Introduction: Red blood cell(RBC), which passes through capillaries and exchanges O2 and CO2 with surrounding tissues, plays a very important in the blood circulation in the human body. Imbalance in blood circulation could lead to various cardiovascular related diseases such as high blood pressure, arteriosclerosis, diabetes, and stroke.[1] This is closely related to RBC aggregation. Negatively charged RBC membrane arranges a compact layer of positive ions around the membrane by electrical attraction with cations in plasma. The rearranged ions layer in plasma forms zeta potential with compact layer. When the electrostatic interaction between RBCs, i.e., zeta potential increases, RBC aggregation is improved. It is known that zeta potential depends on electronegativity of the RBC membrane, the dielectric constant of the medium and ionic strength [2]. Since pulsed magnetic field (PMF) influences human physiology, many clinical studies demonstrated the effect of PMF for wound healing, reducing pain, increasing angiogenesis, as well as improving blood circulation.[3-5] In this study, we have tried to elucidate the effect of PMF on RBC aggregation by indirectly observing zeta potential using negatively-charged magnetic beads(MB). Our hypothesis is that the increase of negative charges on RBC membrane results in the increase of cations in the compact layer, thus the increase of the zeta potential. As a result, the number of MB attached to the RBC increases, as the binding force between MB and compact layer increases. Experimental Method: Blood needed for experiment was obtained from Korean Red Cross after reviewing IRB exemption in Sangji University Bioethics Committee. Whole blood of 6 ml was centrifuged at 3000 rpm for 5 min in 4 oC for separating RBCs and plasma. MB used this experiment has diameter 1 μm with OH- groups (SiMAG-Silanol, Chemicell). To clearly see the MB attached to RBCs and, in addition, to avoid blood clots caused by proteins in plasma, RBC suspension of hematocrit (Ht) of 10% was prepared using PBS(phosphate buffer saline). The amount of MB added to RBC suspension was 0.5 mg. RBCs were also oxidative stressed with tBHP(Tert-Butyl hydroperoxide) in 0.4 mM at 37 oCincubator for 30 min, in order to confirm the change of RBCs membrane charge due to oxidative stress under PMF stimulus. In oxidative stressed RBCs, hemoglobin(Fe2+) is altered to methemoglobin(Fe3+) because of loss an electron, thereby, electronegativity of RBC membrane will be changed.[6] Each sample was exposed to PMF for 3 min. Our PMF stimulator system consisted of single layered coil of 10 turns with an elliptical shape of 12.0 x 4.5 cm. The maximum intensity is 0.27 T at a transition time of 0.102 ms, with pulse intervals of 1 Hz. The numbers of RBC attached MB were compared with control group and PMF treatment group. Only RBCs with a distance of about 1 μm between the MB and the surface of RBC were counted using hemocytometer. Results and Discussion: Fig.1. shows the numbers of RBCs according to the number of attached MB in the control and PMF treatment group. The numbers of RBCs attached MB increased after PMF treatment. The more the number of MBs attached to RBCs, the thicker the compact layer surrounding RBC membranes. This phenomenon might be inferred due to an increase of zeta potential, thereby, increase of negative charges on the RBC membrane. Thus, it can be interpreted that MB having hydroxide OH- group are more attached to RBCs via binding force between MB and compact layer. Also the numbers of MB in the group treated tBHP and the group exposed to PMF after tBHP treatment (tBHP+PMF) were compared in Fig.2. Although the number of MB attached to RBCs was 1~2 in tBHP group, after PMF stimulus, i.e. in tBHP+PMF group it appears the number of MB attached to RBCs increased up to 3~4. This might be explained that PMF affects the increase of RBC membrane charge on not only normal RBCs but also oxidative stressed RBCs. Therefore, it can be deduced that high instantaneous flux change of PMF improves the aggregation of RBCs by increasing the zeta potential, due to the increase of the membrane charge.

**References:**

[1] H. L. Goldsmith, G. R. Cokelet, and P. Gaehtgens, Am. J. Physiol. 257, H1005 (1989) [2] H. P. Fernandes, C. L. Cesar and M. D. L. Barjas-Castro, Rev. Bras. Hematol. Hemoter. 33(4), 297(2011) [3] N. M. Shupak, J. C. McKay, W. R. Nielson, G. B. Rollman, F. S. Prato and A. W. Thomas, Pain Res Manag. 11(2), 85(2006) [4] L. X. Alvarez, J. McCue, N. K. Lam, G. Askin and P. R. Fox, J. Am. Anim. Hosp. Assoc. 55, 83(2019) [5] J. W. Mok, S. H. Han and H. S. Lee, AIP Advances 10, 015005(2020) [6] A. Knishita, Y. Nakayama, T. Kitayama and M. Tomita, The FEBS Journal 274(6), 1449(2007)

**Images:**

![](https://assets.underline.io/uploads/markdown_image/1/image/449981197245053b780d8c1e8a7f02ec.jpg)
The numbers of RBCs according to the number of attached MB in the control and PMF treatment group

![](https://assets.underline.io/uploads/markdown_image/1/image/49f2876eafa8f3d1fcb1c73de892a6f0.jpg)
The numbers of RBCs according to the number of attached MB in the tBHP treated group and tBHP+PMF treatment group

 Observation of Red Blood Cell Membrane Charge using Magnetic Beads under Pulsed Magnetic Field

*Session Co-Chair: Arantxa Fraile Rodríguez, Universitat de Barcelona, Universitat de Barcelona, Barcelona, Catalunya, ES, academic*

*Session Co-Chair: Jonathan Leliaert, Ghent University*

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Observation of Red Blood Cell Membrane Charge using Magnetic Beads under Pulsed Magnetic Field

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