Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes

Abstract

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.Key terms: Coulter electronic cell volume, opacity, erythrocytes, impedance, radio frequency, buoyant density, bovine serum albumin (BSA), density gradient centrifugation Leif and co-workers (9,131 proposed that automated cell analysis should be based on the assumption that there are many descriptors available that permit the easy identification of each type of cell present in a heterodisperse population. However, the use of a combination of two impedance measurements (13,5) together with multidimensional fluorescence and light scattering data on a cell-by-cell basis should allow adequate quantification of cellular parameters for specific identification of cells, For flow analysis to maintain a high level of accuracy, it will be necessary to include redundant information by increasing the dimensionality of data in order to prevent errors.The Coulter volume measurement, which was one of the first parameters to be used for the automated counting and analysis of biological cells (2,3), is an impedance measurement at zero frequency (dc) or a measure of resistance. Besides the abovementioned dc resistance (electronic cell volume) measurements, it is possible to perform simultaneously ac impedance measurements. An instrument for this purpose has been developed by Coulter and Hogg (4). The highest resolution in a twodimensional distribution is achieved by producing orthogonal parameters. In the present case, this was accomplished by dividing the radio frequency ac impedance by the dc resistance, which is defined as the opacity.Hoffman and Britt (6) developed a flow-system technique that simultaneously detected the dc Coulter volume and ac impedance. As pointed out by the authors, the use of the low ac frequency of 1 MHz resulted in "a high correlation between the ac and dc parameters for CHO cells." These authors performed their measurements at the reactance maximum in order to maximize the difference between cell types (Britt, personal communication) rather than at the higher frequency range.The ac impedance changes slowly with frequency above approximately 20 MHz, where, according to the bulk measurements of erythrocyte suspensions by Jenin et al.(71, the conductance of intact erythrocytes plateaus. According to these authors, the erythrocyte membrane appears to be practically short-circuited at frequencies above 50 MHz.The purpose of this study was to determine the relationship of the electrical parameters of Co...

Impedance spectroscopy flow cytometry: On‐chip label‐free cell differentiation

“…The relative opacities found in this system at 5.02 MHz are 0.78 for normal RBCs and 0.91 for fixed RBCs, which are comparable to the values of 0.70 and 0.95, respectively, measured at 4.5 MHz by Hoffmann et al (14). Opacity has been used to characterize erythrocyte aging (24), with measurements at 34 MHz producing an opacity of approximately 0.6 for normal RBCs. Measurements above 20 MHz in this system are likely affected by parasitic capacitances in parallel with the measurement sample; an optimized system will decrease this effect to increase sensitivity in the high frequency range.…”