Poster Y12 - Monitoring Eryptosis using Dielectrophoresis Characterization

Introduction: Apoptosis (programmed cell death) is a biological process that controls cell growth. Eryptosis is a form of programmed cell death akin to apoptosis. Eryptosis is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. Exposed phosphatidylserine is recognized by macrophages that engulf and degrade the affected cells. Phosphatidylserine (PS) is a phospholipid that is flipped from the inside to the outside of dying cells and signal macrophages to degrade the dying cell. Failure or an unbalance in the process can lead to severe consequences such a liver failure or anemia. Other pathological conditions that alter Eryptotic process include malaria, sickle-cell disease, and thalassemia. Though Eryptosis is a well-known process, and effects such a malarial-anemia due to Eryptosis have been established, mechanisms of these processes are still unknown. Current RBC analysis such as blood smears, flow cytometry, and deformability relies on manual observations and often-costly equipment. Moreover, these techniques depend on observable changes in cellular morphology or behaviour.

Materials and

Methods: This work demonstrates DEP’s ability to monitor the Eryptotic process. We followed a recently published step-by-step procedure using the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce Eryptosis. We treated RBCs with 1 μM and 10 μM ionomycin for 2 h and monitored RBC DEP properties for up to 4 days using the 3DEP system. We used microscopy to monitor RBC count and morphology and Annexin-V to monitor PS flipping. We did not observe any significant changes to bulk DEP properties for the 1 μM 2 h treatment. We observed a significant spike in membrane conductance and cytoplasmic conductivity in the 10 μM 2 h treatment group at the 24 h time point. We also saw a slightly significant (p=0.048) decrease in membrane capacitance. From 48-96 h these properties showed no significant trends though they all decreased.

Results, Conclusions, and Discussions: Annexin-V staining revealed that 1 μM 2 h treatment had less than 20% Eryptosis, which could account for our lack of detection via bulk DEP analysis (compared to >50% for the 10 μM treatment). Future work will investigate multi-populations to determine if this small percentage of Eryptosis and possibly hemolysis can be detected. The marked spike of parameters at the 24 h timepoint of the 10 μM treatment presents a promising hypothesis for future analysis. An increasing Ca2+ concentration which stimulates ion pathways such as the Gardos Channels permits the efflux of K+ and Cl- and also water due to osmosis. Therefore, it can be hypothesised that the increasing activity of such channels and shrinkage of the cells may be localized over even shorter periods between 0 and 24 h.The steady decrease in parameters from 24 to 96 h likely reflects the degradation of the cell and ion pathways. Taken together, this work demonstrates the potential of DEP to monitor Eryptosis, however further investigation is needed to examine the significance of the characterization.

Acknowledgements (Optional):