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Basic phenomena of red blood cell rouleaux

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Article in Biorheology · February 1999

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Biorheology 36 (1999) 439–442 439
IOS Press

Basic phenomena of red blood cell rouleaux

formation
H. Bäumler a,∗ , B. Neu a , E. Donath b and H. Kiesewetter a
a Institute
of Transfusion Medicine, Medical Faculty Charité, Humboldt University of Berlin, D-10098
Berlin, Germany
b Max-Planck-Institute of Colloids and Interfaces, Berlin, Germany

Interactions between cellular components of blood such as aggregation, agglutination or adhesion

of cells are observed in a variety of normal and pathological conditions in human or other mammals.
Rouleaux formation of red blood cells (RBCs) in static blood, platelet adhesion and aggregation in both
thrombosis and hemostatic plug formation or leukocyte adhesion to the endothelium are examples of such
interactions. In the case of RBC, the agglutination of cells is caused, for example, by specific binding
of immunoglobulins to the surfaces of adjacent cells [24] whilst the aggregation of RBCs or rouleaux
formation is caused by fibrinogen in blood plasma [18]. But this rouleaux formation can also be seen, if
the RBCs are resuspended in electrolyte solutions containing neutral macromolecules like dextran [11].
The aggregation of RBCs is completely reversible and the disaggregation of these rouleaux is readily
achieved by shearing the suspension. Whilst the fibrinogen mediated RBC aggregation increases steadily
with increasing fibrinogen concentration, the dextran mediated RBC aggregation reaches a maximum at
dextran concentrations of about 4 g/dl. With dextran having a mean molecular weight Mw of 70 kDa,
no RBC aggregation can be found at concentrations higher than 8 g/dl [13]. Interestingly, the rouleaux
formation of RBCs occurs with human or horse RBCs in plasma or dextran electrolyte solutions but no
aggregation of bovine or sheep RBCs could be found. The strength of aggregation depends not only on
the fibrinogen or dextran concentration, and the molecular weight of dextran, but also on the species [2,
22,23]. In the past, two different models were developed to explain the aggregation of RBCs in polymer
solutions – the cross-bridging model and the depletion model.
The bridging model being favored over thirty years was proposed by Merrill et al. [27] for plasma
protein induced RBC aggregation and by Chien and Jan [14] and Brooks [9] for the neutral dextran
macromolecule-induced RBC aggregation (Fig. 1(a)). Non-specifically adsorbed dextran or fibrinogen is
supposed to bridge the adjacent RBCs. Dextran with a molecular weight lower than 40 kDa is not able
to bridge the human RBCs [11]. Adsorption measurements [10,14] demonstrated a very low amount of
dextran at the surface of human RBCs but failed to show adsorption of fibrinogen [10].
Asakura and Oosawa [1] introduced the depletion concept of interaction. This concept was forgotten
and only about thirty years later it developed further in colloid chemistry [20,21] and introduced to
explain RBC aggregation [3,4,6,17]. These authors explained the aggregation of particles in the presence
of polymers, which is effective without surface adsorption of the macromolecules (Fig. 1(b)). Depletion
*
Correspondence to: Dr H. Bäumler, Institute of Transfusion Medicine, Medical Faculty Charité, Humboldt University of
Berlin, D-10098 Berlin, Germany. Fax: +49 30 2802 5167; E-mail: hans.baeumler@charite.de.

