A new method for hemoglobin (Hb) deoxygenation and re-oxygenation, in suspension or within red blood cells, RBCs, is described using the commercial enzyme product, EC-Oxyrase®. This method using EC-Oxyrase has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin, eliminating the need of a sparging deoxygenation gas and airtight vessels, as well as easy re-oxygenation by washing and adding to a normal buffer with dissolved oxygen (DO). Spectra of deoxyHb and metHb from RBCs using three preparation methods: sodium dithionite, sodium nitrite and Oxyrase, show high purity of the deoxyHb using Oxyrase (with little to no methemoglobin or hemichrome production from side reactions). The deoxygenation action of Oxyrase follows first order reaction kinetics. Paramagnetic characteristics of intracellular hemoglobin in RBCs are compared using cell tracking velocimetry for healthy and sickle cell disease (SCD) donors and oxygen dissociation curves show that the function of healthy RBCs is unchanged after Oxyrase treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyhemoglobin, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to the existing methods.

The presence of iron in circulating monocytes is well known as they play an essential role in iron recycling. It has been demonstrated that the iron content of blood cells can be measured through their magnetic behavior; however, the magnetic properties of different monocyte subtypes remain unknown. In this study, we report for the first time, the magnetic behavior of classical, intermediate and non-classical monocytes, which is related to their iron storage capacity. The magnetic properties of monocytes were compared to other blood cells, such as lymphocytes and red blood cells in the oxyhemoglobin and methemoglobin states, and a cancer cell type. For this analysis, we used an instrument referred to as Cell Tracking Velocimetry (CTV), which quantitatively characterizes the magnetic behavior of biological entities. Our results demonstrate that significant fractions of the intermediate and non-classical monocytes have high magnetophoretic mobilities, equivalent to methemoglobin red blood cells and higher than the classical subset, suggesting their higher iron storage capacities. Moreover, our findings have implications for the immunomagnetic separation industry; we demonstrate that negative magnetic isolation techniques for recovering monocytes from blood should be used with caution, as it is possible to lose magnetic monocytes when using this technique.