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DOI 10.1007/s00397-015-0894-3
ORIGINAL CONTRIBUTION
Received: 10 August 2015 / Revised: 23 October 2015 / Accepted: 17 November 2015 / Published online: 5 December 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract Blood is a complex fluid with non-Newtonian characteristics and consists primarily of a concentrated suspension
of red blood cells (RBCs) characterized by high deformability
and aggregability. The majority of both research and clinical
investigations on blood rheology is based on steady shear
measurements aimed at determining blood viscosity as a function of shear rate. On the other hand, investigations of blood
rheology in the linear viscoelastic regime are sparse in the
literature and currently limited. In principle, small amplitude
oscillatory flow is best suited to study blood microstructure
and rheology under quasi-static conditions, which are relevant
in a range of applications, from blood storage to blood aggregability testing. Here, we present the first systematic experimental investigation of blood rheological behavior in the linear viscoelastic regime, by performing oscillatory shear measurements by conventional bulk rheology. The storage (G)
and loss (G) moduli of whole human blood have been measured over an extended range of frequencies [0.130 rad/s].
Data show that G predominates over G across the entire
tested range of frequency. By comparing steady and oscillatory shear viscosity, it was found that the Cox-Merz rule is
followed to a good approximation, with higher deviations at
small shear rate/frequencies. The effects of RBC volume fraction and of aggregating media (i.e., dextran solution at two
different concentrations) have been also investigated. Overall,
This paper belongs to the special issue on the Rheology of blood cells,
capsules and vesicles
* Giovanna Tomaiuolo
g.tomaiuolo@unina.it
1
our results are consistent with the behavior of a weakly attractive suspension, where RBCs form reversible aggregates that
can be broken by the action of flow.
Keywords Blood . Oscillatory . Viscoelasticity . Storage
modulus . Loss modulus
Introduction
Human blood is composed of several kinds of cells, called
corpuscles or formed elements, which are erythrocytes (or
red blood cellsRBCs), leukocytes (white blood cells),
thrombocytes (platelets), and a water-based fluid called plasma. The latter contains 92 % water, 8 % proteins (mainly
serum albumins, globulins, and fibrinogen), and trace
amounts of other materials. Blood is defined as a fluid tissue,
specialized in performing many important functions within
the body, such as supplying oxygen and nutrients to cells
and tissues, removing waste (i.e., carbon dioxide),
transporting hormones and signaling, regulation of body pH
and temperature, immunological functions, and coagulation
(Shattil et al. 2000).
From a rheological point of view, blood is a complex fluid
with non-Newtonian characteristics. It can be defined in several ways: as a two-phase liquid, as a solid-liquid suspension,
where the formed elements represent the solid phase, and as a
liquid-liquid emulsion, considering the high deformability and
aggregability of RBCs under shear (Tomaiuolo et al. 2012b).
The high deformability is due to the excess surface area of
RBCs (which is 40 % larger than the area of a sphere having
the same volume (Guido and Tomaiuolo 2009; Tomaiuolo
2014). Concerning aggregability, RBCs tend to form 2D microstructures, called rouleaux (Tomaiuolo et al. 2012a;
Tomaiuolo et al. 2009b). In static conditions, plasma proteins
486
(e.g., fibrinogen and globulins) foster RBC aggregation leading to rouleaux formation and branching in 3D structures,
which can be broken by the action of flow. Thus, human blood
can be seen as a non-Newtonian, viscoelastic fluid, consisting
primarily of RBCs (99 % of formed elements), which behave
as neutrally buoyant, sticky microcapsules with high
deformability but small area stretchability (Tomaiuolo et al.
2011; Tomaiuolo and Guido 2011; Tomaiuolo et al. 2015;
Tomaiuolo et al. 2009a) suspended in plasma. In turn, plasma
acts as a Newtonian fluid (i.e., viscosity is constant with shear
rate) in continuous shear flow, while it shows a viscoelastic
behavior in pure extensional flow (Brust et al. 2013).
The study of blood rheology or hemorheology (Baskurt
and Meiselman 2003) is relevant both for pathophysiological
applications and bioengineering problems, such as blood storage (Daly et al. 2014; Zheng et al. 2014), testing of artificial
models (Deplano et al. 2014), and design of diagnostic devices (Bose et al. 2013).
