New Analysis of Red Blood Cells (RBC) using the ZetaPALS

By Bruce B. Weiner Ph.D.

image of NanoBrook 90Plus PALS

Introduction: Zeta potential is a useful property to know for colloids, nanoparticles, and proteins. It is a substitute for the otherwise hard to measure surface charge. The zeta potential is not equal to the surface potential but rather it is the electrostatic potential difference between a rotationally averaged point at the shear, i.e., the slip plane, and an average point out in the liquid. Particles with the same sign zeta potential repel each other and thus maintain dispersion stability. Particles with the opposite sign zeta potential attract each other and thus produce aggregation. And particles with zero or nearly zero zeta potential also aggregate because there is no force strong enough to counteract the always present attractive forces (ignoring any steric stabilizsation that may be present).

Two Separate Effects: Zeta potential is determined by two separate effects: charge at the shear plane and free ion concentration (e.g., dissolved salts). In order to monitor the change in zeta potential, one normally adds just enough dissolved salt to keep its effect constant. For many situations, 1 to 10 mM KNO3 is sufficient. However, with many biological samples, it is necessary to work at much higher salt concentration, say physiological saline at 155 mM NaCl (0.9% wt/vol).

Problems at High Salt: At such a high salt concentration two main problems arise with respect to the actual measurement. The high conductivity raises the likelihood of Joule heating due to high current. Then, too, such a high ionic strength collapses the electrical double layer, reducing the zeta potential, making it harder to measure. Using PALS¹, phase analysis light scattering, such as the Brookhaven Instruments ZetaPALS², one has the sensitivity to make measurements under these high salt conditions.

image of red blood cells

Validation at High Salt, RBC’s: The question then becomes how to validate such measurements at high salt concentration. Red blood cells are a convenient answer since we call carry around a ready supply of this reference material and its electrophoretic mobility (from which zeta potential is calculated) has been known for almost 40 years³. The average is -1.1 mob units (µ • cm/V • s, or 10-8 m²/V • s). For pH > 4.75, there is apparently no pH dependence.

While there is no description of whether this was whole blood (as measurements made in Brookhaven’s labs over the years) or plasma, or the blood type, as you will see below, the results are close enough to allow favorable comparisons.

It is noted that -1.1 mob units is close to that reproted for sheep red cells some 52 years ago4. Except for the larger WBC’s present only in low concentrations except in the case of inflammation, RBC’s are the largest particles in whole blood. They would dominate light scattering and be easily discerned by their characteristic shape under a microscope. An average 7 µm in diameter with a donut-hole shape, they easily stand out.

PALS Measurement Nine Years Ago: The first measurement in our lab using the ZetaPALS were made in May 2001 by Alonzo Baker, our lab tech using 1 d whole blood in twice-filtered (0.2 µm), 0.9% wt/vol NaCl. The blood was donated, somewhat reluctantly, by Dr. Walther Tscharnuter, the designer of the ZetaPALS. The blood type was not recorded.

Results: -1.11 ± 0.07 mob units, in excellent agreement with literature values obtained not with phase analysis laser light scattering but with microscopic techniques.

PALS Measurements Four Years Ago: In September of 2006, Brookhaven’s Dr. Jeffery Bodycomb used 1 d of his Type O+ whole blood in filtered, 2x PBS at pH 7.2. The measured conductance was twice that of physiological saline or 1x PBS. The combined measurements yield – 1.09 ± 0.04 mob units using the ZetaPALS. Perhaps steric hindrance ameliorates the effect of salt ions at such concentrations.

PALS Measurements Three Months Ago: In March of 2010, Mr. William Bernt of Particle Characterization Labs (particleanalysis.com) used 1 d of his Type O- whole blood in filtered, 0.9% wt/vol NaCl. The combined measurements yield – 1.09 ± 0.06 mob units using a ZetaPALS.

Summary: There may or may not be a small differences in average zeta potential between whole blood and isolated RBC’s; however, the data reviewed here suggests such differences, if they exist, are smaller than the repeatability in the population and the random measurement error under these difficult conditions (high salt). Nor is there much difference between blood types. With the exception of taking care not to include white blood cells (WBC’s) from someone who is sick, a drop of whole blood in 154 nM NaCl acts like a reasonable reference material for measurements at high salt.

It is worth emphasizing these are measurements under salt concentration that other machines fail to make without frying the sample or electrode assembly. Some thin electrode peel away from the cell after just a few measurements. Not so with the ZetaPALS. And such measurements have been routine for almost a decade using the ZetaPALS.

Footnotes:

1. J.F. Miller, K. Schatzel, and B.J. Vincent, J. Colloid Interface Sci., 143, 532-554, (1991).
2. W.W. Tscharnuter, Applied Optics, 40, 3995-4003, (2001).
3. A. Zeria and D.J. Wilkins, Experientia, 28, 1435, (1972) as reviewed in “Electrical Phenomena at Interfaces: Fundamentals, Measurements, and Applications”, editors A. Kitahara and A. Watanabe, Marcel Dekker publisher, 398-399 (1994).
4. Seaman, et. al., Biochemistry Journal, 69, 12 (1958)

Applications: BiopharmaDiagnosticZeta
Instruments: NanoBrook Series