Methods in Studying Red Blood Cells in Sickle Cell Disease

Sickle cell disease (SCD) is a genetically inherited blood disorder characterized by a single point mutation that produces hemoglobin S within red blood cells (RBCs). When such RBCs are deoxygenated hemoglobin S polymerizes and causes the cells to take on a sickle shape and become more viscous and stiffer than normal RBCs. The hallmark of SCD is painful vaso-occlusive crises in which the flow of blood to parts of the body is obstructed. Today, millions of people suffer from SCD around the world and life expectancy of individuals with SCD is in the 40’s. We developed several techniques to facilitate and expedite experimental investigation of SCD and in general blood biomechanics. Vaso-occlusion is the result of interactions between blood cells and the endothelium. Atomic force microscopy (AFM) has been employed in single-molecule force spectroscopy (SMFM) and single-cell force spectroscopy (SCFS) assays to study these interactions. In order to process results from these assays quickly, we developed MATLAB-based custom software that expedited manual processing efforts from 1 to 2 hours to 10 to 15 minutes per experiment. We explored microfluidic principles in order to further increase the throughput of the number of cells we can study. We have designed an experimental setup that implements aspiration based assembly for microchannels which enables the use of most functionalization techniques, and a pressure controller that allows instant and precise changes in the microchannel flow. Utilizing this setup, we have quantified SS-RBC adhesion to the integrin αvβ3, a specific adhesion protein expressed on the endothelium which has been implicated in vaso-occlusion, and measured the shear modulus and viscosity of the SS-RBC membrane. We have designed a portable, swift, easy-to-use, small sample, and sterilization-free rheometer capable of measuring the viscosity of Non-Newtonian liquids. This rheometer can quickly measure the viscosity of body fluids such as whole blood and blood plasma, which has been hypothesized to be of value in the assessment of SCD. We explored expediting the development and review of simulations and three-dimensional data. To this end, we have developed a virtual reality environment for interactive multiphysics simulation and data visualization utilizing modern head-mounted displays.