From the pathophysiological point of view, a number of
clinical investigations demonstrate the correlation between alterations of hemorheological parameters and many common
diseases (Baskurt 2003). Blood rheological behavior is especially relevant for diseases affecting the cardiovascular apparatus and hence the circulatory function in microcirculation
and tissue perfusion. Impaired blood flow represents a risk
factor of thrombosis and atherosclerosis in pathologies such
as myocardial infarction, hypertension, strokes, or diabetes. In
a recent paper, the role of blood viscosity on the initiation and
progression of atherosclerosis in peripheral arterial disorders
has been revised, stating that keeping under control blood
viscosity paves the way to noninvasive treatments by pharmacological therapy or hemodilution, eventually helping in the
reduction of peripheral vascular resistance and in increasing
blood flow to the lower extremities (Cho et al. 2014). Diabetes
has been associated with increased whole blood and plasma
viscosity, as well as complications such as microvascular disorders and microangiopathy (McMillan 1976). Other cardiovascular pathologies are associated with high levels of cholesterol, which can have both direct and indirect effects on blood
flow. Direct effects, such as the growth of atherosclerotic
plaques and blunted endothelium-dependent vasodilation at
the coronary microcirculation, lead to reduction of the coronary artery lumen and to an impairment of myocardial perfusion, respectively. Indirect effects, instead, involve blood rheology since high levels of cholesterol can affect whole blood
viscosity, platelet activation, and RBC deformability, causing
impaired coronary circulation (Kohno et al. 1997).
In light of such pathophysiological relevance, blood flow
behavior has been the subject of a number of studies. From the
pioneering works of Chien (Chien 1970; Chien et al. 1967a;
Chien et al. 1967b) on blood viscosity, the majority of experimental investigations focused on the steady-state rheological
behavior of whole blood. In continuous shear flow, blood
behaves as a non-Newtonian fluid, mostly dependent on plasma rheological properties, RBC volume fraction (or hematocrit, Hct), and RBC properties. In particular, the ability of
RBCs to deform and recover their shape, and to form aggregates at rest and under weak flow fields, is the basis of blood
shear-thinning behavior (Baskurt and Meiselman 2003). Two
other features, which characterize blood non-Newtonian behavior, are yield stress (Apostolidis and Beris 2014; Merrill
et al. 1963; Picart et al. 1998) and thixotropy (Barnes 1997;
Robertson et al. 2009). Yield stress can be seen as the manifestation of attractive forces between particles suspended in a
continuous medium that drive the formation of a 3D network
in absence of flow but are weak enough to allow network
disruption following the application of a sufficient stress
(Merrill et al. 1963). Two approaches have been mostly used
to measure blood yield stress: (i) direct methods, where the
shear stress at which blood starts flowing is measured and (ii)
indirect methods, where the shear stress vs shear rate diagram
is extrapolated to zero, by using a constitutive model or a
linear fitting. Regarding blood thixotropic behavior, shear
stress hysteresis curves have been reported for normal blood
by using a rotational rheometer with the Couette geometry
(Bureau et al. 1979), thus showing a residual non-zero shear
stress at the end of the cycle in line with the viscoelastic nature
of blood. Many models have been also proposed to describe
the complex shear and time-dependent behavior of blood under steady state conditions. The most successful models for
blood viscosity can be considered the power-law model, the
Quemada model and the Casson equation (Apostolidis and
Beris 2014; Robertson et al. 2009) although no one can fully
describe the rheological behavior of blood. Recently,
Apostolidis and Beris undertook a systematic investigation
of the rheology of normal blood under steady-state shear flow
and showed that the Casson viscoplastic model provides the
best approximation of available experimental data. The same
authors developed a parameterization of the Casson model
parameters, the yield stress and the Casson viscosity, in terms
of parameters that define the physiological state of blood
(Apostolidis and Beris 2014; Robertson et al. 2009). Blood
rheology has been also investigated by numerical simulations.
Fedosov et al. (Fedosov et al. 2011) coupled two different
models with coarse-grained molecular dynamics, predicting
the dependence of blood viscosity on shear rate and Hct.
