Development and Modeling of Multi-scale Continuous High Gradient Magnetophoretic Separator for Malaria-infected Red Blood Cells

According to the World Health Organization, nearly 3.2 billion people are at risk of malaria. The most deadliest form of human malaria is caused by the pathogen Plasmodium falciparum, which has claimed over 400,000 lives worldwide in 20151. Even when optimally treated with drug and donor blood therapies, severe malaria has a high mortality rate. The parasites target a patient’s red blood cells and convert them into paramagnetic units before eventually rupturing the host cell, further spreading the infection. Combination drug therapies using quinine and artemisinin derivatives are common but are either expensive or have associated toxicities from mis-dosing. Moreover, antimalarial drugs are becoming increasingly ineffective against the growing number of drug-resistant malaria strains. Combination drug and blood exchange therapies are often implemented to flush out malaria-infected red blood cells (iRBC) but consume a great quantity of donor blood, carry a high risk of transmitting other blood-borne diseases, and have no agreed upon advantage or disadvantage among clinicians. Due to the relative disadvantages of other treatment methods, small scale high gradient magnetic separation (HGMS) devices, used in a variety of biological applications, may be another treatment option to consider. mPharesis (“magnetic apheresis”) is a proposed low-cost, disposable magnetic blood filtration device which continually removes iRBCs from a patient’s whole blood by capitalizing on the iRBC’s unique magnetic properties. The proposed treatment-scale system will provide emergency care with parameters similar to continuous hemofiltration systems in terms of blood flow rates (up to approximately 500 mL min-1), vascular access, and treatment times (up to about 3 hours). A novel medium-scale high gradient magnetic separation device is detailed here. The device consists of a disposable photo-etched embedded wire array and acrylic layered housing on an external permanent magnet set. The magnetic force and flow field design were computationally optimized. In-vitro feasibility experiments were conducted at several flow rates and physiological hematocrits (Hct) using a blood mixture composed of healthy RBCs and a non-pathogenic paramagnetic blood analog called methemoglobin RBCs (metRBCs). The device was able to selectively remove paramagnetic RBCs without excessive loss of healthy RBCs. Simplified experiments were performed with 30% Hct with 20% metRBCs. At steady state, the concentration of metRBCs was reduced by 27.0±2.2% in a single pass at a flow rate of 77 μL min-1 as compared to 1.6±0.7% in control experiments without a magnet present. The experimental paramagnetic RBC removal rate was over 380 times greater than similar published HGMS devices. These successful results were applied to a theoretical transport model. The model was designed to compare the parasite removal and Hct level changes between combination drug and exchange transfusion (ET) therapy versus treatment-scale mPharesis-drug therapy. When the mPharesis flow rate was set to typical continuous dialysis rates, treatment times and donor blood volumes were reduced for all 10 cases. Calculated treatment times were all less than 60% of the reported ET-drug treatments, with times ranging from 47 to 71 minutes. The mPharesis-drug treatment was calculated to need between 4% and 53% less donor blood than the reported ET-drug treatments. Between 775 and 1772 mL of packed donor RBCs (3 to 6 units of whole blood) were estimated for the mPharesis-drug treatments, versus the average 5 to 20 units used during ET2. Treatment reference charts were generated to provide time and donor blood volume estimates for a range of patient sizes and disease severities. Based on the maximum flow rate of 500 mL min-1, a treatment-scale mPharesis system was estimated to be the size of three stacked briefcases, which is a feasible size for deployment in a clinical setting. Finally, the design, fabrication, and microscopic visualization of a simple, benchtop-fabricated continuous HGMS device was detailed. This proof-of-concept microfluidic device was implemented to test the effect of hematocrit and flow rate on the separation of mixtures of metRBCs (heat-treated and un-heated) and transparent ghost RBCs. An automated image processing protocol provided feasible cell concentration profiles for each flow and rheological condition with a 6.5 to 9.7% lower sum than manual counting for three samples. For the no magnet conditions, the average near-magnet concentration of paramagnetic RBCs at the outlet (within 10% of 130 μm channel height adjacent to the wire array) was between 1.3 and 2.4 times greater than the average of the rest of the flow field (degree of separation, DOS). The most effective separation was found to occur at the lowest flow rate 0.4 μL min-1 and with the 0.5% Hct metRBC sample with DOS=26. The addition of 30% ghost RBCs reduced the efficiency for all flow rates, with DOS=7.4 for best flow rate of 0.4 μL min-1. Heat treatment did not significantly affect separation with DOS=7.3, likely due to the low impact of the relatively low concentration of metRBCs (0.5%). The mesoscale fabrication and design process, clearance model, cell counting algorithm, and HGMS fabrication protocol and microscopy study described in this thesis provides a useful framework for future HGMS optimization and the further development of a clinical treatment system for severe malaria patients with often limited treatment options.