate flexible shape change in response to extrinsic forces. While traditional indices of RBC deformability (elongation index) remained unaltered in all shear conditions, following ~100 s of exposure to 60 Pa, the frequency of asymmetrical ellipses and RBC fragments/extracellular vesicles significantly increased These findings indicate RBC structure is sensitive to shear history, where asymmetrical morphology may indicate sublethal blood damage in real-time shear flow. Watanabe et al.[18] reported the use of a counter rotating shear system fit to a microscope for direct RBC observation Using this apparatus, shear induced cell fragmentation and haemolysis was identified in real-time at 40-s of accumulated exposure to 288 Pa. Given the ability of this system to detect haemolytic ‘end-points’ in continuous shear flow, it is plausible that this apparatus could have the sensitivity to investigate markers of blood cell deterioration prior to haemolysis. Valid indicator of accumulated sublethal trauma in shear flow, where decreased cell stability would present with asymmetrical ellipsoidal morphology

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Conclusion