Using a magnetic column separator to determine the magnetophoretic behavior and adhesion characteristics of oxygenated sickle and healthy red blood cells

Abstract Introduction:Sickle cell disease (SCD) patients experiencing severe crises often require red blood cell (RBC) exchange transfusions to reduce sickle RBC levels. However, this approach is limited by alloimmunization risk and donor blood shortages, highlighting the need for strategies that improve transfusion efficiency and reduce the need for antigen-matched units. One promising method is magnetic separation of sickle RBCs from waste apheresis products based on their distinct magnetophoretic behavior linked to oxygen-binding affinity. This study investigates the use of a commercial magnetically-activated cell separation (MACS) column to characterize magnetic behavior and quantify feed, eluted, and captured RBC fractions from both non transfused SCD and healthy donor samples to evaluate the feasibility of this separation method. Methods: Whole blood from healthy and sickle cell donors was collected under IRB-approved protocols at The Ohio State University. RBCs were isolated via three washes with phosphate buffered saline (PBS) by centrifugation at 1300 × g for 5 minutes, and then resuspended in PBS. A suspension of air-saturated RBCs (200 × 10⁶ cells/mL) was processed through a MACS column (Miltenyi Biotech LS column) placed within a Halbach magnet. Eluted cells were collected by washing the column with PBS. The column was then removed from the magnet and washed with PBS to collect the captured cells. Control runs were performed without the magnet. Cell concentrations in the feed, eluted, and captured fractions were measured using a Beckman Multisizer 4e Coulter Counter. Cell Tracking Velocimetry (CTV) was used to determine magnetophoretic mobilities (MM) of individual cells in each fraction under a defined magnetic field gradient[1]. Concentration and MM differences between healthy and SCD samples were analyzed to assess separation performance with the student's t-test. Results and Discussion:Previous findings using CTV demonstrated that RBCs from SCD donors exhibit significantly elevated MM in the 99th percentile compared to healthy donor RBCs[2]. Building on this observation, we used a commercial MACS column to separate these magnetic RBCs from both healthy and SCD donor samples. Student's t-test analysis revealed that the number ratio of captured to feed cells was significantly higher for SCD donor samples processed with a magnet, compared to healthy donor samples (with or without a magnet) and SCD samples processed without a magnet (p < 0.05). The proportions of eluted cells and unaccounted cells relative to the initial feed did not show significant differences, regardless of donor types or magnetic conditions. Interestingly, the MM of the captured fraction from healthy donors (mean MM: 1.3564×10-6mm³/TA·s with magnet, 1.1941×10-7 mm³/TA·s without magnet) was significantly greater than that of SCD donor RBCs under both magnetic and non-magnetic conditions (p < 0.05). This was likely due to the presence of a small subpopulation of methemoglobin-containing RBCs in healthy samples. In contrast, the MM values for SCD donor cells—with the magnet (mean MM: –2 × 10⁻⁶ mm³/TA·s) and without the magnet (mean MM: –1.88 × 10⁻⁶ mm³/TA·s)—were not significantly different and were consistently negative, indicating that the cells exhibit diamagnetic behavior. These findings suggest that under air-saturated conditions, sickle RBCs may adhere to the magnetic beads via non-magnetic mechanisms such as surface interactions. This could also indicate the presence of ghost RBCs or RBCs that have lost their hemoglobin and therefore their magnetic iron content. Conclusions: While SCD samples exhibited significantly higher capture ratios, magnetophoretic mobility data suggest that cell adhesion, rather than magnetic capture alone, is the driving force under air-saturated conditions. While further studies with a larger patient population are needed to validate these results, the development of a column separator optimized for adhesion-based capture shows promise. Additionally, future use of ImageStream analysis may help confirm the capture of ghost cells from SCD samples, providing further insight into the underlying separation mechanism. [1]Weigand et al., IEEE Transactions on Biomedical Engineering,2022,69(12),3582-3590 [2]Chalmers et al., Blood,2023,142(1),3883