Moreover, cell-cell interactions have been described..
Although the rheological response in steady-state conditions can elucidate the mechanisms governing the nonNewtonian, shear-thinning and viscoplastic behavior of blood,
transient conditions are often encountered in vivo, such as in
going from larger to smaller vessels, in vascular bifurcations,
not to talk about the pulsatile nature of cardiovascular circulation. In particular, oscillatory flow is especially relevant to
investigate blood microstructure at small deformations.
However, blood rheological investigations under oscillatory
487
Experimental
Blood samples
Human blood was obtained from healthy volunteers (male and
female, age ranging between 26 and 52 years, Hct ranging
from 40 to 50 %). Venous blood were drawn from the
antecubital vein and collected into Vacutainers (BD,
Franklin Lakes, NJ, USA) containing ethylenediaminetetraacetic acid (EDTA) and stored at room temperature. In order
to obtain rheological reference curves of human blood, the
samples were used as such (i.e., at the hematocrit at the moment of collection).
In order to evaluate the effect of the RBC volume fraction
on blood rheological properties, blood samples have been
centrifuged for 15 min at 1300 rpm and the suspended plasma
was aspirated through a large-bore needle and saved.
Subsequently, RBCs were resuspended in autologous plasma
at fixed hematocrit level (i.e., 60, 75, and 90 %).
RBC aggregation properties have been studied by adding
an aggregating agent, dextran (MW 500 kDa; Sigma
Chemical, St. Louis, MO, USA) to whole blood at two concentrations: 1.5 and 3 mgDextran/mlblood. Each step of the experimental protocol was performed at room temperature.
Rheological measurements
Rheological tests were performed within 6 h from blood collection by using a stress-controlled Physica rheometer MCR
301 (Anton-Paar, Graz, Austria) equipped with a titanium
double-gap measuring system (DG 26.7, inner cup diameter
24.267 mm, inner bob diameter 24.666 mm, outer bob diameter 26.663 mm, outer cup diameter 27.053 mm, and bob
height: 40.000 mm). The bob rotates inside the cup, which is
still and maintained at a constant physiological temperature of
37 C by a Peltier system. This assembly, presenting a high
surface area and requiring small amount of sample (i.e.,
1.8 ml), is ideal for low viscous biological materials, such as
blood.
In each test, blood samples have been gently agitated before loading, in order to homogenize the sample. After loading, the blood surface exposed to air (i.e., in the upper part of
the assembly) was covered by an oil film to avoid drying.
In order to prevent RBC sedimentation and to disrupt any
aggregates, possibly formed during the loading step, each test
was preceded by a pre-conditioning phase, at a constant shear
rate of 200/s for 20 s, followed by a rest phase of 5 s, needed to
allow sample relaxation. Steady shear experiments were performed from low to high shear rates and back (up-down curve)
488
Results
Whole blood steady-state behavior
489
exponent for both G and G being 0.7. b Strain amplitude sweep test (G
and G as a function of strain) at 10 rad/s for strain amplitudes from 0.1 to
100 %
Moreover, the absence of a crossover region likely indicates that no major change in the structure of the RBC network
occurs in the frequency and deformation range investigated.
