Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide, especially in low-resource regions of the world, where a rapid and affordable test to properly diagnose the disease would be highly valued. Magnetophoresis is a technique that could simultaneously analyze, quantify, and potentially separate the patients sickle red blood cells (RBCs) from healthy RBCs, but the magnetic characteristics of sickle RBCs have yet to be reported. In this work, we present the single cell magnetic characterization of RBCs obtained from SCD patients. Sufficient single cells are analyzed from patient samples undergoing transfusion therapy and not yet having transfusion therapy (TP and NTP, respectively), such that means and distributions of these single RBC mobilities are created in the form of histograms which facilitated comparison to RBCs from healthy donors (HD). The magnetic characterization is obtained using a technique known as Cell Tracking Velocimetry (CTV) that quantitatively characterizes the RBC response to magnetic and gravitational fields. The magnetic properties of RBCs containing oxygenated, deoxygenated hemoglobin (Hb) and methemoglobin (oxyHb-RBCs, deoxyHb-RBCs, and metHb-RBCs) are further determined. The NTP samples report the highest magnetic character, especially when compared to oxyHb-RBCs from HD, which implies impaired oxygen binding capabilities. Also, the oxygen-Hb equilibrium curves are obtained to estimate the magnetic character of the cells under intermediate oxygen levels. Our results confirm higher magnetic moment of SCD blood (NTP) under intermediate oxygen levels. These data demonstrate the potential feasibility of magnetophoresis to identify, quantify and separate sickle RBCs from healthy RBCs.

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.