Sickle cell disease (SCD) is a monogenic disorder that affects up to 100,000 people in the United States and one in every 365 black or African American children are born with the sickle cell trait. This results in many complications, including painful vaso-occlusive crises (VOC), acute chest syndrome, organ failure and hemolytic anemia. In the case of HbS (sickled hemoglobin), valine replaces glutamic acid on the βS globin chain, which triggers cell polymerization at low dissolved oxygen (DO) to turn a flexible, bioconcave red blood cell (RBC) into the rigid, sickled conformation. For HbC (crystalized hemoglobin), the lysine substitution in the β6 chain results in crystallization of the hemoglobin (Hb). Patients with homozygous HbSS (and heterozygous HbSC traits, to a lesser degree) have more severe symptoms, more frequent crises and require more frequent transfusions than HbAS patients who possess the sickle cell trait and normal Hb A (hemoglobin A). Random and unpredictable VOC events occur when the elongated HbS RBCs block microvessels and cause pain and inflammation. Currently, there is neither point-of-care (POC) assay to detect nor a treatment stop a VOC. Because crises are treated with intravenous fluids and often opioids, emergency room providers must rely on self-reporting. The ongoing opioid and racial problems in the United States can confound treatment decisions because no other solutions exist.
When heme iron (Fe) is in the oxygenated state, it forms a covalent bond with oxygen and exhibits diamagnetism, where the induced magnetic field opposes the applied magnetic field and creates a weak repulsive force. In contrast, deoxygenated heme Fe has unpaired electrons that exhibit paramagnetism, where the induced magnetic field is aligned with the applied field, and creates an attractive force. This property makes possible magnetophoresis of RBCs through a suspending buffer to estimate the intracellular Hb through a unique characterization technique, Cell Tracking Velocimetry (CTV). A camera is focused on a small window of dilute, suspended RBCs within a carefully designed magnetic induction field that creates a constant, unidirectional magnetic force on paramagnetic entities while they also move downward due to buoyancy. The forces under Stokes drag are well known and particle tracking software records many particles simultaneously between frames and therefore CTV is able to produce a large set of population data rather than a single average magnetic mobility. Recent findings from our lab suggest that both the mean and confidence intervals intracellular Hb between healthy donors can vary from person to person. The CTV device also has the capability to rapidly filter incident light to a fluorescently stained antibody and the emission light that the camera detects. Therefore, RBCs that express reactive oxygen species, phosphatidylserine, or another relevant marker can be selectively tracked. It is noteworthy that the CTV does not quantify fluorescence like a flow cytometer but records trajectories of cells that emit fluorescence beyond a chosen threshold.
SCD, and its multiple genotypes, can alter many biochemical parameters (e.g., total Hb, % of Hb F, A, C, or S, ferritin) and physical properties (e.g., size, density, aspect ratio, Young’s modulus, intracellular Hb, oxygen on/offloading, or surface marker) of a patient’s RBCs differently. Particularly, the abnormality that causes or indicates a VOC may only be apparent in a small subset of all RBCs. During the sickling process, deoxygenated HbS causes a conformational change in a β subunit that allows polymerization of an adjacent deoxygenated β subunit, forming long fibers that dehydrate the cell, increasing its density. A Percoll density gradient can be used to quickly separate a blood sample into several fractions with homogenous densities. Of particular interest are the properties of the densest fraction that contains dehydrated, sickled HbS that cause painful crisis episodes. Additionally, the magnetic mobility (which can determine if Hb is oxygenated or deoxygenated) at intermediate DO reveal which RBCs can onload oxygen and become diamagnetic, and which ones are irreversibly sickled in the deoxygenated state or contain Hb oxidized into paramagnetic methemoglobin due to aging. Although there are many parameters to consider, clinical data from SCD patients during a VOC and routine transfusion can connect these parameters to quantitative test for a crisis.
Patients often have exchange transfusions where they receive a donor’s blood while an apheresis device removes packed RBC and re-transfuses plasma. There may be acute or long-term reactions with transfusions (alloimmunization) and an innovative POC device that builds on existing apheresis may remove a target subpopulation of native RBCs during or before a crisis. That target must first be defined but current magnetic deposition and continuous split-flow systems can separate diamagnetic and paramagnetic populations from a mixture.
There is a great need for a rapid, quantitative and objective test to diagnose VOC and eliminate bias from a treatment decision involving opioids. SCD is prevalent globally and the demand of blood donations is outpacing supply. Analyzing several fractions cells as a population under various oxygenation conditions can better define what happens during a crisis, leading to acute and preventative treatments for VOC have great potential to reduce risks to the recipient and reduce demand for blood donations.
