Shape-Dependent Ordering of Hereditary Spherocytosis Inspired RBCs

Introduction:: Hereditary spherocytosis is a genetic disorder that causes red blood cells (RBCs) to become more rigid and spherical in shape. This apparent change in RBC morphology leads to lifelong health complications such as anemia, jaundice, gallstones, spleen damage, and potential loss of life without surgical intervention. Current clinical diagnostic methods can be prohibitively expensive and nevertheless, due to the mildness of most cases the disorder is not diagnosed. By leveraging the well-studied hexactic packing of spheres, it may be possible to identify structures caused by spherocytes using a minimal cost sedimentation method. This study seeks to quantify the granular structures for mixtures of discocytes (biconcave disc RBCs) and chemically altered spherocytes (spherical RBCs) in a sedimentation system.

Materials and Methods:: Blood draw protocols have been approved by the University of Michigan Internal Review Board (IRB-MED). RBCs were separated from whole blood samples using centrifugation with the plasma and white blood cells being discarded. Spherocytes were made from healthy RBC samples via exposure to sodium salicylate. Rigidification of RBCs was produced through exposure to tert-butyl hydroperoxide. Rigid spherocytes were made through exposure to both of the above. Alteration of RBC shape was confirmed with confocal imaging of fluorescently stained RBC membranes via Wheat Germ Agglutinin, Alexa Fluor™ 488 (Thermo Fisher). Fractions or pure samples of discocytes and spherocytes were then reconstituted in phosphate buffer solution at less than 1% hematocrit. The blood solutions were then pipetted onto the bottom side of a suspended glass slide and allowed to sediment for 10 minutes. The monolayer of RBCs formed at the air-water boundary where then imaged using light microscopy. Centroid locations of each cell were selected via ImageJ. Centroids were analyzed using OVITO software to quantify average hexactic ordering, radial distribution functions, and clustering of cells.

Results, Conclusions, and Discussions:: We describe the radial distribution functions of each cell type, hexactic order, clustering of cell mixtures using advanced analysis computational methods. We find that the presence of spherocytes can cause differences in the ordering and disordering of the sedimentation system. We investigate the role of rigidity and sphering in the differential hexactic ordering in unary systems, and find shape plays a more significant role (Figure 1). We find that spherocytes produce higher long-range order than discocytes. We also investigated binary fractions of discocytes and spherocytes. We find the onset of aggregation of each cell type occur above a fraction of 0.5. This aligns with a random placement simulation on a hexatic lattice, which indicates that sedimentation is likely random. Increases in hexatic order as a function of cell type fraction are more significantly driven by the aggregation of spherocytes. Likewise, global long-range ordering is also driven significantly after the onset of spherocyte aggregation.



Conclusions: This work details how cell shape and rigidity can impact the ordering of red blood cells (RBCs) that have sedimented at an interface without additional external forces. We complete the first ex vivo quantification of RBCs hexatic ordering. Using artificially sphered and rigidified RBCs, inspired by hereditary spherocytosis, we isolate the ordering effects of shape and rigidity. We find that shape plays a significant role in the enhanced local and long-range ordering of RBCs in unary and binary systems. This work highlights how an interdisciplinary lens of routine soft matter analysis and common biological phenomenon can symbiotically produce meaningful results. This work advances granular dynamics of RBCs and lays a foundation for future studies into bio-granular phenomenon for other shape changing RBC diseases. Future works include the inclusion of patient samples, automation, and bridging towards other types of cells.


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References (Optional): : Piegols et al. Manuscript Submitted. (2023)