Previous work on blood viscoelasticity in tube flow (Thurston
and Henderson 2006; Thurston 1972; Thurston 1975) demonstrated that RBC 3D networks can persist only at very small
deformations (about 1 %). The latter are beyond instrumental
sensitivity due to the low viscosity of blood. Thus, it is possible that the higher deformations in our experimental conditions lead to (partial or full) breakage of 3D structures. A
possible interpretation of this behavior is that RBCs become
arranged in 2D structures flowing in layers separated by the
suspending medium, which acts as a lubricant in between. In
this picture, the elastic response of the material might be due
to the fact that two adjacent RBC layers are close enough to be
affected by each other, also thanks to the presence of plasma
proteins, which can enhance RBC interactions. Thus, the extent of RBC aggregation could be related to two opposing
forces: the shear flow, which tends to disaggregate RBC structures, and the adhesion force between adjacent RBCs, promoted by the presence of plasma proteins. In physiological conditions, blood flow is strong enough to break RBC aggregates,
The viscoelasticity of blood has been characterized by applying oscillatory deformations to whole blood samples (Hct
about 45 %) over the frequency range of 300.1 rad/s. Each
oscillatory measurement has been preceded by amplitude
sweep tests (Fig. 2b) in order to evaluate the linear viscoelastic
region and consequently which value of deformation to be
used in the experiments. It can be noticed that for G, the stress
corresponding to the onset of the nonlinear regime is around
5 mPa, i.e., close to the yield stress value. This confirms that
oscillatory shear in the linear viscoelastic region is probing a
microstructure at rest. Figure 2a reports the storage modulus
G and the loss modulus G as a function of angular frequency,
the error bars being the standard deviation. Both dynamic
moduli increase with frequency, the loss modulus G
predominating over the storage one across the entire tested
range of frequency. A similar behavior, i.e., a parallel trend
of the moduli with G higher than G, is found in other complex fluids, such as weak gels and weak attractive
suspensions/emulsions (Datta et al. 2011; Mason 2000;
Trappe and Weitz 2000). The predominance of G over G
indicates the main role played by viscous dissipation in blood
viscoelasticity.
490
Fig. 5 a G and G as a function of angular frequencies for RBC suspensions of different volume fractions; b Strain amplitude sweep test at 10 rad/s for
RBC suspensions of different volume fractions
frequencies could be explained by the proximity to the instrument detection limit due to the low viscosity of blood.
491
In the following, the rheological behavior of concentrated RBC suspensions at different levels of Hct (60,
75, and 90 %) is presented by both steady state and
oscillatory measurements.
As might be expected, the increase of RBC volume fraction
with respect to the physiological value of 45 % (black circles
in Fig. 4) induces an upward shift of the viscosity curves.
Moreover, the dependence of viscosity on RBC volume fraction is shown in the inset of Fig. 4, at four different values of
shear rates (i.e., 0.5, 1.5, 11, and 193/s). The experimental data
can be fitted with a power law with exponent ranging from
2.19 (shear rate 0.5/s) to 2.42 (shear rate 193/s).
The effect of the variation of Htc on RBC suspension moduli is shown in Fig. 5: increasing Hct causes an upward shift of
both the storage and the loss modulus; furthermore, G and G
tend to approach each other and to be less dependent on frequency, suggesting the approach to an elastic-solid-like
behavior.
To compare oscillatory and steady shear conditions, the
Cox-Merz rules have been applied, as shown in Fig. 6, where
viscosity and the magnitude of complex viscosity are reported
as a function of shear rate and angular frequency, respectively.
The two sets of data show a good agreement, which provides
further support to the disruption of the 3D RBC network both
in continuous and oscillatory flow.
By applying the Cox-Merz rule on the data of Figs. 4 and 5,
a little gap between steady state and oscillatory sets of data is
found at low shear rates/frequencies, with complex viscosity
values slightly higher than viscosity ones. This could indicate
that at a higher RBC volume fraction, it is still possible to find
some 3D structures in the low-frequency range.
In Fig. 7a, the plot of loss modulus G against storage
modulus G at three RBC volume fractions (45, 75, and
90 %) investigated is reported. The G = G plot is represented
by the solid line. Figure 7a shows that the values of G are
higher than the ones of G, indicating that the viscous response
of the material prevails on the elastic one, this trend decreasing
at increasing Hct. Moreover, the higher is Hct, the more the
curves are shifted to the right, making the response of the
material closer to the one of an elastic solid.
492
of plasma proteins, primarily fibrinogen, as well as the addition of model polymer, such as dextran (Ami et al. 2001; Brust
et al. 2014). Nevertheless, the mechanism at the base of RBC
aggregation is controversial and not yet fully elucidated. Two
main models have been proposed to describe RBC aggregation: the bridging model, based on the assumption that macromolecules as fibrinogen or dextran act as a bridge between
two adjacent RBCs, and the depletion model, based on the
gradient of osmotic pressure due to the presence of macromolecules in the suspending medium, leading to depletion interaction (Baumler et al. 1999; Steffen et al. 2013).
