Investigating the Impact of Refrigeration and Autologous Transfusions on Human Red Blood Cell Electrical Properties Using Dielectrophoresis and Inductively Coupled Mass Spectroscopy

Abstract

To manage large blood inventories efficiently for transfusions, medical institutions require cost-effective tools to characterize the viability of human red blood cells (RBCs) from the point of collection to transfusion. Blood is typically refrigerated for up to 42 days before transfusion, but the impact of refrigeration on transfusion safety and effectiveness remains unclear. This dissertation aims to improve understanding of storage-induced effects on cellular electrophysiology, with the intention of predicting storage-related consequences prior to transfusion. The primary goals are to develop a portable method to detect improper blood storage in clinical settings and to identify illicit blood transfusions in high-endurance sports.

The growing concern for safe transfusion practices in medical institutions, as well as its misuse in high-endurance sports, has led to academic interest in microelectromechanical systems (MEMS) to address these concerns. These miniature devices integrate mechanical and electrical components and can be utilized to investigate storage lesions, improve storage practices, and detect illicit autologous blood transfusions (ABTs) in sports. MEMS have numerous benefits for point-of-care diagnostics, such as portability, small sample volumes, and signal multiplexing. However, their ability to operate under clinically relevant samples and conditions is limited.

To address this, we leverage dielectrophoresis (DEP), an AC electrokinetic technique that involves the interaction of a polarizable interface with a non-uniform electric field, to develop a portable, affordable, high-accuracy and precision biosensing assay. We propose using a MEMS system known as the 3DEP dielectrophoresis cytometer, along with a unique experimental procedure involving a crosslinking reaction and a dielectrophoresis buffer with a high permittivity. In RBC DEP, a suspension of RBCs in a dielectrophoretic buffer is manipulated to assemble within a well-defined electric field gradient, that can be integrated with an electrokinetic polarizability model to quantify important electrical parameters of the RBCs. DEP is an averaged technique that provides bulk average electrical properties of RBCs in suspension. In this research, we measured cell cytoplasm conductivity and membrane conductance using a 3DEP cytometer to better understand how storage time affects cellular electrical properties. This assay can detect ABTs, monitor personal health, ensure cell purity, and improve storage conditions for blood components.

Project 1 focuses on developing a DEP-based assay to measure storage-induced changes to electrical properties of human red blood cells (RBCs). The project uses a crosslinking method with a high permittivity DEP buffer and the 3D DEP cytometer and compares the results with measurements taken using a 2D quadrupole electrode array. The objective is to determine the necessity of crosslinking to detect storage-induced changes and identify the optimal crosslinking concentration for the assay.

Project 2 aims to investigate the variability among donors using a high permittivity and low electrical conductivity dielectrophoresis buffer. RBCs from four human donors are analyzed using DEP, with additional quantification of diffused ions and hemoglobin during storage using ICP-ms and UV-spectroscopy, respectively. The objective of this project is to investigate the relationship between species-dependent diffusion across the RBC membrane during refrigerated storage and DEP-facilitated measurements of RBCs’ cytoplasm conductivity and membrane conductance. The investigation also considers the physiological mechanisms contributing to storage-induced changes and interpatient variability.

Project 3 provides further insight into the influence of refrigerated storage on RBCs' electrical properties and the ability to detect ABTs using DEP. Both in vitro and in vivo ABTs are investigated, and the effects of intense exercise on the DEP behavior of RBCs are considered.