0006-355X/99/$8.00  1999 – IOS Press. All rights reserved

440 H. Bäumler et al. / Basic phenomena of red blood cell rouleaux formation

(a)

(b)

Fig. 1. (a) Schematic drawing of the bridging model. The adsorption of dextran at single cells and the formation of bridges
during the absence of shear flow lead to the rouleaux formation of RBCs. (b) Schematic drawing of the depletion model.
Depletion of macromolecules from the interface (with or without a weak adsorption) leads to the rouleaux formation if the cells
come into close proximity due to osmotic pressure difference between the pressure in the bulk phase and in the gap.

of polymers takes place if the loss of configurational entropy of a soluble polymer near the interface is
not balanced by a positive interaction energy. A direct repulsion of the polymer from, or incompatibility
with, the surface is not required. A polymer depletion layer is formed where the polymer segment density
decreases towards the surface. As a consequence, this is accompanied by a parallel position dependent
decrease of the osmotic pressure. This leads to the well-known depletion interaction and to the formation,
as a rule, of reversible flocks of colloidal particles [19,30].
The existence of polymer depletion layers near smooth and hairy surfaces has been shown by means
of electrophoretic measurements [5,15]. The reason why electrokinetic experiments are sensitive to the
viscosity in the nanometer range close to the particle surface is because electroosmotic flow obeys poten-
tiality outside the diffuse part of the electric double layer and thus depends only on the viscosity inside the
double layer. By changing the ionic strength of the electrolyte, the double layer thickness can be varied
independently from the depletion layer thickness. If, for instance, the double layer thickness is signif-
 H. Bäumler et al. / Basic phenomena of red blood cell rouleaux formation 441

icantly smaller than the depletion layer thickness, the particle experiences on average a much smaller
viscosity than the bulk value. The electrophoretic mobility is consequently larger than that predicted by
Smoluchowski’s formula.
Both the bridging and the depletion model can explain the aggregation of human RBC in dextran
solutions or in plasma on the basis of two different nonspecific processes. If the concentration of dextran
in the bulk phase reaches a certain level, disaggregation of RBC rouleaux is observed [12]. Both models
cannot explain either the disaggregation of human RBCs or the absence of aggregation of bovine RBCs
in dextran electrolyte solutions or in plasma. There have been speculations for reasons for the lack of
aggregation of bovine RBCs in plasma or in dextran solutions, but a theoretical description of the effect
is not available.
The promoters of the bridging model concluded from the apparently enhanced electrophoretic mobility
of human RBCs in dextran electrolyte solution that the neutral macromolecule dextran increases the
surface potential of RBCs [7,8,13,14,26,28]. The higher electrostatic potential with increasing dextran
concentration would lead to the disaggregation of RBC rouleaux. But horse RBCs show a much higher
zeta potential, derived from sedimentation measurements, than human RBCs in solutions of the same
dextran concentration although the aggregation of horse RBCs is stronger [2].
The conclusion about an increase of the electric surface potential of RBCs in neutral polymer solu-
tions [7,8,13,14] which has been used to explain the disaggregation of human RBCs at high dextran
concentrations is not warranted. It is difficult to understand how dextran, being a neutral polymer, can
induce large changes of the surface potential.
Our conclusion was that the depletion of macromolecules near the surface of biological cells is a
general phenomenon. RBCs are sterically stabilized like other colloidal particles [16,19,25] and do not
aggregate in electrolyte solutions under physiological conditions. The adsorption of plasma proteins is
normally protected by the structure of the glycocalyx of blood cells. It can be shown that, after the
transfusion of homologous red cell concentrates of different blood subgroups, patients can develop an-
tibodies. These antibodies are able to bind to the antigens of the blood cell surface, which is a highly
specific process and much stronger [29] than the weak nonspecific adsorption of plasma proteins.
The adsorption behavior of dextran depends on the cell surface. In the past, highly concentrated dextran
solutions were used as a plasma expander. Adsorption of dextran onto the platelet surfaces was postu-
lated, because serious hemostaseological problems occurred. It was shown by electrophoretic mobility
measurements [5] that dextran indeed adsorbs onto platelets. The dextran adsorption does not induce
aggregation but prevents the normal platelet aggregation induced by various stimuli.
The addition of macromolecules to an RBC suspension leads to an aggregation of RBCs if the deple-
tion effect is stronger than the effect of steric stabilization. The adsorption of dextran into the glycocalyx
of RBC should promote steric stabilization.

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