Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow

Abstract

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Femtogram Resolution of Iron Content on a Per Cell Basis: Ex Vivo Storage of Human Red Blood Cells Leads to Loss of Hemoglobin

“…We have demonstrated, as a proof of concept, the separation of deoxyHb and metHb RBCs from a cell mixture, and theoretical designs indicate that larger scale RBC separation based on intrinsic magnetization is possible. 46 …”

Fundamentals and application of magnetic particles in cell isolation and enrichment: a review

“…In addition to breast cancer cells, QMS has been used for isolation of cancer cells from patients with head and neck cancer (Yang et al, 2009, Balasubramanian et al, 2012) and to separate islet cells for diabetes research (Shenkman et al, 2009). The same research group has illustrated that red blood cell removal, without magnetic tags, is possible using QMS (Moore et al, 2013, Moore et al, 2014, Jin et al, 2012, Zborowski et al, 2003). Using such a negative selection approach, hematopoietic stem cells have been enriched from blood (Jing et al, 2007, Tong et al, 2007, Schneider et al, 2010, Jin et al, 2012).…”

The potential for liquid biopsies in the precision medical treatment of breast cancer

“…Many of these selection technologies are criticized for their reliance on cellsurface expression of EpCAM to capture (and define) CTCs because some tumors down-regulate expression of this marker during EMT. In addition, preanalytic variables, such as time to assay are significant factors in CTC detection and may be more important than EpCAM high status, with the exception of claudin low tumors 23 . To address this limitation, physical property and functional based technologies have been developed that isolate CTCs on the basis of physicochemical properties-such as size, density, surface charges, unique functional characteristics-that distinguish them from other blood cells 24 .…”

“…Our group has focused on pre-analytic variables pertaining to the amplification of picogram quantities of RNA as well as time to CTC assay influencing the number of cells recovered 23,94 .…”

“…They can be captured via several techiniques such as microfluidic devices, including a specifically designed cluster chip which operates at subphysiological flow rates, preventing these highly deformable cell groupings from squeezing through small pores under higher flow pressures 103 , direct precipitation of all blood cells onto specially prepared slides, followed by high-speed microscopic scanning 104 , as well as enrichment based techniques 105,106 . 23 . Furthermore, detection techniques not dependent on epithelial markers may be used for detection of these cells, see Table 1.…”