In Fig. 8, the effect of the addition of dextran on whole
blood (Hct 45 %) viscosity as a function of shear rate is shown
at two different concentrations of dextran (1.5 mgDextran/
mlblood and 3 mgDextran/mlblood). The viscosity of blood samples with the addition of dextran is very close to the viscosity
of the control, the two sets of dextran data superimposing to
each other and also to the control curve. Nevertheless, a peculiar trend is found in samples with dextran: a small plateau
zone, (from 5.6 to 11/s) in which viscosity is nearly independent on shear rate, is present for both dextran concentrations
of 1.5 and 3 mg/ml. This could indicate a change in the samples microstructures, likely related to the breakage of the 3D
networks formed at low shear rates. In the inset of Fig. 8, the
behavior of viscosity at increasing (down-up curve) and then
decreasing (up-down curve) shear rate is shown for the sample
with high dextran concentration. The reduction of viscosity
values in the up-down curve can be attributed to the
sedimentation of larger clusters, which is, in turn, strictly related to an increased RBC tendency to aggregate, due to the
addition of dextran. The discrepancy between the two curves
at low shear rates is more pronounced as compared to the inset
of Fig. 1a (i.e., whole blood samples with physiological aggregation). Once again, such discrepancy can be referred to as
a thixotropic behavior of blood.
Regarding the oscillatory measurements on RBC suspensions with dextran, Fig. 9a shows that the increase of dextran
concentration results in an upward shift of both G and G, and
in the approach of the moduli. As seen in the case of the
increase of RBC volume fraction, the presence of macromolecules which promote RBC adhesion makes G and G more
independent on frequency, thus suggesting an elastic-solidlike behavior.
Also in this case, by applying the Cox-Merz rule on the
data of Figs. 8 and 9, a little gap between steady state and
oscillatory sets of data is found at low shear rates/frequencies,
with complex viscosity values slightly higher than viscosity
ones (Fig. 10). This could indicate that at a higher RBC volume fraction, it is still possible to find some 3D structures in
the low-frequency range.
Regarding the plot of G vs G, the three sets of data lie on
curves of different slopes, indicating that the rheological behavior of the samples, in terms of microstructure, is dependent
on the presence of dextran. In fact, for an elastic network, G
and G depend on material parameters such as the number of
elastically active crosslinks and the concentration of high
Fig. 10 Comparison of viscosity and magnitude of complex viscosity by applying the Cox-Merz rule for a Hct = 45 % and for whole blood added with
dextran at b 1.5 mg/ml and c 3 mg/ml
493
Conclusions
In this work, blood linear viscoelasticity has been characterized by small amplitude oscillatory flow in a Couette rotational rheometer. The G and G moduli of whole blood were
measured over the range of frequencies from 0.1 to 30 rad/s.
The viscous response was found to prevail on the elastic term.
The effects of RBC volume fraction and of the addition of
dextran have been also investigated. The steady blood viscosity was also measured under the same conditions. The CoxMerz rule was essentially valid, with some deviations at lower
frequencies/shear rates. A comparison with results available in
the literature has also been presented.
The source of blood linear viscoelasticity is still to be fully
understood. Our amplitude sweep results suggest that blood
microstructure is not significantly perturbed in the linear viscoelastic regime. This would point to the reversible deformation of the rouleaux network as the main source of blood linear
viscoelasticity. However, some authors have related blood
viscoelastic moduli at high Hct to RBC membrane rheological
properties (Drasler et al. 1987), which is known to exhibit a
viscoelastic behavior (Tran-Son-Tay et al. 1984; Yoon et al.
2008). More work is still needed to fully elucidate this issue
and the related problem of the clinical significance of blood
linear viscoelasticity.
494
Acknowledgments Funding from the Italian Ministry of University
and Research (PRIN Program 20102011, Project No. 20109PLMH2)
and from the Regione Campania (MICROEMA Project, 220 APQRT02 2008) is acknowledged. This study is related to the activity of the
European network action COST MP1106 Smart and green interfaces
from single bubbles and drops to industrial, environmental, and biomedical applications. The authors thank Dr. A. Perazzo for useful discussions
on the literature.
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