Direct Electrochemical Determination of Erythrocyte Concentration and Morphology
Authors: Goldin Mark M. , Evseev Anatoly K. , Borovkova Natalia V. , Batishchev Oleg V. , Goldin Michael M. 
Published: February 2020

Red blood cell (RBC) count in blood is an important clinical test, since erythrocytosis (high RBC count) and erythropenia (low RBC count) are features of severe diseases. For example, erythrocytosis occurs in certain tumors or in hypoxic conditions, while erythropenia accompanies blood loss, iron deficiency, or poisonings by hemolytic toxicants.
In current routine clinical laboratory practice, the RBC count is determined automatically using a hemocytometer or via microscopy. The latter allows for both the RBC count and identification of sizes and morphologies of erythrocytes, although the method is quite complex and time-consuming.
Electrochemical methods for determining erythrocytes concentration (RBC count) are known, although they are few and have not been utilized in routine clinical laboratories. The main advantages of the electrochemical methods are due to the simplicity of measurement procedure, high sensitivity, and high speed of analysis. However, existing electrochemical techniques were developed for use with animal erythrocytes. For example, a proposed electrochemical method [1] of determining red and white blood cell counts using a graphite electrode was developed for measurements in rabbit blood, and the effect of electrode potential on the behavior of blood cells was examined; however, the mechanism of the observed effects was unclear.
Recently, an attempt was made to develop a method to determine the amount of peroxidase activity of sheep red blood cells on the pyrolytic graphite electrode [2]. This is an indirect electrochemical method that used the electroreduction of oxygen formed in a reaction of hydrogen peroxide with a sample of sheep erythrocytes to characterize the RBC sample.
The present work explored the possibility of using direct electrooxidation of an RBC suspension on an optically transparent ITO electrode. Theoretically, the likelihood of the electrochemical activity of the blood cells is quite high, since the surface of RBC membranes contains electron-donor and electron-acceptor functional groups. Moreover, circumstantial evidence of erythrocyte electrochemical activity has been shown [3], manifesting as morphological changes of cells depending on electrode potential.
The goal of the present work was to develop an electrochemical method for determining the number of erythrocytes and their morphologies in real time.
A suspension of erythrocytes containing 8∙109 – 8∙1011 cells/L was prepared by diluting packed RBCs of apparently healthy donors with isotonic physiologic saline (0.15 M NaCl). A three-electrode electrochemical cell was used, and an optically transparent ITO electrode (Sigma-Aldrich, USA) with a surface area of 28.3 mm2formed the bottom of the cell. A carbon black electrode was used as the auxiliary electrode, with a silver/silver chloride electrode as the reference electrode. Polarization measurements were performed using an IPC Pro L potentiostat ("Kronas", Russia) in cyclic scan mode, with a scan rate of 10 mV/s. Erythrocyte morphology depending on the electrode potential was examined in real time using an Eclipse TS100 inverted microscope (Nikon, Japan), with 40X lens magnification.
Polarization measurements on the ITO electrode were carried out in isotonic saline containing a suspension of erythrocytes. It was observed (Fig. 1A) that the suspension of erythrocytes under these conditions is electrochemically active at anodic potentials between +400 mV and +1,000 mV (Ag/AgCl). It should be emphasized that this is the first time that direct evidence of RBC anodic oxidation has been obtained.
This phenomenon was used to determine the number (concentration) of erythrocytes in blood or other biological media, since erythrocyte electrooxidation produced a limiting current on the polarization curves. The dependence of limiting current on RBC concentration (Fig.1B) is linear across ca. two orders of magnitude of erythrocyte concentrations (8∙109 – 8∙1011 cells/L), with a correlation coefficient for the calibration curve in that range R
2= 0.99.
Previously it was reported [3] that simultaneous microscopy studies allow for the morphology of red blood cells in the process of electrode polarization to be evaluated. In the potential range utilized in the present work, the morphological transformation of RBCs with normal discocyte morphology into stomatocytes was observed between electrode potentials of ca. +600 to +1,000 mV.
Thus, the proposed electrochemical method with concurrent microscopy shows promise for the analytical determination of blood cell counts and their morphological analysis. This may have a wide range of applications, including determination of the effects of blood interaction with foreign materials or monitoring of stored blood quality.
1. Ci Y.-X., Li H.-N., Feng J. Electroanalysis 1998 10(13): 921-925.
2. Sepunaru L., Sokolov S.L., Holter J., Neil P. N.P., Compton R.G. Angew. Chem. Int. Ed. 2016 55(33): 9768-9771.
3. Khubutiya M.Sh., Evseev A.K., Mirzaeian M., Borovkova N.V., Goroncharovskaya I.V., Goldin M.M. 229th ECS Meeting, 2016, San Diego: Abs. 1632.

Figure 1