(664d) Leveraging the Ordered Packing of Spherocytes As a Potential Diagnostic

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, and spleen damage. Current clinical diagnostic methods can be prohibitively expensive and nevertheless, due to the mildness of most cases the disorder is not diagnosed. By leveraging hexactic packing of spheres, it may be possible to identify this disorder 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. 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. Furthermore, we investigate the role of rigidity in the differential hexactic ordering of pure discocytes and spherocytes, and find significantly higher hexactic ordering of spherocytes compared to discocytes.

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 and exposed to tert-butyl hydroperoxide for rigidification as needed. Alteration of RBC shape was confirmed with confocal imaging of fluorescently stained RBC membranes via Wheat Germ Agglutinin, Alexa Fluorâ„¢ 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.

Result and Conclusions: This work highlights how an increasing fraction of spherocytes can cause fundamental changes in how both spherocytes and discocytes hexatically order in sedimentation (Figure 1A). A significant jump in hexatic order from low to high following a bulk fraction of spherocytes of 0.6 may be indicative of a phase transition. Having spherocytes does not always mean increased hexatic ordering but instead regions of varying order are produced through the introduction of spherocytes. Investigations into the role of rigidity on hexatic order for pure samples, highlight shape playing a significant role in hexatic order while rigidity being not significant (Figure 1B). Thus, shape likely plays a larger role in the hexactic ordering of the sedimented RBCs. Radial distribution functions show spherocytes having longer range order than that of discocytes. Undoubtedly, spherocytes produce differential granular crystallization from that of just discocytes. Further investigations into the structures spherocytes can produce via sedimentation may provide key insights into developing a cheap and effective method for identifying hereditary spherocytosis.