Quantitative detection of eryptosis in human erythrocytes using tunable resistive pulse sensing and annexin-V-beads

Abstract

Toxicological assessments of human red blood cells (RBCs) are important in human health because RBCs are the most abundant cell type in our body. Erythrotoxicology testing guidelines using hemolysis have been established as a standard (e.g. by the ASTM International). However, many xenobiotics promote eryptosis (apoptosis in human RBCs) without causing hemolysis. Based on the major features of eryptosis, i.e. cell shrinkage and translocation of phosphatidylserine (PS) to the outer lipid bilayer of the plasma membrane, we report here a novel approach utilizing the quantitative tunable resistive pulse sensing (TRPS) technology, a widely adopted technique for characterizing nanoparticles in the field of nanotechnology, to measure the degree of eryptosis in a non-optical manner. With the TRPS system, we were able to determine PS externalization with microbeads functionalized with annexin-V for PS binding, cell swelling and shrinkage in physiological buffers (cell volume: 86 ± 12 fL) and solutions of different osmolarities with or without apoptotic trigger. After setting these standards, we then evaluated the toxicity of Polyphyllin D (PD), a potential anti-cancer drug that kills more liver cancer cells with multi-drug resistance, in erythrocytes to prove our concept. Data revealed that PD induced PS externalization and shrinkage in RBCs in a dose-dependent manner. Moreover, another feature of eryptosis, as small as 5 fL, was detected thus showing the PD-induced erythrotoxicity in human cells. Taken together, our results indicate that our approach using annexin-V-beads and TRPS is simple, safe and convenient, using only a small volume (35 μL) to evaluate the erythrotoxicity of xenobiotics.